Journal name: Drug Design, Development and Therapy Article Designation: Original Research Year: 2015 Volume: 9 Drug Design, Development and Therapy Dovepress Running head verso: He et al Running head recto: UA reverses liver fibrosis in rats open access to scientific and medical research DOI: http://dx.doi.org/10.2147/DDDT.S85426

Open Access Full Text Article Original Research A bioinformatic and mechanistic study elicits the antifibrotic effect of ursolic acid through the attenuation of oxidative stress with the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in human hepatic stellate cells and rat liver

Wenhua He1,* Abstract: NADPH oxidases (NOXs) are a predominant mediator of redox homeostasis in Feng Shi1,* hepatic stellate cells (HSCs), and oxidative stress plays an important role in the pathogenesis Zhi-Wei Zhou2,* of liver fibrosis. Ursolic acid (UA) is a pentacyclic triterpenoid with various pharmacological Bimin Li1 activities, but the molecular targets and underlying mechanisms for its antifibrotic effect in the Kunhe Zhang1 liver remain elusive. This study aimed to computationally predict the molecular interactome Xinhua Zhang1 and mechanistically investigate the antifibrotic effect of UA on oxidative stress, with a focus on NOX4 activity and cross-linked signaling pathways in human HSCs and rat liver. Drug–drug Canhui Ouyang1 interaction via chemical–protein interactome tool, a server that can predict drug–drug interac- Shu-Feng Zhou2 tion via chemical–protein interactome, was used to predict the molecular targets of UA, and 1 Xuan Zhu Database for Annotation, Visualization, and Integrated Discovery was employed to analyze 1Department of Gastroenterology, the the signaling pathways of the predicted targets of UA. The bioinformatic data showed that First Affiliated Hospital of Nanchang there were 611 molecular proteins possibly interacting with UA and that there were over 49 University, Nanchang, Jiangxi, People’s Republic of China; 2Department of functional clusters responding to UA. The subsequential benchmarking data showed that UA Pharmaceutical Sciences, College of significantly reduced the accumulation of type I collagen in HSCs in rat liver, increased the Pharmacy, University of South Florida, Tampa, FL, USA expression level of MMP-1, but decreased the expression level of TIMP-1 in HSC-T6 cells. UA also remarkably reduced the gene expression level of type I collagen in HSC-T6 cells. Fur- *These authors contributed equally to this work thermore, UA remarkably attenuated oxidative stress via negative regulation of NOX4 activity and expression in HSC-T6 cells. The employment of specific chemical inhibitors, SB203580, LY294002, PD98059, and AG490, demonstrated the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in the regulatory effect of UA on NOX4 activity and expression. Correspondence: Xuan Zhu Collectively, the antifibrotic effect of UA is partially due to the oxidative stress attenuating effect Department of Gastroenterology, the First through manipulating NOX4 activity and expression. The results suggest that UA may act as a Affiliated Hospital of Nanchang University, 17 Yongwai Main St, Nanchang 330006, promising antifibrotic agent. More studies are warranted to evaluate the safety and efficacy of Jiangxi, People's Republic of China UA in the treatment of liver fibrosis. Tel +86 791 8869 2505 Keywords: ursolic acid, liver fibrosis, NADPH oxidase, ROS, DDI-CPI, DAVID Fax +86 791 8862 3153 Email [email protected]

Shu-Feng Zhou Introduction Department of Pharmaceutical Sciences, Liver fibrosis remains the major cause of morbidity and mortality worldwide. It causes College of Pharmacy, University of South several notorious complications, including ascites, portal hypertension, encephalopathy, Florida, 12901 Bruce B Downs Boulevard, MDC 30, Tampa, FL 33612, USA and liver failure, and it accelerates the risk of hepatocellular carcinoma, placing a sub- Tel +1 813 974 6276 stantial burden to individual, society, and health care system.1,2 The liver fibrosis-driven Fax +1 813 905 9885 Email [email protected] chronic liver disease and cirrhosis cause 36,427 deaths and with a ratio of 11.5/100,000 in submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 3989–4104 3989 Dovepress © 2015 He et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further http://dx.doi.org/10.2147/DDDT.S85426 permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php He et al Dovepress

2013 in USA.1 This is a complicated etiology of hepatic fibro- anti-inflammatory, and anticancer activities.14 UA exerts a sis. Viral infection is the most common contributing factor to protective effect against ethanol-induced toxicity in isolated liver fibrosis affecting 1%–2% of the US population,3 and liver rat hepatocytes and ethanol-mediated experimental liver fibrosis resultant cirrhosis is projected to reach 45% of those damage in rats.15,16 Our previous studies showed that UA infected with hepatitis C virus patients in 2030.4 Hepatitis significantly inhibited the proliferation of HSCs and induced B virus is the main type of hepatitis virus in the People’s apoptosis in vitro.17 Moreover, Steinkamp-Fenske et al18 Republic of China with a substantial contribution to the inci- showed that UA downregulated the expression level of NOX4 dence of liver fibrosis.5,6 Moreover, nonalcoholic fatty liver and suppressed ROS generation in human endothelial cells. disease and nonalcoholic steatohepatitis are also important However, there is a lack of study on the antifibrotic effect of causes of liver fibrosis, and over 20% of patients with non- UA, and the underlying mechanisms of the antifibrotic effect alcoholic steatohepatitis progress to cirrhosis worldwide.7 In of UA are largely unknown. addition, other etiologies of liver fibrosis and its late-stage Therefore, the present study aimed to evaluate the molec- liver injury include alcohol-induced disease, drug-induced ular targets of UA and analyze the molecular interactome toxicity, other liver infections (eg, schistosomiasis), immune- of UA using computational and bioinformatic approaches mediated liver diseases (eg, autoimmune hepatitis), metabolic and, subsequently, examine the antifibrotic effect of UA and disorders (eg, lipid, glycogen, or metal storage disorders), and delineate the underlying mechanisms in HSCs and Sprague cholestasis (eg, secondary biliary cirrhosis). Dawley rats. To date, the molecular mechanisms that underlie the development of liver fibrosis have been extensively inves- Materials and methods tigated, including epithelial to mesenchymal transition and Prediction of the interactome of UA and pathway inflammatory response.8–10 Notably, increasing evidence sug- analysis by molecular docking and bioinformatic gests that oxidative stress has been implicated in the patho- approach genesis of liver fibrosis, with the intracellular accumulation of The prediction of interactome of UA was performed using excessive reactive oxygen species (ROS), and the compelling the drug–drug interaction via chemical–protein interactome evidence shows the involvement of ROS in the development (DDI-CPI) tool (http://cpi.bio-x.cn/ddi/) as previously of liver fibrosis with a regulatory role in a variety of cellular described.19 DDI-CPI is a web-based server that can be processes.7,10–13 However, the specific targets and signaling used to predict DDI via CPI.20,21 In brief, protein targets pathways have not been fully mapped yet, and the sources were obtained from a third-party protein structure database of ROS have not yet been conclusively determined in the named PDBbind (http://sw16.im.med.umich.edu/databases/ pathogenesis of liver fibrosis. There are several enzymatic pdbbind/index.jsp), which was based on the contents of sources of ROS, including NADPH oxidase (NOX) family Protein Data Bank (PDB).22 There are a total of 1,780 PDB of oxidoreductases, nitric oxide synthases, xanthine oxidase, entries of human proteins available in PDBbind, and a total and cytochrome P450, of which NOX is the most important of 301 nonredundant PDBs corresponding to 353 ligand- ROS-generating enzyme in both plants and animals, espe- binding pockets were identified from it, with 86% of which cially in mammals. In the liver, NOX has been involved in have resolutions ,2.5 Å. The docking boxes for each of the fibrogenesis. In particular, NOX4 is one of seven NOX iso- pockets were defined by expanding the circumscribed cube of forms and is the most widely distributed isoform.11 It has been the pocket with a margin of 8 Å at six directions (up, down, reported that a functionally active form of NOX is expressed front, back, left, and right). For the preparation of the UA, in hepatic stellate cells (HSCs) and that NOX-generated ROS the 2D structure of the UA was downloaded from PubChem. serve as a second messenger for profibrogenic factor signal The hydrogen and Gasteiger charge were added, and the transduction in HSCs. However, regulation of NOX in HSCs PDB file was converted into mol2 format using VEGA ZZ. remains largely elusive. The docking program AutoDock 4.2 was used to dock the Recently, it has been proposed that antioxidants hold prepared UA molecule into all 353 pockets, generating a promise as potential antifibrotic therapies due to the score vector of 353 dimensions. Z′-scores were then calcu- ROS-attenuating effect.12,13 Ursolic acid (UA), a natural lated using the methodologies we applied previously.20,23,24 pentacyclic triterpenoid carboxylic acid, is the major com- Here, an empirical threshold of -0.6 of the Z′-score was set ponent of certain traditional medicinal herbs and possesses to indicate that the binding of UA toward this target was a wide range of biological functions, such as antioxidative, likely to be true.

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Pathway analysis by Database for Annotation, inhibitor 1 (TIMP-1) and matrix metalloproteinase-1 Visualization, and Integrated Discovery (MMP-1) antibodies were purchased from PL Laboratories Following the computational target prediction, the Database Inc. (Port Moody, British Columbia, Canada). The α-smooth for Annotation, Visualization, and Integrated Discovery muscle actin (α-SMA) antibody was purchased from Santa version 6.7 (DAVID, http://david.abcc.ncifcrf.gov/) was Cruz Biotechnology Inc. (Dallas, TX, USA), and the β-Actin employed to interpret the biological function of the potential antibody and goat anti-rabbit secondary antibodies were targets of UA derived from DDI-CPI.19–21 The protein IDs of purchased from Zhongshan Golden Bridge Biotechnology these targets from UniProtKB, NCBI, and other sources were Co., Ltd. (Beijing, People’s Republic of China). converted into gene lists by using the Gene ID Conversion Tool in DAVID. The DAVID database adds biological function Cell culture and treatment annotation including gene ontology, pathway, protein–protein The immortalized rat liver stellate cell line (HSC-T6) was interactions, functional groups of genes (ie, clustering), and kindly provided by Dr L-M Xu, Shanghai University of disease association derived from main public data sources. The Chinese Traditional Medicine (Shanghai, People’s Republic genes of interest were visualized using BioCarta and Kyoto of China). These cells exhibit the phenotype that most closely encyclopedia of Genes and Genomes (KEGG) pathway maps. resembles primary rat stellate cells.25 HSC-T6 cells were cul- The high classification stringency was selected for functional tured in DMEM supplemented with 10% fetal bovine serum, annotation clustering. Enrichment scores and Fisher’s exact penicillin (120 µg/mL), and streptomycin (100 µg/mL) test P-values (and corresponding false discovery rate) were at a humidified atmosphere of 5% CO2 at 37°C. The culture then calculated to identify which functional-related gene medium was replaced with serum-free DMEM (serum starva- groups were significantly enriched in the target list. These tion) for 24 hours before the start of the experiments. significant enriched gene groups could provide clues on how UA interacts with molecular targets in a systematic way. Animals and experimental design A total of 32 male Sprague Dawley rats (weighing between Chemicals and reagents 180 g and 200 g) were experimented in the present study. Recombinant rat leptin was purchased from Peprotech Inc. All animals were housed in plastic cages containing wood (Rocky Hill, NJ, USA). UA, diphenyleneiodonium (DPI), shaving and cotton bedding and maintained in a room at dimethyl sulfoxide (DMSO), Thiazolyl blue tetrazolium 22°C–25°C with a 12-hour day/night cycle and had free bromide (MTT) and protease inhibitor and phosphatase access to standard laboratory diet and water. The animal inhibitor cocktails were purchased from Sigma-Aldrich Co., study protocol was approved by the Institutional Animal (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium Care and Use Committee of the First Affiliated Hospital (DMEM), fetal bovine serum, and AG490 were purchased of Nanchang University. Dimethylnitrosamine (DMN) from Thermo Fisher Scientific (Waltham, MA, USA). and UA were freshly dissolved in saline at predetermined Antioxidant N-acetyl-l-cysteine (NAC) was purchased concentrations. from Solarbio Inc. (Beijing, People’s Republic of China). A liver fibrosis model was established in rats by admin- PD98059, an extracellular signal-regulated kinase (ERK) istering 1% DMN at a dosage of 1 mL/kg body weight by inhibitor, was purchased from Promega Corporation (Fitch- intraperitoneal injection for 3 consecutive days per week burg, WI, USA). SB203580, a p38 mitogen-activated protein for 4 weeks. At week 5, the DMN-treated rats with liver kinase (p38 MAPK) inhibitor, LY294002, a phosphoinositide fibrosis were randomly divided into three groups with or 3-kinase (PI3K) inhibitor, and the ROS assay kit (which without UA treatment for another 4-week treatment. The contains 2′,7′-dichlorofluorescin diacetate [DCF-DA]) were UA groups were administered by intraperitoneal injections purchased from Beyotime Inc. (Jiangsu, People’s Republic at dosages of 20 mg/kg (UA-2) and 40 mg/kg (UA-3) per of China). p47phox antibody was purchased from Bioworld day. The DMN group received DMN alone for 8 weeks. The Inc. (St Louis Park, MN, USA); gp91phox, p22phox, and Rac1 vehicle group was treated with a volume of saline that was antibodies were purchased from Abcam Inc. (Cambridge, equivalent to the volume used per day in UA groups. The MA, USA). p67phox, PI3K (p110α), protein kinase B (Akt), rats were euthanized by saline perfusion and exsanguinated phosphorylated (p)-Akt, p-ERK1/2, p-p38 MAPK, and p38 via the inferior vena cava 1 day after the last treatment. The MAPK antibodies were purchased from Cell Signaling Tech- liver and blood samples were collected and stored at -80°C nology, Inc. (Danvers, MA, USA). TIMP metallopeptidase for further analysis. During the period of experiment, if the

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 3991 Dovepress He et al Dovepress weight loses 10%, mice will be euthanized. Also, the mice emission wavelengths of 488 nm and 525 nm, respectively. will be immediately euthanatized if other symptoms such The intracellular ROS level was calculated as a percentage as pale mucous membranes, shivering, failure to respond to of DCF fluorescence intensity relative to the vehicle controls stimuli, piloerection, matted hair coat, soiled anogenital/vent (untreated HSC-T6 cells). area, and vocalization are observed. Determination of NOX activity MTT assay Following the examination of ROS generation in HSC-T6 To examine the effect of UA on the proliferation of HSC-T6 cells, the NOX activity was determined to address the cells, the MTT assay was performed. Briefly, HSC-T6 cells regulating effect of UA on the enzymatic source of ROS were seeded in to 96-well plates with medium containing in HSC-T6 cells as previously described with some leptin at a confluence of~ 60% following a 24-hour starvation. modifications.27,30,31 Briefly, HSC-T6 cells were incubated The cells were treated with UA (50 μM), AG490 (50 μM), in the culture medium in the absence or presence of lep- or DPI (20 μM) for 12 hours, 24 hours, or 48 hours, respec- tin and treated with UA (50 μM), NAC (10 mM), DPI tively. After the treatment, a volume of 10 μL of thiazolyl (20 μM), or AG490 (50 μM). After the treatment, the cells blue tetrazolium bromide (MTT) (5 mg/mL) was added and were harvested by trypsinization, pelleted by centrifuga- incubated for an additional 4 hours. Then, the medium was tion at 2,500× g for 5 minutes at 4°C and resuspended in carefully aspirated, and the 100 μL of DMSO was added to PBS. Subsequently, the cells were incubated with 250 µM dissolve the formazan particle. The absorption intensity was of NADPH. NADPH consumption was monitored by the measured using a microplate reader (Bio-Rad 550; Bio-Rad decrease in absorbance at λ=340 nm for 10 minutes. For Laboratories Inc., Hercules, CA, USA) at 490 nm. the specific analysis of NOX activity, the rate of NADPH consumption specifically inhibited by DPI was measured by Histological evaluation pretreated with 10 µM of DPI for 30 minutes. An aliquot To examine the effect of UA on the general characterization, of cells was lysed by adding sodium dodecyl sulfate, and inflammatory cell infiltration, and disposition of connective the protein concentration of the cell lysate was determined. tissue in rat liver, the hematoxylin and eosin and van Gieson The absorption extinction coefficient used to calculate the staining were performed. amount of NADPH consumed was 6.22 mM−1 cm−1. The data of NOX activity were expressed as picomol per liter Determination of serum maleic of substrate per minute per milligram of protein. dialdehyde and superoxide dismutase levels Western blotting analysis In order to assess the antioxidative effect of UA in rats, the Whole-cell extracts were obtained using Triton lysis blood level of maleic dialdehyde (MDA) and superoxide buffer that contained protease inhibitor and phosphatase dismutase (SOD) activity were examined using the thiobar- inhibitor cocktails. Liver extracts were obtained in modified bituric acid method. radioimmunoprecipitation buffer. The proteins were loaded and separated by sodium dodecyl sulfate/polyacrylamide Measurement of intracellular ROS level gel electrophoresis and transferred to a nitrocellulose To further examine the antioxidative effect of UA in HSC-T6 membrane. Then, the membrane was blocked at room tem- cells, the intracellular ROS level was determined using flow perature for 1 hour with 5% nonfat milk in Tris-buffered cytometry by the oxidation-sensitive probe DCF-DA as saline with Tween (TBST), followed by the incubation previously described.26,27 DCF-DA, a ROS probe that under- with indicated primary antibodies overnight at 4°C. Next, goes intracellular deacetylation followed by ROS-mediated the membranes were washed and incubated with the cor- oxidation to a fluorescent DCF, was used to measure ROS responding secondary antibody conjugated to horseradish generation in the cytoplasm and cellular organelles.28,29 peroxidase (HRP) at room temperature for 1 hour. Visual- HSC-T6 cells were treated with UA (50 μM), NAC (10 mM), ization was performed using Bio-Rad ChemiDoc™ XRS DPI (20 μM), or AG490 (50 μM) in the absence or presence system (Hercules, CA, USA) with enhanced chemilumines- of leptin. After the treatment, cells were incubated with cence substrate, and the blots were analyzed using Image DCF-DA (10 µM) for 20 minutes. Then, cellular fluorescence Lab 3.0 (Bio-Rad Laboratories Inc., Hercules, CA, USA). intensity was measured using a FACScan flow cytometer Protein level was normalized to the matching densitometric (Becton Dickinson, San Jose, CA, USA) at excitation and value of internal control.

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Reverse transcription polymerase chain CHEK1, RB1, CHEK2, SFN, WEE1, CDK2, HDAC2, PLK1, reaction GSK3B, MDM2, and ABL1). Furthermore, as shown in Total RNA was extracted using TRNzol A+ total reagent (Tiangen Table 3, our DAVID analysis showed that there were 49 functional Biotech, Beijing, People’s Republic of China) and subject to clusters that were identified to be significantly enriched (enrich- reverse transcription with dT15-oligonucleotide and Moloney ment score .3) in the target list derived from molecular docking Murine Leukemia Virus (M-MLV) reverse transcriptase (Pro- calculations, including energy metabolism, signal transduction, mega Corporation, Fitchburg, WI, USA). The primers for type I vascular regulation, and carbohydrate metabolism. Furthermore, collagen and β-Actin used in the reverse transcription polymerase there were 76 KEGG pathways significantly enriched in the chain reaction are shown in Table 1. The mRNA levels of the target list (Table 4), such as MAPK, p53, and mTOR signaling type I collagen gene were normalized to the β-Actin mRNA pathways. level. The number of amplification cycles was 30, and the specific Following the bioinformatical prediction of the molecu- amplicons were analyzed by 1% agarose gel electrophoresis and lar interactome of UA, we functionally and mechanistically visualized with ethidium bromide. examined the effect of UA on the liver fibrosis through the assessment of collagen accumulation and ROS generation Statistical analysis in vitro and in vivo. The results are expressed as the mean ± standard deviation. Differences between groups were compared using one-way UA ameliorates DMN-induced hepatic analysis of variance followed by the Tukey’s test. Data with fibrosis in rats a skewed distribution or heterogeneity of variance were First, we established the model of liver fibrosis and examined analyzed by the Kruskal–Wallis nonparametric test followed the effect of UA on liver fibrosis in rats through examining the by a Nemenyi test. A P-value ,0.05 was considered to be accumulation of collagen in rat HSCs. As shown in Figure 1A statistically significant. and B and Table 5, there is marked difference in the collagen disposition in rat HSCs. Compared to the vehicle group, rats Results that received 1% DMN treatment remarkably increased the UA likely interacts with a number of important accumulation of collagen in HSCs (Figure 1B). Administra- functional proteins tion of rats with UA at 20 mg/kg and 40 mg/kg decreased First, we computationally predicted the molecular targets the collagen accumulation in rat HSCs (Figure 1C and D). of UA using our web-based DDI-CPI tool. There were 611 Notably, high dose (40 mg/kg) of UA normalized the level proteins that possibly interacted with UA (Table 2), includ- of collagen in rat HSCs (Figure 1D). ing those involved in MAPK signaling pathway (FGFR2, Furthermore, we examined the expression level of α-SMA, TRAF2, FGFR1, HRAS, GRB2, MAPKAPK3, MAPKAPK2, a marker of HSCs activation and fibrogenesis in rat liver. AKT1, CDC42, TNFRSF1A, CASP3, RAC1, PRKACA, Treatment of rats with 1% DMN gave a remarkable increase PAK1, TRAF6, AKT2, EGFR, PRKCA, MAP2K1, BRAF, (5.7-fold) in the expression lever of α-SMA in rat liver, com- TGFBR1, TP53, RAF1, MAPK10, PRKCB, MAPK1, DUSP3, pared to the vehicle control (Figure 2A and B, P,0.001). RPS6KA1, MAPK14, MAPK3, PLA2G2A, MAPK9, and However, administration of rats with UA at 20 mg/kg MAPK8), apoptosis (PIK3CG, TRAF2, XIAP, TP53, BCL2L1, and 40 mg/kg markedly decreased UA-induced expression AKT1, IRAK4, TNFRSF1A, CASP3, CASP7, BCL2, CASP8, level of α-SMA by 54.9% and 70.6% in rat liver, respec- PRKACA, and AKT2), energy metabolism (PPARA, PDPK1, tively (Figure 2A and B, P,0.001). Higher dose (40 mg/ PPARD, CHKB, RXRA, PPARG, FABP3, FABP4, FABP7, kg) of UA exhibited a more potent inhibitory effect on the MMP-1, PCK1, NR1H3, NAMPT, CD38, NT5M, PNP, and expression level of α-SMA than that of low dose (20 mg/ NNMT), and cell proliferation (YWHAZ, TP53, TTK, CDK6, kg) of UA in rat liver (Figure 2A and B). Taken together,

Table 1 Primer sequences for type I collagen and β-Actin Gene Sequence Amplicon length (bp) Type I Collagen Sense: 5′-GGGGCAAGACAGTCATCGAA-3′ 144 Antisense: 5′-GGATGGAGGGAGTTTACACGAA-3′ β-Actin Sense: 5′-TCAGGTCATCACTATCGGCAAT-3′ 432 Antisense: 5′-AAAGAAAGGGTGTAAAACGCA-3′ Abbreviation: bp, base pair.

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 3993 Dovepress He et al Dovepress 6 7.5 - - 6.5 7.3 7.7 9.1 7.3 8.4 3.2 7.7 to 9.4 to - - - - Docking score ------+ 2

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δ / σ ζ , mitochondrial Target name (Pyruvate dehydrogenase [lipoamide]) kinase isozyme 1, mitochondrial (Pyruvate dehydrogenase [lipoamide]) kinase isozyme 3, mitochondrial (Pyruvate dehydrogenase [lipoamide]) kinase isozyme 4, mitochondrial 14-3-3 protein 14-3-3 protein 2-Oxoisovalerate dehydrogenase subunit α 3- H ydroxy-3-methylglutaryl- C o A reductase 3- H ydroxyacyl- C o A dehydrogenase type 2 3-Phosphoinositide-dependent protein kinase 1 PK PK PD Class PD PD PD PD PD PD Predicted molecular targets of U A 4F, 2 R 4F, 2Q6B_2, 2Q6B, 3 CC Z 2O23, 1U7T 1W1 G Table 2 PDB ID 2Q8 G 1Y8O 2ZKJ 3 I QU 1QJ A 1O LS

3994 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 7.2 - Continued ( 9.4 6.9 6.7 8.9 7.2 2.9 9 9.3 6.5 6.9 8.5 to ------

)-phosphates of uracil and thymine deoxyribonucleotides ′ (3 ′ and 2 ′ may be involved in the provision of malonyl- C o A or regulation fatty acid oxidation, rather β Dephosphorylates specifically the 5 and so protects mitochondrial D NA replication from excess dTTP. H as only marginal activity toward d I MP and d G MP. C leaves aggrecan, a cartilage proteoglycan, and may be involved in its turnover. May play an important role in the destruction of aggrecan arthritic diseases. C ould also be a critical factor exacerbation neurodegeneration in A D. C leaves aggrecan at the “392- G lu-|- la-393” site. C leaves aggrecan, a cartilage proteoglycan, and may be involved in its turnover. May play an important role in the destruction of aggrecan arthritic diseases. May play a role inproteolytic processing mostly during peri-implantation period. ACC - than fatty acid biosynthesis. C arries out three functions: biotin carboxyl carrier protein, carboxylase, and carboxyl transferase. N onreceptor tyrosine-protein and serine/threonine–protein kinase that is implicated in cell spreading migration, cell survival, growth, and proliferation. Transduces extracellular signals to cytosolic nuclear effectors. Phosphorylates AKT1, AR, MCF2, WASL, and WWOX. Implicated in trafficking clathrin-mediated endocytosis through binding to EG F R and clathrin. Binds both poly- monoubiquitin and regulates ligand-induced degradation of EG F R , thereby contributing to the accumulation at limiting membrane of early endosomes. Downstream effector C D 42 which mediates 42-dependent and plasticity and function synaptic adult in both involved be May B CAR 1. of phosphorylation via migration cell in brain development. A ctivates KT1 by phosphorylating it on “Tyr-176”. Phosphorylates AR “Tyr-267” and “Tyr-363”, thereby promoting its recruitment to androgen-responsive enhancers. Phosphorylates WWOX on “Tyr-287”. Phosphorylates M C F2, thereby enhancing its activity as a GE F toward R ho family proteins. C ontributes to the control of A X L receptor levels. onfers metastatic properties on cancer cells and promotes tumor growth by negatively regulating suppressor such as WWOX positively regulating pro-survival factors such as A KT1 and AR . A TP-dependent phosphorylation of adenosine and other related nucleoside analogs to monophosphate derivatives. S erves as a potential regulator of concentrations extracellular adenosine and intracellular adenine nucleotides. R eceptor for adenosine. The activity of this receptor is mediated by G proteins that activate adenylyl cyclase. S ynthesizes cyclic A DP-ribose, a second messenger for glucose-induced INS secretion. lso has c DPr hydrolase activity. A lso moonlights as a receptor in cells of the immune system. Plays a role in protein sorting and trafficking between the TGN endosomes. Mediates ARF-dependent recruitment of clathrin to the T GN and binds ubiquitinated proteins membrane cargo molecules with a cytosolic AC - LL motif. Belongs to an adhesion system, probably together with the E -cadherin–catenin which plays a role in the organization of homotypic, interneuronal, and heterotypic cell–cell AJs. Nectin- actin-filament-binding protein that connects nectin to the actin cytoskeleton. )-Deoxyribonucleotidase, ′ (3 ′ 5 mitochondrial A disintegrin and metalloproteinase with thrombospondin motifs 4 A disintegrin and metalloproteinase with thrombospondin motifs 5 A cetyl- C o carboxylase 2 A ctivated C D 42 kinase 1 A denosine kinase A denosine receptor 2a A DP-ribosyl cyclase 1 A DP-ribosylation factor-binding protein GGA 1 A fadin A lcohol dehydrogenase 1 PD PD PD PD PD PD PD PD PD PD PK 1Q91 2 R JP 3 H Y G 3J R X 3 E Q R 2 I 6 A 3 E M L 2P G J 1PY1 2 E X G 1 HS O, 1U3T, 1U3T_2, 1 HS O_2

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B are κ B activation, probably through IRS 1 and the activation -nitrobenzaldehyde) compounds are the best κ m -(hydroxymethyl) glutathione. S essential for the autocrine growth and survival signaling of MDK. Function N / A N / A C ould function in retinol oxidation for the synthesis of retinoic acid, a hormone important cellular differentiation. Medium-chain (octanol) and aromatic ( substrates. E thanol is not a good substrate but at the high ethanol concentrations reached in digestive tract, it plays a role in the ethanol oxidation and contributes to first-pass metabolism. C lass 3 A D H is remarkably ineffective in oxidizing ethanol, but it readily catalyzes the oxidation of long-chain primary alcohols and the oxidation of ALDHs play a major role in the detoxification of alcohol-derived acetaldehyde. They are involved metabolism of corticosteroids, biogenic amines, neurotransmitters, and lipid peroxidation. This protein preferentially oxidizes aromatic aldehyde substrates. I t may play a role in the oxidation of toxicaldehydes. N / A Neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems plays an important role in genesis differentiation of the nervous system. Transduces signals from ligands at cell surface, through specific activation MAPK pathway. Phosphorylates almost exclusively at the first tyrosine of Y-x-x-x-Y-Y motif. Following activation by ligand, AL K induces tyrosine phosphorylation of C B L , F RS 2, IRS 1, and SHC 1 as well the M A PKs PK1/ ER K2 and PK3/ K1. cts as a receptor for ligands PT N , secreted growth factor, and MDK, a PT N -related factor, thus participating in MDK signal transduction. binding induces M A PK pathway activation, which is important for the anti-apoptotic signaling of PT N and regulation cell proliferation. MDK binding induces phosphorylation of the AL K target IRS 1, activates M A PKs and P I 3K, resulting also in cell proliferation induction. Drives N F- of the A KT serine/threonine kinase. R ecruitment IRS 1 to activated AL K and activation N F- C atalyzes the degradation of compounds such as putrescine, histamine, spermine, and spermidine, substances involved in allergic and immune responses, cell proliferation, tissue differentiation, tumor formation, possibly apoptosis. Placental D A O is thought to play a role in the regulation of female reproductive function. chain σ / μ Target name A lcohol dehydrogenase 1B A lcohol dehydrogenase 1 C A lcohol dehydrogenase 4 A lcohol dehydrogenase class 4 A lcohol dehydrogenase class 3 A ldehyde dehydrogenase, dimeric NA DP- preferring A ldehyde dehydrogenase, mitochondrial AL K tyrosine kinase receptor A miloride-sensitive amine oxidase (copper-containing) ) Class PK PK PK PK PK PK PK PD PK Continued ( Table 2 PDB ID 1U3U, 1U3U_2, 1U3V_2, 1U3V, 1 HS Z 1 H T0, 1U3W, 1U3W_2 3 C O S 1D1T, 1D1 S , 1 AGN 2FZ E , 3QJ5, 3QJ5_2, 2FZW 3 S ZB 3 N 80, 2V LE , 3 IN J, 1O04 2XB A 3MP H , 3 HIG , 3 HI 7, 3 HIG _2

3996 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 1.9 1.6 - - Continued ( 10.3 to 6.9 to 5.3 8 9.7 - - - - - C atalyzes the oxidative deamination of biogenic and xenobiotic amines has important functions in metabolism of neuroactive and vasoactive amines in the CNS peripheral tissues. M A O preferentially oxidizes biogenic amines such as 5- H T, norepinephrine, and epinephrine. C atalyzes the oxidative deamination of biogenic and xenobiotic amines has important functions in metabolism of neuroactive and vasoactive amines in the CNS peripheral tissues. M A OB preferentially degrades benzylamine and phenylethylamine. S teroid hormone receptors are ligand-activated transcription factors that regulate eukaryotic gene expression and affect cellular proliferation differentiation in target tissues. Transcription factor activity is modulated by bound coactivator and corepressor proteins. Transcription activation is downregulated NR 0B2. A ctivated, but not phosphorylated, by HI PK3 and Z I PK/D PK3. Tyrosine-protein kinase that acts as cell-surface receptor for ANG PT1, PT2, and PT4 regulates angiogenesis, EC survival, proliferation, migration, adhesion and cell spreading, reorganization of the actin cytoskeleton, but also maintenance of vascular quiescence. Has anti-inflammatory effects by preventing the leakage of proinflammatory plasma proteins and leukocytes from blood vessels. Required for normal angiogenesis and heart development during embryogenesis. R equired for postnatal hematopoiesis. A fter birth, activates or inhibits angiogenesis, depending on the context. I nhibits angiogenesis and promotes vascular stability in quiescent vessels, where EC s have tight contacts. I n ANG PT1 oligomers recruit T E K to cell–cell contacts, forming complexes with molecules from adjoining cells, and this leads preferential activation of phosphatidylinositol 3-kinase and the A KT1 signaling cascades. I n migrating EC s that lack cell–cell adhesions, ANG T1 recruits T E K to contact with the EC M, leading formation of focal adhesion complexes, activation of PTK2/F A K and the downstream kinases M PK1/ ER K2 PK3/ ER K1, and ultimately to the stimulation of sprouting angiogenesis. ANG PT1 signaling triggers receptor dimerization and autophosphorylation at specific tyrosine residues that then serve as binding sites for scaffold proteins and effectors. Signaling is modulated by ANGPT2 that has lower affinity for TEK, can promote TEK autophosphorylation in the absence of ANG PT1, but inhibits PT1-mediated signaling by competing for the same binding site. S ignaling is also modulated by formation of heterodimers with T IE 1 and proteolytic processing that gives rise to a soluble T E K extracellular domain. The domain modulates signaling by functioning as decoy receptor for angiopoietins. T E K phosphorylates DOK2, GR B7, B14, P I K3 R 1; SHC 1, and T IE 1. C onverts angiotensin- I to angiotensin-2 by release of the terminal H is- L eu; this results in an increase vasoconstrictor activity of angiotensin. A lso able to inactivate bradykinin, a potent vasodilator. H as also glycosidase activity that releases G P I -anchored proteins from the membrane by cleaving mannose linkage in the G P I moiety. Amine oxidase (flavin-containing) A Amine oxidase (flavin-containing) B AN D R A ngiopoietin-1 receptor A ngiotensin-converting enzyme PK PK PD PD PD 2BX R _2, 2BX R , 2Z5X, 2Z5Y, 2Z5Y_2, 2Z5X_2 2V5Z, 2XF N , 1 S 3 E , 2XF N _2, 2V5Z_2, 1 S 3 E _2 3B66 2OO8 1O86

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- or - or N N -aspartate. NAA occurs in high concentration l -arginine bioavailability to N O synthase. S ince l -acetylation of various arylamine and heterocyclic amine substrates is able to bioactivate several known -acetylation of various arylamine and heterocyclic amine substrates is able to bioactivate several known O carcinogens. O carcinogens. Function I nvolved in the transport of antigens from cytoplasm to endoplasmic reticulum for association with MHC class I molecules. Also acts as a molecular scaffold for the final stage of folding, namely binding of peptide. N ascent M HC class I molecules associate with T A P via tapasin. nhibited by the covalent attachment of herpes simplex virus IC P47 protein, which blocks the peptide-binding site T A P. I nhibited by human cytomegalo virus U S 6 glycoprotein, which binds to the lumenal side of T A P complex and inhibits peptide translocation by specifically blocking ATP binding to TAP1 and prevents the conformational rearrangement of T A P induced by peptide binding. I nhibited human adenovirus E 3-19K glycoprotein, which binds the T A P complex and acts as a tapasin inhibitor, preventing M HC class I /T association. E xpression of T A P1 is downregulated by human E pstein–Barr virus v IL -10 protein, thereby affecting the transport peptides into the endoplasmic reticulum and subsequent peptide loading by M HC class I molecules. Most important serine protease inhibitor in plasma that regulates the blood coagulation cascade. A T- III inhibits thrombin, matriptase-3/TMP RSS 7 as well factors I Xa, and X a. ts inhibitory activity is greatly enhanced in the presence of heparin. S ubunit of novel type clathrin- or nonclathrin-associated protein coat involved in targeting proteins from the T GN to endosomal–lysosomal system. A po(a) is the main constituent of L p(a). I t has serine proteinase activity and able autoproteolysis. I nhibits tissue-type plasminogen activator 1. L p(a) may be a ligand for megalin/ G p 330. S uppresses apoptosis in a variety of cell systems including factor-dependent lympho hematopoietic and neural cells. R egulates cell death by controlling the mitochondrial membrane permeability. A ppears to function in a feedback loop system with caspases. I nhibits caspase activity either by preventing the release of cytochrome c from the mitochondria and by binding to A P F-1. N / A May play a role in the regulation of extra-urea cycle arginine metabolism and also downregulation N O synthesis. E xtrahepatic arginase functions to regulate synthase is found in the penile corpus cavernosum smooth muscle, clitoral and vagina, arginase II plays a role in both male and female sexual arousal. I t is therefore potential target for the treatment of male and female sexual arousal disorders. R equired for activity of the A h (dioxin) receptor. This protein is required ligand-binding subunit to translocate from the cytosol to nucleus after ligand binding. The complex then initiates transcription of genes involved in the activation P AH procarcinogens. The heterodimer with HI F1 A or E AS 1/ F2 functions as a transcriptional regulator of the adaptive response to hypoxia. Participates in the detoxification of a plethora hydrazine and arylamine drugs. Catalyzes Participates in the detoxification of a plethora hydrazine and arylamine drugs. Catalyzes H ydrolyzes cerebroside sulfate. C atalyzes the deacetylation of NAA to produce acetate and in brain and its hydrolysis. NAA plays a significant part the maintenance of intact white matter. In other tissues, it acts as a scavenger of NAA from body fluids. -acetyltransferase 1 -acetyltransferase 2 N N Target name A ntigen peptide transporter 1 A ntithrombin- III A P-4 complex subunit mu-1 A polipoprotein(a) A poptosis regulator Bcl-2 A rginase-1 A rginase-2, mitochondrial A ryl hydrocarbon receptor nuclear translocator A rylamine A rylamine A rylsulfatase A spartoacylase ) Class PK PD PD PD PD PD PD PK PK PK PK PD Continued ( Table 2 PDB ID 1JJ7 1 A ZX 3 L 81 3K I V 2O22 2 AE B 1PQ3 3 H 82 2PQT, 2 I J A 2PF R 1 E 2 S 2O4 H

3998 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 6.7 6.7 - - Continued ( 7.6 to 7.8 to 6.7 7.7 7 7.1 6.5 ------

′ -. A TT GG -3 ′ direction. A lteration of secondary structure may ′ to 5 ′ box. Binds to nucleosomes (by similarity). chromatin and interacts ′ -TT A TT -3 ′ combined with a 5 selectively with histone H 3 that is not methylated at “ L ys-4”, phosphorylated “Thr-3”, and epigenetic the for important are that modifications H3 histone of sensor a as Functions “Arg-2”. at methylated regulation of gene expression. Functions as a transcriptional activator and promotes the expression otherwise tissue-specific self-antigens in the thymus, which is important for self-tolerance and avoidance of autoimmune reactions. May mediate critical mitochondrial transport functions related to heme biosynthesis (by similarity). Binds heme and porphyrins functions in their A TP-dependent uptake into the mitochondria. Plays a crucial role in heme synthesis. Unwinds double-stranded D NA and RNA in a 3 subsequently influence interactions with proteins or other nucleic acids. Functions as a transcriptional activator. C omponent of the CR D-mediated complex that promotes MY m RNA stability. I nvolved with LAR P6 in the stabilization of type I collagen m RNA s for C O1 A 1 and 2. Mitotic serine/threonine kinases that contribute to the regulation of cell-cycle progression. A ssociates with the centrosome and spindle microtubules during mitosis plays a critical role in various mitotic events including the establishment of mitotic spindle, centrosome duplication, separation as well as maturation, chromosomal alignment, SAC , and cytokinesis. R equired for initial activation of C DK1 at centrosomes. Phosphorylates numerous target proteins, including ARHGE F2, BO RA , B RCA 1, C D 25B, D LG P5, H AC 6, K I F2 A , LA T S 2, N EL 1, P AR D3, PPP1 R L K1, RASS F1, ACC 3, p53/TP53, and TPX2. R egulates K I F2 A tubulin depolymerase activity. equired for normal axon formation. Plays a role in microtubule remodeling during neurite extension. I mportant for formation and/or stabilization. A lso acts as a key regulatory component of the p53/TP53 pathway, and particularly checkpoint-response pathways critical for oncogenic transformation of cells, by phosphorylating and stabilizating p53/TP53. Phosphorylates its own inhibitors, the PP1 isoforms, to inhibit their activity. N ecessary for proper cilia disassembly prior to mitosis. Transcriptional regulator that binds to D NA as a dimmer or tetramer, but not monomer. Binds to G -doublets in an A /T-rich environment; the preferred motif is a tandem repeat of 5 A poptotic regulator capable of exerting proapoptotic and anti-apoptotic activities plays crucial roles in apoptosis, cell proliferation, and cell-cycle control. I ts anti-apoptotic activity is mediated through the inhibition of CAS P3, P7, and P9 as well by its E 3 ubiquitin-protein ligase activity. A s it is a weak caspase inhibitor, its anti-apoptotic activity is thought to be due ability ubiquitinate D IA B L O/ S M AC targeting it for degradation, thereby promoting cell survival. May contribute to caspase inhibition, by blocking the ability of D IA B L O/ S M AC to disrupt X P/B IRC 4–caspase interactions. Protects against apoptosis induced by T N F or by chemical agents such as adriamycin, etoposide, or staurosporine. S uppression of apoptosisis mediated activation of M A PK8/J N K1, and possibly also PK9/J K2. This depends on T B1 NR 2 C 2/ T A K1. I n vitro, inhibits CAS P3 and proteolytic activation of pro- P9. soform 1 blocks staurosporine- induced apoptosis. I soform 2 blocks etoposide-induced protects against N K cell killing whereas isoform 1 augments killing. Transcriptional repressor that is required for germinal center formation and antibody affinity maturation. Probably plays an important role in lymphoma genesis. P repeat-containing protein 7 protein repeat-containing IA P TP-binding cassette subfamily B member 6, 6, member B subfamily cassette TP-binding TP-binding cassette subfamily B member 10, 10, member B subfamily cassette TP-binding A mitochondrial A mitochondrial A TP-dependent RNA helicase A urora kinase A utoimmune regulator Baculoviral B-cell lymphoma 6 protein PK PK PD PD PD PD PD 4 A YT, 4 A YW, 4 A YX 3 NHA , 3 NH 9 3 LL M 3FD N 2K E 1 2 I 3 H 3 L BZ

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/M 2

pathways. Through Through pathways. ER J and B and J NHE checkpoint and progression to cytokinesis during 2 -nitrophenyl phosphate. backbone.D NA the of sugar ribose the on groups -hydroxyl ′ p termini are compatible with extension and ligation either by by either ligation and extension with compatible are termini NA damage, functioning as part of both the the both of part as functioning damage, NA K ensures that D that ensures K N -phosphates from or by phosphorylating by or 5 from -phosphates ′ phase of the cell-cycle. I nvolved in transcriptional regulation P21 response to D NA damage. 2 to mitosis. R equired for appropriate cell-cycle arrests after ionizing irradiation in both the S phase 2 checkpoints on D NA damage via B RCA 1-mediated ubiquitination of R BBP8. Plays a role in process governing chromosomal dynamics during mitosis (by similarity). together with RA MP3 adrenomedullin for CGR P together with RA MP1 and receptor R eceptor for is mediated The activity of this receptor together with RA MP2. adrenomedullin R eceptor for similarity). (by cyclase. that activates adenylyl G proteins by Function Potent inhibitor of cell death. I nhibits activation caspases (by similarity). A ppears to regulate death by blocking the VD AC binding to it and preventing release of caspase activator, C Y 1, from mitochondrial membrane. A lso acts as a regulator of G mitosis. I soform Bcl-X( S ) promotes apoptosis. Bifunctional enzyme. The C -terminal domain has epoxidehydrolase activity and acts on epoxides (alkene oxides, oxiranes) and arene oxides. Plays a role in xenobiotic metabolism by degrading potentially toxic epoxides. A lso determines steady-state levels of physiological mediators. The N -terminal domain has lipid phosphatase activity, with the highest activity toward threo-9,10-phosphonooxy-hydroxy-octadecanoic acid, followed by erythro-9,10-phosphonooxy-hydroxy-octadecanoic acid, 12-phosphonooxy-octadec-9Z-enoic acid, 12-phosphonooxy-octadec-9 E -enoic and D of repair the in role key a Plays P activities, catalytic two its 3 removing Bifunctional enzyme that catalyzes two steps in purine biosynthesis. L igand-activated transcription factor. R eceptor for bile acids such as chenodeoxycholic acid, lithocholic and deoxycholic acid. R epresses the transcription of C YP7 A 1 through induction NR 0B2 or F G F19 expression, via two distinct mechanisms. A ctivates the I B BP. transcription of bile salt export pump A B C B11 by directly recruiting histone methyltransferase CAR M1 to this locus. S ulfotransferase that utilizes P A as sulfonate donor to catalyze the sulfonation of steroids and bile acids in the liver and adrenal glands. A symmetrically hydrolyzes p4 to yield MP and TP. Plays a major role in maintaining homeostasis. E3 ubiquitin-protein ligase that specifically mediates the formation of “Lys-6”-linked polyubiquitin chains and plays a central role in D NA repair by facilitating cellular responses to damage. I t is unclear whether it also mediates the formation of other types polyubiquitin chains. The E 3 ubiquitin-protein ligase activity is required for its tumor suppressor function. The B RCA 1-B AR D1 heterodimer coordinates a diverse range of cellular pathways such as D NA damage repair, ubiquitination, and transcriptional regulation to maintain genomic stability. R egulates centrosomal microtubule nucleation. equired for normal cell-cycle progression from G and the G R equired for F ANC D2 targeting to sites of D NA damage. May function as a transcriptional regulator. I nhibits lipid synthesis by binding to inactive phosphorylated ACACA and preventing its dephosphorylation. Contributes to HRR via its direct interaction with PALB2, fine-tunes recombinational repair partly through its modulatory role in the P AL B2-dependent loading of B RCA 2- RA D51 repair machinery at D NA breaks. C omponent of the B RCA 1- R BBP8 complex which regulates CHE K1 activation and controls cell-cycle G -nucleosyl)-tetraphosphatase ′ Target name Bcl-2-like protein 1 Bifunctional epoxide hydrolase 2 Bifunctional polynucleotide phosphatase/ kinase Bifunctional purine biosynthesis protein PU RH Bile acid receptor Bile salt sulfotransferase Bis(5 (asymmetrical) Breast cancer type 1 susceptibility protein Bromodomain-containing protein 4 CGR P type 1 receptor ) PD PD Class PD PK PD PD PD PK PD PD Continued ( 3P5O 3 N 7 S Table 2 PDB ID 2YXJ 3 I 28, 1ZD3, 3 ANS 2W3O 1PKX 3OK I 3F3Y_2, 3F3Y, 1 E F H 1X SC 3K0K

4000 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 7.4 - Continued ( 8.2 8.4 8.3 8.6 8.5 7 6.6 6.6 9.3 to 8.9 ------. C an bind glutathione, α -difluoromethylornithine in α influx, essential for the + 2 waves, despite reduced wave amplitude + 2 ( N FKB IA ), but thrombin and collagen α B- B p50-p50 D NA binding. I nvolved in embryonic κ κ B ( N FKB1 and FKB2) I - κ . T R P C 1 activation by phosphorylation promotes a + 2 , leading to reduced amplitude and increased frequency of S O ICR characterized by an release and propagation velocity of spontaneous C a + + 2 2 -nitrosoglutathione. disrupt these complexes and free active P R K ACA stimulates platelets leads to platelet aggregation by phosphorylating V AS P. Prevents the antiproliferative and anti-invasive effects of breast cancer cells when activated. R Y 2 channel activity is potentiated by phosphorylation in presence of luminal C a rate of C a and resting cytosolic C a increase in permeability induced by thrombin confluent endothelial monolayers. PSMC5/RPT6 activation by phosphorylation stimulates proteasome. R egulates negatively TJs in ovarian cancer cells via CL D N 3 phosphorylation. N FKB1 phosphorylation promotes F- development by downregulating the H h signaling pathway that determines embryo pattern formation and morphogenesis. Isoform 2 phosphorylates and activates ABL1 in sperm flagellum to promote spermatozoa capacitation. Prevents meiosis redumption in prophase-arrested oocytes via C D 25B inactivation by phosphorylation. May also regulate RE M sleep in the PPT. which explains its higher affinity for glutathione-conjugated substrates. Catalyzes the reduction of S Phosphorylates a large number of substrates in the cytoplasm and nucleus. R egulates abundance of compartmentalized pools its regulatory subunits through phosphorylation PJ A 2 that binds and ubiquitinates these subunits, leading to their subsequent proteolysis. Phosphorylates C D 25B, A B L 1, N FKB1, CL D N 3, P S M C 5/ R PT6, PJ A 2, Y O RA , T 1, and V AS P. is activated by phosphorylation. R equired for glucose-mediated adipogenic differentiation increase and osteogenic inhibition from osteoblasts. I nvolved in the regulation of platelets response to thrombin and collagen; maintains circulating platelets in a resting state by phosphorylating proteins numerous platelet inhibitory pathways when in complex with N F- H ydrolyzes the second messenger c A MP, which is a key regulator of many important physiological processes. H ydrolyzes the second messenger c A MP, which is a key regulator of many important physiological processes. May be involved in mediating CNS effects of therapeutic agents ranging from antidepressants to antiasthmatic and anti-inflammatory agents. H ydrolyzes the second messenger c A MP, which is a key regulator of many important physiological processes. R eversible hydration of carbon dioxide. C an hydrate scyanamide to urea. R eversible hydration of carbon dioxide. E ssential for bone resorption and osteoclast differentiation (by similarity). R eversible hydration of carbon dioxide. Can hydrate cyanamide to urea. Involved in the regulation of fluid secretion into anterior chamber of the eye. R eversible hydration of carbon dioxide. May stimulate the sodium/bicarbonate transporter activity SLC 4 A that acts in p H homeostasis. I t is essential for acid overload removal from the retina and epithelium, and acid release in the choriocapillaris choroid. NADPH-dependent reductase with broad substrate specificity. Catalyzes the reduction of a wide variety of carbonyl compounds including quinones, prostaglandins, menadione, plus various xenobiotics. C atalyzes the reduction of antitumor anthracyclines doxorubicin and daunorubicin to cardiotoxic compounds doxorubicinol and daunorubicinol. C an convert prostaglandin E 2 to F2- H as low NA DP -dependent oxidoreductase activity toward 4-benzoylpyridine and menadione (in vitro). -cyclic -cyclic -cyclic ′ ′ ′ ,5 ,5 ,5 ′ ′ ′ α c A MP-dependent protein kinase catalytic subunit cAMP-specific 3 phosphodiesterase 4 A cAMP-specific 3 phosphodiesterase 4B cAMP-specific 3 phosphodiesterase 4D C arbonic anhydrase 1 C arbonic anhydrase 13 C arbonic anhydrase 2 C arbonic anhydrase 4 C arbonyl reductase ( NA DP H ) 1 C arbonyl reductase ( NA DP H ) 3 PD PD PD PD PD PD PD PD PK PK 3 AG M 2QYK 3F RG 1Y2K 2FOY 3 C ZV 2FOU 3FW3 2PF G _2, 2PF G , 3B H J_3, 3B H J, 3B H J_2, 1WM A 2 HR B

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4001 Dovepress He et al Dovepress 7 7.5 7.5 7.6 8.1 7.2 Docking score ------

B, S T A T1, CRE B1, B, S T A T1, CRE B1, κ κ arrest in response to spindle damage. A lso required for p53/TP53- 2 between an A sp and la, releasing the mature cytokine that is involved β IR F1, F2, A TF1, SR F, M X, JU N , FO S MY C and MYB. Phosphorylates H sp90 its co-chaperones FKBP4 and C D 37, which is essential for chaperone function. R egulates Wnt signaling by phosphorylating T NN B1 and the transcription factor LE F1. A cts as an ectokinase that phosphorylates several extracellular proteins. During viral infection, phosphorylates various proteins involved in the life cycles of E BV, HS V, H HC HI V, C MV and H PV. IR F1, F2, A TF1, SR F, M X, JU N , FO S MY C and MYB. Phosphorylates H sp90 its co-chaperones FKBP4 and C D 37, which is essential for chaperone function. R egulates Wnt signaling by phosphorylating T NN B1 and the transcription factor LE F1. A cts as an ectokinase that phosphorylates several extracellular proteins. During viral infection, phosphorylates various proteins involved in the life cycles of E BV, HS V, H HC HI V, C MV, and H PV. Function C arboxypeptidase that catalyzes the release of a -terminal amino acid, but has little or no action with - A sp, G lu, rg, L ys, or -Pro. Metalloprotease that could be involved in the histone hyperacetylation pathway. C leaves -terminal arginine or lysine residues from biologically active peptides such as kinins anaphylatoxins in the circulation, thereby regulating their activities. Downregulates fibrinolysis by removing C-terminal lysine residues from fibrin that has already been partially degraded by plasmin. C atalytic subunit of a constitutively active serine/threonine–protein kinase complex that phosphorylates large number of substrates containing acidic residues C -terminal to the phosphorylated serine or threonine. R egulates numerous cellular processes, such as cell-cycle progression, apoptosis, and transcription well viral infection. May act as a regulatory node that integrates and coordinates numerous signals, leading to an appropriate cellular response. During mitosis, functions as a component of the p53/TP53-dependent SAC that maintains cyclin-B- C DK1 activity and G mediated apoptosis, phosphorylating “ S er-392” of p53/TP53 following UV irradiation. C an also negatively regulate apoptosis. Phosphorylates the caspases CAS P9 and P2 apoptotic regulator N O L 3. Phosphorylation protects CAS P9 from cleavage and activation by P8 inhibits the dimerization of CAS P2 and activation of P8. R egulates transcription by direct phosphorylation RNA polymerases I , II III , and I V. A lso phosphorylates regulates numerous transcription factors including N F- C atalytic subunit of a constitutively active serine/threonine–protein kinase complex that phosphorylates large number of substrates containing acidic residues C -terminal to the phosphorylated serine or threonine. R egulates numerous cellular processes, such as cell-cycle progression, apoptosis, and transcription well viral infection. May act as a regulatory node which integrates and coordinates numerous signals, leading to an appropriate cellular response. During mitosis, functions as a component of the p53/TP53-dependent SAC that maintains cyclin-B- C DK1 activity and G 2 arrest in response to spindle damage. A lso required for p53/ TP53-mediatedapoptosis, phosphorylating “ S er-392” of p53/TP53 following UV irradiation. C an also negatively regulate apoptosis. Phosphorylates the caspases CAS P9 and P2 apoptotic regulator N O L 3. Phosphorylation protects CAS P9 from cleavage and activation by P8, inhibits the dimerization of CAS P2 and activation of P8. R egulates transcription by direct phosphorylation RNA polymerases I , II III and I V. A lso phosphorylates regulates numerous transcription factors including N F- Thiol protease that cleaves IL -1 in a variety of inflammatory processes. Important for defense against pathogens. Cleaves and activates SREBPs. C an also promote apoptosis. α α′ Target name C arboxypeptidase A 1 C arboxypeptidase A 4 C arboxypeptidase B2 C asein kinase II subunit C asein kinase II subunit C aspase-1 ) Class PD PD PD PD PD PD Continued ( Table 2 PDB ID 2V77 2P C U 3D67 3 H 30 3 E 3B 1 R WX

4002 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 6.4 - Continued ( 8.4 8.3 9.3 7 to 7.1 7.8 7.1 7.6 7.9 7.1 7.6 ------methyl DOP A , α -DOP A , l , antibacterial activity is inhibited by L P S Peudomonas aeruginosa , Z-Gly-Leu-Phe-CH2Cl, and phenylmethylsulfonyl fluoride. -methylation, and thereby the inactivation, of catecholamine neurotransmitters catechol O P. aeruginosa and isoproterenol. from I nvolved in the activation cascade of caspases responsible for apoptosis execution. A t onset apoptosis, it proteolytically cleaves P AR at a “216- A sp-|- G ly-217” bond. C leaves and activates SRE BPs between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. C leaves activates caspase-6, -7 and -9. I nvolved in the cleavage of huntingtin. Triggers cell adhesion sympathetic neurons through RE T cleavage. I nvolved in the activation cascade of caspases responsible for apoptosis execution. C leaves and activates SRE BPs. Proteolytically cleaves P AR at a “216- A sp-|- G ly-217” bond. Overexpression promotes programmed cell death. Most upstream protease of the activation cascade caspases responsible for T N F RS F6/F AS -mediated and T N F RS F1 A -induced cell death. Binding to the adapter molecule, DD recruits it either receptor. The resulting aggregate called D ISC performs CAS P8 proteolytic activation. active dimeric enzyme is then liberated from the D ISC and is free to activate downstream apoptotic proteases. Proteolytic fragments of the N -terminal propeptide (termed CA P3, P5, and P6) are likely retained in D ISC . C leaves and activates CAS P3, P4, P6, P7, P9, P10. May participate in the G ZMB apoptotic pathways. C leaves A DP R T. H ydrolyzes the small-molecule substrate, c- sp- G lu-Val- sp M . L ikely target for the cow pox virus CR M A death inhibitory protein. I soforms 5–8 lack catalytic site and may interfere with the pro-apoptotic activity of complex. C atalyzes the hormones. A lso shortens the biological half-lives of certain neuroactive drugs like Thiol protease that is believed to participate in intracellular degradation and turnover of proteins. H as also been implicated in tumor invasion and metastasis. A cid protease active in intracellular protein breakdown. I nvolved the pathogenesis of several diseases such as breast cancer and possibly A D. Serine protease with trypsin- and chymotrypsin-like specificity. Cleaves complement C3. Has antibacterial activity against the G ram-negative bacterium C losely involved in osteoclastic bone resorption and may participate partially the disorder of remodeling. Displays potent endoprotease activity against fibrinogen at acid pH. May play an important role in EC M degradation. I mportant for the overall degradation of proteins in lysosomes. Thiol protease. Key protease responsible for the removal of invariant chain from M HC class II molecules. The bond specificity of this proteinase is in part similar to the specificities cathepsin L and N. Plasma membrane-associated small G TPase that cycles between an active TP-bound and inactive G DP-bound state. I nactive state binds to a variety of effector proteins regulate cellular responses. I nvolved in epithelial cell polarization processes. R egulates the bipolar attachment of spindle microtubules to kinetochores before chromosome congression in metaphase. Plays a role the extension and maintenance of the formation thin, actin-rich surface projections called filopodia. Mediates CDC42-dependent cell migration. -methyltransferase O C aspase-3 C aspase-7 C aspase-8 C atechol C athepsin B C athepsin D C athepsin G C athepsin K C athepsin L 1 C athepsin S C ell division control protein 42 homolog PD PD PD PK PD PD PD PD PD PD PD 2 H 5 I 2Q L 9 1QT N 3BWM_2, 3BWY_2, 3BWM, 3BWY, 3 A 7 E , 3 A 7 E _2 1 G MY 1 L YB 1T32 3KWZ 3 HHA 3OVX 1 A 4 R

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4003 Dovepress He et al Dovepress 7.4 6.3 8.6 9.7 9.7 9.2 8.9 9 10.8 7.8 9.8 7.3 Docking score ------

antigen antigen

AS X and F and X A ions, phospholipids, and factor + 2 -processing. I nvolved in association ′ - G MP. ′ nvolved in cell-cycle regulation as a trans-activator that acts to to acts that trans-activator a as regulation cell-cycle in nvolved I -end poly( A ) site cleavage and addition. N UDT21/ C P S F5 binds ′ complex is activated by RA P GE F3 and which involved in angiogenesis. γ elements within the pre-m RNA . Binds to, but does not hydrolyze mono- and ′ poptosis induction seems to be mediated either by stimulation of B of stimulation by either mediated be to seems induction poptosis A -U G U A -3 ′ DKs. DKs. C Function Transports retinoic acid to the nucleus. R egulates access of nuclear acid receptors. A cts as a tumor suppressor in many types; induces growth arrest or apoptosis depending on the type. cell and circumstances physiological genes activated the of One process. this for required genes of set a controlling by division cell regulate negatively of inhibitor an is expression or by repression of Bcl-2 expression. I nduces the transcription linc RNA -p21 and -Mkln1. L inc RNA -p21 participates in TP53-dependent transcriptional repression, leading to apoptosis and seem have to effect on cell-cycle regulation. I mplicated in N otch signaling crossover. Prevents C DK7 kinase activity when associated with CA K complex in response to D NA damage, thus stopping cell-cycle progression. I soform 2 enhances the transactivation activity of isoform 1 from some but not all TP53-inducible promoters. I soform 4 suppresses transactivation activity and impairs growth suppression mediated by isoform 1. I soform 7 inhibits isoform 1-mediated apoptosis. Cyclic nucleotide phosphodiesterase with a dual-specificity for the second messengers cAMP and cGMP, which are key regulators of many important physiological processes. Cyclic nucleotide phosphodiesterase with a dual-specificity for the second messengers cAMP and cGMP, which are key regulators of many important physiological processes. May play a role in fat metabolism. R egulates c A MP binding of RA P GE F3. Through simultaneous to F3 and I K3 6 assembles a signaling complex in which the P I 3K Plays a role in signal transduction by regulating the intracellular concentration of cyclic nucleotides. This phosphodiesterase catalyzes the specific hydrolysis of cGMP to 5 V III a. Degrades chitin, chitotriose, and chitobiose. May participate in the defense against nematodes other pathogens. I soform 3 has no enzymatic activity. Has a key role in phospholipid biosynthesis and may contribute to tumor cell growth. Catalyzes the first step in phosphatidylcholine biosynthesis. C ontributes to phosphatidylethanolamine Phosphorylates choline and ethanolamine. H as higher activity with choline. Has a key role in phospholipid biosynthesis. Catalyzes the first step phosphatidylethanolamine Phosphorylates ethanolamine and can also act on choline (in vitro). H as higher activity with ethanolamine. May not significantly contribute to in vivo phosphatidylcholine biosynthesis. Esterase with broad substrate specificity. Contributes to the inactivation of neurotransmitter acetylcholine. C an degrade neurotoxic organophosphate esters. Major secreted protease of mast cells with suspected roles in vasoactive peptide generation, EC M degradation, and regulation of gland secretion. C omponent of the F I m complex that plays a key role in pre-m RNA 3 with C P S F6 or F7 in pre-M RNA 3 to cleavage and polyadenylation RNA substrates. The homodimer mediates simultaneous sequence-specific recognition of two 5 di-adenosine nucleotides. May have a role in m RNA export. Factor I X is a vitamin K-dependent plasma protein that participates in the intrinsic pathway of blood coagulation by converting factor X to its active form in the presence of C a -cyclic ′ -cyclic ,5 ′ ′ -cyclic ′ ,5 ′ ,5 ′ α Target name C ellular retinoic acid-binding protein 2 C ellular tumor antigen p53 c G MP-dependent 3 phosphodiesterase c G MP-inhibited 3 phosphodiesterase B cGMP-specific 3 phosphodiesterase C hitotriosidase-1 C holine kinase C holine/ethanolamine kinase C holinesterase C hymase Cleavage and polyadenylation specificity factor subunit 5 C oagulation factor I X ) Class PD PD PD PD PD PD PD PD PD PD PD PD Continued ( Table 2 PDB ID 2F R 3 2VUK 3 I TU 1 S O2 1XOZ 1WB0 3 G 15 3F EG 2W I J 3 N 7O 3B H O 3 LC

4004 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 8 5.5 8 7.6 7.6 6.9 7 6 6.5 9.9 ------. C

2 2 1 (by (by 1 H Z S LES E – B 1 CA phase. phase. 2 1 in germ cells) cells) germ in 1 A during the early stages stages early the during from initiating mitosis; mitosis; initiating from E NA 2 (cyclin (cyclin 2 2, this phosphorylation is low in in low is phosphorylation this 2, A RCA DK1 at the centrosome and in the nucleus. nucleus. the in and centrosome the at DK1 C -dependent C 1r(2)- 1s(2) proenzyme + as-induced senescence by phosphorylating MY phosphorylating by senescence as-induced 2 R ; activated by interaction with cyclin cyclin with interaction by activated ; 2 transition, and subsequently activated by cyclin cyclin by activated subsequently and transition, S – 1 3K27me3 maintenance and epigenetic gene silencing. Phosphorylates Phosphorylates silencing. gene epigenetic and maintenance 3K27me3 damage checkpoint that prevents cells with damaged D damaged with cells prevents that checkpoint damage H NA DK2 prevents oxidative stress-mediated stress-mediated oxidative prevents DK2 C / E phase D phase S – 1 yclin yclin C DK1 by phosphorylation and coordinates the activation of cyclin B/ cyclin of activation the coordinates and phosphorylation by DK1 erine/threonine–protein kinase involved in the control of the cell cycle; essential for meiosis, but dispensable dispensable but meiosis, for essential cycle; cell the of control the in involved kinase erine/threonine–protein regulates homologous recombination-dependent repair by phosphorylating B phosphorylating by repair recombination-dependent homologous regulates S phase when recombination is active, but increases as cells progress toward mitosis. I nitiates the extrinsic pathway of blood coagulation. S erine protease that circulates in a zymogen form. Factor V II is converted to factor a by factors Xa, X a, I or thrombin minor proteolysis. n the presence of tissue factor and calcium ions, V II a then converts X to Xa by limited proteolysis. Factor V II a will also convert factor I X to Xa in the presence of tissue and calcium ions. Factor V III , along with calcium ions and phospholipid, acts as a cofactor for factor I Xa when it converts X to the activated form, factor Xa. Factor Xa is a vitamin K-dependent glycoprotein that converts prothrombin to thrombin in the presence of factor Va, calcium ions, and phospholipid during blood clotting. Factor X I triggers the middle phase of intrinsic pathway blood coagulation by activating factor X. Degrades type I collagen. Does not act on gelatin or casein. C ould have a role in tumoral process. C1q associates with the proenzymes C1r and C1s to yield C1, first component of serum complement system. The collagen-like regions of C 1q interact with the a complex, and efficient activation of C1 takes place on interaction the globular heads C1q with Fc regions of I g G or gM antibody present in immune complexes. Factor D cleaves factor B when the latter is complexed with C 3b, activating 3bbb complex, which then becomes the C 3 convertase of alternate pathway. I ts function is homologous to that 1s in classic pathway. May supply copper to copper-requiring proteins within the secretory pathway, when localized in T GN . Under conditions of elevated extracellular copper, it relocalized to the plasma membrane where functions in the efflux of copper from cells. R eceptor for the C -X- chemokine X CL 12/ S DF-1 that transduces a signal by increasing intracellular calcium ion levels and enhancing M A PK1/M PK3 activation. cts as a receptor for extracellular ubiquitin, leading to enhanced intracellular calcium ions concentrations and reduced cellular c A MP levels. I nvolved in hematopoiesis and in cardiac ventricular septum formation. A lso plays an essential role vascularization of the gastrointestinal tract, probably by regulating vascular branching and/or remodeling processes in EC s. I nvolved in cerebellar development. n the CNS , could mediate hippocampal-neuron survival. A cts as a co- receptor ( C D4 being the primary receptor) for HI V-1 X4 isolates and as a some V-2 isolates. Promotes E nv-mediated fusion of the virus. S for mitosis. Phosphorylates C T NN B1, U S P37, p53/TP53, N PM1, DK7, R B RCA 2, MY , P A T, and E Z H 2. I nteracts with cyclins A , B1, B3, D, or E . Triggers duplication of centrosomes and D NA cts at the G progression; controls the timing of entry into mitosis/meiosis by controlling subsequent activation cyclin B/ hESCs. in repair DNA C and death, cell proliferation, cellular between balance fine a orchestrating in role Crucial A ctivity of C DK2 is maximal during S phase and G of D NA synthesis to permit G during the late stages of D NA replication to drive transition from S phase mitosis, G promotes phosphorylation similarity). I nvolved in G transition to promote the E 2F transcriptional program and initiation of D NA synthesis modulates G

C oagulation factor V II C oagulation factor V III C oagulation factor X C oagulation factor X I C ollagenase 3 C omplement 1q subcomponent subunit A C omplement factor D C opper-transporting A TPase 1 C -X- chemokine receptor type 4 C DK2 PD PD PD PD PD PD PD PD PD PD 2BZ6 3 HN B 2JK H 3B G 8 830 C 2J G 8 1B I O 2KMX 3ODU 2 R 3 I

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4005 Dovepress He et al Dovepress 7.7 Docking score - – S 1 – S transition and until prophase 1 -catenin signaling pathway. A ctivator of the GAI T ( I F N - β -dependent manner, the initial event in assembly of GAI T complex. Phosphorylation γ -activated inhibitor of translation) pathway, which suppresses expression a posttranscriptional regulon Function I n response to D NA damage, S B repair by homologous recombination a reduction of C DK2-mediated B RCA 2 phosphorylation. Phosphorylation of R B1 disturbs its interaction with E 2F1. N PM1 phosphorylation by cyclin E / C DK2 promotes its dissociation from unduplicated centrosomes, thus initiating centrosome duplication. C yclin E / DK2-mediated phosphorylation of N P A T at G stimulates the N P A T-mediated activation of histone gene transcription during S phase. R equired for vitamin D-mediated growth inhibition by being itself inactivated. I nvolved in the N O-mediated signaling a nitrosylation/activation-dependent manner. U S P37 is activated by phosphorylation and thus triggers G transition. C T NN B1 phosphorylation regulates INS internalization. Proline-directed serine/threonine–protein kinase essential for neuronal cell-cycle arrest and differentiation may be involved in apoptotic cell death neuronal diseases by triggering abortive cell-cycle re-entry. I nteracts with D1- and D3-type G 1 cyclins. Phosphorylates SRC , N O S 3, V M/vimentin, p35/ C DK5 R 1, M E F2 A , SI P 1 L 1, SH 3 GL B1, PX N K1, CA M/MU C 18, SE PT5, S Y D M1, MP H J1, DK16, RAC 1, RH O A , C D 42, TO NE BP/ N F T5, M PT/T U, P1B, histone H p53/TP53, AC P E X1, PTK2/F A K1, huntingtin/ H TT, TM, M P2, NE F , and FM. R egulates several neuronal development physiological processes including neuronal survival, migration and differentiation, axonal neurite growth, synaptogenesis, O L s differentiation, synaptic plasticity, and neurotransmission, by phosphorylating key proteins. A ctivated by interaction with C DK5 R 1(p35) and TP6V0D1(p39), especially in postmitotic neurons, and promotes C DK5 R 1(p35) expression in an autostimulation loop. Phosphorylates many downstream substrates such as R ho and family small G TPases (eg, P A K1, RAC 1, RH O , C D 42) or microtubule- binding proteins (eg, M A PT/T U, P2, P1B), and modulates actin dynamics to regulate neurite growth and/or spine morphogenesis. Phosphorylates also exocytosis-associated proteins such as M CA M/MU C 18, SE PT5, S Y N 1, and P C T AIRE 1/ DK16 as well endocytosis-associated proteins such D M1, A MP H , S Y N J1 at synaptic terminals. I n the mature CNS , regulates neurotransmitter movements by phosphorylating substrates associated with neurotransmitter release and synapse plasticity; synaptic vesicle exocytosis, vesicles fusion with the presynaptic membrane, and endocytosis. Promotes cell survival by activating anti-apoptotic proteins B CL 2 and S T A T3, negatively regulating of J N K3/M PK10 activity. Phosphorylation p53/TP53 in response to genotoxic and oxidative stresses enhances its stabilization by preventing ubiquitin ligase- mediated proteasomal degradation, and induces transactivation of p53/TP53 target genes, thus regulating apoptosis. Phosphorylation of p35/ C DK5 R 1 enhances its stabilization by preventing calpain-mediated proteolysis producing p25/ C DK5 R 1 and avoiding ubiquitin ligase-mediated proteasomal degradation. During aberrant cell-cycle activity and D NA damage, p25/ C DK5 elicits double-strand D NA breaks that precedes neuronal death by deregulating H AC 1. damage-triggered phosphorylation of huntingtin/ H TT in nuclei neurons protects against polyglutamine expansion as well D NA damage-mediated toxicity. Phosphorylation of PX N reduces its interaction with PTK2/F A K1 in M C F s during differentiation of O L s. N egative regulator Wnt/ γ proinflammatory genes in myeloid cells; phosphorylates the linker domain of glutamyl-prolyl tRNA synthetase ( E P RS ) in a I F N - SH 3 GL B1 is required for autophagy induction in starved neurons. Phosphorylation of TO NE BP/ N F A T5 response to osmotic stress mediates its rapid nuclear localization. M E F2 is inactivated by phosphorylation in Target name C DK5 ) Class PD Continued ( Table 2 PDB ID 1U NG

4006 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 5.4 - Continued ( 7.5 6.5 7.9 7 9.7 to 8.1 7.2 8.7 ------

– S

to 1 1

B activation in a T CR / C D3- κ -d C for UMP synthesis. ′ length. Promotes, at least in astrocytes, changes patterns of gene expression, 1 -cells in pancreatic islets of L angerhans. β transition. Phosphorylates p R B/ B1 and N PM1. I nteracts with D-type G 1 cyclins during interphase at form a p R B/ B1 kinase and controls the entrance into cell cycle. I nvolved in initiation maintenance of cell-cycle exit during cell differentiation; prevents proliferation and regulates negatively differentiation, but is required for the proliferation of specific cell types (eg, erythroid and hematopoietic cells). Essential for cell proliferation within the dentate gyrus of hippocampus and subventricular zone lateral ventricles. R equired during thymocyte development. Promotes the production of newborn neurons, probably by modulating G the actin cytoskeleton including loss of stress fibers, and enhances motility during cell differentiation. Prevents myeloid differentiation by interfering with R U N X1 and reducing its transcription transactivation activity, but promotes proliferation of normal myeloid progenitors. Delays senescence. Promotes the of I nvolved in the transport of chloride ions. May regulate bicarbonate secretion and salvage epithelial cells by regulating the SLC 4 A 7 transporter. This enzyme scavenges exogenous and endogenous cytidine 2 dependent manner. I ts interaction with A D also regulates lymphocyte–epithelial cell adhesion. nvolved in association with F A P in the pericellular proteolysis of EC M, migration and invasion s into EC M. May be involved in the promotion of lymphatic adhesion, migration, and tube formation E cs. nucleus in response to neurotoxin, thus leading neuronal apoptosis. A P E X1 P-endodeoxyribonuclease is repressed by phosphorylation, resulting in accumulation of D NA damage and contributing to neuronal death. N O S 3 phosphorylation downregulates 3-derived nitrite ( O) levels. SRC mediates its ubiquitin-dependent degradation and thus leads to cytoskeletal reorganization. May regulate EC migration angiogenesis via the modulation of lamellipodia formation. I nvolved in dendritic spine morphogenesis by mediating the E F NA 1- P HA 4 signaling. S erine/threonine–protein kinase involved in the control of cell cycle and differentiation; promotes G A P that associates with tyrosine-phosphorylated growth factor receptors or their cellular substrates. Maintains low levels of EI F2 S 1 phosphorylation by promoting its dephosphorylation PP1. Plays a role in EL K1-dependent transcriptional activation in response to activated R as signaling. R equired for the phosphorylation of deoxyribonucleosides d C , G and A . H as broad substrate specificity and does not display selectivity based on the chirality of substrate. It is also an essential enzyme for the phosphorylation of numerous nucleoside analogs widely employed as antiviral and chemotherapeutic agents. This enzyme is involved in nucleotide metabolism: it produces dUMP, the immediate precursor of thymidine nucleotides and it decreases the intracellular concentration of dUTP so that uracil cannot be incorporated into D NA . Key enzyme in folate metabolism. C ontributes to the de novo mitochondrial thymidylate biosynthesis pathway. C atalyzes an essential reaction for de novo glycine and purine synthesis D NA precursor synthesis. Binds its own m RNA and that of D H F RL 1. C ell surface glycoprotein receptor involved in the costimulatory signal essential for T CR -mediated T-cell activation. A cts as a positive regulator of T-cell coactivation, by binding at least D , CA V1, IG F2 R and PTP RC . I ts binding to CA V1 and CAR D11 induces T-cell proliferation N F- -triphosphate ′ C DK6 Cystic fibrosis transmembrane conductance regulator C ytidine deaminase C ytoplasmic protein NC K2 d C kinase Deoxyuridine 5 nucleotidohydrolase, mitochondrial Dihydrofolate reductase Dipeptidyl peptidase 4 PD PK PD PD PK PD PD PD 2PZ E 1MQ0 1XO2 2 CIA 2Z I 5_2, 2 N O7, 3 I PX, 2Z I 5, 1P5Z, 2 N O7_2 2 H QU 1KMV 2 G 63

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4007 Dovepress He et al Dovepress 6.6 7.2 6.5 7.6 8 8 8.7 Docking score ------to its mature soluble form. R esponsible for the α -selectin, GH receptor, MU C 1, and the A PP. lso involved in activation of N otch l , α Function When overexpressed, enhanced cell proliferation, a process inhibited by G P C 3. A cts also as serine exopeptidase with a dipeptidyl peptidase activity that regulates various physiological processes by cleaving peptides in the circulation, including many chemokines, mitogenic growth factors, neuropeptides, and peptide hormones. R emoves N -terminal dipeptides sequentially from polypeptides having unsubstituted -termini provided that the penultimate residue is proline. C leaves the membrane-bound precursor of T N F- proteolytical release of soluble J A M3 from EC s surface. R esponsible for the proteolytic several other cell-surface proteins, including p75 T N F-receptor, IL -1 receptor type II , p55T transforming growth factor- pathway (by similarity). E ssential multidomain scaffolding protein required for normal development (by similarity). R ecruits channels, receptors, and signaling molecules to discrete plasma membrane domains in polarized cells. May play a role A J assembly, signal transduction, cell proliferation, synaptogenesis, and lymphocyte activation. R egulates the excitability of cardiac myocytes by modulating the functional expression Kv4 channels. Functional regulator of Kv1.5 channel. May act as a B-cell-associated adapter that regulates B-cell antigen receptor-signaling downstream of P I 3K. Dual specificity protein kinase that acts as an essential component of the MAP signal transduction pathway. Binding of extracellular ligands such as growth factors, cytokines, and hormones to their cell-surface receptors activates Ras and this initiates RAF1 activation. then further the dual-specificity protein kinases M A P2K1/M E K1 and P2K2/M K2. Both K2 function specifically in the MAPK/ERK cascade and catalyze concomitant phosphorylation of a threonine tyrosine residue in a Thr- G lu-Tyr sequence located the extracellular signal-regulated kinases M A PK3/ ER K1 and M A PK1/ K2, leading to their activation further transduction of the signal within PK/ ER K cascade. Depending on the cellular context, this pathway mediates diverse biological functions such as cell growth, adhesion, survival and differentiation, predominantly through the regulation of transcription, metabolism, and cytoskeletal rearrangements. One target of the M A PK/ ER K cascade is PP ARG , a nuclear receptor that promotes differentiation and apoptosis. M A P2K1/M E K1 has been shown to export PP ARG from the nucleus. The M A PK/ ER K cascade is also involved in regulation of endosomal dynamics, including lysosome processing and endosome cycling through the P NRC as well in fragmentation of G olgi apparatus during mitosis. Phosphorylates proteins on serine, threonine, and tyrosine. Probably associated with cell proliferation. E ssential for chromosome alignment by enhancing A U R KB activity (via direct C D CA 8 phosphorylation) at the centromere, and for the mitotic checkpoint. S hows activity both for tyrosine-protein phosphate and serine-protein but displays a strong preference toward phosphotyrosines. Specifically dephosphorylates and inactivates ERK1 ERK2. May play a role in signaling pathway regulating nuclear functions of cell proliferation. Phosphorylates serine, threonine, and tyrosine residues in its sequence exogenous substrates. Target name Disintegrin and metalloproteinase domain- containing protein 17 Disks large homolog 1 Dual adapter for phosphotyrosine and 3-phosphotyrosine and 3-phosphoinositide Dual specificity MAPK1 Dual specificity protein kinase TTK Dual specificity protein phosphatase 3 Dual specificity tyrosine-phosphorylation- regulated kinase 1 A ) Class PD PD PD PD PD PD PD Continued ( Table 2 PDB ID 2DDF 2OQ S 1F A O 3 E 8 N 3 H MP 3F81 3 AN Q

4008 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 8.5 7.3 6.8 6.1 8.7 10.1 7 ------A cts B and J N K activation. A cts κ -catenin to T C F7 L 2/T F4 that is required β outer membrane detachment, alteration of P9 by keeping it in a monomeric, inactive state. monomeric, it in a keeping by CAS P9 Escherichia coli B signaling and the target proteins for its E 3 ubiquitin-protein κ -1,2-linked mannose residue from Man(9) G lc NA c(2) to produce Man(8) c(2), but at high α P7 and obstructs substrate entry. I nactivates substrate obstructs and CAS P7 as an important regulator of innate immune signaling via regulation NLR s. Protects cells from spontaneous formation of the ripoptosome, a large multiprotein complex that has capability to kill cancer cells in caspase-dependent and caspase-independent manner. S uppresses ripoptosome formation by ubiquitinating RI PK1 and CAS P8. A cts as a positive regulator of Wnt signaling ubiquitinates T LE 1, 2, 3, 4, and AES . Ubiquitination of T LE 3 results in inhibition its interaction with C F7 L 2/T F4, thereby allowing efficient recruitment and binding of the transcriptional coactivator to initiate a Wnt-specific transcriptional program. the overall cell shape and partial loss of content. E 3 ubiquitin-protein ligase that mediates ubiquitination of p53/TP53, leading to its degradation by the proteasome. I nhibits p53/TP53- and p73/TP73-mediated cell-cycle arrest apoptosis by binding its transcriptional activation domain. A lso acts as an ubiquitin ligase E 3 toward itself and ARR B1. Permits the nuclear export of p53/TP53. Promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma R B1 protein. I nhibits D A XX-mediated apoptosis by inducing its ubiquitination and degradation. C omponent of the T RI M28/K A P1-MDM2-p53/TP53 complex involved in stabilizing p53/TP53. A lso component of the T RI M28/K P1- ER BB4-MDM2 complex that links growth factor and D NA damage response pathways. Mediates ubiquitination and subsequent proteasome degradation of DY R K2 in nucleus. Ubiquitinates IG F1 R and promotes it to proteasomal degradation. E 3 ubiquitin-protein ligase that is a component of the N -end rule pathway. R ecognizes and binds to proteins bearing specific N-terminal residues that are destabilizing according to the N-end rule, leading their ubiquitination and subsequent degradation. Plays a critical role in chromatin inactivation chromosome- wide transcriptional silencing during meiosis via ubiquitination of histone H 2 A . Binds leucine and is a negative regulator of the leucine-mTO R signaling pathway, thereby controlling cell growth. Multifunctional protein that not only regulates caspases and apoptosis but also modulates inflammatory signaling and immunity, copper homeostasis, mitogenic kinase signaling, cell proliferation as well invasion and metastasis. A cts as a direct caspase inhibitor. Directly binds to the active site pocket of CAS P3 and as an E 3 ubiquitin-protein ligase regulating N F- ligase activity include: RI PK1, CAS P3, P7, P8, P9, M A P3K2/M E KK2, D IA B L O/ S AC , AI FM1, CCS , and B IRC 5/survivin. Ubiquitinion of leads to enhancement its chaperone activity toward physiologic target, S OD1, rather than proteasomal degradation. Ubiquitinion of M A P3K2/M E KK2 and AI FM1 does not lead to proteasomal degradation. Plays a role in copper homeostasis by ubiquitinating C OMMD1 and promoting its proteasomal degradation. C an also function as E 3 ubiquitin-protein ligase of the NE DD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. R egulates the BMP signaling pathway and the S M A D- P3K7/T K1-dependent pathways leading to N F- Binds phospholipid vesicles containing phosphatidylinositol 3-p hosphate and participates in endosomal trafficking. I nvolved in glycoprotein quality control targeting of misfolded glycoproteins for degradation. t primarily trims a single enzyme concentrations, as found in the ER Q C components, it further trims carbohydrates to Man(5-6) G lc NA c(2). C onverts big endothelin-1 to endothelin-1. Cytotoxin and helminthotoxin with low-efficiency ribonuclease activity. Possesses a wide variety of biological activities. E xhibits antibacterial activity, including cytoplasmic membrane depolarization of preferentially G ram- negative, but also G ram-positive strains. Promotes -mannosidase α E 3 ubiquitin-protein ligase Mdm2 E 3 ubiquitin-protein ligase UB R 2 E 3 ubiquitin-protein ligase X IA P E arly endosome antigen 1 E ndoplasmic reticulum mannosyl- oligosaccharide 1,2- E ndothelin-converting enzyme 1 E osinophil cationic protein PD PD PD PD PD PD PD 3 L B 3 N Y3 3 HL 5 1JO C 1X9D 3DWB 1 H

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4009 Dovepress He et al Dovepress 8.5 7.9 10.5 6.7 4.9 Docking score - - - - -

2 , B α κ CCL

igand-dependent igand-dependent L sequence or association association or sequence ERE -cx lacks ligand-binding ability and ligand-binding lacks -cx β B response element of the the of element response B κ binding and/or heterodimerization heterodimerization and/or binding B signaling cascade. A lso directly κ NA I soform - HS D activity. Uses preferentially NA H . B components RELA /p65 and N FKB1/p50 on α κ B in a cell-type specific manner. Decreases NF- κ soforms lacking one or several functional domains are are domains functional several or one lacking soforms I B to activate transcription involving respective recruitment recruitment respective involving transcription activate to B κ C in the absence of ligand while presence ° O production. production. O B-mediated transcription from the IL 6 promoter and displaces RELA / -catenin. I soform 2 may act as an antagonist of EG F action. N κ β cellulin, epiregulin/ EREG and H B EG F/heparin-binding F. L igand binding β igand binding induces a conformational change allowing subsequent or combinatorial combinatorial or subsequent allowing change conformational a induces binding igand L s in an estrogen-dependent manner. estrogen-dependent in an ERE s 3 and endothelial endothelial and 3 S O -PK C , and S T A Ts modules. May also activate the N F- γ N -estradioland 4-hydroxy-tamoxifen loss in D NA binding at elevated temperature is more gradual. -independent signaling. signaling. -independent β uclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of of regulation the in involved are receptors their and hormones steroid The receptor. hormone uclear amphiregulin, epigen/ E P GN , BT C / triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits A Ps like GR B2 that in turn activates complex downstream signaling cascades. A ctivates at least four major downstream signaling cascades including the R as- RA F-M E K- ER K, P I 3K- A KT, LC phosphorylates other proteins like RGS 16, activating its G TPase activity and probably coupling the EG F receptor signaling to the G protein-coupled signaling. A lso phosphorylates MU C 1 and increases its interaction with SRC and C T NN B1/ D NA -binding activity and inhibits N F- p65 and associated coregulators from the promoter. R ecruited to N F- and IL 8 promoters can displace CRE BBP. Present with N F- ERE sequences. C an also act synergistically with N F- adjacent response elements; the function involves CRE BBP. C an activate transcriptional activity of TFF1. A lso mediates membrane-initiated estrogen signaling involving various kinase cascades. I soform 3 is involved of activation in D or ligand competitive by activity transcriptional modulate to thought with the full length receptor. I soform 3 can bind to ERE and inhibit isoform 1. has no or only very low binding activity resulting in the loss of ligand-dependent transactivation ability. D NA binding by ESR 1 and 2 is rapidly lost at 37 17 Function R eceptor tyrosine kinase that binds promiscuously transmembrane ephrin-B family ligands residing on adjacent cells, leading to contact-dependent bidirectional signaling into neighboring cells. The pathway downstream of the receptor is referred to as forward signaling while pathway ephrin ligand is referred to as reverse signaling. Together with its cognate ligand/functional E F N B2 plays a central role in heart morphogenesis and angiogenesis through regulation of cell adhesion migration. E P H B4-mediated forward signaling controls cellular repulsion and segregation form F N B2-expressing cells. Plays also a role in postnatal blood vessel remodeling, morphogenesis and permeability is thus important in the context of tumor angiogenesis. R eceptor tyrosine kinase-binding ligands of the EG F family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EG F, T G F A /T F- Favors the reduction of estrogens and androgens. A lso has 20- N tissues. target in differentiation and proliferation cellular affect and expression gene eukaryotic palindromic a to binding homodimer direct either involves transactivation nuclear with other D NA -binding transcription factors, such as A P-1/c-JU N , c-FO S TF-2, p1, and p3, to mediate ERE association with multiprotein coactivator complexes through L XX LL motifs of their respective components. Mutual transrepression occurs between the ER and N F- Nuclear hormone receptor. Binds estrogens with an affinity similar to that of ESR1 and activates expression containing genes reporter -dehydrogenase 1 β β Target name E phrin type-B receptor 4 EG F R E stradiol 17- ER ER ) Class PD PD PD PD PD Continued ( Table 2 PDB ID 2VWX 2 RG P 1 I 5 R 1XP C 1YY E

4010 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 6.4 - Continued ( 7.8 to 5.6 7.7 8.8 5.9 6.3 6.7 8.3 9 ------cap and mediates translational translational mediates and cap RNA cap during an early step in the initiation of of initiation the in step early an during cap RNA 1 complex that binds to the m the to binds that complex 1 R -FM E F4 EI P1- I YF C omponent of the the of omponent ecognizes and binds the 7-methyl guanosine-containing m guanosine-containing 7-methyl the binds and ecognizes -estradiol and estrone at concentrations of 20 nM. A lso sulfates dehydroepiandrosterone, pregnenolone, S ulfotransferase that utilizes P A as sulfonate donor to catalyze the sulfate conjugation of estradiol and estrone. May play a role in the regulation of ER activity by metabolizing free estradiol. Maximally sulfates β ethinylestradiol, equalenin, diethylstilbesterol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone, and dopamine are not sulfated. Orphan receptor that acts as transcription activator in the absence of bound ligand. Binds specifically to an ERE and activates reporter genes controlled by (by similarity). I ts translation stimulation activity is repressed by binding to the C YF P1-FM R 1 complex (by similarity). R protein synthesis and facilitates ribosome binding by inducing the unwinding of m RNA s secondary structures. C repression. I n the C YF P1- EI F4 E -FM R 1 complex, this subunit mediates binding to them RNA cap. Key enzyme in isoprenoid biosynthesis that catalyzes the formation of FPP, a precursor for several classes essential metabolites including sterols, dolichols, carotenoids, and ubiquinones. FPP also serves as substrate for protein farnesylation and geranylgeranylation. C atalyzes the sequential condensation of isopentenyl pyrophosphate with the allylic pyrophosphates, dimethylallyl pyrophosphate, and then resultant geranylpyrophosphate to the ultimate product farnesyl pyrophosphate. L ipid transport protein in adipocytes. Binds both long-chain fatty acids and retinoic acid. Delivers fatty acids and retinoic acid to their cognate receptors in the nucleus (by similarity). B-F A BP could be involved in the transport of a so far unknown hydrophobic ligand with potential morphogenic activity during CNS development. It is required for the establishment of radial glial fiber system in developing brain, a that is necessary for the migration of immature neurons to establish cortical layers (by similarity). F A BP is thought to play a role in the intracellular transport of long-chain fatty acids and their acyl- C o esters. Tyrosine-protein kinase that acts as cell-surface receptor for fibroblast growth factors and plays an essential role in the regulation of embryonic development, cell proliferation, differentiation, and migration. R equired for normal mesoderm patterning and correct axial organization during embryonic development, skeletogenesis, and normal development of the G n RH neuronal system. Phosphorylates P LCG 1, F RS 2, GA B1, and SH B. L igand binding leads to the activation of several signaling cascades. A ctivation P LCG 1 the production of cellular signaling molecules D AG and inositol 1,4,5-trisphosphate. Phosphorylation of F RS 2 triggers recruitment GR B2, GA B1, P I K3 R 1, and S O 1 mediates activation as, M A PK1/ ER K2, M A PK3/ K1, and the PK signaling pathway as well of KT1 pathway. Promotes phosphorylation of SHC 1, S T A T1, and PTP N 11/ SH P2. I n the nucleus, enhances R P 6K 1 CRE B1 activity and contributes to the regulation of transcription. F G R 1 signaling is downregulated by IL -17 D/ SE F G R 1 ubiquitination, internalization, and degradation. Tyrosine-protein kinase that acts as cell-surface receptor for fibroblast growth factors and plays an essential role in the regulation of cell proliferation, differentiation, migration, and apoptosis of embryonic development. R equired for normal patterning, trophoblast function, limb bud development, lung morphogenesis, osteogenesis, and skin development. Plays an essential role in the regulation of osteoblast differentiation, proliferation and apoptosis, is required for normal skeleton development. Promotes cell proliferation in keratinocytes and immature osteoblasts, promotes apoptosis in differentiated osteoblasts. Phosphorylates P LCG 1, F RS 2, and A K4. L igand binding leads to the activation γ E strogen sulfotransferase E strogen-related receptor E ukaryotic translation initiation factor 4 Farnesyl pyrophosphate synthase F A BP, adipocyte F A BP, brain F A BP, heart Fibroblast growth factor receptor 1 Fibroblast growth factor receptor 2 PK PD PD PD PD PD PD PD PD 1 G 3M, 1 G 3M_2, 1 H Y3 2 E R 2W97 3 N 45 2 NN Q 1FDQ 1 H M R 3 C 4F 3B2T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4011 Dovepress He et al Dovepress 7.6 7 6.6 6.4 Docking score - - - -

-galactoside and a wide array β 12-induced cell chemotaxis (By similarity). similarity). (By chemotaxis cell 12-induced CL X C 4 that is critical for for critical is that 4 CR X C eems to be involved in the desensitization of D2-like dopamine receptors instriatum and and instriatum receptors dopamine D2-like of desensitization the in involved be to eems S -arrestin-mediated receptor desensitization, internalization, and signaling events leading to their β Function of several signaling cascades. A ctivation P LCG 1 leads to the production cellular molecules D AG and inositol 1,4,5-trisphosphate. Phosphorylation of F RS 2 triggers recruitment GR B2, GA B1, P I K3 R 1, and S O 1 mediates activation of R as, M A PK1/ ER K2, PK3/ K1, the PK signaling pathway as well as of the A KT1 signaling pathway. F G R 2 is downregulated by ubiquitination, internalization, and degradation. Mutations that lead to constitutive kinase activation or impair normal F G R 2 maturation, internalization, and degradation lead to aberrant signaling. Over-expressed F G R 2 promotes activation of S T A T1. N onreceptor PTK that plays an essential role in regulating cell migration, adhesion, spreading, reorganization of the actin cytoskeleton, formation and disassembly focal adhesions cell protrusions, cell-cycle progression, cell proliferation, and apoptosis. R equired for early embryonic development placenta development. R equired for embryonic angiogenesis, normal cardiomyocyte migration and proliferation, and normal heart development. R egulates axon growth neuronal cell migration, branching synapse formation; required for normal development of the nervous system. Plays a role in osteogenesis and differentiation of osteoblasts. Functions in integrin signal transduction, but also signaling downstream of numerous growth factor receptors, ( G P CR s), E HA 2, netrin and L D receptors. Forms multisubunit signaling complexes with SRC and family members upon activation; this leads to the phosphorylation of additional tyrosine residues, creating binding sites for scaffold proteins, effectors, and substrates. R egulates numerous signaling pathways. Promotes activation of P I 3K and the A KT1signaling cascade. Promotes activation of M A PK1/ ER K2, PK3/ K1, and the PK signaling localized and transient activation of GE Fs GA Ps, thereby modulates the activity R ho family G TPases. S ignaling via CAS family members mediates activation of RAC 1. R ecruits the ubiquitin ligase MDM2 to P53/TP53 in the nucleus, and thereby regulates activity, ubiquitination, proteasomal degradation. Phosphorylates SRC ; this increases kinase activity. AC T N 1, ARHGE F7, GR B7, RE T, and W ASL . Promotes phosphorylation of PX N S T A T1; most likely S T A T1 are phosphorylated by a SRC family kinase that is recruited to autophosphorylated PTK2/F K1, rather than by PTK2/F A K1 itself. Promotes phosphorylation of B CAR 1; GI T2 and SHC this requires both SRC PTK2/F A K1. Promotes phosphorylation of BMX and P I K3 R 1. soform 6 (F RN K) does not contain a kinase domain and inhibits PTK2/F A K1 phosphorylation signaling. I ts enhanced expression can attenuate the nuclear accumulation of L PX N and limit its ability to enhance SR F-dependent gene transcription. phosphorylation receptor Such receptors. protein-coupled G of forms activated the phosphorylates Specifically initiates desensitization. receptor chemokine Phosphorylates RH O (in vitro) and a non G -protein-coupled receptor: LR P6 during Wnt signaling vitro). May regulate apoptosis, cell proliferation, and differentiation. Binds of complex carbohydrates. I nhibits C D45 protein phosphatase activity and therefore the dephosphorylation of L Y N kinase. Target name Focal adhesion kinase 1 Fructose-1,6-bisphosphatase 1 G protein-coupled receptor kinase 6 G alectin-1 ) Class PD PD PD PD Continued ( Table 2 PDB ID 3BZ3 2JJK 3 N Y 3OY8

4012 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 8.4 7.9 7.3 8.3 8.0 - - - - - 5.7 - Continued ( 6.3 8.7 7 to 9.4 to 8.6 9.5 8.3 8.5 to 9.6 to 11.4 to 8 8.5 to ------(5)-pregnene- δ -glutamate peptides. I n the α -1 integrin the stimulation by CS P G 4 β -amyloid. May be involved in the N -terminal β -3, (5)-androstene-3,17-dione and α δ peptides [ G lu-3]- β of EC s migration. Together with DMBT1, required for terminal differentiation columnar epithelial cells during early embryogenesis (by similarity). I n the nucleus: acts as a pre-m RNA splicing factor. nvolved in acute inflammatory responses including neutrophil activation and adhesion, chemoattraction of monocytes macrophages, opsonization of apoptotic neutrophils, and activation mast cells. intestine, required for the uptake of folate. I n brain, modulates excitatory neurotransmission through hydrolysis of the neuropeptide, NAAG , thereby releasing glutamate. I soform P S M-4 and isoform M-5 would appear to be physiologically irrelevant. I nvolved in prostate tumor progression. A lso exhibits a dipeptidyl- peptidase I V type activity. n vitro, cleaves G ly-Pro- A M C . pyroglutamate formation of several amyloid-related plaque-forming peptides. 3,20-dione precursors to testosterone and progesterone, respectively. Galactose-specific lectin that binds IgE. May mediate with the C atalyzes the trans-addition of three molecules I PP onto DM A to form geranylgeranyl pyrophosphate, an important precursor of carotenoids and geranylated proteins. R eceptor for GC s. H as a dual mode of action: transcription factor that binds to GRE s and modulator of other transcription factors. Affects inflammatory responses, cellular proliferation, and differentiation in target tissues. C ould act as a coactivator for S T A T5-dependent transcription upon GH stimulation and could reveal an essential role of hepatic GR in the control body growth. I nvolved chromatin remodeling. Plays a significant role in transactivation. Involved nuclear translocation (by similarity). Produces pentose sugars for nucleic acid synthesis and main producer of NA DP H reducing power. N / A H as both folate hydrolase and NAALA Dase activity. a preference for tri- R esponsible for the biosynthesis of pyroglutamyl peptides. H as a bias against acidic and tryptophan residues adjacent to the N -terminal glutaminyl residue and a lack of importance chain length after second residue. A lso catalyzes N -terminal pyroglutamate formation. I n vitro, formation of N -terminally truncated form of A PP amyloid- C onjugation of reduced glutathione to a wide number exogenous and endogenous hydrophobic electrophiles. C onjugation of reduced glutathione to a wide number exogenous and endogenous hydrophobic electrophiles. C onjugation of reduced glutathione to a wide number exogenous and endogenous hydrophobic electrophiles. C atalyzes isomerization reactions that contribute to the biosynthesis of steroid hormones. Efficiently catalyze obligatory double-bond isomerizations of C onjugation of reduced glutathione to a wide number exogenous and endogenous hydrophobic electrophiles. This isozyme has a high catalytic efficiency with 4-hydroxyalkenals such as 4-HNE. Significant glutathione conjugating activity is found only with the model substrate, CDNB. 1 κ -transferase A 1 -transferase A 2 -transferase A 3 -transferase A 4 -transferase S S S S S G alectin-3 G eranylgeranyl pyrophosphate synthase GC receptor G lucose-6-phosphate 1-dehydrogenase G lucosylceramidase G lutamate carboxypeptidase 2 G lutaminyl-peptide cyclotransferase G lutathione G lutathione G lutathione G lutathione G lutathione PD PD PK PK PD PD PD PK PK PK PK PK 2X G 3 2Q80 1 NH Z, 3K22_2, 3K22, 3 E 7 C 2B HL , 2B H 9, 2B H 9_2, 1QK I , 1QK I _2 2V3D 3B I 1 2 A FW 1K3 L , 1K3Y, 3KT L , 1YDK 2V C T, 4 ACS , 2WJU 2V C V, 2V C V_2, 1TD I 3 I K7 3 R P N , 1YZX

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4013 Dovepress He et al Dovepress 7.1 7.0 7.3 7.7 7.9 7.2 ------7.7 to 7.9 to 7.7 to 7.9 to 8.1 to 6.8 6.9 7.6 to 8.5 8.9 Docking score ------(phenacyl) S -methylglycine (sarcosine) with the -catenin, A P C , X IN 1, DPY SL 2/ CR MP2, β N -catenin and phosphorylates the N -terminus β -transferase activity. Participates in the biotransformation S - A doMet to form S cy). Possible crucial role in the regulation of tissue of regulation the in role crucial Possible ( A do H cy). homocysteine -adenosyl S glutathione reductase activity. H as also of inorganic arsenic and reduces MM A dimethylarsonic acid. of production concomitant concentration of A doMet and metabolism methionine. JU N , F A T C 1/ M PT/T U, and AC F1. R equires primed phosphorylation of the majority its substrates. I n skeletal muscle, contributes to INS regulation of glycogen synthesis by phosphorylating and inhibiting G Y S 1 activity and hence glycogen synthesis. May also mediate the development of INS resistance by regulating activation of transcription factors. R egulates protein synthesis controlling the activity initiation factor 2B ( EI F2B E / F2B5) in the same manner as glycogen synthase. I n Wnt signaling, GS K3B forms a multimeric complex with A P C , X IN 1, and T NN B1/ of C T NN B1, leading to its degradation mediated by ubiquitin/proteasomes. Phosphorylates JU N at sites proximal to its DNA-binding domain, thereby reducing affinity for DNA. Phosphorylates NFATC1/NFATC on conserved serine residues promoting N F A T C 1/ nuclear export, shutting off Function C onjugation of reduced glutathione to a wide number exogenous and endogenous hydrophobic electrophiles. C onjugation of reduced glutathione to a wide number exogenous and endogenous hydrophobic electrophiles. E xhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities. H as E xhibits glutathione-dependent thiol transferase activity. H as high dehydroascorbate reductase activity and may contribute to the recycling of ascorbic acid. Participates in biotransformation inorganic arsenic and reduces MM A . C onjugation of reduced glutathione to a wide number exogenous and endogenous hydrophobic electrophiles. R egulates negatively C DK5 activity via p25/p35 translocation to prevent neurodegeneration. C onjugation of reduced glutathione to a wide number exogenous and endogenous hydrophobic electrophiles. A cts on 1,2-epoxy-3-(4-nitrophenoxy) propane, phenethylisothiocyanate 4-nitrobenzyl chloride and 4-nitrophenethyl bromide. Displays glutathione peroxidase activity with cumene hydroperoxide. C atalyzes the biosynthesis of guanidinoacetate, immediate precursor creatine. reatine plays a vital role in energy metabolism muscle tissues. May play a role embryonic and CNS development. be involved in the response to heart failure by elevating local creatine synthesis. C atalyzes the methylation of glycine by using Phosphorylase is an important allosteric enzyme in carbohydrate metabolism. E nzymes from different sources differ in their regulatory mechanisms and natural substrates. H owever, all known phosphorylases share catalytic and structural properties. C onstitutively active protein kinase that acts as a negative regulator in the hormonal control of glucose homeostasis, Wnt signaling, and regulation of transcription factors microtubules, by phosphorylating inactivating glycogensynthase ( G Y S 1 or 2), EI F2B, C T NN B1/ β 1 2 1 2 1 μ μ ω ω θ -transferase -transferase -transferase -transferase -transferase P -transferase S S S S S S -methyltransferase N Target name G lutathione G lutathione G lutathione G lutathione G lutathione G lutathione G lycine amidinotransferase, mitochondrial G lycine G lycogen phosphorylase, liver form G lycogen synthase kinase-3 ) Class PK PK PK PK PK PK PD PK PD PD Continued ( Table 2 PDB ID 1XWK, 1YJ6, 1XW6 1 HNA , 3 G U R , 2 C 4J, 1XW5 1 EE M, 3 L F , 3V LN 3Q AG , 3Q19 3 CSH _2, 2 A R , 3 G U S , 3 CSH , 3 G U S _2 2 C 3Q 3JDW 2 A ZT, 1 R 74 1 L 5 R 1Q5K

4014 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 9.7 7.7 7.5 6.4 7.3 7.3 7.4 8.3 6.9 6.1 7 ------eems to S eems

-catenin. I s β

A sp. (T N F/T F A ). α B ( N FKB1) at the κ -cells and diabetes. β t cleaves after cleaves I t B-mediated anti-apoptotic response to T N F- κ -cells, resulting in apoptosis, loss of β -adrenergic stimuli. The inactive G DP-bound form prevents β transcriptional level and is required for the N F- N egatively regulates replication in pancreatic Phosphorylates MU C 1 in breast cancer cells, decreasing the interaction of with T NN B1/ necessary for the establishment of neuronal polarity and axonout growth. Phosphorylates M AR K2, leading to inhibit its activity. Phosphorylates SI K1 at “Thr-182”, leading to sustain Z C 3 HA V1 that enhances its antiviral activity. gene regulation, and thereby opposing the action of calcineurin. Phosphorylates M A PT/T U on “Thr-548”, decreasing significantly MAPT/TAU ability to bind and stabilize microtubules. is the principal component of neurofibrillary tangles in AD. Plays an important role ERBB2-dependent stabilization microtubules at the cell cortex. Phosphorylates M AC F1, inhibiting its binding to which is critical for its role in bulge stem cell migration and skin wound repair. Probably regulates N F- A ccelerates the intermembrane transfer of various glycolipids. C atalyzes glycosphingolipids between membranes but does not catalyze the transfer of phospholipids. May be involved in the intracellular translocation of glucosylceramides. This enzyme is necessary for target cell lysis in cell-mediated immune responses. I t cleaves after L ys or A rg. C leaves A P E X1 after “ L ys-31” and destroys its oxidative repair activity. I nvolved in apoptosis. responses. immune cell-mediated in lysis cell target for necessary is enzyme This be linked to an activation cascade of caspases (aspartate-specific cysteine proteases) responsible for apoptosis execution. C leaves caspase-3, -7, -9, and 10 to give rise active enzymes mediating apoptosis. A P that provides a critical link between cell surface growth factor receptors and the R as signaling pathway. I soform 2 does not bind to phosphorylated EG F R but inhibits F-induced transactivation of a as-responsive element. I soform 2 acts as a dominant negative protein over GR B2 and, by suppressing proliferative signals, may trigger active programmed cell death. R as proteins bind G DP/ TP and possess intrinsic TPase activity. N / A N / A G uanine nucleotide-binding proteins ( proteins) are involved as modulators or transducers in various transmembrane signaling systems. The G (i) proteins are involved in hormonal regulation of adenylate cyclase: they inhibit the cyclase in response to the association of RGS 14 with centrosomes and is required for translocation from the cytoplasm to plasma membrane. May play a role in cell division. Proton-coupled chloride transporter. Functions as antiport system and exchanges ions against protons. Important for normal acidification of the endosome lumen. May play an important role in renal tubular function. R equired for normal development and maintenance of cochlear hair cell bundles. A nchoring/scaffolding protein that is a part of the functional network formed by U SH 1 C , G D H 23, and MYO7 A which mediates mechanotransduction in cochlear hair cells. R equired for normal hearing (by similarity). Molecular chaperone that promotes the maturation, structural maintenance, and proper regulation of specific target proteins involved for instance in cell-cycle control and signal transduction. Undergoes a functional cycle that is linked to its A TPase activity. This cycle probably induces conformational changes in the client proteins, thereby causing their activation. I nteracts dynamically with various co-chaperones that modulate its substrate recognition, A TPase cycle, and chaperone function. α -methyltransferase N -1 α exchange transporter 5 - / C l + G lycolipid transfer protein G ranzyme A G ranzyme B G rowth factor receptor-bound protein 2 G TPase HR as G TPase I M A P family member 2 G uanidinoacetate G uanine nucleotide-binding protein (i) subunit H H armonin H eat shock protein HS P 90- PD PK PD PD PD PD PK PD PD PD PD 2 E UK 1O R F 1 IA U 2W0Z 1 CL U 2XT N 3O RH 2 G 83 2J9 L 2KB S 3 E KO

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4015 Dovepress He et al Dovepress 8.2 - 8 8.2 9.9 8.5 8.1 9.5 9.8 to 8.7 7.5 Docking score ------PK C . The R as- ER K activation is associated with the γ nhibits cell-cycle progression and counteracts the mitogenic mitogenic the counteracts and progression cell-cycle nhibits I 4) and also with nucleosome core particles. particles. core nucleosome with also and 4) H Function Molecular chaperone that promotes the maturation, structural maintenance, and proper regulation of specific target proteins involved for instance in cell-cycle control and signal transduction. Undergoes a functional cycle that is linked to its A TPase activity. This cycle probably induces conformational changes in the client proteins, thereby causing their activation. I nteracts dynamically with various co-chaperones that modulate its substrate recognition, A TPase cycle, and chaperone function. Bifunctional enzyme that catalyzes both the conversion of P GH 2 to G D2, a prostaglandin involved in smooth muscle contraction/relaxation and a potent inhibitor of platelet aggregation, the conjugation glutathione with a wide range of aryl halides and organic isothiocyanates. A lso exhibits low glutathione-peroxidase activity toward cumene hydroperoxide. R eceptor tyrosine kinase that transduces signals from the extracellular matrix into cytoplasm by binding to hepatocyte growth factor/ HG F ligand. R egulates many physiological processes including proliferation, scattering, morphogenesis, and survival. L igand binding at the cell surface induces autophosphorylation of M E T on its intracellular domain that provides docking sites for downstream signaling molecules. Following activation by ligand, interacts with the P I 3K subunit K3 R 1, LCG SRC , GR B2, S T A T3, or adapter GA B1. R ecruitment of these downstream effectors by M E T leads to the activation several signaling cascades including the R as- ER K, P I 3K- A KT, or LC morphogenetic effects while P I 3K/ A KT coordinates prosurvival effects. During embryonic development, M E T signaling plays a role in gastrulation, development and migration of muscles neuronal precursors, angiogenesis, and kidney formation. I n adults, participates in wound healing as well organ regeneration tissue remodeling. Promotes also differentiation and proliferation of hematopoietic cells. A cts as a receptor for L isteria internalin in lB, mediating entry of the pathogen into cells. H ydrolyzes the second messenger c A MP, which is a key regulator of many important physiological processes. May have a role in muscle signal transduction. H ydrolyzes the second messenger c A MP, which is a key regulator of many important physiological processes. May be involved in maintaining basal levels of the cyclic nucleotide and/or c A MP regulation germ cell development. H ydrolyzes the second messenger c G MP, which is a key regulator of many important physiological processes. I nactivates histamine by N -methylation. Plays an important role in degrading and regulating the airway response to histamine. This protein is the basis of A BO blood group system. The histo-blood involves three carbohydrate antigens: A , B, and H . B individuals express a glycosyl transferase activity that converts the H antigen to A (by addition of UDP- G al NA c) or B UDP- G al), whereas O individuals lack such activity. (H3 histones core with activity HAT significant Has activation. transcriptional promote to HAT a as Functions and activity of the adenoviral oncoprotein E 1 A . I n case HI V-1 infection, it is recruited by viral protein Tat. R egulates Tat’s transactivating activity and may help inducing chromatin remodeling of proviral genes. -cyclic -cyclic ′ ′ ,5 ,5 ′ ′ β -cyclic phosphodiesterase ′ ,5 -methyltransferase ′ N Target name H eat shock protein HS P 90- H ematopoietic prostaglandin D synthase H epatocyte growth factor receptor High affinity cAMP-specific 3 phosphodiesterase 7 A High affinity cAMP-specific and IBMX- insensitive 3 8 A High affinity cGMP-specific 3 phosphodiesterase 9 A H istamine H isto-blood group A BO system transferase HA T K A T2B ) Class PD PD PD PD PD PD PK PD PD Continued ( Table 2 PDB ID 3 N MQ 2 C VD 3F66 1ZK L 3 ECN 2 H D1 2 A OT, 2 A OT_2, 1JQD, 1JQ E , 1JQD_2 3 I O 1WU G

4016 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 7.2 8.4 6.8 6.8 8.4 8.4 ------transition in cell 1 phase, it probably 0 – G 0 contributes to silencing of MY C - and E 2F-responsive genes, suggesting a role in G cycle. I n addition to the histone methyltransferase activity, also methylates nonhistone proteins: mediates dimethylation of “ L ys-373” p53/TP53. Histone methyltransferase that specifically mono- and dimethylates “Lys-9” of histone H3 (H3K9me1 H3K9me2, respectively) in euchromatin. H3K9me represents a specific tag for epigenetic transcriptional repression by recruiting H P1 proteins to methylated histones. A lso mediates monomethylation of “ L ys-56” of histone H 3 ( 3K56me1) in G 1 phase, leading to promote interaction between and P CNA regulating D NA replication. A lso weakly methylates “ L ys-27” of histone H 3 ( 3K27me). required for D NA methylation, the histone methyltransferase activity is not required for suggesting that these two activities function independently. Probably targeted to histone H 3 by different D NA -binding proteins like E 2F6, M GA , A X, and/or DP1. May also methylate histone H 1. I n addition to the methyltransferase activity, also methylates non-histone proteins: mediates dimethylation of “ L ys-373” p53/ TP53. A lso methylates C DY L , W I Z, ACIN 1, D N MT1, H AC ERCC 6, K F12 and itself. R esponsible for the deacetylation of lysine residues on N -terminal part core histones ( H 2 A , 2B, H 3, and 4). istone deacetylation gives a tag for epigenetic repression plays an important role in transcriptional regulation, cell-cycle progression, and developmental events. H istone deacetylases act via the formation of large multiprotein complexes. Forms transcriptional repressor complexes by associating with M A D, SIN 3, YY1, and N - C O R . I nteracts in the late S phase of D NA replication with MT1 other transcriptional repressor complex composed of D N MT1, DM A P1, P CNA , CA F1. Deacetylates T SH Z3 and regulates its transcriptional repressor activity. C omponent of a RC O R - G F I -KDM1 A H D AC complex that suppresses, via H D AC recruitment, a number of genes implicated in multilineage blood cell development. R esponsible for the deacetylation of lysine residues on N -terminal part core histones ( H 2 A , 2B, H 3, and 4). istone deacetylation gives a tag for epigenetic repression plays an important role in transcriptional regulation, cell-cycle progression, and developmental events. H istone deacetylases act via the formation of large multiprotein complexes. I nvolved in muscle maturation via its interaction with the myocyte enhancer factors such as M E F2 A , C and F2D. R esponsible for the deacetylation of lysine residues on N -terminal part core histones ( H 2 A , 2B, H 3, and 4). istone deacetylation gives a tag for epigenetic repression plays an important role in transcriptional regulation, cell-cycle progression, and developmental events. H D AC s act via the formation of large multiprotein complexes. I nvolved in muscle maturation by repressing transcription of myocyte enhancer factors such as M E F2 A , F2B, and C . During muscle differentiation, it shuttles into the cytoplasm, allowing the expression of myocyte enhancer factors (by similarity). May be involved in E BV latency, possibly by repressing the viral BZ L F1 gene. R esponsible for the deacetylation of lysine residues on N -terminal part core histones ( H 2 A , 2B, H 3, and 4). istone deacetylation gives a tag for epigenetic repression plays an important role in transcriptional regulation, cell-cycle progression, and developmental events. H D AC s act via the formation of large multiprotein complexes. May play a role in smooth muscle cell contractility. Histone methyltransferase that specifically mono- and dimethylates “Lys-9” of histone H3 (H3K9me1 H3K9me2, respectively) in euchromatin. H3K9me represents a specific tag for epigenetic transcriptional repression by recruiting H P1 proteins to methylated histones. A lso weakly methylates “ L ys-27” of histone 3 ( H 3K27me). A lso required for D NA methylation, the histone methyltransferase activity is not D NA methylation, suggesting that these two activities function independently. Probably targeted to histone H 3 by different D NA -binding proteins like E 2F6, M GA , A X, and/or DP1. During G -methyltransferase -methyltransferase N N H D AC 2 H D AC 4 H D AC 7 H D AC 8 H istone–lysine EH MT1 H istone–lysine EH MT2 PD PD PD PD PD PD 3K5K 3M A X 2VQM 3 C 10 3MZ6 3MO5

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4017 Dovepress He et al Dovepress 5.9 - 8.4 8.1 8.2 8.6 8.9 7.3 to 7.5 8 6.6 9.2 Docking score ------R equired 3 acetylated on “ L ys-9”. on acetylated H 3 at “ A sp-803” in the CA D. Functions as an oxygen sensor and, under normoxic α Function H istone methyltransferase that plays an essential role in early development and hematopoiesis. C atalytic subunit of the M LL 1-M complex, a multiprotein complex that mediates both methylation “ L ys-4” histone H 3 ( 3K4me) complex and acetylation of “ L ys-16” 4 4K16ac). I n the M LL 1-M complex, it specifically mediates H3K4me, a specific tag for epigenetic transcriptional activation. Has weak methyltransferase activity by itself and requires other component of the M LL 1-M complex to obtain full methyltransferase activity. H as no activity toward histone 3 phosphorylated on “Thr-3”, less toward activity higher has while it “ L ys-9”, or “ A rg-8” on dimethylated H 3 for transcriptional activation of H OX A 9. Promotes PPP1 R 15 -inducedapoptosis. I nvolved in the presentation of foreign antigens to immune system. I nvolved in the presentation of foreign antigens to immune system. I nvolved in the presentation of foreign antigens to immune system. Preferably binds to a peptide derived from the signal sequence of most HLA - A , -B, C and G molecules. C onverts guanine to guanosine monophosphate, and hypoxanthine inosine monophosphate. Transfers the 5-phosphoribosyl group from 5-phosphoribosylpyro phosphate onto the purine. Plays a central role in generation of purine nucleotides through the salvage pathway. H ydroxylates HI F-1 conditions, the hydroxylation prevents interaction of HI F-1 with transcriptional coactivators including C bp/ p300-interacting transactivator. I nvolved in transcriptional repression through interaction with HI F1 A , VHL, and HDACs. Hydroxylates specific Asn residues within ARDs of NFKB1, NFKBIA, NOTCH1, ASB4, PPP1 R 12 A , and several other AR D-containing proteins. lso hydroxylates sp H is residues within AR Ds of AN K1 and T N K S 2, respectively. egatively regulates OT CH 1 activity, accelerating myogenic differentiation. Positively regulates AS B4 activity, promoting vascular N / A N / A R eceptor tyrosine kinase that mediates the pleiotropic actions of INS . Binding leads to phosphorylation of several intracellular substrates, including, IRS s ( 1, 2, 3, 4), SHC , GA B1, C B L and other signaling intermediates. E ach of these phosphorylated proteins serve as docking for other signaling that contain Src-homology-2 domains (SH2 domain) specifically recognize different phosphotyrosines residues, including the p85 regulatory subunit of P I 3K and SH P2. Phosphorylation IRS s proteins lead to the activation of two main signaling pathways: P I 3K- A KT/PKB pathway, which is responsible for most of the metabolic actions INS , and R as-M A PK pathway, which regulates expression some genes cooperates with the P I 3K pathway to control cell growth and differentiation. Binding of SH 2 domains P I 3K to phosphotyrosines on IRS 1 leads the activation of and generation P3, a lipid second messenger, which activates several P I P3-dependent serine/threonine kinases, such as PDPK1 and subsequently A KT/PKB. The net effect of this pathway is to produce a translocation the glucose transporter SLC 2 4/ GL UT4 from cytoplasmic vesicles to the cell membrane facilitate glucose transport. Moreover, upon INS stimulation, activated A KT/PKB is responsible for: anti-apoptotic effect of INS by inducing phosphorylation B A D; regulates the expression of gluconeogenic and lipogenic enzymes by controlling activity

inhibitor α -methyltransferase M LL N chain chain chain α α α chain C region -1 chain C region κ γ chain E Target name H istone–lysine HLA class I histocompatibility antigen, A -11 HLA class I histocompatibility antigen, A -2 HLA class I histocompatibility antigen, B-15 HLA class I histocompatibility antigen, α H ypoxanthine–guanine phosphoribosyl transferase H ypoxia-inducible factor 1- I g I g INS receptor ) Class PD PD PD PD PD PK PD PD PD PD Continued ( Table 2 PDB ID 3 L QJ 1X7Q 3D25 1X R 9 3BZ E 1 H MP, 1BZY 2W0X 1D6V 1 GA F 3 E K N

4018 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 9.5 7.4 - -

F2, isoform IG isoform F1, INSR PK pathway. IG A INSR n particular, I and R as-M and R F1 R Ks can also be activated IG F1 N IG hc leads to recruitment T proteins. S A T S 1 or hort are activated by S IRS -protein by neurons and microglia. β . When present in a hybrid receptor with R F1 isoform KT/PKB pathway and the IG A INSR 3K- I K activation via phosphorylation and inhibition of and by phosphorylated N R S F1 O S IG rb2/ G K proteins can lead to phosphorylation/activation of A F1 exerts inhibiting activities on J F1. PubMed:12138094 shows that hybrid receptors composed of IG . K1, which is able to directly associate with the IG R n contrast, PubMed:16831875 shows that hybrid receptors composed of AS F1 I . IG , binds 3K-driven signaling, recruitment of INS I , and that hybrid receptors composed of P3K5/ A winged helix or forkhead (FOX) class of transcription factors. A nother pathway regulated by P I 3K- KT/PKB activation is mTO RC 1 signaling pathway that regulates cell growth and metabolism integrates signals from INS . A KT mediates -stimulated protein synthesis by phosphorylating, T SC 2 thereby activating mTO RC 1 pathway. The R as/ RA F/M A P2K/M PK pathway is mainly involved in mediating cell growth, survival, and cellular differentiation of INS . Phosphorylated IRS 1 recruits GR B2- S O complex, which triggers the activation of the R as/ RA F/M A P2K/M PK pathway. I n addition to binding INS , receptor can bind IG Fs ( F and IGFII). Isoform Short has a higher affinity for IGFII binding. When present in hybrid receptor with IGF1R, binds IG F1. PubMed:12138094 shows that hybrid receptors composed of F1 R and INSR isoform L ong are activated with a high affinity by IGF1, low IGF2 and not significantly INS, that hybrid receptors composed of IG F1 R and INSR isoform S hort are activated by F1, F2, INS . I n contrast, PubMed:16831875 shows that hybrid receptors composed of IG F1 R and INSR isoform L ong hybrid receptors composed of IG F1 R and INSR isoform S hort have similar binding characteristics, both bind IGF1 and have a low affinity for INS. Plays a role in the cellular breakdown of INS , IA PP, glucagon, bradykinin, kallidin, and other peptides, thereby plays a role in intercellular peptide signaling. Degrades amyloid formed by A PP and IA PP. May play role in the degradation and clearance of naturally secreted amyloid Receptor tyrosine kinase that mediates actions of IGF1. Binds I GF1 with high affinity and IGF2 INS a lower affinity. The activated IGF1R is involved in cell growth and survival control. crucial for tumor transformation and survival of malignant cell. L igand binding activates the receptor kinase, leading to receptor autophosphorylation and tyrosines phosphorylation of multiple substrates that function as signaling A Ps including, the IRS s ( 1/2), S hc, and 14-3-3 proteins. Phosphorylation of proteins lead to the activation of two main signaling pathways: the P The result of activating the M A PK pathway is increased cellular proliferation, whereas P I 3K pathway inhibits apoptosis and stimulates protein synthesis. Phosphorylated IRS 1 can activate the 85 kDa regulatory subunit of P I 3K (P K3 R 1), leading to activation several downstream substrates, including protein A KT/PKB. KT phosphorylation, in turn, enhances synthesis through mTO R activation and triggers the antiapoptotic effects of IG F IR through phosphorylation inactivation B A D. I n parallel to P of R as and activation the as-M A PK pathway. I n addition to these two main signaling pathways, IG F1 signals also through the Janus kinase/signal transducer and activator of transcription pathway (J A K/ S T T). Phosphorylation of J activation of S T A T3 may be essential for the transforming activity IG F1 R . The J K/ pathway activates gene transcription and may be responsible for the transforming activity. J by the M INSR Long are activated with a high affinity by IGF1, low affinit y IGF2 and not significantly INS and L ong and hybrid receptors composed of IG F1 R INSR isoform S hort have similar binding characteristics, both bind IGF1 and have a low affinity for INS. INS -degrading enzyme IG F1 receptor PD PD 3 E 4 A 2OJ9

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4019 Dovepress He et al Dovepress 10.4 - 6.6 7.9 8.7 6.9 7.9 6.8 8.5 9.7 11.2 to 8.6 7.4 6.2 Docking score ------

B. κ -glucose (2D G ) transport d -kynurenine to form K A . l B nuclear translocation and κ -(seleno) cysteine, resulting in the cleavage l -tryptophan metabolite l -elimination of S -conjugates and e-conjugates β -2 is a receptor for ICA M1, M2, M3, and M4. I t involved in variety of immune β - L / α -lactoylglutathione. I nvolved in the regulation of T N F-induced transcriptional activity F- and glycogen synthesis in osteoblasts. S timulates glucose transport rat bone-derived osteoblastic (PyM ) cells and is effective at much lower concentrations than INS , not only regarding glycogen D NA synthesis but also with regard to enhancing glucose uptake. activation. A lternatively, phosphorylates T IRA P to promote its ubiquitination and subsequent degradation. Phosphorylates NC F1 and regulates NA DP H oxidase activation after L P S stimulation suggesting a similar mechanism during microbial infections. Metabolizes the cysteine conjugates of certain halogenated alkenes and alkanes to form reactive metabolites. C atalyzes the of the C - S or e bond. Function The IG Fs, isolated from plasma, are structurally and functionally related to INS but have a much higher growth-promoting activity. May be a physiologicalregulator of [1-14 C ]-2-deoxy- I ntegrin phenomena including leukocyte- EC interaction, cytotoxic T-cell mediated killing, and antibody-dependent killing by granulocytes and monocytes. S erine/threonine–protein kinase that plays a critical role in initiating innate immune response against foreign pathogens. I nvolved in T LR and IL -1 R signaling pathways. s rapidly recruited by MYD88 to the receptor- signaling complex upon T LR activation to form the Myddosome together with IRA K2. Phosphorylates initially IRA K1, thus stimulating the kinase activity and intensive autophosphorylation of K1. Phosphorylates E 3 ubiquitin ligases pellino proteins (P ELI 1, P 2, and 3) to promote pellino-mediated polyubiquitination of IRA K1. Then, the ubiquitin-binding domain I KBK G / NE MO binds to polyubiquitinated K1 bringing together the IRA K1-M A P3K7/T K1-T RA F6 complex and NE MO- I KK - KKB complex. n turn, M P3K7/ T A K1 activates I KKs ( CH UK/ KK and KBKB/ KKB) leading to N F- Produced by T-cells in response to antigenic or mitogenic stimulation, this protein is required for T-cell proliferation and other activities crucial to regulation of the immune response. C an stimulate B-cells, monocytes, lymphokine-activated killer cells, N K and glioma cells. C leaves collagens of types I , II and III at one site in the helical domain. A lso cleaves V and X. I n case of HI V infection, interacts cleaves the secreted viral Tat protein, leading to a decrease in neuronal Tat’s mediated neurotoxicity. May be involved in desquamation. N / A Motor protein required for establishing a bipolar spindle. Blocking of K I F11 prevents centrosome migration and arrest cells in mitosis with monoastral microtubule arrays. C atalyzes the irreversible transamination of C atalyzes the conversion of hemimercaptal, formed from methylglyoxal and glutathione, to S May play a role in E P S 8 signaling. Binds glutathion. Putative Pc G protein. proteins maintain the transcriptionally repressive state of genes, probably via a modification of chromatin, rendering it heritably changed in its expressibility. Its association with a chromatin- remodeling complex suggests that it may contribute to prevent expression of genes trigger the cell into mitosis. Binds to monomethylated and dimethylated “ L ys-20” on histone H 4. 3 peptides that are monomethylated or dimethylated on “ L ys-4”, ys-9”, ys-27”. - L α Target name IG F I I ntegrin IL -1 receptor-associated kinase 4 IL -2 I nterstitial collagenase Kallikrein-5 Ketohexokinase Kinesin-like protein K I F11 Kynurenine–oxoglutarate transaminase 1 L actoylglutathione lyase L an C -like protein 1 L ethal(3)malignant brain tumor-like protein 2 ) Class PD PD PD PD PD PD PD PD PK PD PD PD Continued ( Table 2 PDB ID 1 I MX 2 ICA 2O IC 1M48 966 C 2P S X 3 NC 9 2P G 2 3FV S , 3FVX, 3FVX_2, 3FVU, 3FVU_2 1Q IN 3.00 E +73 3F70

4020 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 3.0 - Continued ( 8.8 7.2 to 6.5 6.7 9.8 10 ------ys-20” of s and AC 3, nor “ L nor ys-27” of histone H 3, ys-4”, “ L ys-4”, 3, while it has no activity on mono- of H 3, ys-36” residues ys residue generates formaldehyde and succinate. Participates and succinate. generates formaldehyde ys residue ys-9” and “ L ys-9” and 2F-responsive promoters via the recruitment of H D via the recruitment promoters ASCL 2 and E 2F-responsive exosome-mediated m RNA decay. R eleases m7 G MP. C an also degrade DP to H as no ′ 5 . - ′ Epoxide hydrolase that catalyzes the final step in biosynthesis of proinflammatory mediator leukotriene B4. H as also aminopeptidase activity. Involved in the detoxification of xenobiotics and activation ester amide prodrugs. Hydrolyzes aromatic and aliphatic esters, but has no catalytic activity toward amides or a fatty acyl- C o A ester. H ydrolyzes the methyl ester group of cocaine to form benzoylecgonine. Catalyzes transesterification form cocaethylene. Displays fatty acid ethyl ester synthase activity, catalyzing the esterification of oleic acid to ethyloleate. thereby residues of histone H3, “Lys-36” and “Lys-9” demethylates that specifically Histone demethylase “ L Does not demethylate in histone code. a central role playing “ L trimethylated Demethylates histone H 4. of L Demethylation residues. and dimethylated of in transcriptional repression transcriptional activation of the promotes differentiation, muscle C rucial for 2: I soform respectively. NC O R 1, L acks the N -terminal marks at its promoter. chromatin of repressive the removal directing gene by Myog domain. demethylase Histone demethylase that specifically demethylates “Lys-4” residue of histone H3, thereby playing a central role in histone code. Does not demethylate “ L ys-9”, ys-27”, ys-36”, ys-79” of H 3, or ys-20” of histone H 4. Demethylates trimethylated and imethylated but not monomethylated “ L ys” 3. May stimulate transcription mediated by nuclear receptors. May be involved in transcriptional regulation of H ox proteins during cell differentiation. May participate in transcriptional repression of cytokines such as C X CL 12. H istone demethylase that demethylates both “ L ys-4” ( 3K4me) and ys-9” 3K9me) of histone 3, thereby acting as a coactivator or corepressor, depending on the context. A cts by oxidizing substrate by F A D to generate the corresponding imine that is subsequently hydrolyzed. cts as a corepressor mediating demethylation of H3K4me, a specific tag for epigenetic transcriptional activation. Demethylates both mono- ( H 3K4me1) and dimethylated 3K4me2) 3K4me. May play a role in the repression of neuronal genes. A lone, it is unable to demethylate H 3K4me on nucleosomes and requires the presence of RC O R 1/ C o RES T to achieve such activity. A lso acts as a coactivator of AN D -dependent transcription, by being recruited to ANDR target genes and mediating demethylation of H3K9me, a specific tag for epigenetic transcriptional repression. The presence of P R K C B in AN D -containing complexes, which mediates phosphorylation of “Thr-6” histone H3 (H3T6ph), a specific tag that prevents demethylation H3K4me, and prevents H 3K4me demethylase activity of KDM1 A . Demethylates dimethylated “ L ys-370” p53/TP53, which prevents interaction of p53/TP53 with TP53BP1 and represses p53/TP53-mediated transcriptional activation. Demethylates and stabilizes the D NA methylase N MT1. R equired for gastrulation during embryogenesis. C omponent of a RC O R - G F I -KDM1 A H D AC complex that suppresses, via recruitment, number genes implicated in multilineage blood cell development. N ecessary for the complete degradation of m RNA s, both in normal turnover and MD. R emoves the 7-methyl guanine cap structure from m RNA fragments shorter than ten nucleotides that are produced by 3 activity toward m RNA molecules longer than 25 nucleotides. L eukotriene A 4 hydrolase L iver carboxylesterase 1 Lysine-specific demethylase 4A Lysine-specific demethylase 5A Lysine-specific histone demethylase 1A m7 G pppX diphosphatase PD PK PD PD PD PD 3FU N 2 H 7 C , 1MX1 3 N JY 3 GL 6 2UXX 3B L 9

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4021 Dovepress He et al Dovepress 8.1 6.4 7 5.8 7.6 6.9 Docking score ------/M A PK14, α

-amylase activity is reduced because of α site, but has a preference for leucine. A romatic or hydrophobic residues are preferred ′ -butyl and cumene hydroperoxides. I s able to catalyze the glutathione-dependent oxygenation tert Function Tyrosine-protein kinase that acts as cell-surface receptor for CS F1 and IL 34 plays an essential role in the regulation of survival, proliferation and differentiation hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes. Promotes the release of proinflammatory chemokines in response to IL 34 and CS F1, thereby plays an important role innate immunity in inflammatory processes. Plays an important role the regulation of osteoclast proliferation and differentiation, the regulation of bone resorption, and is required for normal tooth development. R equired for normal male and female fertility, development of milk ducts acinar structures in the mammary gland during pregnancy. Promotes reorganization of actin cytoskeleton, regulates formation of membrane ruffles, cell adhesion and migration, promotes cancer invasion. Activates several signaling pathways in response to ligand binding. Phosphorylates P I K3 R 1, LCG 2, GR B2, SLA and C B L . A ctivation of P LCG 2 leads to the production cellular signaling molecules, such as D AG and inositol 1,4,5-trisphosphate, that then lead to the activation of PK C family members, especially P R K D. Phosphorylation of P I K3 R 1, the regulatory subunit phosphatidylinositol 3-kinase, leads to activation A KT1 signaling pathway. ctivated CS F1 R also mediates activation of the M P kinases PK1/ ER K2 and/or M A PK3/ ER K1, and of the SRC family kinases , FY N Y ES 1. ctivated CS F1 R transmits signals both via proteins that directly interact with phosphorylated tyrosine residues in its intracellular domain and via A Ps, such as GR B2. Promotes activation of S T A family members T3, T5 , and/or T5B. tyrosine phosphorylation of SHC 1 and IN PP5D/ SHI P-1. R eceptor signaling is downregulated by protein phosphatases, such as IN PP5D/ SHI P-1, that dephosphorylate the receptor and its downstream effectors by rapid internalization of the activated receptor. May be involved in tissue injury and remodeling. Has significant elastolytic activity. Can accept large small amino acids at the P1 at the P1 site, with small hydrophobic residues (preferably alanine) occupying P3. Proinflammatory cytokine. Involved in the innate immune response to bacterial pathogens. The expression of MIF at sites inflammation suggests a role as mediator in regulating the function macrophages host defense. Counteracts the anti-inflammatory activity of GCs. Has phenyl pyruvate tautomerase and dopachrome tautomerase activity (in vitro), but the physiological substrate is not known. I t clear whether the tautomerase activity has any physiological relevance, and it is important for cytokine activity. Bifunctional enzyme showing minimal glutathione-conjugating activity with ethacrynic acid and 7-chloro-4- nitrobenz-2-oxa-1,3-diazole and maleylacetoacetate isomerase activity. H as also low glutathione peroxidase activity with of dichloroacetic acid to glyoxylic acid. May serve as an alternate pathway for starch digestion when luminal immaturity or malnutrition. May play a unique role in the digestion of malted dietary oligosaccharides used food manufacturing. S tress-activated serine/threonine–protein kinase involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell-cycle control, chromatin remodeling, D NA damage response, and transcriptional regulation. Following stress, it is phosphorylated and activated by M A P kinase p38- leading to phosphorylation of substrates. Phosphorylates serine in the peptide sequence, H yd-X- R -X(2)- S , where H yd is a large hydrophobic residue. Phosphorylates AL OX5, C D 25B, 25 , ELA V L 1, HNRN P A 0, HS F1, P27/ PB1, K R T18, T20, LI MK1, LS P1, P A BP C 1, ARN , PD E 4 RCS D1, S 6K 3, T B3, Target name Macrophage CS F1 receptor Macrophage metalloelastase Macrophage migration inhibitory factor Maleylacetoacetate isomerase Maltase-glucoamylase, intestinal M A PK 2 ) Class PD PD PD PK PD PD Continued ( Table 2 PDB ID 2 I 1M 3F17 3 I J G 1FW1 3 L 4Y 3M2W

4022 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 6.9 7.1 7.4 - - - /M A PK14, leading to phosphorylation of substrates. Phosphorylates serine α /M checkpoint following D NA damage through a process of posttranscriptional m RNA stabilization: 2 and TTP/ZFP36. Mediates phosphorylation of HS P27/ PB1 in response to stress, leading dissociate HS P27/ PB1 from large s H sps oligomers and impair their chaperone activities ability to protect against oxidative stress effectively. Involved in inflammatory response by regulating TNF and IL6 production posttranscriptionally: acts by phosphorylating ARE -binding proteins ELA V L 1, HNRN P A 0, BP C and TTP/ ZFP36, leading to regulate the stability and translation of T N F IL 6 m RNA s. Phosphorylation TTP/ ZFP36, a major posttranscriptional regulator of T N F, promotes its binding to 14-3-3 proteins and reduces ARE mRNA affinity, leading to inhibition of dependent degradation ARE-containing transcript. Also involved in late G following D NA damage, it relocalizes from nucleus to cytoplasm and phosphorylates HNRN P A 0 ARN , leading to stabilize GA DD45 A m RNA . I nvolved in T LR signaling pathway D C s: required for acute - induced macropinocytosis by phosphorylating and activating R P S 6K A 3. S tress-activated serine/threonine–protein kinase involved in cytokines production, endocytosis, cell migration, chromatin remodeling, and transcriptional regulation. Following stress, it is phosphorylated activated by M A P kinase p38- in the peptide sequence, H yd-X- R -X(2)- S , where yd is a large hydrophobic residue. M A PK PK2 and MAPKAPK3 share the same function and substrate specificity, but kinase activity level in protein expression are lower compared to M A PK PK2. Phosphorylates HS P27/ PB1, K R T18, T20, RCS D1, R P S 6K A 3, T B3, and TTP/ZFP36. Mediates phosphorylation of HS P27/ PB1 in response to stress, leading to dissociate HS P27/ PB1 from large s H sps oligomers and impair their chaperone activities ability to protect against oxidative stress effectively. Involved in inflammatory response by regulating TNF and IL 6 production posttranscriptionally: acts by phosphorylating ARE -binding proteins, such as TTP/ZFP36, leading to regulate the stability and translation of T N F IL 6 m RNA s. Phosphorylation TTP/ZFP36, a major posttranscriptional regulator of T N F, promotes its binding to 14-3-3 proteins and reduces ARE mRNA affinity, leading to inhibition of dependent degradation ARE-containing transcript. Involved in TLR signaling pathway in D C s: required for acute T LR -induced macropinocytosis by phosphorylating and activating R P S 6K A 3. lso acts as a modulator of polycomb-mediated repression. Tyrosine-protein kinase that acts as cell-surface receptor for the cytokine K I T LG / SC F and plays an essential role in the regulation of cell survival and proliferation, hematopoiesis, stem maintenance, gametogenesis, mast cell development, migration and function, in melanogenesis. I n response to K T LG / SC F binding, can activate several signaling pathways. Phosphorylates P I K3 R 1, LCG SH 2B2/ A S , and C B L . ctivates the A KT1 signaling pathway by phosphorylation of P I K3 R 1, the regulatory subunit phosphatidylinositol 3-kinase. A ctivated K I T also transmits signals via GR B2 and activation of R as, RA F1, the M P kinases PK1/ ER K2 and/or M A PK3/ ER K1. Promotes activation of S T family members T1, T3, T5 , and T5B. A ctivation of P LCG 1 leads to the production cellular signaling molecules D AG and inositol 1,4,5- trisphosphate. K I T signaling is modulated by protein phosphatases and rapid internalization degradation of the receptor. A ctivated K I T promotes phosphorylation protein phosphatases PTP N 6/ SH P-1and PTP R U, and of the transcription factors S T A T1, T3, T5 , T5B. Promotes phosphorylation P I K3 R 1, C B L , CR K (isoform rk- II ), Y N M A PK1/ ER K2, and/or PK3/ K1, LCG SRC and SHC 1. Degrades casein, gelatins of types I, III, IV, and V, fibronectin. Activates procollagenase. M A PK 3 Mast/stem cell growth factor receptor kit Matrilysin PD PD PD 3F HR 3 G 0 E 1MMQ

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4023 Dovepress He et al Dovepress 7.6 7 7.5 6.3 8.6 8.1 8.2 Docking score ------, F, F, CC K2 K2 ER 3, or or 3, S B CHE K1, K1, F2, D F2, R lso required required lso O EL K cascade. cascade. K A S , , 6, 6, ER N FX. FX. CL TM and and TM ARHGE A PK/ 2 A A H PT, PX PT, TF2, B TF2, A A F. Depending on the cellular cellular the on Depending F. SC P2, M P2, / A pproximately 160 substrates have have substrates 160 pproximately LG A T I 1, M 1, ; as well as in the fragmentation of the the of fragmentation the in as well as ; A J G , , NRC T and K and T N I TT C X, X, er-139” phosphorylation of histone histone of phosphorylation er-139” CAN S PKs that play an important role in the M the in role important an play that PKs A K2 kinases, and stabilization of TP53 and apoptosis. apoptosis. and TP53 of stabilization and kinases, K2 K cascade is also involved in the regulation of the endosomal dynamics, dynamics, endosomal the of regulation the in involved also is cascade K CHE ER damage marked by “ by marked damage Ks. Many of these substrates are localized in the nucleus and seem to participate participate to seem and nucleus the in localized are substrates these of Many Ks. K1 are the two M two the are K1 PK/ NA ER BP1), and a variety of other signaling-related molecules (such as as (such molecules signaling-related other of variety a and BP1), A ER K1, and and K1, E F4 K cascade mediates diverse biological functions such as cell growth, adhesion, survival, survival, adhesion, growth, cell as such functions biological diverse mediates cascade K EI CHE PK3/ A ER TM, TM, PK/ A PZ1), cytoskeletal elements (such as as (such elements cytoskeletal PZ1), S A K2 and M and K2 K cascade plays also a role in initiation and regulation of meiosis, mitosis, and postmitotic functions functions postmitotic and mitosis, meiosis, of regulation and initiation in role a also plays cascade K F4, or or F4, ER ER HS , , PK1/ PK/ S /M phases of the cell cycle. May serve as a scaffold for recruitment D NA repair and signal transduction A A olgi apparatus during mitosis. The substrates include transcription factors (such as as (such factors transcription include substrates The mitosis. during apparatus olgi 2 Function E ndopeptidase that degrades various components of the extracellular matrix, such as collagen type III and fibronectin. Activates progelatinase A. Involved in the matrix remodeling of blood vessels. Isoform Short collagens. V and IV, II, I, type on effect no has It rate. lower at but collagen, III type also and fibronectin cleaves H owever, upon interaction with CS P G 4, it may be involved in degradation and invasion of type I collagen by melanoma cells. Degrades amelogenin, the major protein component of enamel matrix and two macromolecules characterizing the cartilage extracellular matrix: aggrecan and C OMP. May play a central role in tooth enamel formation. May play an essential role in local proteolysis of the extracellular matrix and leukocyte migration. C ould play a role in bone osteoclastic resorption. C leaves Ki SS 1 at G ly-|- L eubond. type I V and collagens into large C -terminal three quarter fragments and shorter N one fragments. Degrades fibronectin but not laminin or Pz-peptide. R equired for checkpoint-mediated cell-cycle arrest in response to D NA damage within both the S phase and G D of foci discrete to proteins for downstream events subsequent to the recruitment of these proteins. These include phosphorylation and the of activation may also be activated independently by a parallel pathway mediated TP53BP1. R emoves the N -terminal methionine from nascent proteins. equired for normal progression through cell cycle. R emoves the N -terminal methionine from nascent proteins. The catalytic activity of human M E T A P2 toward Met-Val peptides is consistently two orders of magnitude higher than that M E T A P1, suggesting it responsible for processing proteins containing N -terminal Met-Val and Met-Thr sequences in vivo. Protects eukaryotic initiation factor EI F2 S 1 from translation-inhibiting phosphorylation by inhibitory kinases such as EI F2 A K2/PK R and K1/ HCR . Plays a critical role in the regulation of protein synthesis. S erine/threonine kinase that acts as an essential component of the M A P signal transduction pathway. M K activated by initiated cascade signaling a in also participate They M the context, The rearrangements. cytoskeletal and translation, transcription, of regulation the through differentiation and M factors. transcription of number a phosphorylating by cells differentiated in for discovered been already in the regulation of transcription upon stimulation. H owever, other substrates are found cytosol as and mitosis, translation, as such processes for responsible are those and organelles, cellular other in as well M the Moreover, apoptosis. P the through cycling endosome and processing lysosome including G FO S TM N 1), regulators of apoptosis (such as B A D, BT G 2, CAS P9, D PK1, IER 3, M CL 1, or PP ARG ), as (such translation of F RS 2, or GR B10). Protein kinases (such as RA F1, R P S 6K A 1/ K1, Target name Matrix metalloproteinase 16 Matrix metalloproteinase 20 Matrix metalloproteinase 9 Mediator of D NA damage checkpoint protein 1 Methionine aminopeptidase 1 Methionine aminopeptidase 2 M A PK1 ) Class PD PD PD PD PD PD PD Continued ( Table 2 PDB ID 1 R M8 2J S D 2OVX 3K05 2 N Q6 2O A Z 1PM E

4024 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 7.1 8.4 - - - A PP/ PP signaling β -induced genes. S eems to γ 200–300 substrates each. S ome of the targets are downstream ~ B isoform RELA / N FKB3, S T A T1, and T3, but can also phosphorylate histone H 3 κ bind to the promoter of CCL 5, DMP1, I F IH 1, TM1, IR F7, F9, LA MP3, O AS 2, 3, and S T A T1. Transcriptional activity is independent of kinase activity. during neuronal differentiation by phosphorylating A PP. Participates also in neurite growth spiral ganglion neurons. R P S 6K A 3/ RS K2, 2/ K3, 6/ K4, YK, MK N K1/M K1, K2/M 5/M R P S 6K A 4/M K2, M PK PK3, or PK5) and phosphatases (such as DU P1, P4, P6, DU S P16) are other substrates that enable the propagation M A PK/ ER K signal to additional cytosolic and nuclear targets, thereby extending the specificity of cascade. Acts as a transcriptional repressor. Binds to a [ GC ] AAA consensus sequence. R epress the expression of interferon S erine/threonine–protein kinase involved in various processes such as neuronal proliferation, differentiation, migration, and programmed cell death. Extracellular stimuli such as proinflammatory cytokines or physical stress stimulate the SAP/JNK signaling pathway. In this cascade, two dual specificity kinases MAP2K4/MKK4 and M A P2K7/MKK7 phosphorylate activate PK10/J N K3. I n turn, K3 phosphorylates a number of transcription factors, primarily components A P-1 such as JU N and TF2 thus regulates A P-1 transcriptional activity. Plays regulatory roles in the signaling pathways during neuronal apoptosis. Phosphorylates the neuronal microtubule regulator S TM N 2. A cts in regulation of S erine/threonine kinase that acts as an essential component of the M A P signal transduction pathway. M A PK14 is one of the four p38 PKs that play an important role in cascades cellular responses evoked by extracellular stimuli such as proinflammatory cytokines or physical stress, leading to direct activation of transcription factors. A ccordingly, p38 M PKs phosphorylate a broad range proteins, and it has been estimated that they may have kinases, which are activated through phosphorylation and further phosphorylate additional targets. R P S 6K A 5/ M S K1 and R P 6K A 4/M K2 can directly phosphorylate activate transcription factors such as CRE B1, A TF1, the N F- and the nucleosomal protein H M GN 1. R P S 6K A 5/M K1 4/M K2 play important roles in rapid induction of immediate-early genes in response to stress or mitogenic stimuli, either by inducing chromatin remodeling or by recruiting the transcription machinery. On other hand, two kinase targets, M A PK PK2/MK2 and PK3/MK3, participate in the control of gene expression mostly at the posttranscriptional level, by phosphorylating ZFP36 (tristetraprolin) and ELA V L 1, regulating EE F2K, which is important for the elongation of m RNA during translation. MK N K1/M K1 and K2/ M N K2, two other kinases activated by p38 A PKs, regulate protein synthesis phosphorylating the initiation factor EI F4 E 2. M A PK14 interacts also with casein kinase II , leading to its activation through autophosphorylation and further phosphorylation of TP53/p53. I n the cytoplasm, p38 M A PK pathway is an important regulator of protein turnover. For example, C F LAR is inhibitor T N F-induced apoptosis whose proteasome-mediated degradation is regulated by p38 M A PK phosphorylation. I n a similar way, M A PK14 phosphorylates the ubiquitin ligase SIAH 2, regulating its activity toward EGLN 3. may also inhibit the lysosomal degradation pathway of autophagy by interfering with intracellular trafficking transmembrane protein A T G 9. nother function of M PK14 is to regulate the endocytosis membrane receptors by different mechanisms that impinge on the small G TPase RA B5 A . I n addition, clathrin-mediated EGFR internalization induced by inflammatory cytokines and UV irradiation depends on MAPK14-mediated phosphorylation of EG F R itself as well RA B5 A effectors. E ctodomain shedding transmembrane proteins is regulated by p38 MAPKs as well. In response to inflammatory stimuli, phosphorylate the membrane-associated metalloprotease A D M17. S uch phosphorylation is required for M17-mediated M A PK10 M A PK14 PD PD 3D A 6 2QD9

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4025 Dovepress He et al Dovepress 6.7 Docking score - 1), 1), N

, S TM S TF2. TF2. K signal to to signal K F, FO F, A B10). Protein Protein B10). ER 3, or or 3, ER K cascade. cascade. K S B GR ER PK/ R K1, K1, A O EL PK/ S YK, MK N K1/M K1,S YK, , , A 2, or or 2, 6, 6, N RS CL F. Depending on the cellular cellular the on Depending F. F2, F F2, PT, PX PT, SC TF2, B TF2, A / A LG T ARHGE R P S 6K A 6/ RS K4, I B-binding sites marking promoters B-binding sites marking promoters P2, M P2, κ A lso plays an essential role in an essential role A lso plays 1, M 1, A T and K and T J I G , , N PKs are emerging as The p38 M A PKs are , and M E F2 A . TT R P S 6K A 2/ RS K3, C X, X, owever, other substrates are found in the cytosol as well as as well as cytosol the in found are substrates other owever, H PKs that play an important role in the M the in role important an play that PKs A CAN following T IAR following Phosphorylates radiation. after ultraviolet delay 2 R P S 6K A 3/ RS K2, P16) are other substrates that enable the propagation the M the propagation the enable that substrates other are P16) S family ligands, which results in the activation of EG F R signaling and cell which results ligands, family α K1 are the two M two the are K1 ER R P S 6K A 1/ RS K1, PK3/ P6, or DU or P6, A S that are required for binding to for Phosphorylates required C D 25B and 25 that are B recruitment. κ RA F1, BP1), and a variety of other signaling-related molecules (like (like molecules signaling-related other of variety a and BP1), T3, TP53/p53, M E F2 C TP53/p53, DD I T3, PTP RH , EL K1, TF6, 1 can be translocated from the extracellular F G R 1 can be translocated from A nother p38 M PK substrate is F G R 1. P4, DU P4, E A S K2 and M and K2 PZ1), cytoskeletal elements (such as as (such elements cytoskeletal PZ1), F4 K cascade plays also a role in initiation and regulation of meiosis, mitosis, and postmitotic functions functions postmitotic and mitosis, meiosis, of regulation and initiation in role a also plays cascade K S EI ER TF2, TF2, ER A P1, DU P1, S PK1/ PK/ F4, or or F4, A A TF1, TF1, Function T G F- of ectodomain shedding proliferation. such as r RNA synthesis and cell processes of target cells and regulates space into the cytosol and nucleus transcription factors are many I n the nucleus, p38 M A PK activation. F G R 1 translocation requires growth. C lassic examples include stimuli. to different phosphorylated p38 M A PKs in response and activated by A The promoters remodelers. modifiers and chromatin regulating by important modulators of gene expression a p38 MAPK- display and IL-12B, IL8, such as IL6, in the inflammatoryresponse, genes involved of several myeloid ( H 3 S 10ph) in L P -stimulated “ S er-10” dependent enrichment of histone H 3 phosphorylation on This phosphorylation enhances the accessibility of cryptic N F- cells. for increased N F- increased for and lead to initiation of a G 14-3-3 proteins The p38 M A PKs m RNA degradation. GA DD45 A m RNA and preventing T IAR from releasing D NA damage, thought to be due the binding targets in absence which are kinase-independent roles, also have may although and, M A PK14, by the O G T is regulated by G lc NA cylation catalyzed Protein of phosphorylation. upon M A PK14 activation their interaction increases O G T does not seem to be phosphorylated M A PK14, by it to specific targets recruiting OGT activity by regulate This interaction may glucose deprivation. induced by for the growth development in mid-fetal Required its GlcNAcylation. stimulating H, such as neurofilament of the placenta. layer in the labyrinth blood vessels of embryo-derived MX I 2 I soform of E PO gene expression. regulation through erythropoiesis, and stress-induced developmental phosphorylates poorly and only EL K1 and stress mitogens and oxidative by activation is stimulated “Thr-113”. Phosphorylates S 100 A 9 at onset of apoptosis. in the early a role play E X I P may I soform S erine/threonine kinase that acts as an essential component of the M A P signal transduction pathway. M K activated by initiated cascade signaling a in also participate They context, the M A PK/ ER K cascade mediates diverse biological functions such as cell growth, adhesion, survival, The rearrangements. cytoskeletal and translation, transcription, of regulation the through differentiation and M in differentiated cells by phosphorylating a number of transcription factors. A pproximately 160 substrates have already been discovered for ER Ks. Many of these substrates are localized in the nucleus and seem to participate stimulation. upon transcription of regulation the in apoptosis. and mitosis, translation, as such processes for responsible are those and organelles, cellular other in Moreover, the M A PK/ ER K cascade is also involved in regulation of endosomal dynamics, including lysosome processing and endosome cycling through the P NRC as well in fragmentation of G olgi as (such factors transcription include substrates The mitosis. during apparatus HS regulators of apoptosis (such as B A D, BT G 2, CAS P9, D PK1, IER 3, M CL 1, or PP ARG ), translation as (such as (such kinases MK N K2/M K2, R P S 6K A 5/M K1, 4/M M PK PK3, or PK5) and phosphatases (such as DU additional cytosolic and nuclear targets, thereby extending the specificity of cascade. Target name M A PK3 ) Class PD Continued ( Table 2 PDB ID 2ZOQ

4026 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 6.9 8.1 7.1 - - - -2 β -2, and α

-1, α -2 bind to A TF2. H owever, there is β -1 and β -1 preferentially binds to c-Jun, whereas β -2, and JU N D binds only weakly to it. α -catenin/ C T NN B1 degradation and inhibits the canonical Wnt signaling pathway. β -2 preferentially bind to JU N , whereas α -1 and α have a similar low level of binding to both c-Jun or A TF2. H owever, there is no correlation between and phosphorylation, which is achieved at about the same efficiency by all isoforms. no correlation between binding and phosphorylation, which is achieved at about the same efficiency by all isoforms. JU N B is not a substrate for J K2 S erine/threonine–protein kinase involved in various processes such as cell proliferation, differentiation, migration, transformation, and programmed cell death. Extracellular stimuli such as proinflammatory cytokines or physical stress stimulate the SAP/JNK signaling pathway. In this cascade, two dual specificity kinases M A P2K4/MKK4 and P2K7/MKK7 phosphorylate activate PK8/J N K1. I n turn, PK8/ J N K1 phosphorylates a number of transcription factors, primarily components A P-1 such as JU , JDP2, and A TF2 thus regulates P-1 transcriptional activity. Phosphorylates the replication licensing factor C DT1, inhibiting the interaction between DT1 and histone H 4 acetylase BO1 to replication origins. L oss of this interaction abrogates the acetylation required for replication initiation. Promotes stressed cell apoptosis by phosphorylating key regulatory factors including p53/TP53 and Yes-associates protein Y A P1. I n T-cells, M A PK8 and PK9 are required for polarized differentiation of T-helper cells into Th1 cells. C ontributes to the survival of erythroid cells by phosphorylating antagonist cell death B A D upon E PO stimulation. Mediates starvation-induced B CL 2 phosphorylation, dissociation from ECN 1, and thus activation of autophagy. Phosphorylates S TM N 2 and hence regulates microtubule dynamics, controlling neurite elongation in cortical neurons. I n the developing brain, through its cytoplasmic activity on S TM N 2, negatively regulates the rate of exit from multipolar stage and radial migration ventricular zone. Phosphorylates several other substrates including HS F4, the deacetylase SIR T1, EL K1, or E 3 ligase I T CH . J N K1 isoforms display different binding patterns: S erine/threonine–protein kinase involved in various processes such as cell proliferation, differentiation, migration, transformation, and programmed cell death. Extracellular stimuli such as proinflammatory cytokines or physical stress stimulate the SAP/JNK signaling pathway. In this cascade, two dual specificity kinases M A P2K4/MKK4 and P2K7/MKK7 phosphorylate activate PK9/J N K2. I n turn, PK9/ J N K2 phosphorylates a number of transcription factors, primarily components A P-1 such as JU and A TF2 and thus regulates P-1 transcriptional activity. I n response to oxidative or ribotoxic stresses, inhibits rRNA synthesis by phosphorylating and inactivating the RNA polymerase 1-specific transcription initiation factor RRN 3. Promotes stressed cell apoptosis by phosphorylating key regulatory factors including TP53 and Y A P1. I n T-cells, M PK8 and PK9 are required for polarized differentiation of T-helper cells into Th1 cells. Upon T CR stimulation, is activated by CAR M A 1, B CL 10, P2K7, and P3K7/T K1 to regulate JU N protein levels. Plays an important role in the osmotic stress-induced epithelial tight-junctions disruption. When activated, promotes Participates also in neurite growth spiral ganglion neurons. M A PK9 isoforms display different binding patterns: S erine/threonine kinase that acts as an essential component of the M A P signal transduction pathway. Plays an important role in the cascades of cellular responses evoked by changes environment. Once activated, acts as an upstream activator of the MKK/J N K signal transduction cascade through phosphorylation of M A P2K4/MKK4 and P2K7/MKK7 which in turn activate the J N Ks. The MKK/J K signaling pathway regulates stress response via activator protein-1 (JU N ) and GA T A 4 transcription factors. Plays also a role in mitochondrial death signaling pathway, including the release cytochrome c, leading to apoptosis. M A PK8 M A PK9 M A PK9 PD PD PD 3 EL J 3 E 7O 3DT C

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4027 Dovepress He et al Dovepress 9.0 7.3 - - 6.8 5.9 8.7 8.7 to 8.7 10 10.0 to 8.2 8.9 5.8 Docking score ------arginine, which act as inhibitors of l -o-glucuronide, methotrexate, antiviral β -monomethyl- G -arginine ( A DM ) and N l -acetylaspartyl glutamate. -dimethyl- ′ N G , N -carboxylation of glutamate residues in prothrombin synthesis. G γ drugs and other xenobiotics. Confers resistance to anticancer drugs. Hydrolyzes ATP with low efficiency. Function Mediates export of organic anions and drugs from the cytoplasm. A TP-dependent transport glutathione and conjugates, leukotriene C 4, estradiol-17- H ydrolyzes N N O S . H as therefore a role in the regulation of generation. H as NAALA Dase activity. A lso exhibits a dipeptidyl-peptidase I V type nactivate the peptide neurotransmitter The enzyme apparently serves as a quinone reductase in connection with conjugation reactions of hydroquinones involved in detoxification pathways as well biosynthetic processes such the vitamin K-dependent N / A NAD-dependent lysine demalonylase and desuccinylase that specifically removes malonyl succinyl groups on target proteins. A ctivates C P S 1 and contributes to the regulation of blood ammonia levels during prolonged fasting: acts by mediating desuccinylation of C P S 1, thereby increasing 1 activity in response to elevated NA D levels during fasting. H as weak D-dependent protein deacetylase activity; however, this activity may not be physiologically relevant in vivo. Deacetylates cytochrome c ( C Y CS ) and a number of other proteins in vitro. This enzyme is required for electron transfer from NA DP to cytochrome P450 in microsomes. I t can also provide electron transfer to heme oxygenase and cytochrome B5. Regulatory subunit of the dimeric UBA3-NAE1 E1 enzyme. activates NEDD8 by first adenylating its C -terminal glycine residue with A TP, thereafter linking this to the side chain of catalytic cysteine, yielding a NEDD8-UBA3 thioester and free AMP. E1 finally transfers NEDD8 to the catalytic cysteine of UB E 2M. N ecessary for cell-cycle progression through the S –M checkpoint. Overexpression of NAE 1 causes apoptosis through deregulation of NE DD8 conjugation. Thermolysin-like specificity, but is almost confined on acting polypeptides of up to 30 amino acids. Biologically important in the destruction of opioid peptides such as Met- and L eu-enkephalins by cleavage a G ly-Phe bond. A ble to cleave angiotensin-1, angiotensin-2 and angiotensins 1–9. I nvolved in the degradation of ANF. Displays UV-inducible elastase activity toward skin preelastic and elastic fibers. in system, of the cardiovascular in the development involved 1 is a receptor The membrane-bound isoform and in organogenesis outside the nervous system. circuits of certain in the formation neuronal angiogenesis, the P LG F-2 isoform 3 A , I t binds to semaphorin activity of semaphorins. I t mediates the chemo-repulsant in increased with KD R results C oexpression V EG F-B. V EG F and of V EG F-165 isoform and the of P G F, The V EG F-induced angiogenesis. regulate I t may chemotaxis. as increased V EG F-165 binding to KD R as well also induce apoptosis I t may to inhibit its binding cells. V EG F-165 and appears 2 binds soluble isoform has an I ts expression family. various members of this maphorin bind as well May V EG F-165. sequestering by and integrity. number on blood vessel effect adverse -linked acidic dipeptidase 2 α ( G )-dimethylarginine N ( G ), Target name Multidrug resistance-associated protein 1 N dimethylaminohydrolase 1 N -acetylated- NA D(P) H dehydrogenase (quinine) 1 NA D-dependent malic enzyme, mitochondrial NA D-dependent protein deacylase sirtuin-5, mitochondrial NA DP H -cytochrome P450 reductase NE DD8-activating enzyme E 1 regulatory subunit N eprilysin N europilin-1 ) Class PK PD PD PK PD PD PK PD PD PD Continued ( Table 2 PDB ID 2 C BZ 3 I 4 A 3F E D 1KBQ_2, 1 H 69, 1KBQ, 1D4 A 1 G Z4 2 N Y R 3Q E 2, 3Q E 2_2, 3Q E 2_3, 3QF S _2, 3QFT, 3QFT_2, 3QF S 3 G Z N 1 R H 3 I 97

4028 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 7.5 8.6 7 9.2 6.3 to 14.0 6.8 7.8 7 8.4 7.3 ------′ -cap of pre-m RNA s and is involved in ′ chain. direction through the nuclear pore. The C B is also involved ′ α –3 ′ receptor β / α -methyltransferase that monomethylates both histones and nonhistone proteins. Specifically N various processes such as pre-m RNA splicing, translation regulation, N MD, -mediated genesilencing ( RNA i) by mi s, and m export. The C B is involved in export from the nucleus via its interaction with AL Y RE F/T H O C 4/ Y, leading to the recruitment of m RNA export machinery 5 end of m RNA and to export in a 5 in mediating U sn RNA and intron less m s export from the nucleus. The C B is essential for a pioneer round of m RNA translation, before steady-state translation when the C B is replaced by cytoplasmic cap- binding protein e I F4 E . The pioneer round of m RNA translation mediated Can degrade fibrillar type I, II, and III collagens. C omponent of the NA DP H -oxidase, a multicomponent enzyme system responsible for oxidative burst in which electrons are transported from NA DP H to molecular oxygen, generating reactive oxidant intermediates. I t may be important for the assembly and/or activation of NA DP H -oxidase complex. Modifies the functions of NK cells, monocytes, and granulocytes. Inhibits C5a-dependent neutrophil enzyme release and chemotaxis. Iron-trafficking protein involved in multiple processes such as apoptosis, innate immunity, and renal development. Binds iron through association with 2,5-D H B A , a side rophore that shares structural similarities with bacterial enterobactin, and delivers or removes iron from the cell, depending on context. I ron-bound form (holo-24p3) is internalized following binding to the SLC 22 A 17 (24p3 R ) receptor, leading release of iron and subsequent increase intracellular concentration. I n contrast, association the iron-free form (apo-24p3) with the SLC 22 A 17 (24p3 R ) receptor is followed by association an intracellular side rophore, iron chelation, and transfer to the extracellular medium, thereby reducing intracellular concentration. I nvolved in apoptosis due to IL 3 deprivation: iron-loaded form increases intracellular iron concentration without promoting apoptosis, while iron-free form decreases intracellular iron levels, inducing expression of the proapoptotic protein B CL 2 L 11/B I M, resulting in apoptosis. nvolved innate immunity, possibly by sequestrating iron, leading to limit bacterial growth. C atalyzes the N -methylation of nicotinamide and other pyridines to form pyridinium ions. This activity is important for biotransformation of many drugs and xenobiotic compounds. C atalyzes the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield mononucleotide, an intermediate in the biosynthesis of NA D. I t is rate-limiting component mammalian NA D biosynthesis pathway (by similarity). Protein-lysine monomethylates “ L ys-20” of histone H 4 ( 4K20me1). 4K20me1 is enriched during mitosis and represents a specific tag for epigenetic transcriptional repression. Mainly functions in euchromatin regions, thereby playing a central role in the silencing of euchromatic genes. R equired for cell proliferation, probably by contributing to the maintenance of proper higher order structure D NA during mitosis. I nvolved in chromosome condensation and proper cytokinesis. N ucleosomes are preferred as substrate compared to free histones. Mediates monomethylation of p53/TP53 at “ L ys-382”, leading to repress p53/TP53-target genes. Probably involved in intracellular signal transduction by being the initiation of type I F N signaling. Phosphorylates the interferon- This is a nonsecretory ribonuclease. It pyrimidine-specific nuclease with slight preference for U. C ytotoxin and helminthotoxin. S electively chemotactic for D s. Possesses a wide variety of biological activities. C omponent of the B , which binds cotranscriptionally to 5 -methyltransferase N - L ysine methyltransferase SE TD8 N eutrophil collagenase N eutrophil cytosol factor 4 N eutrophil elastase N eutrophil gelatinase-associated lipocalin N icotinamide N icotinamide phosphoribosyl transferase N N onreceptor tyrosine-protein kinase TYK2 N onsecretory ribonuclease N uclear cap-binding protein subunit 1 PD PD PD PD PK PD PD PD PD PD 1Z S 0 1 H 6 1 H 1B 3D S Z 2 II P, 3 R OD, 3 R OD_2 3DKJ 3F9Y 3 N YX 2BZZ 1 H 2T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4029 Dovepress He et al Dovepress 8.1 6.5 - - 9.4 to 6.8 to 6.3 5.8 8.9 7.8 7.4 7.1 Docking score ------

2 and alcohol β and 4-nt spacing (D R -4). egulates cholesterol uptake through MY LI P- ′ - AGG T CA -3 ′ exonuclease activities. I nvolved in cell proliferation, differentiation and development, signal ′ -5 ′ dehydrogenase 3 genes. Transactivates both the phenobarbital responsive element module of human C YP2B6 gene and the YP3 A 4 xenobiotic response element. Function by the C B plays a central role in N MD, MD only taking place m RNA s bound to , but not one I F4 E -bound m RNA s. The C B enhances N MD in s containing at least one J via its interaction with UPF1, promoting the interaction between UPF1 and UPF2. The C B is also involved in “failsafe” N MD, which is independent of the E J C , while it does not participate in S MD. During cell proliferation, B complex also involved in mi RNA s biogenesis via its interaction with SRR T/ ARS 2 and is required for -mediated RNA interference. The C B also acts as a negative regulator of P ARN , thereby acting an inhibitor m RNA deadenylation. I n the C B , NC BP1/ BP80 does not bind directly capped s (m7 G ppp -capped RNA ) but is required to stabilize the movement of N -terminal loop NC BP2/ C BP20 and lock B into a high affinity cap-binding state with the cap structure. N uclear receptor that binds and is activated by variety of endogenous xenobiotic compounds. Transcription factor that activates the transcription of multiple genes involved in metabolism and secretion of potentially harmful xenobiotics, drugs, and endogenous compounds. A ctivated by the antibiotic rifampicin and various plant metabolites, such as hyperforin, guggulipid, colupulone, isoflavones. Response to specific ligands is species specific. Activated by naturally occurring steroids, such as pregnenolone and progesterone. Binds to a response element in the promoters of C YP3 A 4 B B1/ MD R 1 genes. Binds and transactivates the RARE s that control expression of retinoic acid receptor I nvolved in the nuclear export of m RNA species bearing retroviral C T E s and from nucleus to the cytoplasm. The N XF1– XT1 heterodimer is involved in export of HS P70 m RNA in conjunction with AL Y RE F/T H O C 4 and T 5. Major role in the synthesis of nucleoside triphosphates other than A TP. Possesses nucleoside-diphosphate kinase, serine/threonine-specific protein geranyl and farnesylpyrophosphate histidine kinase, and 3 transduction, G protein-coupled receptor endocytosis, and gene expression. R equired for neural development including neural patterning and cell fate determination. Major role in the synthesis of nucleoside triphosphates other than A TP. N egatively regulates R ho activity by interacting with AKAP13/LBC. Acts as a transcriptional activator of the MYC gene; binds DNA nonspecifically (PubMed:8392752). E xhibits histidine protein kinase activity. N / A Orphan receptor. I nteraction with R X shifts fromits role as a silent D NA -binding partner to an active ligand-binding subunit in mediating retinoid responses through target genes defined by LXRES. LXRES are D R 4-type response elements characterized by direct repeats of two similar hexanuclotide half-sites spaced four nucleotides. Plays an important role in the regulation of cholesterol homeostasis, regulating uptake through MY LI P-dependent ubiquitination of L D LR , V and P8 (by similarity). Orphan receptor. Binds preferentially to double-stranded oligonucleotide direct repeats having the consensus half-site sequence 5 dependent ubiquitination of L D LR , V and P8 (by similarity). α β Target name N uclear receptor subfamily 1 group I member 2 N uclear receptor subfamily 1 group I member 3 N uclear RNA export factor 1 N ucleoside diphosphate kinase A N ucleoside diphosphate kinase B Ornithine decarboxylase Oxysterols receptor L X R - Oxysterols receptor L X R - ) Class PD PK PD PD PD PD PD PD Continued ( Table 2 PDB ID 1M13, 1 NRL , 3 H V L 1XV9, 1XVP 1O AI 1U CN 3BBB 2O N 3 3 I PQ 3KF C

4030 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 5.8 6.1 6.4 - - - Continued ( 9.6 7 7.3 7.9 8 5.6 10 6.9 7.2 to 8.3 to 7.6 to 8.8 ------

-linoleic acid and γ -oxidation pathway of fatty acids. Functions as β -oxidation pathway of fatty acids. Key regulator β type I serine/threonine kinase receptor, preventing β -oxidation pathway for fatty acids. C atalyzes the formation of β -oxidation pathway of fatty acids. Functions as transcription activator for the acyl- β -glycerol-3-phosphocholine (16:0/18:1- G P C ). A ctivated by oleylethanolamide, sn B-mediated proinflammatory responses. κ receptor activation in absence of ligand. R ecruits S M A D7 to AC V 1B which prevents the association β transcription activator for the AC OX1 and P450 genes. Transactivation activity requires heterodimerization with R X RA and is antagonized by NR 2 C 2. eicosapentanoic acid. Once activated by a ligand, the receptor binds to promoter elements of target genes. R egulates the peroxisomal C o A oxidase gene. Decreases expression of N P 1 L once activated by a ligand. N / A Putative A TP-dependent zinc metalloprotease. PP I ases accelerate the folding of proteins. t catalyzes cis–trans isomerization proline imidic peptide bonds in oligopeptides. Keeps in an inactive conformation T G FB R 1, the F- T G F- of S M A D2 and D3 with the activin receptor complex, thereby blocking signal. May modulate the R Y 1 calcium channel activity. PP I ases accelerate folding of proteins. t catalyzes cis–trans isomerization of proline imidic peptide bonds in oligopeptides. FKBPs and rapamycin-binding proteins constitute a family of receptors for the two immunosuppressants that inhibit T-cell proliferation by arresting two distinct cytoplasmic signal transmission pathways. PP I ases accelerate the folding of proteins. E ssential PP I ase that regulates mitosis presumably by interacting with NI M A and attenuating its mitosis- promoting activity. Displays a preference for an acidic residue N -terminal to the isomerized proline bond. C atalyzes p S er/Thr-Pro cis–trans isomerizations. Downregulates kinase activity of BTK. an transactivate multiple oncogenes and induce centrosome amplification, chromosome instability, cell transformation. Required for the efficient dephosphorylation and recycling of RAF1 after mitogen activation. Bifunctional enzyme acting on the peroxisomal 3-ketoacyl- C o A intermediates from both straight-chain and 2-methyl-branched-chain fatty acids. Binds to the C -terminal PT S 1-type tripeptide peroxisomal targeting signal ( K L -type) and plays an essential role in peroxisomal protein import. L igand-activated transcription factor. Key regulator of lipid metabolism. A ctivated by the endogenous ligand 1-palmitoyl-2-oleoyl- a naturally occurring lipid that regulates satiety (by similarity). R eceptor for peroxisome proliferators such as hypolipidemic drugs and fatty acids. R egulates the peroxisomal R eceptor that binds peroxisome proliferators such as hypolipidemic drugs and fatty acids. Once activated by a ligand, the receptor binds to promoter element in gene for acyl- C o A oxidase and activates its transcription. I t therefore controls the peroxisomal of adipocyte differentiation and glucose homeostasis. A cts as a critical regulator gut homeostasis by suppressing N F- L igand-activated transcription factor. R eceptor that binds peroxisome proliferators such as hypolipidemic drugs and fatty acids. H as a preference for poly-unsaturated acids, such Catalyzes direct attachment of p-Tyr (Tyr) to tRNAPhe. Permits also, with a lower efficiency, the of m-Tyr to t RNA Phe, thereby opening the way for delivery misacylated ribosome and incorporation of R O S -damaged amino acid into proteins. isomerase A isomerase FKBP3 isomerase NI M A - isomerase FKBP1 A isomerase cis–trans cis–trans cis–trans cis–trans -amylase α α δ Pancreatic Paraplegin Peptidyl-prolyl Peptidyl-prolyl Peptidyl-prolyl Peptidyl-prolyl interacting 1 Peroxisomal multifunctional enzyme type 2 Peroxisomal targeting signal 1 receptor Peroxisome proliferator-activated receptor PP ARG Peroxisome proliferator-activated receptor Phenylalanine–t RNA ligase, mitochondrial PD PK PD PD PD PD PD PD PK PK PK PD 1U33 2QZ4 1Y N D 1FKB 1PBK 3 I 6 C 1 I KT 1F CH 3KDU, 2Z NN , 2P54 3B1M, 3 L MP, 3U9Q, 3V9V, 3 H 0 A 2 A W H , 3TKM, 3 G Z9 3 H FV

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4031 Dovepress He et al Dovepress 6.7 to 19.9 6.9 9.2 Docking score - - -

. -adrenergic receptor endocytosis. May also have a β -adrenergic receptor internalization by binding to A D R BK1 β -3 integrins ( I T GA 2B/ G B3) adhesive function in platelets downstream of β - II b/ I ns(1,3,4,5)P4. The lipid phosphatase activity is critical for its tumor suppressor α . complex is activated by RA P GE F3 and which involved in angiogenesis. γ Ptd I ns3P . Function C onverts noradrenaline to adrenaline. Tumor suppressor. Acts as a dual-specificity protein phosphatase, dephosphorylating tyrosine-, serine- and threonine-phosphorylated proteins. A lso acts as a lipid phosphatase, removing the phosphate in D3 position of the inositol ring from P I P3, phosphatidylinositol 3,4-diphosphate, 3-phosphate, and inositol 1,3,4,5-tetrakisphosphate with order of substrate preference in vitro Ptd I ns(3,4,5)P3 Ptd I ns(3,4)P2 function. A ntagonizes the P I 3K- KT/PKB signaling pathway by dephosphorylating phosphoinositides, and thereby modulating cell-cycle progression and cell survival. The unphosphorylated form cooperates with AI P1 to suppress A KT1 activation. Dephosphorylates tyrosine-phosphorylated focal adhesion kinase and inhibits cell migration and integrin-mediated spreading focal adhesion formation. Plays a role as a key modulator of the A KT-mTO R signaling pathway controlling tempo process newborn neurons integration during adult neurogenesis, including correct neuron positioning, dendritic development, and synapse formation. May be a negative regulator of INS signaling glucose metabolism in adipose tissue. The nuclear monoubiquitinated form possesses greater apoptotic potential, whereas the cytoplasmic nonubiquitinated form induces less tumor suppressive ability. P I 3K that phosphorylates Ptd ns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) to generate P3. P3 plays a key role by recruiting P H domain-containing proteins to the membrane, including A KT1 and PDPK1, activating signaling cascades involved in cell growth, survival, proliferation, motility, and morphology. L inks GPCR activation to PIP3 production. Involved in immune, inflammatory, and allergic responses. Modulates leukocyte chemo taxis to inflammatory sites and in response attractant agents. May control leukocyte polarization and migration by regulating the spatial accumulation of P I P3 organization of F-actin formation and integrin-based adhesion at the leading edge. C ontrols motility D s. Together with PIK3CD is involved in NK cell development and migration toward the sites of inflammation. Participates in T-lymphocyte migration. R egulates proliferation and cytokine production. Together with P I K3 C D participates in T-lymphocyte development. R equired for B-lymphocyte development and signaling. Together with P I K3 C D participates in neutrophil respiratory burst. is involved in neutrophil chemotaxis and extra vasation. Together with P I K3 C B promotes platelet aggregation and thrombosis. R egulates P2Y12 through a lipid kinase activity-independent mechanism. May have also activity-dependent function in platelet aggregation. I nvolved endothelial progenitor cell migration. N egative regulator of cardiac contractility. Modulates cardiac contractility by anchoring PK A and PD E 3B activation, reducing c MP levels. R egulates cardiac contractility also by promoting and by nonmuscle tropomyosin phosphorylation. A lso exhibits has serine/threonine protein kinase activity: both lipid and protein kinase activities are required for scaffolding role in modulating cardiac contractility. C ontributes to hypertrophy under pathological stress. Through simultaneous binding of PD E 3B to RA P GE F3 and I K3 R 6 is assembled in a signaling complex in which the P I 3K isoform γ -methyltransferase N Target name Phenylethanolamine PIP3-phosphatase and dual-specificity protein phosphatase PT EN Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit ) Class PK PD PD Continued ( Table 2 PDB ID 2 G 8 N , 3KQ S , 2 G 72, 2 G 72_2, 2 G 8 N _2, 3KQ S _2 1D5 R 3 L J3

4032 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 7.1 8 8.6 8.1 8.4 7.2 6.6 7.3 10.4 6.2 ------

-UT R , possibly via its ′ -phosphoglycerides. sn -end cap structure during ′ -endpoly( A ) tail and the 5 ′ / S cell-cycle progression without interfering with centrosome duplication. Binds D NA . May 1 -Exoribonuclease that has a preference for poly(A) tails of mRNAs, thereby efficiently degrading ′ degradation, the interaction with cap structure being required for an efficient degradation of poly(A) tails. I nvolved in N MD, a critical process of selective degradation m RNA s that contain premature stop codons. A lso involved in degradation of inherently unstable m RNA s that contain ARE their 3 interaction with K HSR P. Probably mediates the removal of poly( A ) tails ARE s m RNA s, which constitutes the first step of destabilization. C atalyzes the conversion of O AA to P E P, rate-limiting step in metabolic pathway that produces glucose from lactate and other precursors derived the citric acid cycle. Thought to participate in the regulation of phospholipid metabolism biomembranes including eicosanoid biosynthesis. C atalyzes the calcium-dependent hydrolysis of 2-acyl groups in 3- I nvolved in the maintenance of ubiquitin levels (by similarity). Possible transcriptional coregulator. May contribute to the regulation of cellular processes via its interaction with BCL3. May be required for efficient terminal myeloid maturation of hematopoietic cells. play a role in the regulation of cell migration. May promote apoptosis when overexpressed. H as quercetin 2,3- dioxygenase activity (in vitro). Plasmin dissolves the fibrin of blood clots and acts as a proteolytic factor in variety other processes including embryonic development, tissue remodeling, tumor invasion, and inflammation. In ovulation, weakens several and collagenases activator, plasminogen urokinase-type the activates It follicle. Graafian the of walls the complement zymogens, such as C1 and C5. Cleavage of fibronectin laminin leads to cell detachment and apoptosis. Also cleaves fibrin, thrombospondin, von Willebrand factor. Its role in tissue remodeling and tumor invasion may be modulated by CS P G 4. Binds to cells. A ngiostatin is an angiogenesis inhibitor that blocks neovascularization and growth of experimental primary metastatic tumors in vivo. S erine protease inhibitor. This inhibitor acts as “bait” for tissue plasminogen activator, urokinase, protein C , and matriptase-3/TMP RSS 7. I ts rapid interaction with P LA T may function as a major control point in the regulation of fibrinolysis. Major PK C substrate of platelets. I nvolved in the B ER pathway, by catalyzing poly( A DP-ribosyl)ation of a limited number acceptor proteins involved in chromatin architecture and DNA metabolism. This modification follows damages and appears as an obligatory step in a detection/signaling pathway, leading to the reparation of D NA strand breaks. I nvolved in the B ER pathway, by catalyzing poly( A DP-ribosyl)ation of a limited number acceptor proteins involved in chromatin architecture and DNA metabolism. This modification follows damages and appears as an obligatory step in a detection/signaling pathway, leading to the reparation of D NA strand breaks. May link the DNA damage surveillance network to mitotic fidelity checkpoint. Negatively influences the G be involved in the regulation of P RC 2 and 3 complex-dependent gene silencing. 3 tails. Exonucleolytic degradation of the poly(A) tail is often first step in decay eukaryotic mRNAs and is also used to silence certain maternal m RNA s translationally during oocyte maturation early embryonic development. I nteracts with both the 3 P E carboxykinase, cytosolic ( G TP) Phospholipase A 2, membrane associated Phospholipase A 2-activating protein Pirin Plasminogen Plasminogen activator inhibitor 1 Pleckstrin P AR 2 P AR 3 Poly(A)-specific ribonuclease PARN PD PD PD PD PD PD PD PD PD PD 1 NH X 1KVO 3 E BB 3 ACL 1 CEA 1 A 7 C 2 I 5F 3KJD 3 C 4 H 3 C T R

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4033 Dovepress He et al Dovepress 11.3 6.7 6 7.8 8.2 6.9 8.2 10.3 Docking score ------30 amino ~ to directly regulate its γ subunit is responsible for peptide binding. β subunits ( SCNN 1 G ). γ -nucleotidase activity in D RG neurons. G enerates adenosine from A MP that acts as a ′ Function I nvolved in the B ER pathway, by catalyzing poly( A DP-ribosyl)ation of a limited number acceptor proteins involved in chromatin architecture and DNA metabolism. This modification follows damages and appears as an obligatory step in a detection/signaling pathway leading to the reparation of D NA strand breaks. Mediates the poly( A DP-ribosyl)ation of P L F and CH R . Positively regulates transcription MTU S 1 and negatively regulates the transcription of 2/T I P150. With EE F1 A TXK, forms a complex that acts as a Th1 cell-specific transcription factor and binds the promoter of IFN- transcription, and is thus involved importantly in Th1 cytokine production. Pc G protein. C omponent of the P RC 2/ EE D- E Z H 2 complex, which methylates “ L ys-9” and ys-27” histone H 3, leading to transcriptional repression of the affected target gene. A lso recognizes “ L ys-26” trimethylated histone H 1 with the effect of inhibiting P RC 2 complex methyltransferase activity on nucleosomal 3 “ L ys-27”, whereas H 3 ys-27” recognition has the opposite effect, enabling propagation of this repressive mark. The P RC 2/ EE D- E Z H 2 complex may also serve as a recruiting platform for D NA methyltransferases, thereby linking two epigenetic repression systems. G enes repressed by the P RC 2/ EE D- E Z H 2 complex include H OX C 8, A 9, MYT1, and DK N 2 . The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression affect cellular proliferation and differentiation in target tissues. P R B is involved activation of c- SRC /M A PK signaling on hormone stimulation. I soform A is inactive in stimulating c- S rc/M PK stimulation. C lass E VP S protein involved in concentration and sorting of cargo proteins the MVB for incorporation into IL Vs that are generated by invagination and scission from the limiting membrane of endosome. Binds to the phospholipid L BP A which is abundant in MVBs internal membranes. The MVB pathway appears require the sequential function of ESCR T-O, - I , II and III complexes. The T machinery also functions in topologically equivalent membrane fission events, such as the terminal stages of cytokinesis and enveloped virus budding ( HI V-1 and other lentiviruses). A ppears to be an adapter for a subset of ESCR T- III proteins, such as CH MP4, to function at distinct membranes. R equired for completion of cytokinesis. I nvolved in HI V-1 virus budding. C an replace T SG 101 it its role of supporting HI V-1 release; this function implies the interaction with CH MP4B. May play a role in the regulation of both apoptosis and cell proliferation. C leaves peptide bonds on the -terminal side of prolyl residues within peptides that are up to acids long. Possesses a trypsin-like cleavage specificity with preference for poly-basic substrates. Stimulates ENaC activity through activating cleavage of the A nonspecific tyrosine phosphatase that dephosphorylates a diverse number of substrates under acidic conditions (p H 4–6) including alkyl, aryl, and acyl orthophosphate monoesters phosphorylated proteins. H as lipid phosphatase activity and inactivates lysophosphatidic acid in seminal plasma. I soform 2: the cellular form also has ecto-5 pain suppressor. A cts as a tumor suppressor of prostate cancer through dephosphorylation ER BB2 and deactivation of M A PK-mediated signaling. C atalyzes the transfer of a farnesyl moiety from pyrophosphate to cysteine at fourth position from the C -terminus of several proteins. The β Target name P AR 1 Polycomb protein EE D Progesterone receptor Programmed cell death 6-interacting protein Prolyl endopeptidase Prostasin Prostatic acid phosphatase Protein farnesyl transferase subunit ) Class PD PD PD PD PD PD PD PD Continued ( Table 2 PDB ID 3 G JW 3K27 1 S Q N 2 R 03 3DDU 3.00 E +16 1 N D5 1 L D8

4034 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 8.8 7.9 - - B and JU N , which in turn is essential κ B and JU N pathways. I n platelets, B, JU N , F A T C 1, and 2. I n CR - κ κ B ( N FKB2) pathway. I n the signaling pathway κ protein-induced apoptosis by interrupting cell death β B pathway ( N FKB1) by direct phosphorylation of CAR D11 on κ B activation, cell survival, differentiation and polarity, contributes to κ B and neuronal cell survival. Functions in the organization of apical domain epithelial cells by κ C D3/ D28-co-stimulated T-cells, is required for the activation of N F- for IL 2 production, and participates to the calcium-dependent N F A T C 1 transactivation. Mediates the activation of canonical N F- several serine residues, inducing CAR D11 association with lipid rafts and recruitment of the B CL 10-M AL T1 complex, which then activates I KK resulting in nuclear translocation and activation of N FKB1. May also play an indirect role in activation of the noncanonical N F- leading to JU N activation, acts by phosphorylating the mediator S TK39/ P A K and may not act through M A P kinases signaling. Plays a critical role in T CR / C D28-induced N F 1 and 2 transactivation by participating in the regulation of reduced inositol 1,4,5-trisphosphate generation and intracellular calcium mobilization. A fter costimulation of T-cells through C D28 can phosphorylate B L and is required for the ubiquitination and subsequent degradation of C B L B, which is a prerequisite for activation T CR . During T-cells differentiation, plays an important role in the development of Th2 cells following immune and inflammatory responses, and, in the development of autoimmune diseases, is necessary for the activation of IL -17-producing Th17 cells. May play a minor role in Th1 response. Upon T CR stimulation, mediates T-cell protective survival signal by phosphorylating B A D, thus protecting T-cells from D-induced apoptosis, and by upregulating B CL -X( L )/B 2 1 levels through N F- regulates signal transduction downstream of the I T GA 2B, C D36/ G P4, F2 R /P AR 1 and RL 3/P 4 receptors, playing a positive role in “outside-in” signaling and granule secretion signal transduction. May relay signals from the activated I T GA 2B receptor by regulating uncoupling of W AS P and PF1, thereby permitting regulation of actin filament nucleation and branching activity the Arp2/3 complex. May mediate inhibitory effects of free fatty acids on insulin signaling by phosphorylating IRS 1, which in turn blocks 1 tyrosine phosphorylation and downstream activation of the P I 3K/ A KT pathway. Phosphorylates M SN in presence of phosphatidylglycerol or phosphatidylinositol. Phosphorylates PDPK1 at “ S er-504” and er-532” negatively regulates its ability to phosphorylate PKB/ A KT1. C alcium- and D AG -independent serine/threonine–protein kinase, that plays a general protective role against apoptotic stimuli, is involved in N F- the regulation of microtubule dynamics in early secretory pathway. I s necessary for B CR - A L oncogene- mediated resistance to apoptotic drug in leukemia cells, protecting cells against drug-induced apoptosis. I n cultured neurons, prevents amyloid process at a very early step. I n glioblastoma cells, may function downstream of phosphatidylinositol 3-kinase (P I [3]K) and PDPK1 in the promotion of cell survival by phosphorylating inhibiting pro-apoptotic factor B A D. C an form a protein complex in NSCLC cells with P AR D6 and EC T2 regulate oncogenic activity by phosphorylation, which in turn promotes transformed growth and invasion. I n response to NG F, acts downstream of SRC phosphorylate and activate IRA K1, allowing the subsequent activation of N F- phosphorylating E Z R . This step is crucial for activation and normal distribution of at the early stages intestinal epithelial cell differentiation. Forms a protein complex with LLGL 1 and P AR D6B independently of P AR D3 to regulate epithelial cell polarity. Plays a role in microtubule dynamics the early secretory pathway through interaction with RA B2 A and GA PD H recruitment to VT C s. I n HCAEC s, is activated by saturated fatty acids and mediates lipid-induced apoptosis. C alcium-independent, phospholipid- and D AG -dependent serine/threonine–protein kinase that mediates nonredundant functions in T CR signaling, including T-cells activation, proliferation, differentiation, and survival, by mediating activation of multiple transcription factors such as N F- type θ PK C iota type PK C PD PD 1Z R Z 1XJD

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4035 Dovepress He et al Dovepress 7.3 9.1 Docking score - - A F ratio, ratio, EG

NA F1, which which F1, 1, which which 1, -dependent -dependent G A 4, which which 4, RA F4 -1 G P , which facilitates facilitates which , EI IL A CS 1 B pathway ( N FKB1) N κ DK phosphorylation. phosphorylation. E C F4 EI , phosphorylates phosphorylates , treatment, is required to induce induce to required is treatment, A A nvolved in the stabilization of V of stabilization the in nvolved I T, or activating signaling cascade involving involving cascade signaling activating or T, B-induced genes, through through genes, B-induced N κ T P4 receptor for granule release, and activates activates and release, granule for receptor P4 P pathway for adhesion. During response to to response During adhesion. for pathway P C G GA T2/ P1 D36/ NN C F1, is translocated to the nucleus and is associated with with associated is and nucleus the to translocated is F1, RA CS 4, T 4, G P ). Upon stimulation with TP with stimulation Upon ). CS A 2, 2, s highly expressed in a number of cancer cells where it can act as as act can it where cells cancer of number a in expressed highly s KB I I / K signaling cascade, and/or by upregulating upregulating by and/or cascade, signaling K CL B3 through the the through B3 IA an promote cell growth by phosphorylating and activating activating and phosphorylating by growth cell promote an ER G C F1, B F1, K1 and may be involved in the regulation of of regulation the in involved be may and K1 T I FKB PK/ N A N RA 2B- ( B activation. Mediates the activation of canonical N F- α fter wounding, translocates from focal contacts to lamellipodia and participates participates and lamellipodia to contacts focal from translocates wounding, fter κ 4 with extensive lamellipodia at the cell periphery and polarization of the cell cell the of polarization and periphery cell the at lamellipodia extensive with 4 GA A G T I P K1/2)-dependent signaling pathway, and upon PM upon and pathway, signaling K1/2)-dependent CS ER B inhibitor 1 binding to MK to binding 1 κ G PK1/3 ( PK1/3 F4 A EI egatively regulates myocardial contractility and positively regulates angiogenesis, platelet aggregation, aggregation, platelet angiogenesis, regulates positively and contractility myocardial regulates egatively N Function C alcium-activated, phospholipid- and D AG -dependent serine/threonine–protein kinase that is involved adhesion, and migration differentiation, apoptosis, proliferation, cell of regulation negative and positive in directly by inflammation, and function, platelet angiogenesis, hypertrophy, cardiac tumorigenesis, as such targets phosphorylating M A PK1/3 ( ER K1/2) and RA P1 GA P. I nvolved in cell proliferation growth arrest by positive cycle. cell the of regulation negative M the of activation the mediates active C DK complex formation in glioma cells. I n intestinal cells stimulated by the phorbol ester PM A , can hyperphosphorylated the of accumulation the with associated is which program arrest cell-cycle a trigger growth-suppressive form of R B1 and induction the C DK inhibitors N 1 A 1B. E xhibits anti- p53/TP53-mediated the suppressing by apoptosis from them protects and cells glioma in function apoptotic activation of IG FBP3, and in leukemia cells mediates anti-apoptotic action by phosphorylating B CL 2. During macrophage by induced differentiation macrophage development. macrophage phosphorylating by motility cell in role a Plays adhesion. desmosomal of modulation the in of association induces motility. cell in increases by accompanied breast and gliomas as such tumors several of phenotypes malignant in implicated is and promoter tumor a cancers. part in cardiomyocytes, neonatal of growth hypertrophic Mediates arteries. in formation thrombus and M a through protein–D synthesis, protein increasing by death, and failure heart to up hypertrophy cardiomyocyte and cell surface area. R egulates cardiomyocyte function by phosphorylating cardiac troponin T (T NN T2/ sensitivity, calcium myofilament activity, ATPase actomyosin in reduction significant induces which CTNT), and myocardial contractility. I n angiogenesis, is required for full EC migration, adhesion to VT N , V EG F A - formation. tube vascular and activation, kinase of regulation dependent m RNA at posttranscriptional level and mediates V EG F A -induced cell proliferation. I n the regulation of calcium- the from signals mediates aggregation, platelet induced heterodimer integrin the inflammation. and defense host in involved functions macrophage LPS-induced selective regulate may LPSs, But in some inflammatory responses, may negatively regulate NF- induction of N F- modulates Phosphorylates K I T, leading to inhibition of T activity. C alcium-activated, phospholipid- and D AG -dependent serine/threonine–protein kinase involved in various cellular processes such as regulation of the B CR signalosome, oxidative stress-induced apoptosis, AN D R - dependent transcription regulation, INS signaling, and EC s proliferation. Plays a key role in B-cell activation by regulating B CR -induced N F- by direct phosphorylation of CAR D11/ M A 1 at “ S er-559”, er-644”, and er-652”. Phosphorylation induces CAR D11/ M A 1 association with lipid rafts and recruitment of the B CL 10-M AL T1complex as well as M A P3K7/T K1, which then activates I KK complex, resulting in nuclear translocation and activation of N FKB1. Plays a direct role in the negative feedback regulation of B CR signaling, by downmodulating type type α β Target name PK C PK C ) Class PD PD Continued ( Table 2 PDB ID 3 I W4 2 I 0 E

4036 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 6.6 6.3 6.7 6.9 8.1 9 8.3 ------

CE

, ould ould C CE R

O L 2) and and 2) RR P S M1 onto the growing growing the onto M1 G after myocardial infarction infarction myocardial after 1 and and 1 RR P S AGER nvolved in the formation of the the of formation the in nvolved I nteraction with with nteraction I K1/2 and p53/TP53 signaling pathways (by similarity). similarity). (by pathways signaling p53/TP53 and K1/2 ER atalyzes small proline-rich proteins ( proteins proline-rich small atalyzes C n hair follicles, involved in cross-linking structural proteins to hardening the inner root root inner the hardening to proteins structural cross-linking in involved follicles, hair n I -cells, is probably involved in the inhibition of INS gene transcription, via regulation MY C β atalyzes the calcium-dependent formation of isopeptide cross-links between glutamine and lysine residues residues lysine and glutamine between cross-links isopeptide of formation calcium-dependent the atalyzes BTK function via direct phosphorylation of at “ S er-180”, which results in the alteration plasma membrane localization and in turn inhibition of BTK activity. I nvolved apoptosis following oxidative damage: in case of oxidative conditions, specifically phosphorylates “Ser-36” isoform p66Shc SHC1, leading to mitochondrial accumulation of p66 S hc, where hc acts as a R O producer. A cts coactivator of ANDR-dependent transcription, by being recruited to ANDR target genes and specifically mediating phosphorylation of “Thr-6” histone H3 (H3T6ph), a specific tag for epigenetic transcriptional activation that prevents demethylation of histone H 3 “ L ys-4” ( 3K4me) by LS D1/KDM1 A . I n INS signaling, may function downstream of IRS 1 in muscle cells and mediate INS -dependent D NA synthesis through the RA F1-M A PK/ ER K signaling cascade. May participate in the regulation of glucose transport adipocytes by negatively modulating the INS -stimulated translocation of glucose transporter SLC 2 A 4/ GL UT4. Under high in pancreatic expression. I n EC s, activation of P R K C B induces increased phosphorylation B1, V EG F A -induced cell proliferation, and inhibits P I 3K/ A KT-dependent N O synthase ( S 3/e ) regulation by INS , which causes endothelial dysfunction. A lso involved intriglyceride homeostasis (by similarity). A cts as an adapter for the receptor ER BB2, in epithelia. By binding unphosphorylated “Tyr-1248” of receptor ER BB2, it may contribute to stabilize this unphosphorylated state. I nhibits p53/TP53- and TP73/p73-mediated cell-cycle arrest apoptosis by binding its transcriptional activation domain. I nhibits degradation of MDM2. C an reverse MDM2-targeted TP53 while maintaining suppression of TP53 transactivation and apoptotic functions. N / A ions both for exist affinities different with sites binding distinct tightly very zinc binds but calcium binds Weakly on each monomer. Physiological concentrations of potassium ion antagonize the binding both divalent cations, releasing by STK38 of activation the initiates and to Binds sites. calcium-binding high-affinity affecting especially kinase. the within interactions molecular intra autoinhibitory activating by apoptosis myocyte in role a play may assist A T D3 cytoplasmic processing, preventing aggregation, and favoring mitochondrial localization. C atalyzes the cross-linking of proteins and conjugation polyamines to proteins. C proteins. to polyamines of conjugation the as well as proteins various in of membrane plasma the beneath proteins of cross-links covalent of consisting component specialized a keratinocytes. differentiated terminally T by cross-linked further are which oligomers, interchain small form to cross-linking similarity). (by scaffold sheath. N onreceptor PTK that regulates reorganization of the actin cytoskeleton, cell polarization, migration, adhesion, spreading, and bone remodeling. Plays a role in the regulation of humoral immune response is required for normal levels of marginal B-cells in the spleen and migration splenic B-cells. R equired for normal macrophage polarization and migration toward sites of inflammation. Regulates cytoskeleton rearrangement and cell spreading in T-cells contributes to the regulation of T-cell responses. Promotes osteoclastic bone resorption; this requires both PTK2B/PYK2 and SRC . May inhibit differentiation activity of osteoprogenitor cells. Functions in signaling downstream integrin and collagen receptors, immune receptors, G P CR s, and cytokine, chemokine, growth factor receptors; mediates responses to cellular stress. Forms multisubunit signaling complexes with SRC and its family members upon activation; this leads E -glutamyltransferase 2 -glutamyltransferase γ γ β Protein LA P2 Protein Mdm4 Protein S 100- A 7 Protein S 100-B Protein–glutamine Protein–glutamine PTK 2- PD PD PD PD PD PD PD 1MF G 3F EA 2WO R 3 HC M 2Q3Z 1VJJ 3FZ S

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4037 Dovepress He et al Dovepress 8 6.5 6 Docking score - - -

cts cts A ctive in the absence absence the in ctive egulates nociceptor nociceptor egulates A R F. F. N D G F in somatotrophs (within pituitary), promotes promotes pituitary), (within somatotrophs in F N levels. May also regulate potassium ion transport + D 2 G mechanoreceptors. Mediator of several diseases such as as such diseases several of Mediator mechanoreceptors. RA nvolved in the development of the neural crest. crest. neural the of development the in nvolved I production, but triggers apoptosisin absence of of absence apoptosisin triggers but production, GH B3)-dependent manner. manner. B3)-dependent equired for the molecular mechanisms orchestration during intestine organogenesis; involved in in involved organogenesis; intestine during orchestration mechanisms molecular the for equired G R T I B1 and and B1 G eceptor tyrosine-protein kinase involved in numerous cellular mechanisms including cell proliferation, proliferation, cell including mechanisms cellular numerous in involved kinase tyrosine-protein eceptor T Function to the phosphorylation of additional tyrosine residues, creating binding sites for scaffold proteins, effectors, and substrates. R egulates numerous signaling pathways. Promotes activation of phosphatidylinositol 3-kinase and of the A KT1 signaling cascade. Promotes activation N O S 3. R egulates production cellular messenger c G MP. Promotes activation of the M A P kinase signaling cascade, including PK1/ ER K2, M A PK3/ K1, and PK8/J N K1. Promotes activation of R ho family G TPases, such as RH O RAC 1. R ecruits the ubiquitin ligase MDM2 to P53/TP53 in nucleus, and thereby regulates activity, P53/TP53 ubiquitination and proteasomal degradation. A cts as a scaffold, binding to both PDPK1 and SRC , thereby allowing to phosphorylate PDPK1 at “Tyr-9”, “Tyr-373”, “Tyr-376”. Promotes phosphorylation of N MD A receptors by SRC family members, and thereby contributes to the regulation N MD A receptor ion channel activity and intracellular C a by phosphorylation of potassium channel subunits. Phosphorylates SRC ; this increases kinase activity. Phosphorylates ASA P1, N P H K CNA 2, and SHC 1. Promotes phosphorylation of P2, RH OU, PX ; this requires both SRC and PTK2/PYK2. Thrombin, which cleaves bonds after Arg and Lys, converts fibrinogen to fibrin activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. Functions blood homeostasis, inflammation, and wound healing. R trophic neuro derived cell glial with binding upon differentiation cell and migration, cell navigation, neuronal factor family ligands. Phosphorylates PTK2/F A K1. R egulates both cell death/survival balance and positional information. the promotes and life, embryonic during organogenesis renal and system nervous enteric of development the Modulates tissue. lymphoid gut-associated the of component major a structures, patch-like Peyer’s of formation (eg, integrin an in migration cell mediates and neurons sympathetic in caspase by cleavage its via adhesion cell I cleavage. caspase intracellular receptor requires that mechanism a through apoptosis triggering ligand, of ligand the of presence the in receptor; dependence a as downregulates and survival of differentiation the Triggers size. and survival neuroendocrine cancers; these diseases are characterized by aberrant integrins-regulated cell migration. N onreceptor PTK that is activated following engagement of many different classes cellular receptors including immune response receptors, integrins and other adhesion receptor PTKs, G protein- coupled receptors as well cytokine receptors. Participates in signaling pathways that control a diverse spectrum of biological activities including gene transcription, immune response, cell adhesion, cell-cycle progression, apoptosis, migration, and transformation. Due to functional redundancy between members of the SRC kinase family, identification of specific role each is very difficult. appears to be one of the primary kinases activated following engagement receptors and plays a role in activation of other PTK families. R eceptor clustering or dimerization leads to recruitment SRC the receptor complexes where it phosphorylates the tyrosine residues within receptor cytoplasmic domains. Plays an important role in the regulation of cytoskeletal organization through phosphorylation specific substrates such as A F P1. Phosphorylation of P1 allows the SRC SH 2 domain to bind and localize actin filaments. Cytoskeletal reorganization is also controlled through the phosphorylation of cortactin (CTTN). Target name Prothrombin Proto-oncogene tyrosine-protein kinase receptor R et Proto-oncogene tyrosine-protein kinase S rc ) Class PD PD PD Continued ( Table 2 PDB ID 2BV R 2X2 L 1O4 A

4038 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 7.2 7.5 7 4.7 8.2 - - - - - . 2 X3 X3 N -elicited -elicited U I - R RIG B2) desensitization through through desensitization B2) F-mediated calcium-activated calcium-activated F-mediated , which phosphorylates phosphorylates which , nhances DDX58/ nhances EG ARR E SRC B1 and and B1 ARR -catenin ( C T NN D1), and plakoglobin δ on “Tyr-731”. “Tyr-731”. on L F-mediated tyrosine phosphorylation of both both of phosphorylation tyrosine F-mediated F1. Plays a role in in role a Plays F1. B G C -arrestin ( -arrestin -arrestin phosphorylation and internalization. H as a β β RASGR 1 and and 1 internalization through phosphorylation of clathrin heavy heavy clathrin of phosphorylation through internalization R F -catenin ( C T NN B1), β EG RASA lso plays a role in PD in role a plays lso K2 on “Tyr-284” and and “Tyr-284” on K2 A N 1. 1. S B R equired for for equired D R H nother type of cell–cell junction, the gap junction, is also a target for for target a also is junction, gap the junction, cell–cell of type nother -glucosylmoieties. A α OX2 on tyrosine residues, T residues, tyrosine on OX2 2 ontyrosine residues, thereby promoting its subsequent autophosphorylation. Phosphorylates Phosphorylates autophosphorylation. subsequent its promoting thereby residues, ontyrosine 2 C R as pathway through phosphorylation of of phosphorylation through pathway as -(deoxy)ribonucleoside molecules, with the formation of corresponding free purine bases and pentose- When cells adhere via focal adhesions to the extracellular matrix, signals are transmitted by integrins into the the into integrins by transmitted are signals matrix, extracellular the to adhesions focal via adhere cells When cell resulting in tyrosine phosphorylation of a number focal adhesion proteins, including PTK2/F A K1 and PX N . I n addition to phosphorylating focal adhesion proteins, SRC is also active at the sites of cell–cell contact A Js and phosphorylates substrates such as (JUP). connexin-43 ( G J A 1). SRC is implicated in regulation of pre-m RNA -processing and phosphorylates - K as such proteins binding S T A T1 and T3, leading to increased D NA -binding activity of these transcription factors. I nvolved in the R activation. channel chloride chains ( CL T C and 1) at “Tyr-1477”. I nvolved in phosphorylation and activation of A D R BK1, leading to critical role in the stimulation of C DK20/M A PK3 M PK cascade by EG F. Might be involved not only controlling in also but membrane plasma the of level the at signals mitogenic of transduction the mediating important an Plays nucleus. the in proteins regulatory with interaction via cycle cell the through progression role in osteoclastic bone resorption conjunction with PTK2B/PYK2. Both the formation of a SRC -PTK2B/ PYK2 complex and SRC kinase activity are necessary for this function. R ecruited to activated integrins by PTK2B/PYK2, thereby phosphorylating C B L , which in turn induces the activation and recruitment of function. osteoclast for critical is that pathway signaling a in membrane cell the to 3-kinase phosphatidylinositol Promotes energy production in osteoclasts by activating mitochondrial cytochrome C oxidase. Phosphorylates DD and antiviral signaling. Phosphorylates PDPK1 at “Tyr-9”, “Tyr-373”, and “Tyr-376”. C ontributes to the lung’s defense against inhaled microorganisms. May participate in extracellular reorganization or turnover of pulmonary surfactant. Binds strongly maltose residues and to a lesser extent other The purine nucleoside phosphorylases catalyze the phosphorolytic breakdown of N -glycosidic bond in β 1-phosphate. I nvolved in signal transduction through the Wnt pathway. The pyruvate dehydrogenase complex catalyzes the overall conversion of to acetyl- C o A and O I t contains multiple copies of three enzymatic components: pyruvate dehydrogenase ( E 1), dihydrolipoamide acetyltransferase ( E 2), and lipoamide dehydrogenase 3). G lycolytic enzyme that catalyzes the transfer of a phosphoryl group from P E to A DP, generating TP. S timulates POU5F1-mediated transcriptional activation. Plays a general role in caspase-independent cell death of tumor cells. The ratio between the highly active tetrameric form and nearly inactive dimeric determines whether glucose carbons are channeled to biosynthetic processes or used for glycolytic A TP production. The transition between the two forms contributes to control of glycolysis and is important for tumor cell proliferation and survival. , somatic form, mitochondrial α Pulmonary surfactant-associated protein D Purine nucleoside phosphorylase Pygopus homolog 1 Pyruvate dehydrogenase E 1 component subunit Pyruvate kinase isozymes M1/M2 PD PD PD PD PD 2O S 9 3B GS 2VP E 3 E X 1T5 A

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4039 Dovepress He et al Dovepress 6.7 Docking score - D A (3)K) to to (3)K) I er-939” er-939” S er-50” negatively negatively er-50” TPase-activating TPase-activating S G 2 at “ at 2 KT3) called the the called KT3) -induced translocation translocation -induced A SC ho ho 1 at “ at 1 INS R N Y), thereby potentially potentially thereby Y), B, playing an important role in in role important an playing B, KT2, and and KT2, F1, and nuclear translocation. translocation. nuclear and F1, 1 and promotes its nuclear nuclear its promotes and 1 ACL H A receptor and the attenuation of of attenuation the and receptor RASS ACIN KT1, KT1, B-dependent gene transcription and and transcription gene B-dependent INS MP levels and inhibition of lipolysis. lipolysis. of inhibition and levels MP A κ A P3K5 (apoptosis signal-related kinase). kinase). signal-related (apoptosis P3K5 (3)P-5 activity. The The activity. (3)P-5 A I signaling pathway controlling the tempo of of tempo the controlling pathway signaling F2 and and F2 er-9”, resulting in inhibition of its kinase activity. activity. kinase its of inhibition in resulting er-9”, R S SRS TP citrate lyase ( lyase citrate TP A T2 at “Thr-117” and “Thr-384” leading to inhibition inhibition to leading “Thr-384” and “Thr-117” at T2 S KT regulates also the storage of glucose in the form of of form the in glucose of storage the also regulates KT K3B at “ at K3B KT-mTO A A GS TK3/M ctivates the 3B isoform of cyclic nucleotide phosphodiesterase phosphodiesterase nucleotide cyclic of isoform 3B the ctivates S er-454” on on er-454” A ignals downstream of phosphatidylinositol 3-kinase (P 3-kinase phosphatidylinositol of downstream ignals S S MP-response element-binding protein). The phosphorylation of of phosphorylation The protein). element-binding MP-response A er-21” and and er-21” er-259” and negatively regulates its activity. Phosphorylation of B of Phosphorylation activity. its regulates negatively and er-259” S KT is also thought to be one mechanism by which cell proliferation is is proliferation cell which by mechanism one be to thought also is KT S 13-mediated regulation of cell migration and adhesion assembly and and assembly adhesion and migration cell of regulation 13-mediated -stimulated protein synthesis by phosphorylating T phosphorylating by synthesis protein -stimulated A B1 (c B1 1D4 triggers the binding of this effector to inhibitory 14-3-3 proteins, proteins, 14-3-3 inhibitory to effector this of binding the triggers 1D4 A at “ at C T INS A er-273”, resulting in reduced cyclic cyclic reduced in resulting er-273”, A CRE F1 at “ at F1 S P K3 S er-318”, which results in increased P increased in results which er-318”, RA S GS KT phosphorylates “ phosphorylates KT A -stimulated glucose transport. transport. glucose -stimulated KT is involved in the phosphorylation of members of the FOXO factors (Forkhead (Forkhead factors FOXO the of members of phosphorylation the in involved is KT on “ on E A 1. 1. K3 isoforms by by isoforms K3 KT mediates mediates KT INS KT is responsible of the regulation of glucose uptake by mediating mediating by uptake glucose of regulation the of responsible is KT CL A A GS UT4 glucose transporter to the cell surface. Phosphorylation of PTP of Phosphorylation surface. cell the to transporter glucose UT4 PK2 and enhances its kinase activity toward toward activity kinase its enhances and PK2 KFYV I 6KB1. 6KB1. ssential for the the for ssential SR S GL E Y activity and fatty acid synthesis. synthesis. acid fatty and activity Y P KT plays a role as key modulator of the the of modulator key as role a plays KT . . 4/ I 1 is another substrate and its phosphorylation is implicated in the regulation cell proliferation and and proliferation cell regulation the in implicated is phosphorylation its and substrate another is 1 R A 2 and M and 2 F A 2 ACL LC IG CL KT regulates also cell survival via the phosphorylation of M of phosphorylation the via survival cell also regulates KT A SLC 3B) via phosphorylation of “ of phosphorylation via 3B) E signaling. Phosphorylation of TB of Phosphorylation signaling. KT1 is one of three closely related serine/threonine–protein kinases ( kinases serine/threonine–protein related closely three and of one growth, is KT1 survival, cell proliferation, metabolism, including processes many regulate which and kinase, KT Function A downstream A of range a of phosphorylation threonine and/or serine through mediated is This specificity isoform angiogenesis. no them, of most for but far, so reported been have candidates substrate 100 Over substrates. reported. been has the of the of dephosphorylation preventing activity phosphatase its modulates INS for required is which phosphorylating by glycogen of Phosphorylation driven. Phosphorylation of “ S er-83” decreases M A P3K5 kinase activity stimulated by oxidative stress, and thereby apoptosis. prevents and “Thr-1462”, thereby activating mTO RC 1 signaling leading to both phosphorylation of 4 E -BP1 in of activation family of transcription factors), leading to binding 14-3-3 proteins and cytoplasmic localization. I n particular, FOXO1 is phosphorylated at “Thr-24”, “ S er-256”, and er-319”. FOXO3 FOXO4 are on equivalent sites. A KT has an important role in the regulation of N F- of activity the regulates positively CRE B1 induces the binding of accessory proteins that are necessary for transcription pro-survival genes B as such regulating (PD P Phosphorylates D protein growth. positioning, cell neuron correct including neurogenesis, adult during integration neurons newborn of process the formation. synapse and development, dendritic mediate the effects of various growth factors such as PD G F, EG INS , and IG F I . A KT mediates antiapoptotic of effects and disassembly. May be involved in the regulation of placental development. Phosphorylates S TK4/M T1 at “Thr- autophosphorylation, translocation, nuclear activity, kinase its: of inhibition to leading “Thr-387” and 120” Phosphorylates FOXO3. phosphorylate to ability to binding Thr-180, at autophosphorylation activity, kinase cleavage, its: of Phosphorylates Phosphorylates PALLD, including translocation. identified, recently been have substrates AKT1-specific activity. pro-apoptotic its stimulates P motility; cell and ganization or cytoskeletal modulates phosphorylation which specific more a cell metabolism and proliferation; C DK N 1 A , for which phosphorylation at “Thr-145” induces its release from has isoform AKT1 the that indicate findings recent These relocalization. cytoplasmic and CDK2 rolein cell motility and proliferation. Phosphorylates CL K2, thereby controlling survival to ionizing radiation. serine/threonine–protein kinase α Target name RAC - ) Class PD Continued ( Table 2 PDB ID 2UVM

4040 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 8.4 -

er-319”. er-319”. er-9”, er-9”, S S receptor receptor ssential for the the for ssential KT3) called the the called KT3) INS E . . A I K3B at “ at K3B P3K5 kinase activity activity kinase P3K5 KT phosphorylates phosphorylates KT F- A GS A IG er-256”, and “ and er-256”, -stabilizing complex thus thus complex -stabilizing 1. 1. S 1 signaling and leading leading and signaling 1 CL KT2, and and KT2, RC RNA A er-21” and and er-21” er-318”, which results in increased increased in results which er-318”, S S D1 m D1 KT1, KT1, 2 and M and 2 A CL at “ at on “ on A CCN KT is involved in the phosphorylation of of phosphorylation the in involved is KT E er-83” decreases M decreases er-83” S A KT plays a role as key modulator of the the of modulator key as role a plays KT K3 B1 induces the binding of accessory proteins proteins accessory of binding the induces B1 A GS KFYV 1D4 triggers the binding of this effector to to effector this of binding the triggers 1D4 I CRE 6KB1. 6KB1. C S P R [3]K) to mediate the effects of various growth growth various of effects the mediate to [3]K) I KT mediates the antiapoptotic effects of of effects antiapoptotic the mediates KT A . . I F IG KT2 seems also to be the principal isoform responsible of the regulation regulation the of responsible isoform principal the be to also seems KT2 A , and and , TX2 impairs its association with the the with association its impairs TX2 D1. D1. er-939” and “Thr-1462”, thereby activating mTO activating thereby “Thr-1462”, and er-939” I -BP1 and in activation of of activation in and -BP1 n particular, FOXO1 is phosphorylated at “Thr-24”, “ “Thr-24”, at phosphorylated is FOXO1 particular, n S E I INS signaling. Phosphorylation of TB of Phosphorylation signaling. F, F, CCN 2 at “ at 2 INS EG SC F, F, G signaling pathway controlling the tempo of the process of newborn neurons integration during during integration neurons newborn of process the of tempo the controlling pathway signaling R B-dependent gene transcription and positively regulates the activity of CRE B1 (cyclic A MP [c MP]- MP levels and inhibition of lipolysis. Phosphorylates P Phosphorylates lipolysis. of inhibition and levels MP 13-mediated regulation of cell migration and adhesion assembly and disassembly. May be involved in in involved be May disassembly. and assembly adhesion and migration cell of regulation 13-mediated κ P3K5 (apoptosis signal-related kinase). Phosphorylation of “ of Phosphorylation kinase). signal-related (apoptosis P3K5 A A A T TX2. Phosphorylation of P of Phosphorylation TX2. I A er-50” negatively modulates its phosphatase activity preventing dephosphorylation of the the of dephosphorylation preventing activity phosphatase its modulates negatively er-50” KT2 is one of three closely related serine/threonine–protein kinases ( kinases serine/threonine–protein related closely three of one is KT2 KT-mTO ignals downstream of phosphatidylinositol 3-kinase (P 3-kinase phosphatidylinositol of downstream ignals P S A A KT kinase, and which regulate many processes including metabolism, proliferation, cell survival, growth, downstream of range a of phosphorylation threonine and/or serine through mediated is This angiogenesis. isoform no them, of most for but far, so reported been have candidates substrate 100 Over substrates. specificity has been reported. AKT is responsible of the regulat ion glucose uptake by mediating INS-induced translocation of the SLC 2 A 4/ GL UT4 glucose transporter to cell surface. Phosphorylation PTP N 1 at “ of attenuation the and inhibitory 14-3-3 proteins, which is required for INS -stimulated glucose transport. A KT regulates also the phosphorylating by glycogen of form the in glucose of storage resulting in inhibition of its kinase activity. Phosphorylation GS K3 isoforms by A KT is also thought to be one mechanism by which cell proliferation is driven. A KT regulates also survival via the phosphorylation M of stimulated by oxidative stress, and thereby prevents apoptosis. A KT mediates INS -stimulated protein synthesis T phosphorylating by 4 of phosphorylation both to members of the FOXO factors (Forkhead family transcription factors), leading to binding 14-3-3 proteins localization. cytoplasmic and FOXO3 and FOXO4 are phosphorylated on equivalent sites. A KT has an important role in the regulation of N F- of phosphorylation The protein). element-binding response B as such genes prosurvival of transcription the for necessary are that “ S er-454” on ACL Y, thereby potentially regulating Y activity and fatty acid synthesis. A ctivates the 3B isoform of cyclic nucleotide phosphodiesterase (PD E 3B) via phosphorylation “ S er-273”, resulting in reduced cyclic P I (3)P-5 activity. The R ho G TPase-activating protein D LC 1 is another substrate and its phosphorylation growth. cell and proliferation cell regulation the in implicated is A formation. synapse and development, dendritic positioning, neuron correct including neurogenesis, adult S PD as such factors S recently identified AKT2 of substrates specific few the of One development. placental the of regulation the P is of half-life the shortening RNA by Downregulation differentiation. muscle skeletal in involved specifically also is AKT2 uptake. glucose of interference reduces the expression of phosphorylated form B A D, resulting in induction caspase- dependent apoptosis. Phosphorylates CL K2 on “Thr-343”. serine/threonine–protein kinase β RAC - PD 2JDO

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4041 Dovepress He et al Dovepress 7.8 5.9 6.8 Docking score - - - 2 N ssential for for ssential E timulates PK timulates S t is able to bind to to bind to able is t I TP-dependent manner, suggesting that the the that suggesting manner, TP-dependent TPase-activating proteins. proteins. TPase-activating G G K in a a in K A oxidase activity in macrophages (by similarity). similarity). (by macrophages in activity oxidase H DP NA K but not full-length P full-length not but K A TP hydrolysis, which is restored partially by by partially restored is which hydrolysis, TP G 13-mediated regulation of cell migration and adhesion assembly and disassembly. disassembly. and assembly adhesion and migration cell of regulation 13-mediated A T A TPase-binding domain of P of domain TPase-binding P S G Function Plasma membrane-associated small G TPase that cycles between active TP-bound and inactive DP-bound states. I n its active state, binds to a variety of effector proteins regulate cellular responses such as secretory processes, phagocytosis of apoptotic cells, epithelial cell polarization, and growth factor-induced formation sigma1, factor cytosolic the of component active the is heterodimer GDI p21/rho Rac1 ruffles. membrane of the of stimulation in involved is which the kinase activity. I n concert with RA B7 A , plays a role in regulating the formation of R Bs osteoclasts. glioma cells, promotes cell migration and invasion. I soform B has an accelerated GE F-independent G DP/ TP exchange impaired an and the insertion does not completely abolish effector interaction. Tyrosine-protein kinase that plays an essential role as cell surface receptor for neuregulins and EG F family members and regulates development of the heart, CNS , mammary gland, gene transcription, cell proliferation, differentiation, migration, and apoptosis. R equired for normal cardiac muscle differentiation during embryonic development, and for postnatal cardiomyocyte proliferation. R equired normal development of the embryonic CNS , especially for normal neural crest cell migration and axon guidance. R equired for mammary gland differentiation, induction of milk proteins and lactation. A cts as cell- surface receptor for the neuregulins NRG 1, 2, 3, and 4 EG F family members BT C , EREG, and HBEGF. Ligand-binding triggers receptor dimerization autophosphorylation at specific tyrosine residues that then serve as binding sites for scaffold proteins and effectors. Ligand specificity signaling is modulated by alternative splicing, proteolytic processing, and the formation of heterodimers with other ER BB family members, thereby creating multiple combinations of intracellular phosphotyrosines that trigger ligand- and context-specific cellular responses. Mediates phosphorylation of SHC1 activation the MAP kinases M A PK1/ ER K2 and PK3/ K1. I soforms JM- C YT-1 JM-B phosphorylate P K3 R 1, leading to the activation of phosphatidylinositol 3-kinase and A KT1 protect cells against apoptosis. I soforms JM- A C YT-1 and JM-B mediate reorganization of the actin cytoskeleton promote cell migration in response to NRG 1. I soforms JM- A C YT-2 and JM-B lack the phosphotyrosine that mediates interaction with P I K3 R 1 and hence do not phosphorylate 1, protect cells against apoptosis, and do not promote reorganization of the actin cytoskeleton cell migration. Proteolytic processing isoforms JM- A C YT-1 and YT-2 gives rise to the corresponding soluble intracellular domains (4 IC D) that translocate to the nucleus, promote nuclear import of S T A T5 , activation mammary epithelium differentiation, cell proliferation, and activation of gene expression. The ER BB4-soluble intracellular domains (4 IC D) colocalize with S T A T5 at the CSN 2 promoter to regulate transcription of milk proteins during lactation. The ER BB4-soluble intracellular domains can also translocate to mitochondria and promote apoptosis. Plays an important role in blood vessel remodeling and angiogenesis. N ot necessary for the initial formation of blood vessels, but is essential for their maintenance and remodeling. C an induce dephosphorylation of T E K/ T IE 2, C D H 5/V E -cadherin, and KD R /V EG F -2. egulates angiopoietin-T 2 signaling in EC s. A cts as a negative regulator of T IE 2, and controls 2-driven EC proliferation, which in turn affects blood vessel remodeling during embryonic development and determines blood vessel size perinatal growth. E ssential for the maintenance of EC contact integrity and for the adhesive function V E -cadherin in s this requires presence of plakoglobin (by similarity). β Target name R as-related C 3 botulinum toxin substrate 1 R eceptor tyrosine-protein kinase ER BB4 R eceptor-type tyrosine-protein phosphatase ) Class PD PD PD Continued ( Table 2 PDB ID 1 R YF 2 R 4B 2 H 03

4042 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 4.6 - Continued ( 9.9 7.3 6.2 6.2 to 7.6 6.9 ------

O α RH /PP AR sites known sites sites known ′ ′ ′ α transition when 1 – G 0 - AGG T CA -3 - AGG T CA -3 - AGG T CA -3 ′ ′ ′ retinoic acid. R X RA serves as a common heterodimeric cis -retinaldehyde, from the inactive 11-trans products of the the of products 11-trans inactive the from -retinaldehyde, retinoic acid, and regulate gene expression in various biological retinoic acid, and regulate gene expression in various biological retinoic acid and regulate gene expression in various biological cis cis cis cis or 9- or 9- or 9- trans trans trans -retinol and 11- cis sites known as D R 1–D 5. I n the absence of ligand, X - RAR heterodimers ′ - AGG T CA -3 ′ oluble retinoid carrier is essential for proper function of both rod and cone photoreceptors. Participates in the the in Participates photoreceptors. cone and rod both of function proper for essential is carrier retinoid oluble photocycle and in the de novo synthesis of these retinoids from 11-trans metabolic precursors. The cycling of of cycling The precursors. metabolic 11-trans from retinoids these of synthesis novo de the in and photocycle retinoids between photoreceptor and adjacent pigment epithelium cells is known as the “visual cycle”. phosphorylated by C DK3/cyclin- . A cts as a transcription repressor of E 2F1 target genes. The under phosphorylated, active form of R B1 interacts with E 2F1 and represses its transcription activity, leading to cell-cycle arrest. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histonemethylation. R ecruits and targets histone methyltransferases S UV39 H 1, UV420 2, leading to epigenetic transcriptional repression. C ontrols histone H 4 “ L ys-20” trimethylation. I nhibits the intrinsic kinase activity of T A F1. Mediates transcriptional repression by S M ARCA 4/B RG 1 recruiting a H D AC complex to the c-FO S promoter. I n resting neurons, transcription of the promoter is inhibited by B RG 1-dependent recruitment of a phospho-RB1-HDAC1 repressor complex. Upon calcium influx, RB1 is dephosphorylated by calcineurin, which leads to release of the repressor complex (by similarity). I n case viral infections, interactions with S V40 large T antigen, H PV E 7 protein or adenovirus 1 A induce the disassembly of R B1- E 2F1 complex, thereby disrupting B1’s activity. R eceptor for retinoic acid. etinoic acid receptors bind as heterodimers to their target response elements in response to their ligands, all- processes. The RAR / R X heterodimers bind to the RARE s composed of tandem 5 as DR1–DR5. The high-affinity ligand for RXRs is 9- partner for a number of nuclear receptors. The R X / RAR heterodimers bind to the RARE s composed tandem 5 associate with a multiprotein complex containing transcription corepressors that induce histone acetylation, chromatin condensation, and transcriptional suppression. On ligand binding, the corepressors dissociate from the receptors and associate with coactivators, leading to transcriptional activation. The R X heterodimer is required for PP ARA transcriptional activity on fatty acid oxidation genes such as AC OX1 and the P450 system genes. known as D R 1–D 5. I n the absence or presence of hormone ligand, acts mainly an activator gene expression due to weak binding corepressors. I n concert with RARG , required for skeletal growth, matrix homeostasis, and growth plate function. as D R 1–D 5. I n the absence of ligand, acts mainly an activator gene expression due to weak binding to corepressors. R equired for limb bud development. I n concert with RARA or RAR B, required skeletal growth, matrix homeostasis, and growth plate function (by similarity). Renin is a highly specific endopeptidase, with the only known function to generate angiotensin I from angiotensinogen in the plasma, initiating a cascade of reactions that produce an elevation blood pressure and increased sodium retention by the kidney. S regeneration of active 11- Key regulator of entry into cell division that acts as a tumor suppressor. Promotes G R eceptor for retinoic acid. etinoic acid receptors bind as heterodimers to their target response elements in response to their ligands, all- processes. The R X / RAR heterodimers bind to the RARE s composed of tandem 5 R eceptor for retinoic acid. etinoic acid receptors bind as heterodimers to their target response elements in response to their ligands, all- processes. The RAR / R X heterodimers bind to the RARE s composed of tandem 5 α β γ R enin R etinaldehyde-binding protein 1 R etinoblastoma-associated protein R etinoic acid receptor X R etinoic acid receptor R etinoic acid receptor PK PD PD PD PD PD 1MZ N , 2P1T 1 HRN 3 H X3 1 G UX 1X A P 1F C Y

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. I s involved in cell-cycle 4 progression. 1 -carboxylation of glutamate residues in prothrombin synthesis. γ Function Delivers retinol from the liver stores to peripheral tissues. I n plasma, R BP–retinol complex interacts with transthyretin, this prevents its loss by filtration through the kidney glomeruli. G TPase activator for the R ho-type TPases by converting them to an inactive DP-bound state. H as a substantial GA P activity toward C D 42 and RAC 1 less RH O A . H as a role in regulating adhesion of hematopoietic cells to the extracellular matrix. Protein kinase that is a key regulator of actin cytoskeleton and cell polarity. I nvolved in regulation smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion and motility via phosphorylation of D A PK3, G F P, LI MK1, MK2, MY L 9/M LC 2, PF N 1, and PPP1 R 12 A . Phosphorylates F H OD1 acts synergistically with it to promote SRC -dependent nonapoptotic plasma membrane blebbing. Phosphorylates J I P3 and regulates the recruitment of N K to JIP3 upon UVB-induced stress. Acts as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability. A cts as a negative regulator of V EG F-induced angiogenic EC activation. R equired for centrosome positioning and centrosome-dependent exit from mitosis. Plays a role in terminal erythroid differentiation. May regulate closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles. Promotes keratinocyte terminal differentiation. S erine/threonine–protein kinase that acts downstream of ER K (M A PK1/ K2 and M PK3/ K1) signaling mediates mitogenic and stress-induced activation of the transcription factors CRE B1, E TV1/ ER 81, NR 4 A 1/ N U R 77, regulates translation through P S 6 and EI F4B phosphorylation, mediates cellular proliferation, survival, and differentiation by modulating mTO R signaling repressing pro-apoptotic function of B A D and DAPK1. In fibroblast, is required for EGF-stimulated phosphorylation of CREB1, which results in the subsequent transcriptional activation of several immediate-early genes. I n response to mitogenic stimulation ( EG F and PM A ), phosphorylates activates NR 4 1/ N U R 77 E TV1/ ER 81 transcription factors the cofactor CRE BBP. Upon INS -derived signal, acts indirectly on the transcription regulation of several genes by phosphorylating GS K3B at “ S er-9” and inhibiting its activity. Phosphorylates R P 6 in response to serum or EG F via an mTO R -independent mechanism and promotes translation initiation by facilitating assembly of the preinitiation complex. In response to INS, phosphorylates EIF4B, enhancing EIF4B affinity for EIF3 complex and stimulating cap-dependent translation. I s involved in the mTO R nutrient-sensing pathway by directly phosphorylating T SC 2 at “ S er-1798”, which potently inhibits ability to suppress mTO R signaling and mediates phosphorylation of R PTO , which regulates mTO RC 1 activity may promote rapamycin- sensitive signaling independently of the P I 3K/ A KT pathway. Mediates cell survival by phosphorylating pro- apoptotic proteins B A D and PK1 suppressing their pro-apoptotic function. Promotes the survival of hepatic stellate cells by phosphorylating CE BPB in response to the hepatotoxin CC l regulation by phosphorylating the C DK inhibitor N 1B, which promotes 1B association with 14-3-3 proteins and prevents its translocation to the nucleus inhibition of G The enzyme apparently serves as a quinone reductase in connection with conjugation reactions of hydroquinones involved in detoxification pathways as well biosynthetic processes such the vitamin K-dependent N / A -1 α - A doMet decarboxylase proenzyme Target name R etinol-binding protein 4 R ho G TPase-activating protein 9 R ho-associated protein kinase 1 R ibosomal protein S 6 kinase R ibosyldihydronicotinamide dehydrogenase (quinine) S ) Class PD PD PD PD PK PD Continued ( Table 2 PDB ID 1 R BP 2P0D 2 E T R 2Z7 R 3T E M, 1 SG 0, 1X I 2, 1X I 2_2, 3T E M_2, 1 SG 0_2 3 H 0W

4044 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 7.5 7.5 7.3 9.5 6.8 - - - - -

er-178” er-178” S at “ at er-279”, er-279”, A S 2F family of of family 2F 2 and enhances enhances and 2 E 25 signaling, may C β D RCA C er-178”, “ er-178”, S er-76” primes the the primes er-76” S er-743”, which prevents prevents which er-743”, S D51 from B from D51 at “ at . This may enhance chromatin chromatin enhance may This . A A RA er-124”, “ er-124”, 25 S F1 K1 at “ at K1 C 3T11ph), which leads to epigenetic epigenetic to leads which 3T11ph), L AS D H C . Phosphorylation of of Phosphorylation . 25 leads to increased inhibitory tyrosine tyrosine inhibitory increased to leads 25 repair by homologous recombination. recombination. homologous by repair C C er-76”, “ er-76”, S 25 D NA C C D at “ at C A 3.1 (to form form (to 3.1 25 H B1 to promote its interaction with the the with interaction its promote to B1 C R er-216” create binding sites for 14-3-3 proteins which which proteins 14-3-3 for sites binding create er-216” D S /M phase transition. 2 C nhibition of of nhibition 25B, and and 25B, I . . . Phosphorylation of of Phosphorylation . C at “ at A A C D C 25 25 , , C D25 C A -terminus of TP53, which promotes activation of TP53 by acetylation acetylation by TP53 of activation promotes which TP53, of -terminus C D . Binds to and phosphorylates T phosphorylates and to Binds . D C 25 C D C proteins are themselves degraded. By acting on T G F- C C family members. This activity requires the integrity of catalytic site, β D β ANCE C . Phosphorylation of of Phosphorylation . C -adenosyl methionine from and A TP. . May regulate the transcription of genes that regulate cell-cycle progression through through progression cell-cycle regulate that genes of transcription the regulate May . S 25 D51 at “Thr-309”, which promotes the release of of release the promotes which “Thr-309”, at D51 C CNA D RA D51 with chromatin, thereby promoting D promoting thereby chromatin, with D51 C RA and and A 25 C D C K1-dependent phosphorylation of the chromatin assembly factor factor assembly chromatin the of phosphorylation K1-dependent er-293” promotes proteolysis of of proteolysis promotes er-293” L S erine/threonine–protein kinase which is required for checkpoint-mediated cell-cycle arrest and activation of of activation and arrest cell-cycle checkpoint-mediated for required is which kinase erine/threonine–protein phosphorylates and to Binds /T]. regulate many physiological processes, including retinal angiogenesis and neuronal survival maturation during development. I ntracellularly, degrades T SC 2, leading to the activation of 2 downstream targets. C atalyzes the formation of Plays an essential role in cell growth and maintenance of morphology. May mediate the activating cleavage of HG F and M S T1/ L . Serine protease with a variety of targets, including extracellular matrix proteins such as fibronectin. HTRA1- generated fibronectin fragments further induce synovial cells to upregulate MMP-1 and MMP-3 production. May also degrade proteoglycans, such as aggrecan, decorin, and fibromodulin. Through cleavage of proteoglycans, may release soluble F G F-glycosaminogly can complexes that promote the range and intensity of F G signals in the extracellular space. R egulates availability IG Fs by cleaving F-binding proteins. I nhibits signaling mediated by T G F- although it is unclear whether T G F- role in the play a the nucleus. May to the cell membrane from mitogenic signals of in the transduction I nvolved postsynaptic responses of hippocampal neuron. S D NA repair in response to the presence of damage or unreplicated . May also negatively regulate cell-cycle progression during unperturbed cell cycles. This regulation is achieved by a number of mechanisms that together help to preserve the integrity of genome. R ecognizes substrate consensus sequence [ -X-X- S of phosphorylation and “Thr-507” and inhibit “ and protein for subsequent phosphorylation at “ S er-79”, er-82”, and er-88” by NE K11, which is required of degradation and polyubiquitination phosphorylation of C DK-cyclin complexes and blocks cell-cycle progression. A lso phosphorylates NE K6. Binds phosphorylates and to of association the the within sites multiple Phosphorylates and promotes cell-cycle arrest suppression of cellular proliferation. A lso repair D NA cross- F of phosphorylation through links T the assembly both in the presence or absence of D NA damage. May also play a role replication fork maintenance P of regulation through histone Phosphorylates histones. of phosphorylation the phosphorylate also May genes. of subset a of inhibition transcription factors and subsequent cell-cycle arrest. I soform 2: E ndogenous repressor of isoform 1, interacts with, and antagonizes CH K1 to promote the S G - A doMet synthase isoform type 1 S S erine protease hepsin S erine protease H T RA 1 S erine/threonine–protein kinase B- RA F S erine/threonine–protein kinase CH K1 PK PD PD PD PD 2OBV 1O5 E 2JO A 3 C 4 3P A 3

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/ S -arrested 5/ I T G B5 1 β /M cell-cycle arrest. 2 Function S erine/threonine–protein kinase that is required for checkpoint-mediated cell-cycle arrest, activation of D NA repair and apoptosis in response to the presence of S Bs. May also negatively regulate cell- cycle progression during unperturbed cell cycles. Following activation, phosphorylates numerous effectors preferentially at the consensus sequence [ L -X- R -X-X- S /T]. egulates cell-cycle checkpoint arrest through phosphorylation of C D 25 A , 25B, and inhibiting their activity. I nhibition phosphatase activity leads to increased inhibitory tyrosine phosphorylation of C DK-cyclin complexes and blocks cell-cycle progression. May also phosphorylate NE K6 which is involved in G R egulates D NA repair through phosphorylation of B RCA 2, enhancing the association RA D51 with chromatin that promotes D NA repair by homologous recombination. A lso stimulates the transcription of genes involved in D NA repair (including B RCA 2) through the phosphorylation and activation transcription factor FOXM1. R egulates apoptosis through the phosphorylation of p53/TP53, MDM4, and PM L . Phosphorylation of p53/TP53 at “ S er-20” by CHE K2 may alleviate inhibition MDM2, leading to accumulation of active p53/TP53. Phosphorylation MDM4 may also reduce degradation A lso controls the transcription of pro-apoptotic genes through phosphorylation factor E 2F1. Tumor suppressor, it may also have a D NA damage-independent function in mitotic spindle assembly by phosphorylating B RCA 1. I ts absence may be a cause of the chromosomal instability observed in some cancer cells. Protein kinase that is involved in the control of centrosome separation and bipolar spindle formation mitotic cells and chromatin condensation in meiotic cells. R egulates centrosome separation (essential for the formation of bipolar spindles and high-fidelity chromosome separation) by phosphorylating centrosomal proteins such as CR O CC , CE P250, and NINL resulting in their displacement from the centrosomes. R egulates kinetochore microtubule attachment stability in mitosis via phosphorylation of N D C 80. I nvolved in regulation of mitotic checkpoint protein complex via phosphorylation C D 20 and M A D2 L 1. Plays an active role in chromatin condensation during the first meiotic division through phosphorylation of HMGA2. Phosphorylates: PPP1 CC , SG O L 1, NECA B3, and N PM1. E ssential for localization of M A D2 1 to kinetochore and M A PK1 N PM1 to the centrosome. I soform 1 phosphorylates activates NE K11 in G cells. I soform 2, which is not present in the nucleolus, does not. The activated kinase acts on a variety of targets. L ikely to be the G TPase effector that links R ho- related G TPases to the J N K M A P kinase pathway. ctivated by C D 42 and RAC 1. I nvolved in dissolution of stress fibers and reorganization focal complexes. Involved in regulation microtubule biogenesis through phosphorylation of TB C B. DV L 1 and P A K1 form aternary complex with MU S K which is important for MU S K-dependent regulation of AC h R clustering during the formation N MJ. A ctivity is inhibited in cells undergoing apoptosis, potentially due to binding of C D 2 L 1 and 2. Phosphorylates MY 9/ M LC 2. Phosphorylates RA F1 at “ S er-338” and er-339” resulting in: activation of F1, stimulation translocation to mitochondria, phosphorylation of B A D by RA F1, and F1 binding CL 2. S erine/threonine protein kinase that plays a role in variety of different signaling pathways including cytoskeleton regulation, cell migration, growth, proliferation, or survival. A ctivation by various effectors including growth factor receptors or active C D 42 and RAC 1 results in a conformational change a subsequent autophosphorylation on several serine and/or threonine residues. Phosphorylates and inactivates the protein phosphatase SSH 1, leading to increased inhibitory phosphorylation of actin binding/depolymerizing factor cofilin. Decreased cofilin activity may lead to stabilization of actin filaments. Phosphorylates LIMK1, a kinase that also inhibits the activity of cofilin. integrin N e k 2 Target name S erine/threonine–protein kinase CH K2 S erine/threonine–protein kinase S erine/threonine–protein kinase P A K 1 S erine/threonine–protein kinase P A K 4 ) Class PD PD PD PD Continued ( Table 2 PDB ID 2W0J 2XKD 3FXZ 2X4Z

4046 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 8.1 8.2 - - – S 1

-tubulin ring complex (gTu RC ) γ , results in the relocation of C DK N 1 A to cytoplasm and 1 –M transition of mitotic cell cycle by phosphorylating CCN B1, C D 25 , FOXM1, 2 –M transitions by phosphorylation of C D 25 A and . Phosphorylation DK N 1 , 2 / C BX3. A cts also as a regulator of homing and migration bone marrow cells involving functional γ and G a regulator of cell-cycle progression at G enhanced C DK N 1 A protein stability. Promotes cell-cycle progression and tumorigenesis by downregulating expression of a regulator cell-cycle progression, C DK N 1B, at both transcriptional and posttranslational levels. Phosphorylation of C DK N 1B induces binding 14-3-3-proteins, permits nuclear export, and inhibits proteasome-dependent degradation. May affect the structure or silencing of chromatin by phosphorylating H P1 interaction with the C X CL 12- CR 4 signaling axis. S erine/threonine–protein kinase that performs several important functions throughout M phase of the cell cycle, including the regulation of centrosome maturation and spindle assembly, removal cohesins from chromosome arms, the inactivation of an aphase-promoting complex/cyclosome ( A P C / ) inhibitors, and the regulation of mitotic exit and cytokinesis. Polo-like kinase proteins act by binding phosphorylating proteins are that already phosphorylated on a specific motif recognized by the POLO box domains. Phosphorylates BO RA , BUB1B/BUB R 1, CCN B1, C D 25 CE P55, EC T2, ERCC 6 L FBXO5/ E M I FOXM1, K I F20 A /MK L P2, M F1 P, NE DD1, NINL , N PM1, UD C PKMYT1/MYT1, P K1 S 1/K Z, PPP1 R 12 /MYPT1, P RC 1, RACGA P1/ C YK4, SG O L S T AG 2/ SA 2, E X14, TOPO RS , p73/TP73, TPT1, and W EE 1. Plays a key role in centrosome functions and the assembly of bipolar spindles by phosphorylating P L K1 S 1/K I Z, NE DD1, and NINL . NE DD1 phosphorylation promotes subsequent targeting of the to the centrosome, an important step for spindle formation. Phosphorylation of NINL component centrosome leads to NINL dissociation from other centrosomal proteins. I nvolved in mitosis exit and cytokinesis by phosphorylating CE P55, EC T2, K I F20 A /MK L P2, M F1 P, P RC 1, and RACGA P1. R ecruited at the central spindle by phosphorylating and docking P RC 1 K I F20 A /MK L P2; creates its own sites on P RC 1 and K I F20 A /MK L P2 by mediating phosphorylation of sites subsequently recognized the PO O box domains. Phosphorylates RACGA P1, thereby creating a docking site for the R ho G TP exchange factor EC T2 that is essential for the cleavage furrow formation. Promotes central spindle recruitment of T2. Plays a central role in G M L F1 I P, PKMYT1/MYT1, PPP1 R 12 A /MYPT1, and W EE 1. Part of a regulatory circuit that promotes the activation of C DK1 by phosphorylating the positive regulator D 25 and inhibiting negative regulators and thus regulates cell motility. Phosphorylates ARHGE F2 activates the downstream target RH O A that plays a role in the regulation of assembly focal adhesions and actin stress fibers. Stimulates cell survival by phosphorylating the B CL 2 antagonist of cell death A D. lternatively, inhibits apoptosis preventing caspase-8 binding to death domain receptors in a kinase-independent manner. Plays role cell-cycle progression by controlling levels of the cell-cycle regulatory protein C DK N 1 A and phosphorylating RAN . Proto-oncogene with serine/threonine kinase activity involved in cell survival and proliferation thus providing a selective advantage in tumorigenesis. E xerts its oncogenic activity through: the regulation of MY C transcriptional activity, the regulation of cell-cycle progression, and by phosphorylation inhibition of proapoptotic proteins (B A D, M P3K5, FOXO3). Phosphorylation MY C leads to an increase protein stability, and thereby an increase of transcriptional activity. The stabilization MY C exerced by P I M1 might explain partly the strong synergism between these two oncogenes in tumorigenesis. Mediates survival signaling through phosphorylation of B A D, which induces release the anti-apoptotic protein Bcl- X(L)/BCL2L1. Phosphorylation of MAP3K5, an other proapoptotic protein, by PIM1, significantly decreases M A P3K5 kinase activity and inhibits P3K5-mediated phosphorylation of J N K K/p38M PK subsequently reducing caspase-3 activation and cell apoptosis. S timulates cell-cycle progression at the G 1 p i m S erine/threonine–protein kinase S erine/threonine–protein kinase P L K1 PD PD 3FV H 3F2 A

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4047 Dovepress He et al Dovepress 7.2 - Docking score /dicarboxylate + exchange and + / H + B. S timulates sodium κ channels: SCNN 1 A / ENAC , + 19; creatine transporter: SLC 6 A 8; N a SLC 6 A 19; creatine transporter: -dependent phosphate cotransporter: SLC 34 A 2/ NA P I -2B; and + channels: K CN J1/ R OMK1, CNA 1-5, Q1-5, and CNE 1; epithelial + elimination, salt appetite, gastric acid secretion, intestinal N a + A TPase, and transcription factors: C T NN B1 nuclear factor N F- + /K + 1, and SLC 1 A 5/ ASC T2, 38 1/ SN 1, retention, renal K

+ channels: T R PV5 and PV6; chloride B SN D, CLCN 2, C FT ; glutamate transporters: + 2 SCN 5 A , and ASIC 1/ ACCN 2; K C a SLC 1 A 3/ EAA T1, 2/ T2, 1/ T3, 6/ T4, and 7/ T5; amino acid transporters: cotransporter: SLC 13 A 2/ NA D C 1; N a glutamate receptor: GRI K2/ GL U R 6. Upregulates carriers: SLC 9 A 3/ NHE 3, 12 1/ N K CC 2, NCC , SLC 5 A 3/ S M I T, 2 1/ GL UT1, SGL T1, and 15 2/P E PT2. R egulates enzymes: GS K3 /B, PMM2, and N a transport into epithelial cells by enhancing the stability and expression of SCNN 1 A / ENAC . This is achieved by phosphorylating the NE DD4 L ubiquitin E 3 ligase, promoting its interaction with 14-3-3 proteins, thereby preventing it from binding to SCNN 1 A / ENAC and targeting for degradation. R egulates S O CE by stimulating O RAI 1 and S T I M1. R egulates K CN J1/ OMK1directly via its phosphorylation or indirectly increased interaction with SLC 9 A 3 R 2/ NHER F2. Phosphorylates MDM2 and activates MDM2-dependent ubiquitination of p53/TP53. Phosphorylates M A PT/T U and mediates microtubule depolymerization neurite formation in hippocampal neurons. Phosphorylates SLC 2 A 4/ GL UT4 and upregulates its activity. PBB1/ F E 65 and promotes its localization to the nucleus. Phosphorylates M A PK1/ ER K2 activates it by enhancing its interaction with M A P2K1/M E K1 and P2K2/M K2. Phosphorylates FBXW7 plays an inhibitory role in the N OT CH 1 signaling. Phosphorylates FOXO1 resulting its relocalization from nucleus to the cytoplasm. Phosphorylates FOXO3, promoting its exit from nucleus and interference with FOXO3- Phosphorylates activity. their inhibits and M A P3K3/M E KK3 and B RA F Phosphorylates transcription. dependent SLC 9 A 3/ NHE 3 in response to dexamethasone, resulting its activation and increased localization at the cell Function W EE 1 and PKMYT1/MYT1. A lso acts by mediating phosphorylation of cyclin-B1 ( CCN B1) on centrosomes in prophase. Phosphorylates FOXM1, a key mitotic transcription regulator, leading to enhance FOXM1 transcriptional activity. I nvolved in kinetochore functions and sister chromatide cohesion by phosphorylating BUB1B/BUB R 1, FBXO5/ E M I and S T AG 2/ SA 2. P L K1 is high on nonattached kinetochores suggesting a role of P L K1 in kinetochore attachment or SAC regulation. R equired for localization BUB1B. R egulates the dissociation of cohesin from chromosomes by phosphorylating subunits such as S T AG 2/ SA 2. Phosphorylates SG O L 1: required for spindle pole localization of isoform 3 1 and plays a role in regulating its centriole cohesion function. Mediates phosphorylation of FBXO5/ E M I 1, a negative regulator the A P C / during prophase, leading to FBXO5/ E M I 1 ubiquitination and degradation by proteasome. cts as a negative regulator of p53 family members: phosphorylates TOPO RS , leading to inhibit the sumoylation of p53/TP53 and simultaneously enhance the ubiquitination subsequent degradation p53/TP53. Phosphorylates the transactivation domain of transcription factor p73/TP73, leading to inhibit p73/TP73- mediated transcriptional activation and pro-apoptotic functions. Phosphorylates BO RA , thereby promotes the degradation of BO RA . C ontributes to regulation A U R K function. lso required for recovery after D NA damage checkpoint and entry into mitosis. S erine/threonine–protein kinase that is involved in the regulation of a wide variety ion channels, membrane transporters, cellular enzymes, transcription factors, neuronal excitability, cell growth, proliferation, survival, migration, and apoptosis. Plays an important role in cellular stress response. C ontributes to regulation of renal N a nutrient transport, INS -dependent salt sensitivity of blood pressure, peripheral glucose uptake, cardiac repolarization, and memory consolidation. Upregulates N a S g k 1 Target name S erine/threonine–protein kinase ) Class PD Continued ( Table 2 PDB ID 3 H DM

4048 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 6.8 - Continued ( 9.5 to –6.1 7.6 6.8 7.6 7.2 to - - - - - , fatty acids, + , K transport than + + , N a + 2 /calmodulin-dependent protein + 2 /calmodulin-dependent protein kinase II . C omponent of + 2 hormones, bilirubin, and drugs. I ts main function is the regulation of colloidal osmotic pressure blood. Major zinc transporter in plasma, typically binds approximately 80% of all plasma zinc. isoform 1. The P R 65 subunit of protein phosphatase 2 A serves as a scaffolding molecule to coordinate the assembly the catalytic subunit and a variable regulatory B subunit. R equired for proper chromosome segregation for centromeric localization of SG O L 1 in mitosis. Protein phosphatase that associates with over 200 regulatory proteins to form highly specific holoenzymes which dephosphorylate hundreds of biological targets. PP1 is essential for cell division and participates in the regulation of glycogen metabolism, muscle contractility, and protein synthesis. I nvolved in ionic conductances and long-term synaptic plasticity. May play an important role in dephosphorylating substrates such as the postsynaptic density-associated C a the PTW/PP1 phosphatase complex, which plays a role in control of chromatin structure and cell-cycle progression during the transition from mitosis into interphase. R egulates NE K2 function in terms of kinase activity and centrosome number splitting, both in the presence absence of radiation-induced D NA damage. Regulator of neural tube and optic fissure closure, ENCCs migration during development. Protein phosphatase that associates with over 200 regulatory proteins to form highly specific holo enzymes which dephosphorylate hundreds of biological targets. PP1 is essential for cell division and participates in the regulation of glycogen metabolism, muscle contractility, and protein synthesis. Dephosphorylates R P S 6KB1. I nvolved in regulation of ionic conductances and long-term synaptic plasticity. May play an important role dephosphorylating substrates such as the postsynaptic density-associated C a kinase II . C omponent of the PTW/PP1 phosphatase complex, which plays a role in control chromatin structure and cell-cycle progression during the transition from mitosis into interphase. N / A S erum albumin, the main protein of plasma, has a good binding capacity for water, C membrane. Phosphorylates CRE B1. N ecessary for vascular remodeling during angiogenesis. S ustained high levels and activity may contribute to conditions such as hypertension diabetic nephropathy. I soform 2 exhibited a greater effect on cell plasma membrane expression of SCNN 1 A / ENAC and N isoform α catalytic subunit catalytic subunit α γ S erine/threonine-protein phosphatase 2 A 65 kDa regulatory subunit A S erine/threonine-protein phosphatase PP1- S erine/threonine-protein phosphatase PP1- S erine–pyruvate aminotransferase S erum albumin PD PD PD PK PK 2 IE 4 3 E 7 A 1 I T6 1J04, 3 R 9 A , 1 H 0 C 3 A 73, 3 A 73_2, 3 S QJ_7, 3 S QJ, 3 A 73_4, 3 A 73_8, 3 S QJ_8, 3 S QJ_4, 3 S QJ_3, 3 A 73_6, 3 A 73_5, 3 A 73_7, 3 S QJ_6, 3 S QJ_2, 3 S QJ_5, 3 A 73_3

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4049 Dovepress He et al Dovepress 5.5 5.3 - - 6.3 7.4 6.3 6.5 7.2 8.1 7.1 9.4 7.7 6.5 8.2 2.8 6.4 to 6.7 7.7 6.9 to Docking score ------estradiol. -2,3-linked β α -AdoMet. Has broad substrate specificity S -hydroxy-2-acetylamino-fluorene (N-OH-2AAF). N -dihydrotestosterone, testosterone, and 17- -hydroxyarylamines to D NA -binding products and could α N -nitrophenol and p -2,6-linked sialic acid. The acid recognition site maybe masked by cis interactions with acids α -hydroxyarylamines to D NA -binding products and so it could participate as modulating factor of cancer risk. Function C an interact with D NA and histones may scavenge nuclear material released from damaged circulating cells. May also function as a calcium-dependent lectin. Putative Pc G protein. proteins act by forming multiprotein complexes, which are required to maintain the transcriptionally repressive state of homeotic genes throughout development (by similarity). Functions as an androgen transport protein, but may also be involved in receptor-mediated processes. E ach dimer binds one molecule of steroid. Specific for 5- R egulates the plasma metabolic clearance rate of steroid hormones by controlling their concentration. Putative adhesion molecule that mediates sialic-acid dependent binding to cells. Binds equally and on the same cell surface. C atalyzes the reversible phosphorylation of MT A to adenine and 5-methylthioribose-1-phosphate. I nvolved in the breakdown of MTA, a major by-product polyamine biosynthesis. Responsible for first step methionine salvage pathway after MT A has been generated from with 6-aminopurine nucleosides as preferred substrates. Functions as an UsnRNP-specific nuclear import adapter. Involved in the m3G-cap-dependent of UsnRNPs. Binds specifically to the terminal m3G-cap UsnRNAs. I nhibits PF1/PDX1 transactivation of established target promoters, such as INS , may be by recruiting a repressor complex (by similarity). I n with C U L 3, involved in ubiquitination of BM 1, H 2 A FY, and D A XX, and probably also in ubiquitination proteasomal degradation of G li2 or li3. N / A Functions as a bridging factor between S T A T6 and the basal transcription factor. Plays role in P I M1 regulation of MYB activity. Functions as a transcriptional coactivator for the E B NA 2. Mediates the association of molecular chaperones HSC 70 and HS P90 ( P CA C B). Can degrade fibronectin, laminin, gelatins of type I, III, IV, and V; X, IX collagens, cartilage proteoglycans. A ctivates procollagenase. C atalyzes one step in the degradation of inhibitory neurotransmitter GA B A . S ulfotransferase that utilizes P A as sulfonate donor to catalyze the sulfate conjugation of catecholamines, phenolic drugs, and neurotransmitters. H as also estrogen sulfotransferase activity. R esponsible for the sulfonation and activation of minoxidil. I t mediates metabolic carcinogenic N S ulfotransferase that utilizes P A as sulfonate donor to catalyze the sulfate conjugation of catecholamines, phenolic drugs, and neurotransmitters. I s also responsible for the sulfonation activation of minoxidil. Mediates the metabolic activation of carcinogenic so participate as modulating factor of cancer risk. S ulfotransferase that utilizes P A as sulfonate donor to catalyze the sulfate conjugation of drugs, xenobiotic compounds, hormones, and neurotransmitters. May be involved in the activation of carcinogenic hyroxylamines. S hows activity toward S ulfotransferase that utilizes P A as sulfonate donor and has low sulphotransferase activity toward various substrates with alcohol groups (in vitro). May catalyze the sulfate conjugation of xenobiotic compounds and endogenous substrates. Target name S erum amyloid P-component S ex comb on midleg-like protein 2 S ex hormone-binding globulin S ialic acid-binding I g-like lectin 5 MT A phosphorylase S nurportin-1 S peckle-type POZ protein S qualene synthase S taphylococcal nuclease domain-containing protein 1 S tress-induced-phosphoprotein 1 S tromelysin-1 S uccinate-semialdehyde dehydrogenase, mitochondrial S ulfotransferase 1 A S ulfotransferase 1 A 2 S ulfotransferase 1 C 2 S ulfotransferase 1 C 3 ) Class PD PD PD PD PD PD PD PD PD PD PD PK PK PK PK PK Continued ( Table 2 PDB ID 2W08 2VYT 1KDK 2Z G 1 1K27 1XK5 3 I VV 1 E ZF 3OM C 1 ELR 1 H Y7 2W8Q 3U3O_2, 1Z28, 2D06, 1 LS 6_2, 1 LS 6, 3U3O 1Z29 3BFX 2 H 8K, 2 RE O

4050 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 4.9 6.2 - - Continued ( 10.4 to 8.1 7.5 6.9 7.5 6.3 to 6.1 9.1 ------regulated genes. This β -nitrophenol and thyroid hormones, p type II serine/threonine kinase receptor, β cytokines T G FB1, FB2, and FB3. Transduces β -catenin destruction complex: poly- A DP-ribosylated β signaling cascade. A lso involved in noncanonical, S M D- β signaling pathways. For instance, T G FB R 1 induces RA F6 autoubiquitination which in turn β -methylation of thiopurine drugs such as 6-mercaptopurine. S -diiodothyronine, triidothyronine, reverse triiodothyronine, and thyroxine. ′ including 3,3 T G FB R 2, thenon-promiscuous receptor for the F- the T G FB1, FB2, and FB3 signals from cell surface to cytoplasm is thus regulating a plethora of physiological and pathological processes including cell-cycle arrest in epithelial hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression, and carcinogenesis. The formation of the receptor complex composed two T G FB R 1 and two T G FB R 2 molecules symmetrically bound to the cytokine dimer results in phosphorylation the activation of T G FB R 1 by constitutively active 2. A ctivated phosphorylates S M D2 that dissociates from the receptor and interacts with S M A D4. The D2- D4 complex is subsequently translocated to the nucleus where it modulates transcription of T G F- constitutes the canonical S M A D-dependent T G F- independent T G F- results in M A P3K7 ubiquitination and activation to trigger apoptosis. lso regulates epithelial mesenchymal transition through a S M A D-independent signaling pathway P AR D6 phosphorylation and activation. S ulfotransferase that utilizes P A as sulfonate donor to catalyze the sulfate conjugation of many hormones, neurotransmitters, drugs, and xenobiotic compounds. S ulfonation increases the water solubility of most compounds, and therefore their renal excretion, but it can also result in bioactivation to form active metabolites. S ulfates dopamine, small phenols such as 1-naphthol and Degrades extracellular matrix. Proposed to play a role in breast cancer invasion and metastasis. E xhibits trypsin-like activity as defined by cleavage of synthetic substrates with Argor Lys the P1 site. S eems to function as an A P. I n Js, may couple syndecans cytoskeletal proteins or signaling components. S eems to couple transcription factor OX4 the IL 5 receptor ( RA ). May also play a role in vesicular trafficking. Seems to be required for the targeting of TGFA cell surface in early secretory pathway. Poly- A DP-ribosyl transferase involved in various processes such as Wnt signaling pathway, telomere length, and vesicle trafficking. Acts as an activator of the Wnt signaling pathway by mediating poly-ADP-ribosylation of A X IN 1 and 2, two key components the target proteins are recognized by RN F146, which mediates their ubiquitination and subsequent degradation. A lso mediates poly- DP-ribosylation of B L ZF1 and CASC 3, followed by recruitment RN F146 subsequent ubiquitination. Mediates poly- A DP-ribosylation of T ER F1, thereby contributing to the regulation of telomere length. May also regulate vesicle trafficking and modulate the subcellular distribution SLC2A4/ GL UT4-vesicles. Transmembrane serine/threonine kinase forming with the T G F- C atalyzes the May have a role in maintaining the integrity of blood vessels. H as growth promoting activity on EC s, angiogenic activity in vivo and chemotactic on EC s vitro. C atalyzes the reversible phosphorolysis of thymidine. The produced molecules are then utilized as carbon and energy sources or in the rescue pyrimidine bases for nucleotide synthesis. C ontributes to the de novo mitochondrial thymidylate biosynthesis pathway. -methyltransferase S receptor type 1 β S ulfotransferase family cytosolic 1B member 1 S uppressor of tumorigenicity 14 protein S yntenin-1 Tankyrase-2 T G F- Thiopurine Thymidine phosphorylase Thymidylate synthase PK PD PD PD PD PK PD PD 2Z5F, 3 C K L , 3 C K L _2, 1Q20_2, 1Q22_2, 1Q22, 1Q1Z, 1Q20 2 G V6 1OBX 3K R 8 3 H MM 2BZ G , 2 H 11 1UOU 1 I 00

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4051 Dovepress He et al Dovepress 6.9 - 5.9 6.8 9.7 to 8.5 7.6 7.4 6.3 7.1 7.8 10.1 Docking score ------B and κ B-regulated gene κ B, in response to RAN K- L stimulation. κ domain might be involved in stabilizing junctions. Plays a role the α B and J N K plays a central role in the regulation of cell survival apoptosis. κ JU N . May be essential for the formation of functional osteoclasts. S eems to also play a role in D C s maturation and/or activation. R epresses c-Myb-mediated transactivation in B-lymphocytes. A P that seems to play a role in signal transduction initiated via T N F, IL -1, and -17 receptors. R egulates osteoclast differentiation by mediating the activation of A P complex 1 ( P-1) and N F- expression. I nhibits the transcriptional activation mediated by FOX A 2 and C T NN B1 F family members in Wnt signaling. The effects of full-length T LE family may be modulated by association with dominant-negative AES . Unusual function as coactivator for ESRRG Function Nuclear hormone receptor. High-affinity receptor for triiodothyronine. High-affinity receptor for triiodothyronine. Major thyroid hormone transport protein in serum. The N -terminal may be involved in transducing a signal required for TJ assembly, while the C have specific properties of TJs. The regulation of cell migration by targeting C D 42BPB to the leading edge migrating cells. C onverts the abundant, but inactive, zymogen plasminogen to plasmin by hydrolyzing a single A rg-Val bond in plasminogen. By controlling plasmin-mediated proteolysis, it plays an important role tissue remodeling and degradation, in cell migration many other physiopathological events. Plays a direct role facilitating neuronal migration. Modulates the activity of RH O-like proteins and connects extracellular signals to cytoskeletal activities. A cts as a G DP-dissociation stimulator protein that stimulates the DP- TP exchange activity of RH O-like G TPases and activates them. A ctivates RAC 1, C D 42, to a lesser extent RH O . R egulates activation of N F- R equired for normal antibody isotype switching from I gM to g G . H as E 3 ubiquitin-protein ligase activity and promotes “ L ys-63”-linked ubiquitination of target proteins, such as B IRC 3, RI PK1, and T ICA M1. I s an essential constituent of several E 3 ubiquitin-protein ligase complexes, where it promotes the ubiquitination target proteins by bringing them into contact with other E 3 ubiquitin ligases. R egulates B IRC 2 and protein levels by inhibiting their autoubiquitination and subsequent degradation; this does not depend on the T RA F2 RING-type zinc finger domain. E 3 ubiquitin ligase that, together with UB 2 N and 2V1, mediates the synthesis of “ L ys-63”-linked- polyubiquitinchains conjugated to proteins, such as I KBK G , A KT1, and KT2. lso mediates ubiquitination of free/unanchored polyubiquitin chain that leads to M A P3K7 activation. L eads the activation of N F- Transcriptional corepressor that binds to a number of transcription factors. I nhibits N F- N ecessary for the fragmentation of G olgi stacks during mitosis and their reassembly after mitosis. I nvolved in the formation of t ER . The transfer membranes from endoplasmic reticulum to G olgi apparatus occurs via 50–70 nm transition vesicles that derive from part-rough, part-smooth transitional elements of the t ER . Vesicle budding from is an A TP-dependent process. The ternary complex containing UFD1 L , V C P, and N P O 4 binds ubiquitinated proteins is necessary for the export of misfolded proteins from the ER to cytoplasm, where they are degraded by proteasome. α β Target name Thyroid hormone receptor Thyroid hormone receptor Thyroxine-binding globulin TJ protein ZO-1 Tissue-type plasminogen activator T-lymphoma invasion and metastasis- inducing protein 1 T N F receptor-associated factor 2 T N F receptor-associated factor 6 Transducin-like enhancer protein 1 t ER A TPase ) Class PD PD PK PD PD PD PD PD PD PD Continued ( Table 2 PDB ID 3JZB 1 N 46 2X N 6_2, 2X N 7_2, 2X N 7, 2 CE O, 2X N 6 3 C YY 1 A 5 H 3KZ E 1Q SC 1 L B6 2 CE 9 3 H U3

4052 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 6.9 6.7 7.5 6.8 6.6 6.9 8.2 10.6 6.8 6.5 7.5 ------

. The adapter molecule F A DD α infection (by similarity). and homotrimeric T N F S F1/lymphotoxin- (7)-methylguanine at position 46 (m7 G 46) in t RNA . α N Klebsiella pneumoniae recruits caspase-8 to the activated receptor. The resulting D ISC performs proteolytic activation that initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases) mediating apoptosis. C ontributes to the induction of noncytocidal T N F effects including anti-viral state and activation acid sphingomyelinase. The N P L O C 4-UFD1 -V complex regulates spindle disassembly at the end of mitosis and is necessary for the formation of a closed nuclear envelope. R egulates E 3 ubiquitin-protein ligase activity RN F19 A (by similarity). Component of the VCP/p97-AMFR/gp78 complex that participates in final step sterol- mediated ubiquitination and ERA D of H M GCR . A lso involved in NA damage response: recruited to S Bs sites in a RN F8- and F168-dependent manner promotes the recruitment of TP53BP1 at D NA damage sites. Thyroid hormone-binding protein. Probably transports thyroxine from the blood stream to brain. N / A N / A C atalyzes the formation of Troponin is the central regulatory protein of striated muscle contraction. Tn consists three components: Tn- I , which is the inhibitor of actomyosin A TPase; Tn-T, contains binding site for tropomyosin; and Tn-C. The binding of calcium to Tn-C abolishes the inhibitory action Tn on actin filaments. Digestive protease specialized for the degradation of trypsin inhibitors. I n ileum, may be involved in defensing processing, including D E F A 5. Tryptase is the major neutral protease present in mast cells and secreted upon coupled activation- degranulation response of this cell type. H as an immunoprotective role during bacterial infection. R equired to efficiently combat N / A R eceptor for T N F S F2/T F- Component of the ESCRT-I complex, a regulator vesicular trafficking process. Binds to ubiquitinated cargo proteins and is required for the sorting of endocytic ubiquitinated cargos into MVBs. Mediates association between the ESCR T-0 and T- I complex. R equired for completion of cytokinesis; function requires CE P55. May be involved in cell growth and differentiation. A cts as a negative regulator. I nvolved the budding of many viruses through an interaction with viral proteins that contain a late-budding motif P-[ S T]- A -P. This interaction is essential for viral particle budding of numerous retroviruses. survival and growth cell to linked processes key many in role a plays that kinase tyrosine-protein N onreceptor such as cytoskeleton remodeling in response to extracellular stimuli, cell motility and adhesion, receptor endocytosis, autophagy, and D NA damage response apoptosis. C oordinates actin remodeling through tyrosine phosphorylation of proteins controlling cytoskeleton dynamics like W AS F3 (involved in branch formation); AN X A 1 (involved in membrane anchoring); DB N 1, NL , C TT RA P H and ENAH in signaling); or M A PT and PX N (microtubule-binding proteins). Phosphorylation of W AS F3 is critical for the stimulation of lamellipodia formation and cell migration. I nvolved in the regulation adhesion motility through phosphorylation of key regulators these processes such as B CAR 1, CR K, K L , DOK1, E F S or NE DD9. Phosphorylates multiple receptor tyrosine kinases and more particularly promotes endocytosis of EG F R , facilitates the formation neuro muscular synapses through MU S K, inhibits PD G B-mediated chemotaxis, and modulates the endocytosis of activated B CR complexes. Other substrates that are involved in endocytosis regulation are the caveolin ( CA V1) and RIN 1. Moreover, A B L 1 regulates (7)-]-methyltransferase N -2 β Transthyretin Trifunctional purine biosynthetic protein adenosine-3 Triosephosphate isomerase t RNA [guanine- Troponin C , slow skeletal and cardiac muscles Trypsin-3 Tryptase Tryptophan 5-hydroxylase 1 T N F receptor super family member 1 A Tumor susceptibility gene 101 protein Tyrosine-protein kinase A B L 1 PD PD PD PK PD PD PD PD PD PD PD 3 H J0 1 N J S 1 H T I 3 C KK 1 IH 0 1 H 4W 2FPZ 3 H F6 1FT4 3OBQ 2 H Z I

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4053 Dovepress He et al Dovepress 7.5 8.9 Docking score - - , or B. Transiently κ -catenin). R egulates δ Helicobacter pylori . Pathogens can highjack A B L 1 -catenin) and C T NN D1 ( β B, which is involved in regulating the expression of κ Anaplasma phagocytophilum , C ag A (cytotoxin-associated gene ) of Citrobacter and possibly E. coli Function C B L family of ubiquitin ligases that drive receptor downregulation and actin remodeling. Phosphorylation CBL leads to increased EGFR stability. Involved in late-stage autophagy by regulating positively the trafficking and function of lysosomal components. A B L 1 targets to mitochondria in response oxidative stress, thereby mediates mitochondrial dysfunction and cell death. A B L 1 is also translocated in the nucleus where it has D NA -binding activity and is involved in damage response apoptosis. Many substrates are known mediators of D NA repair: DDB1, DDB2, ERCC 3, 6, RA D9 A , D51, D52, and W RN . ctivates the proapoptotic pathway when the D NA damage is too severe to be repaired. Phosphorylates TP73, a primary regulator for this type of damage-induced apoptosis. Phosphorylates P S M A 7 that leads to an inhibition proteasomal activity and cell cycle transition blocks. A B L 1 acts also as a regulator of multiple pathological signaling cascades during infection. S everal known tyrosine-phosphorylated microbial proteins have been identified as ABL1 substrates. This is the case of A36R vaccinia virus, Tir (translocated intimin receptor) of pathogenic A nk (ankyrin repeat-containing protein ) of kinase signaling to reorganize the host actin cytoskeleton for multiple purposes, like facilitating intracellular movement and host cell exit. Finally, functions as its own regulator through autocatalytic activity well through phosphorylation of its inhibitor, A B I 1. N onreceptor tyrosine kinase in dispensable for B lymphocyte development, differentiation, and signaling. Binding of antigen to the B-cell receptor triggers signaling that ultimately leads activation. A fter B CR engagement and activation at the plasma membrane, phosphorylates P LCG 2 several sites, igniting the downstream signaling pathway through calcium mobilization, followed by activation of PK C family members. P LCG 2 phosphorylation is performed in close cooperation with the A B-cell linker protein B LN K. BTK acts as a platform to bring together diverse array of signaling proteins and is implicated in cytokine receptor signaling pathways. Plays an important role the function of immune cells of innate as well adaptive immunity, a component the T LR s pathway. The pathway acts primary surveillance system for the detection of pathogens and is crucial to activation host defense. E specially, is a critical molecule in regulating T LR 9 activation splenic B-cells. Within the pathway, induces tyrosine phosphorylation of T IRA P which leads to degradation. BTK plays also a critical role in transcription regulation. I nduces the activity of N F- hundreds of genes. BTK is involved in the signaling pathway linking T LR 8 and 9 to N F- phosphorylates transcription factor G TF2 I on tyrosine residues in response to B CR . then translocates to the nucleus bind regulatory enhancer elements modulate gene expression. ARI D3 A and N F T are other transcriptional target of BTK. BTK is required for the formation functional ARI D3 A D NA -binding complexes. There is however no evidence that BTK itself binds directly to D NA . has a dual role in the regulation of apoptosis. N on-receptor tyrosine-protein kinase that plays a role in many biological processes including regulation of cell growth and survival, adhesion, integrin-mediated signaling, cytoskeletal remodeling, motility, immune response and axon guidance. I nactive FY N is phosphorylated on its C -terminal tail within the catalytic domain. Following activation by PK A , the protein subsequently associates with PTK2/F K1, allowing PTK2/F A K1phosphorylation, activation and targeting to focal adhesions. I nvolved in the regulation of cell adhesion and motility through phosphorylation of C T NN B1 ( cytoskeletal remodeling by phosphorylating several proteins including the actin regulator W AS and F y n Target name Tyrosine-protein kinase BTK Tyrosine-protein kinase ) Class PD PD Continued ( Table 2 PDB ID 3 GEN 2DQ7

4054 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 8 6 7.3 7.4 - - - - Fc region γ , and multiple IL s. Following γ , IL 2, 6, and 8, γ , I F N - β , I F N - signal pathway. Kinase partner for the IL 2 α γ / β / α integrins, such as I T G B1 and B2. During the phagocytic process, mediates mobilization of secretory lysosomes, degranulation, and activation of NA DP H oxidase to bring about the respiratory burst. Plays a role in the release of inflammatory molecules. Promotes reorganization actin cytoskeleton and polymerization, formation of podosomes and cell protrusions. I nhibits TP73-mediated transcription activation and TP73-mediated apoptosis. Phosphorylates C B L in response to activation of immunoglobulin receptors. Phosphorylates A D M15, B CR , EL MO1, F CGR 2 GA B1, B2, RA P GE F1, S T T5B, TP73, V A V1, and W AS . receptor. ligand binding to cell surface receptors, phosphorylates specific tyrosine residues on the cytoplasmic tails of the receptor, creating docking sites for S T A proteins. ubsequently, phosphorylates proteins once they are recruited to the receptor. Phosphorylated S T A Ts then form homodimer or heterodimers and translocate to the nucleus activate gene transcription. For example, cell stimulation with E PO during erythropoiesis leads to J A K2 autophosphorylation, activation, and its association with E PO R that becomes microtubule-associated proteins M A P2 and PT. Promotes cell survival by phosphorylating AGA P2/P I K E - and preventing its apoptotic cleavage. Participates in signal transduction pathways that regulate the integrity of the glomerular slit diaphragm (an essential part filter kidney) by phosphorylating several slit diaphragm components including N P HS 1, K IRREL and T R C 6. Plays a role in neural processes by phosphorylating DPY SL 2, a multifunctional A P within the CNS , ARHGA P32, regulator for R ho family G TPases implicated in various neural functions, and SNCA , a small pre-synaptic protein. Participates the downstream signaling pathways that lead to T-cell differentiation and proliferation following T CR stimulation. A lso participates in negative feedback regulation of T CR signaling through phosphorylation P AG 1, thereby promoting interaction between P AG 1 and CS K recruitment of to lipid rafts. maintains LC FY N in an inactive form. Promotes C D28-induced phosphorylation of V A V1. N onreceptor tyrosine-protein kinase found in hematopoietic cells that transmits signals from cell surface receptors and plays an important role in the regulation of innate immune responses, including neutrophil, monocyte, macrophage and mast cell functions, phagocytosis, survival proliferation, adhesion and migration. A cts downstream of receptors that bind the Fc region immunoglobulins, such as F CGR 1 A and 2 , but also CS F3 R P LA U the receptors for I N - Tyrosine kinase that plays an essential role in regulation of the adaptive immune response. R egulates development, function, and differentiation of conventional T-cells nonconventional N KT-cells. When A P C s activate T CR , a series of phosphorylation lead to the recruitment I TK cell membrane, in vicinity of the stimulated T CR receptor, where it is phosphorylated by LC K. Phosphorylation leads to I TK autophosphorylation and full activation. Once activated, phosphorylates P LCG 1, leading to the activation of this lipase and subsequent cleavage its substrates. I n turn, the endoplasmic reticulum releases calcium ions in the cytoplasm and N F A T translocates into nucleus to perform its transcriptional duty. Phosphorylates 2 essential A Ps: the linker for activation of T-cells/ LA T protein and LC P2. Then, a large number of signaling molecules such as V A V1 are recruited and ultimately lead to lymphokine production, T-cell proliferation and differentiation. Tyrosine kinase of the nonreceptor type, involved in I F N - N onreceptor tyrosine kinase involved in various processes such as cell growth, development, differentiation, or histone modifications. Mediates essential signaling events in both innate and adaptive immunity. In the cytoplasm, plays a pivotal role in signal transduction via its association with type I receptors such as GHR , P RLR , LE R E PO and T H PO; or type II receptors including I F N - Tyrosine-protein kinase HC K Tyrosine-protein kinase I TK/T S K Tyrosine-protein kinase J A K1 Tyrosine-protein kinase J A K2 PD PD PD PD 2 H K5 3M I Y 3 E Y G 3.00 E +64

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4055 Dovepress He et al Dovepress 10 6.4 8.8 Docking score - - - , R T CR . -15 β

IL , , α activation activation R R -9 T5B are are T5B -2 IL A IL , , T R S ) from chromatin. -7 α IL , , and and R A -4 T5 IL A , , T R S ) subunits inducing the tyrosine tyrosine the inducing subunits ) -2 IL RG -2 IL chain ( chain γ subunits such as as such subunits γ n the cytoplasm, plays a pivotal role in signal transduction via via transduction signal in role pivotal a plays cytoplasm, the n I ( IL -2 R B) and chains and C D3 subunits, initiating the T CR / signaling β γ onreceptor tyrosine kinase involved in various processes such as cell growth, development, or differentiation. differentiation. or development, growth, cell as such processes various in involved kinase tyrosine onreceptor phosphorylation of both receptor subunits on their cytoplasmic domain. Then, Then, domain. cytoplasmic their on subunits receptor both of phosphorylation recruited, phosphorylated, and activated by J A K1 K3. Once activated, dimerized S T T5 translocates to the nucleus and promotes transcription of specific target ge nes in acytokine-specific fashion. Phosphorylates other substrates including R U N X3, PTK2B/PYK2, the microtubule-associated protein M A PT, RH O H , or TY R OBP. Function phosphorylated in its cytoplasmic domain. Then, S T A T5 ( or T5B) is recruited, phosphorylated, and activated by J A K2. Once activated, dimerized S T T5 translocates into the nucleus promotes transcription of several essential genes involved in the modulation erythropoiesis. I n addition, J A K2 mediates angiotensin-2-induced ARHGE F1 phosphorylation. Plays a role in cell cycle by phosphorylating C DK N 1B. ooperates with T EC through reciprocal phosphorylation to mediate cytokine-driven activation of FOS transcription. In the nucleus, plays a key role in chromatin by specifically mediating phosphorylation “Tyr-41” of histone H3 (H3Y41ph), a specific tag that promotes exclusion CBX5 (HP1 N in role crucial a plays and immunity adaptive and innate both in events signaling essential Mediates T-cells. of development the during hematopoiesis its association with type I receptors sharing the common on residues tyrosine specific phosphorylates receptors, surface cell to binding ligand Following IL-21R. and the cytoplasmic tails of receptor, creating docking sites for S T A proteins. ubsequently, phosphorylates the S T A proteins once they are recruited to receptor. Phosphorylated Ts then form homodimer or upon example, For transcription. gene activate to nucleus the to translocate and heterodimers by IL 2, J A K1, and K3 molecules bind to -2 R N onreceptor tyrosine-protein kinase that plays an essential role in the selection and maturation of developing T-cells in the thymus and function of mature T-cells. Plays a key role T-cell antigen receptor-linked signal transduction pathways. C onstitutively associated with the cytoplasmic portions of D4 and D8 surface receptors. A ssociation of the T CR with a peptide antigen-bound M HC facilitates interaction of C D4 and D8 with M HC class II I molecules, respectively, thereby recruiting the associated LC K protein to the vicinity of T CR / C D3 complex. then phosphorylates tyrosines residues within the I T A Ms of cytoplasmic tails CR - pathway. Once stimulated, the T CR recruits tyrosine kinase Z A P70, which becomes phosphorylated and activated by LC K. Following this, a large number of signaling molecules are recruited, ultimately leading to lymphokine production. LC K also contributes to signaling by other receptor molecules. A ssociates directly with the cytoplasmic tail of C D2, which leads to hyperphosphorylation and activation LC K. A lso plays a role in the IL 2 receptor-linked signaling pathway that controls T-cell proliferative response. Binding of to its receptor results in increased activity of LC K. I s expressed at all stages thymocyte development and is required for the regulation of maturation events that are governed by both pre-T CR and mature N onreceptor tyrosine-protein kinase that transmits signals from cell surface receptors and plays an important role in the regulation of innate and adaptive immune responses, hematopoiesis, responses to growth factors and cytokines, integrin signaling, but also responses to D NA damage genotoxic agents. Functions primarily as negative regulator, but can also function activator, depending on the context. R equired not only for initiation of the B-cell response, but also for its downregulation and termination. Plays an important role in the regulation of B-cell differentiation, proliferation, survival, and apoptosis is important for immune self- tolerance. A cts downstream of several immune receptors, including the B CR , C D79 D79B, D5, D19, Target name Tyrosine-protein kinase J A K3 Tyrosine-protein kinase LC K Tyrosine-protein kinase L Y N ) Class PD PD PD Continued ( Table 2 PDB ID 3PJ C 1 L KK 3 A 4O

4056 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) Continued ( 8.1 7.8 - - 1 LCG signaling. Plays also a crucial crucial a also Plays signaling. K, an adapter linking the the linking adapter an K, M domains. The associationThe domains. I T A M CR LN M independent. Direct downstream downstream Direct independent. M A T I t also phosphorylates and activates P activates and phosphorylates also t I plays also a role in T in role a also plays CR YK could be be could YK S engagement, it phosphorylates and activates B activates and phosphorylates it engagement, CR 2 domains and the receptor tyrosine-phosphorylated receptor the and domains SH 2 to downstream signaling adapters and effectors. effectors. and adapters signaling downstream to ctivated upon B upon ctivated CR A CD22, FCER1, FCGR2, FCGR1A, TLR2, and TLR4. Plays a role in the inflammatory response to bacterial LPS. Mediates the responses to cytokines and growth factors in hematopoietic progenitors, platelets, erythrocytes, and in mature myeloid cells, such as D C s, neutrophils, eosinophils. A cts downstream of E PO R , K I T, MP L the chemokine receptor C X CR 4 as well receptors for IL 3, 5, and CS F2. Plays an important role in integrin signaling. R egulates cell proliferation, survival, differentiation, migration, adhesion, degranulation, and cytokine release. Downregulates signaling pathways by phosphorylation of I T Ms, which then serve as binding sites for phosphatases, such as PTP N 6/ SH P-1, 11/ P-2, and IN PP5D/ SHI which modulate signaling by dephosphorylation of kinases and their substrates. Phosphorylates LI M E 1 in response to C D22 activation. Phosphorylates BTK, C B L , D5, D19, D72, D79 A D79B, CS F2 R B, DOK1, HCLS 1, LILR B3/P IR -B, M S 4 A 2/F CER 1B, PTK2B/PYK2, YK, and T EC . Promotes phosphorylation of SIR P , PTP N 6/ SH P-1, 11/ SH P-2, and IN PP5D/ SHI P-1. Mediates phosphorylation of the B CR - A L fusion protein. R equired for rapid phosphorylation of F ER in response to CER 1 activation. Mediates K I T phosphorylation. A cts as an effector E PO R in controlling K I T expression and may play a role erythroid differentiation during the switch between proliferation and maturation. Depending on the context, activates or inhibits several signaling cascades. R egulates phosphatidylinositol 3-kinase activity and A KT1 activation. activation of the M P kinase signaling cascade, including activation of M A P2K1/M E K1, PK1/ ER K2, PK3/ PK8/J N and M A PK9/J N K2. Mediates activation of S T T5 and/or T5B. Phosphorylates L PX on “Tyr-72”. R eceptor tyrosine kinase that transduces signals from the extracellular matrix into cytoplasm by binding to several ligands including LGALS 3, TUB, TU L P1, or GAS 6. R egulates many physiological processes cell survival, migration, differentiation, and phagocytosis of apoptotic cells (efferocytosis). L igand binding at the cell surface induces autophosphorylation of M ER TK on its intracellular domain that provides docking sites for downstream signaling molecules. Following activation by ligand, interacts with GR B2 or P LCG 2 and induces phosphorylation of M A PK1, PK2, F K/PTK2, or RAC 1. ER TK signaling plays a role in various processes such as macrophage clearance of apoptotic cells, platelet aggregation, cytoskeleton reorganization, and engulfment. Functions in the R P E as a regulator of rod outer segments fragments phagocytosis. Plays also an important role in inhibition of T LR -mediated innate immune response by activating S A T1, which selectively induces production of suppressors cytokine signaling S O CS 1 and 3. N onreceptor tyrosine kinase that mediates signal transduction downstream of a variety transmembrane receptors including classic immunoreceptors like the B CR . R egulates several biological processes vascular and activation, platelet maturation, osteoclast adhesion, cell immunity, adaptive and innate development. A ssembles into signaling complexes with activated receptors at the plasma membrane via its between interaction with the receptor can also be indirect and mediated by A Ps containing I T M or partial hem domains. The phosphorylation of the I T A M domains is generally mediated by SRC subfamily kinases upon engagement via transduction signal rarely More receptor. the of as identified Initially BLNK. and LCP2, PI-3-kinase, PLCG1, VAV1, include SYK by phosphorylated effectors essential in B CR signaling, it is necessary for the maturation of B-cells most probably at pro-B to pre-B transition. B activated and the PK C signaling pathway. I t also phosphorylates BTK regulates its activity in B-cell antigen receptor- B of downstream function, its Besides signaling. coupled role in the innate immune response to fungal, bacterial, and viral pathogens. I t is for instance activated by membrane lectin CLEC 7 A . Upon stimulation by fungal proteins, together with S YK activates immune Mer Tyrosine-protein kinase Tyrosine-protein kinase S YK PD PD 3BP R 1XBB

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K. May EI F2 A K3/p- ER K. B-mediated transcription κ K; inactivating the protein kinase activity of activity kinase protein the inactivating EI F2 A K3/p- ER K; Function cells inducing the production of ROS. Also activates inflammasome and NF- of chemokines and cytokines in presence pathogens. R egulates neutrophil degranulation phagocytosis through activation of the M A PK signaling cascade. lso mediates D C s by cell necrosis stimuli. A lso involved in mast cells activation. functions downstream of receptors mediating cell adhesion. R elays for instance, integrin-mediated neutrophils and macrophages activation P-selectin receptor/ SEL P G - mediated recruitment of leukocytes to inflammatory loci. Plays also a role in nonimmune processes. It is for instance involved in vascular development where it may regulate blood and lymphatic separation. I t is also required for osteoclast development and function. Functions in the activation of platelets by collagen, mediating P LCG 2 phosphorylation and activation. May be coupled to the collagen receptor by I T A M domain-containing F CER 1 G . A lso activated by the membrane lectin CLEC 1B that is required for activation of platelets by PDPN/podoplanin. Involved in platelet adhesion being activated ITGB3 engaged fibrinogen. Tyrosine kinase that plays an essential role in regulation of the adaptive immune response. R egulates motility, adhesion, and cytokine expression of mature T-cells as well thymocyte development. C ontributes also to the development and activation of primary B-lymphocytes. When A P C s activate T CR , a series phosphorylations lead to the recruitment of Z A P70 doubly phosphorylated T CR component C D247/ C D3Z through I T A M motif at the plasma membrane. This recruitment serves to localization stimulated T CR and to relieve its autoinhibited conformation. R elease of Z A P70 active conformation is further stabilized by phosphorylation mediated LC K. S ubsequently, Z A P70 phosphorylates at least two essential Ps: LA T and LC P2. I n turn, a large number of signaling molecules are recruited ultimately lead to lymphokine production, T-cell proliferation, and differentiation. Furthermore, ZAP70 controls cytoskeleton modifications, adhesion, and mobility of T-lymphocytes, thus ensuring correct delivery effectors to the A P C . Z P70 is also required for T CR - C D247/ D3Z internalization and degradation through interaction with the E 3 ubiquitin-protein ligase C B L and A Ps SLA 2. Thus, Z P70 regulates both T-cell activation–switch on and switch off–by modulating T CR expression at the T-cell surface. During thymocyte development, Z A P70 promotes survival and cell-cycle progression of developing thymocytes before positive selection (when cells are still C D4/ D8 double negative). A lso, Z P70-dependent signaling pathway may also contribute to primary B-cells formation and activation through B CR . Tyrosine-protein phosphatase that acts as a regulator of endoplasmic reticulum unfolded protein response. of dephosphorylation Mediates play an important role in C K II - and p60c-src-induced signal transduction cascades. May regulate the E F NA 5- E P HA 3 signaling pathway which modulates cell reorganization and cell–cell repulsion. A cts downstream of various receptor and cytoplasmic PTKs to participate in the signal transduction from cell surface to the nucleus. Dephosphorylates R O C K2 at Tyr-722 resulting in stimulation of its ho A -binding activity. E xcises uracil residues from the D NA which can arise as a result of misincorporation dUMP by D NA polymerase or deamination of cytosine. N / A Specifically cleave the zymogen plasminogen to form active enzyme plasmin. -monophosphate synthase ′ Target name Tyrosine-protein kinase Z A P-70 Tyrosine-protein phosphatase nonreceptor type 1 Tyrosine-protein phosphatase nonreceptor type 11 Uracil-D NA glycosylase Uridine 5 Urokinase-type plasminogen activator ) Class PD PD PD PD PD PD Continued ( Table 2 PDB ID 1U59 2F71 3O5X 3F CI 3M I 2 3M H W

4058 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 6.6 - Continued ( 8.1 9.1 7.9 7.7 9.3 to 14.2 7.2 9.2 8.7 to ------

-barrel domain. β s. Promotes reorganization Promotes EC s. as also low oxidase activity toward activity oxidase low also H as R O S . –M transition) by protecting the nucleus from 2 phase, probably due to its degradation. 1 ontributes to the generation of generation the to C ontributes phases and decreases at M phase when it is hyperphosphorylated. A 2 phase and a minimum as cells enter M phase. Phosphorylation of cyclin 2 -barrel domain. Also binds synthetic drugs and influences their distribution availability. Appears to Also binds synthetic drugs and influences their distribution availability in the body. Appears to function modulating the activity of immune system during acute-phase reaction. Tyrosine-protein kinase that acts as a cell-surface receptor for V EG F A , C and FD. Plays an essential role in the regulation of angiogenesis, vascular development, permeability, and embryonic of differentiation and migration, survival, proliferation, Promotes hematopoiesis. of the actin cytoskeleton. I soforms lacking a transmembrane domain, such as isoforms 2 and 3, may function as decoy receptors for V EG F A , C and/or FD. I soform 2 plays an important role negative regulator of V EG F A - and C -mediated lymph angiogenesis by limiting the amount free and/or V EG F C and preventing their binding to L T4. Modulates T1 T4 signaling by forming heterodimers. Binding of vascular growth factors to isoform 1 leads the activation several signaling cascades. A ctivation of P LCG 1 leads to the production cellular signaling molecules D AG and inositol 1,4,5-trisphosphate and the activation of PK C . Mediates M A PK1/ ER K2, PK3/ K1, M A PK signaling pathway as well of the KT1 pathway. Mediates phosphorylation P I K3 R 1, regulatory subunit of phosphatidylinositol 3-kinase, reorganization the actin cytoskeleton and activation PTK2/F A K1. R equired for V EG F -mediated induction of N O S 2 and 3, leading to the production signaling molecule N O by EC s. Phosphorylates P LCG 1. Promotes phosphorylation of FY , NC K1, S 3, P I K3 R 1, PTK2/F A K1, and SRC . N uclear hormone receptor. Transcription factor that mediates the action of vitamin D3 by controlling expression of hormone-sensitive genes. R egulates transcription genes via its association with the W INAC complex, a chromatin-remodeling complex. R ecruited to promoters via its interaction the W INAC complex subunit B A Z1B/W S TF, which mediates interaction with acetylated histones, an essential step for VD R –promoter association. Plays a central role in calcium homeostasis. Contributes to histone modification. May position the N-terminus of H3 for efficient trimethylation at “ L ys-4”. A s part of the M LL 1-M complex, it is involved in methylation and dimethylation ys-4” histone H3. H3 “Lys-4” methylation represents a specific tag for epigenetic transcriptional activation. As part of the NSL complex, it may be involved in acetylation nucleosomal histone H 4 on several lysine residues. May regulate osteoblasts differentiation. A cts as a negative regulator of entry into mitosis ( G cytoplasmically activated cyclin B1-complexed C DK1 before the onset of mitosis by mediating phosphorylation of CDK1 on “Tyr-15”. Specifically phosphorylates and inactivates cyclin B1-complexed reaching a maximum during G B1- C DK1 occurs exclusively on “Tyr-15” and phosphorylation of monomeric does not occur. I ts activity increases during S and G correlated decrease in protein level occurs at M/ G Key enzyme in purine degradation. C atalyzes the oxidation of hypoxanthine to xanthine. acid. uric to xanthine of oxidation aldehydes (in vitro). N / A Functions as transport protein in the blood stream. Binds various ligands interior of its Functions as transport protein in the blood stream. Binds various hydrophobic ligands interior of its β function in modulating the activity of immune system during acute-phase reaction. -1- A cid glycoprotein 1 -1- A cid glycoprotein 2 Vascular endothelial growth factor receptor 2 Vitamin D3 receptor WD repeat-containing protein 5 Wee1-like protein kinase Xanthine dehydrogenase/oxidase Zinc finger CW-type PWWP domain protein 1 α α PD PD PD PD PK PD PK PK 3 E W H 1DB1 2 H 6Q 2 IN 6 2 E 1Q, 2 E 1Q_3, 2 E 1Q_2 2 RR 4 3KQ0 3 A PV, 3 A PU, 3 A PW

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HRR , CA D, EG F R , S O CE , I T A Ms, AN F, atrial NAALA Dase, -adenylylsulfate; ′ 9.9 7.5 7.6 9.9 Docking score - - - - -aminobutyric acid; ERA D, endoplasmic γ , leptin receptor; leptin LE P R , -phospho-5 GA B A , ′ HNE , hydroxynonenal; -protein coupled receptors; coupled G -protein -tetradecanoylphorbol-13-acetate; O EG F, epidermal growth factor; G P CR s, , insulin receptor substrate; receptor insulin IRS , -aceylaspartyl glutamate; -aceylaspartyl C T R s, constitutive transport elements; S taufen-mediated m RNA decay; H edgehog; N AN D R , androgen receptor; N MD, nonsense-mediated m RNA decay; ER , estrogen receptor; H h, peptide S MD, β , B-cell receptor; B-cell B CR , antithrombin; A T, NAAG , , insulin; INS , , lysobisphosphatidic acid; lysobisphosphatidic L BP A , EC M, extracellular matrix; , interleukin; IL , NLR s, nod-like receptors; -acetylaspartic acid; -acetylaspartic N ; PP1, protein phosphatase type 1; PPT, pedunculopontine tegmental; P R B, AL D H , aldehyde dehydrogenase; γ NAA , E PO R , erythropoietin receptor; PP, amyloid precursor protein; precursor amyloid A PP, -adrenergic and closely related receptors, , gonadotropin-releasing hormone; gonadotropin-releasing G n RH , β E r, t receptor; T-cell T CR , transcription; of activators and transducers signal S T A T, E pstein–Barr virus; CS F1 R , colony-stimulating factor 1 receptor; N K, natural killer; E BV, , kynurenic acid; kynurenic K A , kinase; N -terminal -secretase. I t has also been shown that it can cleave A PP -cleaved soluble A PP, and a corresponding cell-associated γ F, insulin-like growth factor; growth insulin-like IG F, β A J, adherens junction; which was based on the contents of PDB. E PO, erythropoietin; 20,21 ), p F, serum response factor; response serum SR F, s, antigen-presenting cells; antigen-presenting A P C s, H D AC , histone deacetylase; h ESC s, human embryonic stem cells; , growth hormone receptor; hormone growth GHR , SA P, stress-activated protein kinase; s H sps, small heat shock protein; -thioadenosine; MVB, multivesicular body; multivesicular MVB, -thioadenosine; CS F1, colony-stimulating factor; 1; ′ NHE J, nonhomologous end-joining; that is a web-based server used to predict drug–drug interaction via chemical–protein interactome. The protein targets were E pstein–Barr virus nuclear antigen 2; K, c-Jun J N K, vesicles; intralumenal IL Vs, -methyl-5 20,21 A D H , alcohol dehydrogenase; ) S / ENCC s, enteric neural crest cell; E B NA 2, , growth hormone; growth GH , BP, intestinal bile acid-binding protein; acid-binding bile intestinal I B A BP, -cleaved soluble A PP, and a corresponding cell-associated C -terminal fragment which is later released by -secretase. probably inducing a desensitization of them. Key regulator L P AR 1 signaling. C ompetes with RALA for binding to L P AR 1, thus affecting the signaling properties of receptor. Desensitizes 1 and 2 in a phosphorylation-independent manner. sequence, between residues 671 and 672 of A PP, leads to the generation extracellular release β γ Function Multifunctional enzyme mediating important protective effects. Metabolizes betaine aldehyde to betaine, an important cellular osmolyte and methyl donor. Protects cells from oxidative stress by metabolizing a number of lipid peroxidation-derived aldehydes. I nvolved in lysine catabolism. Specifically phosphorylates the agonist-occupied form of R esponsible for the proteolytic processing of A PP. C leaves at N -terminus - R esponsible for the proteolytic processing of A PP. C leaves PP, between residues 690 and 691, leading to the generation and extracellular release of C -terminal fragment which is later released by between residues 671 and 672. NG F, nerve growth factor; A lzheimer's disease; SAC , spindle assembly checkpoint; http://cpi.bio-x.cn/ddi C P I , chemical–protein interactome; A D, ER quality control compartment; F A BP, fatty acid-binding protein; FKBPs, FK506-binding proteins; FPP, farnesyl diphosphate; EC s, endothelial cells; L P S , lipopolysaccharide; M A PK, mitogen-activated protein kinase; M C F A , matrix-cell focal adhesions; MDK, midkine; m3 G , trimethylguanosine; M HC , major , anaphase-promoting complex/cyclosome; anaphase-promoting A P C / , http://sw16.im.med.umich.edu/databases/pdbbind/index.js HCAEC s, human coronary artery endothelial cells; BPs, sterol regulatory element-binding proteins; element-binding regulatory sterol SRE BPs, T, hydroxytryptamine; hydroxytryptamine; H T, ER Q C , EN a C , epithelial sodium channel; release; + 2 C a C ; P NRC , perinuclear recycling compartment; ARG , PP peroxisome proliferator-activated receptor A TP citrate lyase; F, guanine nucleotide exchange factor; exchange nucleotide guanine GE F, L p(a), lipoprotein(a); ACL Y, M, immunoreceptor tyrosine-based inhibitory motifs; inhibitory tyrosine-based immunoreceptor I T M, R P E , retinal pigment epithelium; , 12- TP A , factor; necrosis tumor T N F, receptor; toll-like T LR , junction; tight TJ, thrombopoietin; T H PO, 2; T-helper Th2, 1; T-helper Th1, network; - G olgi RNA p21; trans N F A T, nuclear factor of activated T-cells; HA T, histone acetyltransferase; F, apoptosis-activating factor; apoptosis-activating A P F, E J C , exon junction complex; F4, heat shock factor protein 4; protein factor shock heat HS F4, , glucocorticoids; GC , ERE , estrogen response element; A ; PK C , protein kinase C OMP, cartilage oligomeric matrix protein; - A minoadipic semialdehyde - A drenergic receptor kinase 1 - S ecretase 1 - S ecretase 2 Target name α dehydrogenase β β β , store overload-induced store S O ICR , -hydroxylase gene; d A , deoxyadenosine; AG , D diacylglycerol; d C , deoxycytidine; C s, D dendritic cells; DD I , drug–drug interaction; d G , deoxyguanosine; D H B A , dihydroxybenzoic acid; D ISC , death-inducing ) α entry;

+ 2 P, adapter protein; adapter A P, R O S , reactive oxygen species; AC - LL , acidic cluster-dileucine; Class PK PD PD PD C o A , coenzyme;

C a -linked-acidic dipeptidase; α Continued ( The prediction of targets of U A was performed using the I - C P DD tool ( TPase-activating proteins; G TPase-activating -acetylated- GA Ps, GRE , glucocorticoid response element; repair; recombination homologous motifs; activation tyrosine-based immunoreceptor linc RNA -p21, long intergenic noncoding MT A , moesin; M SN , acid; monomethylarsonic MM A , micro RNA s; mi RNA s, complex; histocompatibility N NMJ, neuromuscular junction; NO, nitric oxide; NSCLC, non-small-cell lung cancer; NSL, nonspecific lethal; OAA, oxaloacetate; OLs, oligodendrocytes; PAK, p21-activated kinase; PALLD, paladin; PAPS, 3 P AR P, poly( A DP-ribose) polymerase; Pc G , polycomb group; PDB, Protein Data Bank; G F, PD platelet-derived growth factor; P E P, phosphoenolpyruvate; P H B, prohibitin; P I 3K, phosphoinositide-3-kinase; P I P3, phosphatidylinositol-3,4,5- triphosphate; PK A , protein kinase movement; eye rapid REM, borders; ruffled RBs, elements; response acid retinoic RAREs, adapting; rapidly RA, paxillin; PXN, pleiotrophin; PTN, kinase; protein-tyrosine PTK, receptor; prolactin PRLR, B; isoform receptor progesterone RH O, rhodopsin; store-operated T GN , reticulum; endoplasmic transitional U A , ursolic acid; VD AC voltage-dependent anion channel; V EG F, vascular endothelial growth factor; VT C s, vesicular tubular clusters; N vitronectin. Table 2 PDB ID 2J6 L 3K R X 2QP8 2 E WY Notes: obtained from a third-party protein structure database named PDBBind ( Abbreviations: factor; natriuretic central CNS, peptide; calcitonin-gene-related CGRP, Im; factor cleavage CFIm, envelope; cornified CE, 1-chloro-2,4-dinitrobenzene; CDNB, kinase; cyclin-dependent CDK, complex; cap-binding CBC, domain; transactivation C-terminal nervous system; C YP7 A 1, cholesterol 7- signaling complex; D RG , dorsal root ganglion; D S Bs, double-strand breaks; epidermal growth factor receptor; reticulum-associated degradation;

4060 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) FDR 2.87 E -40 7.68 E -36 1.68 E -34 9.04 E -33 Continued ( Benjamini 9.33 E -41 1.40 E -36 9.68 E -35 2.20 E -33 Fold enrichment 6.42 5.33 6.05 6.28

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LCK , , PRKCB JAK3 , , , MAT1A , , , , ERBB2 , DYRK1A RET , MAP2K1 , , TNK2 ADK ZAP70 , , LYN , , WEE1 WEE1 , , , MAPKAPK2 KIT KIT EGFR , MET , , , , EGFR KHK JAK1 ITK , EPHB4 PRKCB , , , , MAP3K9 MAP3K9 MAP3K9 , FGFR1 FGFR1 CDK2 CSF1R TEK , , , RET , , JAK3 , , SGK1 , CHKB , , , , MAPK3 GSK3B CDK5R1 , NME1 RPS6KA1 , , CDK5 CDK5 , , , , CSNK2A1 KDR PDK4 , , , PRKCA CHEK1 , , PDK1 GRK6 CDK2 , , PRKCA , , SGK1 EGFR CHEK2 FGFR2 FGFR2 WEE1 ALK ALK , , NEK2 PTK2B ZAP70 , , , , KDR , , , , , ABCC1 , NME1 , CHKA MAPKAPK2 MAPKAPK2 , , NME2 , , SYK , CSNK2A1 CSNK2A1 CSNK2A1 , , , , MAPK14 KIT PDPK1 , , , TEK CSF1R TGFBR1 EGFR , WEE1 , , , MAPK10 PLAU PTK2 , JAK1 CDK5 CDK6 CDK6 , , ADRBK1 , PIK3CG PRKCA , , , , DYRK1A , , , ERBB4 , MAPK8 MAPK8 , , , FGFR1 NME2 PNKP AKT2 FYN , , , , , MAPK10 CHEK1 CHEK1 , PDPK1 PDPK1 PDPK1 , , AKT1 , , , , ALK SYK SYK , PDK3 ABL1 RAF1 , , CDK5 , PTK2B , , ZAP70 GRK6 IGF1R , , PRKCA , PRKCI PRKCI , , , , , INSR FGFR1 PLK1 GSK3B , , , , FGFR2 RAF1 MAPKAPK2 MAPK9 MAPK9 , , PRKCQ PRKCQ , , , AKT1 AKT1 AKT1 , , , , , ALK HCK TEK MET , SYK AKT2 AKT2 CDK6 , , , , MAPKAPK3 , , , PTK2 ADRBK1 ADRBK1 , , , , PIM1 PIM1 IRAK4 , , MERTK , PAK1 LCK LCK , CSNK2A2 , MET , , , CHKA , , DYRK1A , MAPK8 INSR INSR CHEK1 DCK CDK6 MAPK14 AKT2 PDK4 , , , , MAPK1 MAPK1 , RPS6KA1 , , , , SRC PRKCI , , , PTK2B , , IGF1R , BTK , , MAP2K1 CSNK2A2 CSNK2A2 CSNK2A2 TNK2 , ROCK1 ROCK1 , , , , , , FGFR2 MAPK3 MAPK3 , FYN PRKACA , , GSK3B , , ABL1 TGFBR1 PAK1 PAK1 INSR KHK , , , , , , , TYK2 TYK2 MAPKAPK3 MAPKAPK3 PRKCI PIM1 PTK2 , , ITK , , , ERBB2 , , , MAPK9 , BTK BTK BTK IRAK4 , EPHB4 , , , JAK3 , TGFBR1 , ADRBK1 , , BRAF BRAF , AURKA , , , PLK1 PAK4 KDR HCK , , , , PAK1 ABL1 , MAPK8 LCK PRKCQ PRKCQ PIM1 ,

SRC , , , , , PDK1 , , IGF1R PRKCB PRKCB MERTK CHKB ERBB2 ERBB2 , JAK1 , LYN LYN , , , , , , PRKACA PRKACA TTK , ROCK1 MAPK14 , , , , PDK3 , , , , AURKA AURKA AURKA , , , CHEK2 , Genes SPG7 MAP3K9 SGK1 CDK2 MAPK9 MAPKAPK3 IRAK4 HCK RPS6KA1 TNK2 PAK4 SGK1 CDK2 MAPK3 NEK2 KIT CSF1R MAPK10 GRK6 PAK4 BRAF MAPK1 NEK2 EPHB4 TTK TTK PRKACA ROCK1 MAPK1 NEK2 EPHB4 MAP2K1 PLK1 MERTK TTK -value P 1.71 E -43 5.29 E -39 1.08 E -37 5.38 E -36 % 18.20 19.06 16.49 15.42 derived from molecular docking calculations 3) by the D A V I database for target list of U derived from molecular . Count 85 89 77 72

Term Enrichment score: 31.55 A TP binding site Kinase Protein kinase, A TP binding site Protein kinase The top enriched clusters (enrichment score Table 3 Category Annotation cluster 1 UP_ SE Q_F EA TU RE S P_P IR _K E YWO R D IN T ER P R O UP_ SE Q_F EA TU RE

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4061 Dovepress He et al Dovepress 3.26 E -32 2.14 E -29 2.13 E -26 1.13 E -23 FDR 9.38 E -33 4.17 E -30 3.79 E -26 6.19 E -24 Benjamini 5.71 4.08 4.00 3.90 Fold enrichment

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4062 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 5.41 E -22 2.78 E -21 2.78 E -21 1.83 E -26 1.42 E -21 (Continued) 3.20 E -22 1.23 E -21 1.23 E -21 1.33 E -27 2.73 E -22 3.41 3.09 3.09 13.36 11.79

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MERTK LCK , , , , , , , RET TGFBR1 , , , TEK PPP2CA , , EGFR , BTK PTPN1 , BTK PRKCA BTK , ZAP70 , , BRD4 BIRC7 , PDK3 , , , KIT CHEK2 , , WEE1 CDK2 MAP2K1 , , , ZAP70 , , , PIK3CG JAK3 , KDR , , , PAK4 DUSP3 LYN ERBB4 MAPKAPK2 , , , , , PNKP , EGFR BCL2 TEK NME1 FGFR1 FGFR1 FGFR1 ITK CDK5R1 , , , , , , , PRKCB , PTPN1 , TNK2 AKT2 MET HCK PPP1CA PRKCQ , , PTK2B PAK1 TEK , , , , SYK , , ALK , , , , , MAPK14 MAPK14 AURKA AURKA AURKA JAK1 CDK5 CDK5 PIK3CG , , , , , , MAP2K1 , , , , TTK MAP3K9 PRKCA ALK NME2 CDK5R1 MAPKAPK2 BCL2 PDK4 FYN ABL1 INSR MAPK1 FGFR2 ADRBK1 CSF1R GRK6 PAK4 ALK PTK2B MAPK10 TTK TTK SYK PNKP FGFR1 MAPKAPK2 ITK TNK2 BRD4 CDK2 TYK2 PDK1 FYN FGFR2 PTK2 MAP2K1 LCK FGFR2 PTK2B MET 2.98 E -25 1.53 E -24 1.53 E -24 1.26 E -29 9.13 E -25 19.27 21.20 21.20 7.71 6.85 90 99 99 36 32 Phosphorylation Phosphate metabolic process Phosphorus metabolic process Enrichment score: 23.53 Tyrosine protein kinase Tyrosine protein kinase, active site G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 2 S P_P IR _K E YWO R D IN T ER P R O

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4063 Dovepress He et al Dovepress FDR 5.26 E -21 1.74 E -19 6.10 E -17 2.14 E -29 1.11 E -25 Benjamini 7.57 E -22 2.41 E -20 1.67 E -17 4.17 E -30 6.74 E -27 Fold enrichment 10.30 8.93 6.60 4.08 3.32

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, , , CHKB , ITK ITK , TNK2 TNK2 , , MAT1A MERTK FYN , , CSF1R , , IGF1R IGF1R CHEK2 , , BTK , , ABCB6 , EGFR , MERTK , , PTK2 , PLK1 CDK6 PRKCA DCK , CDK6 , , , , , MET , PAK4 KIT PRKCQ ROCK1 , JAK3 JAK3 , EGFR EGFR , , NEK2 SRC , , , , MAPK8 , TYK2 , TNK2 , , VCP MAP3K9 , JAK3 SYK LCK IRAK4 IRAK4 , MAPK10 , , ZAP70 , , , , , CSF1R , ACACB , IRAK4 MAPKAPK3 , PRKCI PRKCI , , , , CSNK2A1 PDK4 , , JAK1 JAK1 BRAF PNKP , , , KDR , PDPK1 , BTK BTK CSF1R CSF1R , ADK , , ERBB4 SYK , , GRK6 , RAF1 EPHB4 JAK3 , AKT2 , , TAP1 SRC TNK2 , , , , MAPK9 MET LYN , , , , , , MAT1A PIM1 , LCK LCK , RET FARS2 , , , MAPKAPK2 RPS6KA1 , , , SRC SRC SYK SYK , , KIT PDPK1 , , ABL1 , , AKT1 TEK , WEE1 INSR TGFBR1 , , , INSR , MAPK1 JAK1 , CHKA PDK4 , , , , , PRKCQ KIF11 FYN FYN , ABCC1 , , , EPHB4 , , EGFR DYRK1A KHK MAPK3 , , , TTK , TGFBR1 INSR INSR , , ROCK1 ERBB2 ALK , , , AKT1 , , SPG7 , CHEK1 EPHB4 EPHB4 , , PDK3 , , PAK1 CSNK2A1 TYK2 , GRK6 , PTK2B , , PDK3 ZAP70 , MERTK , JAK1 TYK2 , , FGFR1 , TYK2 TYK2 , , , , , GART UBA3 , , MET KIT KIT BRAF NME1 , , , , , ERBB2 PIK3CG CHKB ZAP70 ZAP70 , , , HCK , , TEK , , HCK HSP90AA1 MAP2K1 PTK2 , UBA3 KDR KDR , , , , , , PRKCB TNK2 MAPKAPK2 , , , ADRBK1 KDR FGFR2 PRKCB PRKACA GSK3B , LYN CSNK2A2 PLK1 , , , , HCK , , , , SYK , TEK TEK , HSP90AB1 CSNK2A2 , , , , ITK ERBB2 ERBB2 NME2 NEK2 , , , ALK ALK , , ERBB4 , , , PDK1 SGK1 PTK2B JAK3 , IGF1R , , DCK , VCP BTK , , CDK2 LYN , , MAP2K1 , , , CDK2 INSR BTK KIF11 CHEK2 , , DYRK1A ABCB6 , , , , , MAPK8 , MET MET ABCB10 PNKP , , , MAPK14 , DHX9 ERBB4 ERBB4 NRP1 , TAP1 , ERBB4 JAK1 , , , UBE2E1 PTK2 , , , MAP3K9 MAP3K9 , , , , IRAK4 PRKCA , , ATP7A WEE1 , AURKA SGK1 , CDK5 DHX9 , , , , HCK HCK FYN AURKA , , PAK4 GSK3B MAP2K1 , LCK , , , , PRKACA , ABL1 ABL1 , MAPK9 ADRBK1 , , SRC MAPK10 , , CSF1R MAPKAPK3 FGFR1 FGFR1 FGFR1 CHKA , MAPK1 , IGF1R , , PTK2B , , , , , ITK , , RET AKT2 PTK2B PTK2B TTK ALK , CLCN5 CLCN5 , , , , , , , GART , LYN LYN , CDK5 TTK , , RET , , , Genes FGFR2 PTK2 RET MERTK PTK2 RET MERTK IRAK4 ITK DYRK1A MAP3K9 EGFR ALK MAPK3 ERBB2 EPHB4 PDK1 ACACB MAPK14 ABL1 ABCB10 WEE1 FGFR1 CHEK1 ZAP70 KDR ABL1 PAK1 CFTR IGF1R RPS6KA1 FGFR2 FGFR2 SPG7 IDE -value P 3.37 E -24 1.43 E -22 3.94 E -20 1.27 E -32 7.66 E -29 % 7.28 7.28 8.14 20.56 22.91 Count 34 34 38 96 107 Term Tyrosine protein kinase TyrKc Protein tyrosine kinase activity Enrichment score: 16.58 N ucleotide phosphate- binding region: A TP A TP binding ) Continued ( Table 3 Category IN T ER P R O S M AR T G OT ER M_MF_F A T Annotation cluster 3 UP_ SE Q_F EA TU RE S P_P IR _K E YWO R D

4064 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 2.50 E -23 6.69 E -12 1.03 E -11 (Continued) 1.14 E -24 5.23 E -13 6.26 E -13 2.88 2.15 2.08

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, , , MAP3K9 , MAPK3 VCP KIF11 FARS2 , , , JAK1 , , DYRK1A UBE2E1 MAPK3 , VCP ALK , SRC TYK2 CHKB , , SGK1 , BRAF DHX9 , , , , MAT1A BRAF , UBE2E1 , , SPG7 , PDK1 , IGF1R , , , MAPK14 , TAP1 , , DHX9 TGFBR1 ROCK1 , , MAPK10 , PAK1 , CDK5 NEK2 , , , ITK MAPKAPK2 MAP3K9 , RET RPS6KA1 , , RET KIT UBA3 ATP7A , , MAPKAPK2 CFTR MAPK9 , PDE5A ERBB2 , , , , , , NEK2 MAT1A , , GSK3B ITK ATP7A , , PDE5A , , , PRKCB CLCN5 EPHB4 PRKCA , PYGL , , , , , TGM2 CLCN5 , ALK BRAF , PTK2B RAF1 , , , , PAK4 HSP90AA1 , , KHK CSNK2A1 DCK , , GNAI1 EGFR , PLK1 CSF1R MAP2K1 , , HSP90AB1 GART , , , CDK6 FYN , , CHEK2 CHKB MAT1A , GART , NME1 , ABL1 , , , NUDT2 RET AKT2 CHEK2 ERBB4 CSF1R , , MAPK3 CDK2 , NME1 , TAP1 , , , ABL1 , , , CFTR , , , , IGF1R PDK4 LCK , MAPK14 , SGK1 VCP , , PCK1 CSNK2A1 , , NT5M , , KDR , , KDR ERBB4 , CDA KDM1A DHX9 MAPKAPK2 ZAP70 PRKCI , NEK2 , PIK3CG PAK1 , , , , KIT EGFR EGFR , , , , , CHKA , , NME2 CDK5 , UBE2E1 , FYN CDK6 NME1 ADK , NME2 PTK2B MERTK , , , JAK3 , , , ADRBK1 , PDK3 , , MERTK RPS6KA1 , , ADRBK1 , , , CSNK2A1 ITK ATP7A PRKCA , , , SYK , LYN PDPK1 , , ABCB6 , DCK , ALK RAC1 CHKA , PAK4 ABCB6 MAPK10 , , , , , ZAP70 , , ERBB4 PYGL CSNK2A2 CHEK2 FGFR1 , , HCK , , PRKCI , KHK PIK3CG PIK3CG , TNK2 IGF1R , , NME2 , , MAPK1 , CLCN5 TNK2 , , PRKCQ ABCC1 , MAPK1 , CSNK2A2 , , , , , LCK CHEK1 INSR AKT2 GART , , , CHEK1 TEK , , CSF1R , KIF11 , TEK , , CFTR , RAF1 , BTK , CDC42 ACACB KIT , FGFR1 SYK SYK PDPK1 , ACACB , , XDH , , , , , TP53 CHKA CSNK2A2 , PDK1 , BTK , , , JAK1 , ABL1 GRK6 PNKP ADK , GRK6 , , TYK2 JAK3 PNKP KDR , , , , , , , PAK1 ADRBK1 MAPK1 PTK2 , RPS6KA1 , PTK2 , , MAPK10 , , , PDK4 DCK ZAP70 MET CDK6 PRKACA MET , LYN , , INSR INSR BTK , AKT1 , , , , PNP AKT1 , , , , , , FGFR2 AURKA FGFR2 , , , FGFR1 , MAPKAPK3 MAPKAPK3 GSK3B WEE1 , HSP90AA1 PAK4 WEE1 , , , PRKCB PRKCQ GIMAP2 , , , ABCC1 MERTK PNKP LCK ABCB6 TEK RAF1 , , , , , , , , IRAK4 , , DYRK1A IRAK4 DYRK1A , , , PDE2A PDK3 CHEK1 , SPG7 , , , KIF11 TTK SPG7 PRKCI , , , , , AURKA AURKA TGFBR1 ABCB10 TGFBR1 , , , PRKACA PRKACA , , , , FGFR2 MAPK8 PIM1 MAPK8 PIM1 ADK SRC JAK1 JAK3 , , SRC SGK1 , , FARS2 TAP1 , TYK2 , FARS2 , , , , , PTK2 , , , , CDK2 TNK2 IDE PDK4 , HCK PDPK1 KHK , WEE1 , , , UBA3 , UBA3 , , ACACB , , , , TP53 MAPK14 TTK , TTK , , , ABCB10 CDK5 PCK1 ABCC1 IDE ABCB10 PIM1 HRAS ROCK1 PLK1 HSP90AB1 PRKCA PTK2B MAPK8 MAPKAPK3 GRK6 AKT1 PRKCQ GSK3B EPHB4 MAP2K1 MAPK9 ERBB2 MAP2K1 IRAK4 MET LYN PDK3 IDE MAP3K9 ROCK1 MAPK9 ERBB2 PLK1 HSP90AB1 HSP90AA1 PRKCB PDK1 FYN EPHB4 4.31 E -15 1.72 E -26 6.62 E -15 23.56 25.27 24.84 110 118 116 A TP binding N ucleotide binding N ucleoside binding G OT ER M_MF_F A T S P_P IR _K E YWO R D G OT ER M_MF_F A T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4065 Dovepress He et al Dovepress 1.67 E -11 4.07 E -11 9.08 E -11 FDR 9.11 E -13 2.02 E -12 3.82 E -12 Benjamini 2.12 2.05 2.05 Fold enrichment

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CLCN5 ABCB10 , FGFR1 , TGFBR1 , MAPK9 , , , LYN , UBA3 CDK5 ZAP70 CHKB PRKCQ GSK3B MAPK3 , , , , , , DYRK1A , , BRAF PRKCA WEE1 BRAF MAP2K1 , , , ERBB2 , , , EPHB4 , KDR PYGL SPG7 , , , , PRKCB , HCK TGM2 , ABL1 PTK2B ALK , TEK , , , , CHEK1 HSP90AB1 HSP90AA1 RET MAT1A PLK1 , RET FGFR2 , , , , MAPKAPK2 , KIF11 TYK2 RPS6KA1 , , MAT1A PIM1 NEK2 , , , , , AKT2 UBA3 , , MAPK3 , , PDE5A MAP3K9 , CHKB , , MAP2K1 DHX9 , , , ABCB6 MAPK14 CDK2 TAP1 , , , , VCP PDK1 CFTR PTK2 , , , IGF1R , , KHK EGFR RPS6KA1 SGK1 EGFR , MERTK , , , , ITK PAK1 , PLK1 , , MAPKAPK2 HSP90AB1 , MAPK8 PRKCB , , ERBB4 , UBE2E1 , CHEK2 ROCK1 NME1 , , NEK2 , , NME1 KIT , , PYGL , , DHX9 MAT1A , CSNK2A1 , CDK5 , ACACB , , MAPKAPK3 CSNK2A1 CDK6 PTK2B , ATP7A , , , IRAK4 TGM2 , KHK , HSP90AA1 VCP , , , , , PAK4 TNK2 , , PRKCA CSF1R ITK PIK3CG , BRAF PIK3CG DCK FYN , CDK2 , , , , MAPK10 ALK , CHKA , , , , , CHEK2 MAPK9 NME2 MET CLCN5 , , SRC ERBB4 NME2 UBE2E1 , , , , JAK3 , , , , ADRBK1 GART PRKCI TAP1 FARS2 IGF1R PCK1 , , , , SGK1 , , , , KDM1A SYK RET LCK SYK ATP7A GRK6 , KDM1A , , , , , , AKT2 CSNK2A1 , PDPK1 , , , RAF1 CFTR , CDK5 CSF1R ZAP70 , JAK3 , , ABL1 , KIT , FGFR1 , , , KDR CHKA TP53 , MAPK3 EPHB4 , , , , MAPK1 ABCC1 CLCN5 RPS6KA1 ADK TGFBR1 MAPK1 , , ADRBK1 , , PTK2B INSR ERBB2 , , EGFR INSR , , , , , CHEK1 , , PRKCA GART PAK1 , TEK , ALK , AKT1 , , , , PDK4 MAPK10 , DCK CDK6 , DYRK1A , LYN , XDH , , ZAP70 , ABCC1 CSNK2A2 , JAK1 , , CSNK2A2 KHK MERTK CSNK2A2 , ABCB6 ABL1 FGFR1 , , , , PNKP NME1 , , , PNKP KDR , , CHKB , , IGF1R AKT2 , PRKCQ PAK4 SPG7 , CFTR PTK2 , , LCK , , , , CHEK1 PIK3CG , PDK3 RAF1 , , MAP2K1 PRKACA , , TEK PRKACA , , BTK , , UBA3 PRKCI KIF11 JAK1 , , BTK BTK MAPKAPK2 , , , , , FGFR2 , KIT , TNK2 XDH , , GSK3B , MAPKAPK3 , ACACB SYK WEE1 NME2 ADK WEE1 , NEK2 TYK2 , PAK1 , , , , , , , MERTK JAK3 ABCB6 HCK , CDK6 , , , , IRAK4 MAPK10 , PDPK1 , PDK4 , PTK2 PLK1 , TP53 , , , DHX9 , , DCK , AURKA ABCB10 GSK3B ABCB10 AURKA AURKA , CHEK2 GRK6 , , , , , HSP90AB1 MET INSR , , , , MAPK8 FGFR2 , PIM1 PIM1 SRC , , PAK4 , , FARS2 , MAPKAPK3 VCP , ITK PDK1 , LYN ERBB4 , , TNK2 PRKCB RAF1 , , PRKCQ MAPK1 HSP90AA1 , , AKT1 , PRKCI , , MAPK14 , , , ABCC1 ACACB PDK3 , , LCK , , , , IRAK4 , TTK TTK TTK , , , , MAP3K9 ROCK1 MAPK9 ERBB2 EPHB4 TGFBR1 DYRK1A SPG7 KIF11 TYK2 HCK MAPK14 MAP3K9 ROCK1 MAPK8 FARS2 SRC MET GRK6 PDPK1 TP53 ADK PDK4 JAK1 PYGL IDE IDE CDK2 TGM2 UBE2E1 SGK1 CSF1R ATP7A Genes IDE PRKACA PNKP CHKA ADRBK1 -value 2.63 E -14 5.87 E -14 P 1.07 E -14 24.41 23.98 % 23.56 114 112 Count 110 Purine nucleoside binding A denyl nucleotide binding Term A denyl ribonucleotide binding ) Continued ( G OT ER M_MF_F A T G OT ER M_MF_F A T Table 3 Category G OT ER M_MF_F A T

4066 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats ) 6.26 E -09 1.47 E -08 1.47 E -08 (Continued 2.28 E -10 4.72 E -10 4.72 E -10 1.76 1.85 1.85

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RAF1 PARP1 , , FARS2 , , CDK5 , , CDK5 CHKB CHKB JAK1 , , , , MAP3K9 POR MAPK3 PDK3 VCP PRKCA LYN TYK2 MAPK3 , , , , , ATP7A , PRKCA , , MAP3K9 , , , UBE2E1 , VCP PCK1 , PIK3CG KDM1A BRAF BRAF DHX9 DHX9 PARN , UBE2E1 , , , , DHX9 , , , , , ALK , PTK2B PTK2B ABL1 ALK , , MAPK14 , , , , SIRT5 NEK2 , , MAPK8 , HCK NEK2 NEK2 , , , ITK ITK MAPKAPK2 AKT2 MAPKAPK2 GSK3B MAPKAPK2 SYK AKT2 ITK KIF11 , , , , GART , JAK3 WEE1 , , , , , , ATP7A , PDE5A , , PRKCB , PDE5A MAT1A , , ATP7A , FYN , MAT1A , CFTR CLCN5 , , CFTR , , PDPK1 IGF1R CLCN5 , , IGF1R , NUDT2 , NUDT2 PDK4 , , , MAPK10 , , MERTK GNAI1 PDK1 , CSNK2A2 , KDR INSR PAK1 , , PAK1 , , GNAI1 GNAI1 , MAPK9 , CSF1R CSF1R , , , GART CSF1R , , , CHEK2 CDK2 KIT PYGL , ABCC1 GART CHEK2 CHEK2 NME1 , ABL1 , , , , , , , RET RET NME1 , , ABL1 CDK6 , , , MAPK14 , CDK6 , TYMS , , , PDK3 RAF1 BTK CDC42 , , , , HSP90AA1 TNK2 PPARGC1A CSNK2A1 , PAK4 , , PAK4 , CSNK2A1 KDR ERBB4 ,

, , , , , DCK ABCB6 KDR JAK1 , ERBB4 ERBB4 , , , KIT GIMAP2 ZAP70 ZAP70 , , FYN CDK5 PRKACA MAPK3 , , ZAP70 EGFR , , EGFR , , , , , , , NME2 PRKCI HCK NME2 , PRKCI , , , MERTK PDK4 , ADRBK1 ADRBK1 MERTK , AKT1 ADRBK1 , , , , RPS6KA1 LCK , , PIM1 SGK1 , , LCK GRK6 ME2 , , AURKA , , , , ALK PDPK1 PDPK1 , ABCB6 , PYGL , , RAC1 RAC1 ABCB6 CHKA CHKA , ACACB RAC1 ADH5 , , GSK3B , , , TGM2 , , , , , TP53 TP53 PDE5A CSNK2A2 , PDK1 , , , CSNK2A2 , ADK PIK3CG PIK3CG KHK SPG7 TNK2 ABCB10 PDK3 TNK2 ADK , TTK , , , MAPK1 BRAF , , , , , , , , MAPK1 , , , CHEK1 CHEK1 CHEK1 PRKCA , , TEK TEK TEK CBR3 , , , , , , DCK LYN LYN , BTK TAP1 DYRK1A , , CDC42 NXF1 ACACB CDC42 , , , FGFR1 FGFR1 SYK BTK SYK ACACB , IDE , , , , , , , , , , HCK PCK1 , NME1 GRK6 GRK6 , , HSD17B10 PNKP RPS6KA1 , JAK3 , PNKP MAPK14 , , , PRKCQ , , , GIMAP2 PRKCQ , LCK AKT2 , ROCK1 , PTK2 PTK2 PTK2 , , , , , , CDK6 MAP3K9 , , MET MET KIF11 KIF11 MET INSR INSR , , UBA3 AKT1 AKT1 , , , , , , , , HSP90AB1 PTK2B FGFR2 FGFR2 , AURKA , PDK1 AURKA FYN , , , KHK , , , , MAPKAPK3 MAPKAPK3 NME2 CHKA ADK HMGCR MAPKAPK3 TYK2 TYK2 WEE1 WEE1 , , , , , , , , , , , PAK1 ABCC1 TGM2 , CFTR , IRAK4 IRAK4 IRAK4 DYRK1A MAPK10 , DYRK1A , , , , , , , PLK1 SPG7 SPG7 TTK TTK NUDT2 , , , , , , MAT1A , IGF1R PYGL TGFBR1 PCK1 TGFBR1 TGFBR1 , , CLCN5 , , , , PRKACA PRKACA , , , MAPK8 MAPK8 PIM1 PIM1 G6PD HRAS SRC SRC SRC JAK1 JAK3 , , , , FARS2 FARS2 PAK4 , , FGFR1 , , , GIMAP2 , , TAP1 , , TP53 ERBB2 , VCP PRKCB PRKCB , , IDE IDE RAF1 , MAPK1 HSP90AA1 HSP90AA1 , , , , , RET , , , UBA3 UBA3 , , , , KIT , , , NCBP2 CSNK2A1 EGFR PNKP FGFR2 CHKB EPHB4 MAP2K1 DHFR UBE2E1 NT5M PRKCI PRKCQ ADH4 MAPK10 ABCC1 ABCB10 ROCK1 MAPK9 ERBB2 EPHB4 MAP2K1 PLK1 HSP90AB1 SGK1 CDK2 PDK4 GSK3B ABCB10 ROCK1 MAPK9 ERBB2 EPHB4 MAP2K1 PLK1 HSP90AB1 SGK1 CDK2 TGM2 RPS6KA1 HRAS DCK HRAS 4.05 E -12 9.49 E -12 9.49 E -12 29.34 25.27 25.27 137 118 118 N ucleotide binding R ibonucleotide binding Purine ribonucleotide binding G OT ER M_MF_F A T G OT ER M_MF_F A T G OT ER M_MF_F A T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4067 Dovepress He et al Dovepress FDR 5.32 E -08 3.48 E -16 3.48 E -16 6.39 E -15 1.68 E -14 1.68 E -14 5.40 E -14 1.33 E -13 1.78 E -13 Benjamini 1.45 E -09 5.65 E -17 5.65 E -17 7.36 E -16 1.61 E -15 1.61 E -15 3.73 E -15 6.22 E -15 1.42 E -14 Fold enrichment 1.81 18.19 18.19 15.54 14.90 14.90 13.47 12.91 14.45

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, ESR1 ESR1 ESR1 ESR1 ESR1 ESR1 CLCN5 , , , , , , , , GNAI1 JAK1 , ESR1 , , MAT1A , CHEK2 NUDT2 , , ESR1 PDE5A CSF1R CDK6 DCK , , , , GART , , PAK1 ABL1 PDK4 , NR1H4 NR1H4 NR1H4 NR1H4 NR1H4 NR1H4 NR1H4 , , NR1H4 , , , , , , , , ESRRG ESRRG ESRRG ESRRG ESRRG ESRRG RET ERBB4 , , , , , , GSK3B , , ESRRG , LCK , , KDR , PRKCI NME1 , ESRRG ZAP70 , PAK4 ADRBK1 , , , CSNK2A1 , PDK3 RARB RARB RARB RARB RARB RARB RARB , RARB , , , , , , , , , MERTK EGFR ADK , , CHKA , PPARG PPARG PPARG PPARG PPARG PPARG , , , , , , , PPARG TP53 , , ABCB6 RAC1 MAPK14 , HCK , , NME2 , PPARG , , PDPK1 NR1I2 NR1I2 NR1I2 NR1I2 NR1I2 NR1I2 NR1I2 , CHEK1 NR1I2 TNK2 , , , , , , , , , , KDM1A LYN , RXRA RXRA RXRA RXRA RXRA RXRA , FGFR1 , , , , , , PRKCQ TEK PIK3CG , RXRA , , FYN , , , PDK1 ACACB , , RXRA , MAPK1 GRK6 NR1I3 NR1I3 NR1I3 NR1I3 NR1I3 NR1I3 NR1I3 , SYK CDC42 , NR1I3 , , , , , , , , , , KIF11 TYK2 XDH THRB THRB THRB THRB THRB THRB PTK2 , , , PYGL , , , , , , , THRB , , MET , CSNK2A2 MAPKAPK3 , THRB , VDR VDR VDR VDR VDR VDR VDR VDR , , , , , , , , , PNKP INSR GIMAP2 AKT1 , , , , THRA THRA THRA THRA THRA THRA THRA IRAK4 FGFR2 , , , , , , PRKCB DYRK1A , , , , , BTK , PGR PGR PGR PGR PGR PGR PGR PGR , , , , , , , , RPS6KA1 THRA TGFBR1 FARS2 , , , , HSP90AA1 SPG7 WEE1 , , TGM2 SRC , , , RARG RARG RARG RARG RARG RARG RARG PRKACA , , , , , , , UBA3 , CDK2 , , MAPK8 AURKA , KHK , , AR AR AR AR AR AR RARG AR TAP1 , , , , , , , NR1H2 NR1H2 NR1H2 NR1H2 NR1H2 NR1H2 NR1H2 NR1H2 , SGK1 PIM1 , , , , , , , , , ERBB2 , , , EPHB4 MAP2K1 PLK1 , , TTK , CDK5 , , ABCB10 PCK1 HSP90AB1 , , , MAPK9 ESR2 ESR2 ESR2 ESR2 ESR2 ESR2 ESR2 ESR2 JAK3 , , , , , , , , , PPARD PPARD PPARD PPARD PPARD PPARD PPARD PPARD , VCP PTK2B CHKB , , , , , , , , IDE , PRKCA , , ALK , ROCK1 , , , DHX9 , Genes HRAS MAP3K9 BRAF MAPK3 NEK2 MAPKAPK2 ITK ATP7A UBE2E1 AKT2 CFTR IGF1R MAPK10 ABCC1 NR3C1 NR1H3 NR3C1 NR1H3 NR3C1 NR1H3 NR3C1 NR1H3 NR3C1 NR1H3 NR3C1 NR1H3 NR3C1 NR1H3 NR3C1 NR1H3 PPARA PPARA PPARA PPARA PPARA PPARA PPARA PPARA -value P 3.44 E -11 2.07 E -19 2.07 E -19 4.10 E -18 1.08 E -17 1.08 E -17 4.44 E -17 1.16 E -16 1.59 E -16 % 25.70 4.28 4.28 4.28 4.28 4.28 4.28 4.28 4.07 Count 120 20 20 20 20 20 20 20 19 Term Purine nucleotide binding Enrichment score: 16.40 D NA -binding region: nuclear receptor Zinc finger region: NR C4 type Zinc finger, nuclear hormone receptor type N uclear hormone receptor, ligand binding, core N uclear hormone receptor, ligand binding ZnF_ C 4 H O LI S teroid hormone receptor ) Continued ( Table 3 Category G OT ER M_MF_F A T Annotation cluster 4 UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE IN T ER P R O IN T ER P R O IN T ER P R O S M AR T S M AR T IN T ER P R O

4068 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 1.20 E -12 1.39 E -12 5.01 E -11 7.34 E -22 1.67 E -13 4.12 E -12 7.55 E -12 2.00 E -07 (Continued) 1.31 E -13 7.25 E -14 2.29 E -12 2.43 E -23 3.13 E -14 3.76 E -13 5.17 E -13 1.32 E -08 11.78 13.32 9.95 5.11 3.92 3.12 3.15 2.19

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ST14 C1QB , , GZMB GZMB MMP- , , , NR3C1 CASP8 , , , HPN HPN , , LTA4H ESR1 ESR1 , , , , , CTSG MMP-20 CPB2 HPN PRSS3 , , , , , DPP4 CPA1 SERPINE1 F9 F9 F9 , , , MMP-16 , , , ADAM17 ADAM17 , , , FOLH1 FOLH1 MMP-7 NR1H3 PLG MME , , , MMP-3 MMP-3 MMP-3 , , ADAMTS5 , ESR1 , , , , CPA4 CPA4 BACE2 , NR1H4 NR1H4 PRKCQ REN , , , CASP7 , , , SERPINE1 , , CPA4 , ESRRG ESRRG , CASP3 ADAMTS4 , , , , LTA4H TPSB2 ,

CTSD CASP8 CASP8 CTSD ,

ELANE ELANE ELANE , , , , GZMB , , , , XIAP CTSK CTSK , KLK5 , , , , CTSG ADAMTS5 PLAT PLAT , , , , RARB RARB MMP-8 ECE1 , , C1QA F2 ESRRG PLAT NR1H4 , , MMP-7 MMP-7 MMP-7 F7 , , , , , F9 FOLH1 , , , , , CASP8 , CTSG HTRA1 , PPARG PPARG , , , , CMA1 MMP-1 , CASP1 PREP , HPN CASP7 CASP7 , , BACE1 , IDE GZMA GZMA GZMA , , , , CTSB CTSB , , , , , PRSS3 PRSS3 MME SPOP LPA CTSS RARB , , NR1I2 NR1I2 , , , , , , , CTSK PPARG PLAU , F2 BACE1 , , , , NAALAD2 NAALAD2 RXRA RXRA ELANE MMP-8 MMP-8 MMP-8 CFD CASP7 , , , , , , , , , , F10 F10 F10 UBA3 MMP-12 , , , , CPA4 , , MMP-3 REN REN , , , HTRA1 HTRA1 PREP MMP-9 ADAMTS5 , , , , , IDE IDE IDE TRAF6 CASP3 , , , ST14 NR1I3 NR1I3 CPB2 NR1I2 ST14 F11 F11 F11 PLAU , , , VCP THRB , , , , , , , , ADAMTS4 , , , ADAM17 ADAM17 THRB THRB , , , , , PLAT MMP-16 GZMA , , , LPA LPA HTRA1 PLAU , , , , ECE1 ECE1 NAALAD2 MMP-12 MMP-12 , , , , , CTSB MMP-13 VDR VDR , SERPINE1 MMP-7 , , , , , DPP4 DPP4 DPP4 CPB2 F10 PRSS3 QPCT , , , , CMA1 THRA , NR1I3 LPA , BACE2 , MMP-9 MMP-9 MMP-9 TSG101 CTSD CTSD , BACE2 , THRA THRA , , , , , , , , , , , , F7 ACE ACE ADAMTS4 PRSS3 , MMP-20 GZMB , , , , , , CMA1 CASP3 CASP3 PGR PGR , , , , , F2 F2 F11 , , ADAMTS5 , , REN CASP8 VDR F9 , , , , MMP-12 MMP-13 MMP-13 CMA1 MMP-8 NAE1 TPSB2 TPSB2 TPSB2 CTSK RARG , , , , , RARG RARG , , , , , , REN CTSS , , ADAM17 PLG APP APP CASP3 LTA4H , , , , , , , , CTSG MME MME FOLH1 , , , , METAP2 METAP2 METAP2 METAP2 F7 F7 , , , , MMP-1 , , AR PGR DPP4 AR AR , , CTSG IDE , , , , NR1H2 NR1H2 BACE1 BACE1 , ECE1 , CFD , , PLAU , , , , , CASP7 ELANE , ECE1 , KLK5 CASP1 CASP1 CTSD QPCT LTA4H BRCA1 PREP PREP , , , , CTSK , , , , , , GZMB , , MMP-13 CTSS CTSS MMP-12 SPG7 , , , , , ACE MMP-20 MMP-20 MMP-20 C1QC , CTSB ESR2 ESR2 , , , , F2 ST14 ST14 , , , TPSB2 PPARD PPARD PPARD CPB2 , , , , , , , , ACE PLG PLG PLG CFD CFD CASP1 METAP1 METAP1 METAP1 UBE2E1 NR1H2 MMP-9 ADAMTS5 F7 , HPN , , , , , , , , , , , , , , , GZMA HTRA1 , , UBR2 , PPARA NR3C1 ESR2 F10 MMP-13 PPARA PPARA NR3C1 CTSB SPG7 MMP-1 CASP1 CTSS BACE1 MME CMA1 SPG7 LPA PLAT SPG7 MMP-1 CPA1 MMP-16 BACE2 ADAMTS4 KLK5 SPG7 MMP-1 CPA1 MMP-16 BACE2 ADAMTS4 KLK5 METAP1 MMP-3 CPA1 16 NAALAD2 MDM2 NEDD8 KLK5 PLAU 8.16 E -16 8.68 E -16 3.24 E -14 5.06 E -25 1.01 E -16 2.65 E -15 4.84 E -15 1.10 E -10 4.28 4.07 4.28 12.85 10.92 13.28 12.85 16.27 20 19 20 60 51 62 60 76 -amino acid peptides S teroid hormone receptor activity Zinc finger, NHR/GATA- type L igand-dependent nuclear receptor activity Enrichment score: 15.83 Protease E ndopeptidase activity Peptidase activity Peptidase activity, acting on l Proteolysis G OT ER M_MF_F A T IN T ER P R O G OT ER M_MF_F A T Annotation cluster 5 S P_P IR _K E YWO R D G OT ER M_MF_F A T G OT ER M_MF_F A T G OT ER M_MF_F A T G OT ER M_BP_F A T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4069 Dovepress He et al Dovepress 4.64 E -14 3.44 E -13 6.88 E -13 1.03 E -12 1.32 E -11 1.16 E -08 3.83 E -12 FDR 1.20 E -15 2.49 E -14 4.43 E -14 5.98 E -14 4.57 E -13 3.97 E -10 7.55 E -13 Benjamini Fold enrichment 4.76 5.25 4.44 4.38 4.55 3.15 2.76

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, , BCL2 , , CHEK2 , BIRC7 AURKA CDK2 CASP8 , , , PAK1 CDK5 , MAPK10 , , MAP2K1 , RPS6KA1 RPS6KA1 RPS6KA1 CHEK2 CHEK2 CHEK2 , , , , , , , MAPK8 ALB ERBB2 CDK5 , DYRK1A , PRKCI PRKCI PRKCI , , AKT2 , , , , , PRKACA PRKACA PRKACA , , , MAPK14 MAPK14 , NUDT2 , F7 , MAPK8 MAPK8 MAPK8 CDK5 , , , , , CSNK2A1 , TIAM1 CDK6 SGK1 IL2 , , , TGFBR1 , ADRBK1 , PRKCQ PRKCQ PRKCQ PIM1 PIM1 PIM1 PRKACA , , , , MAPKAPK2 , , , IGF1R PAK1 , MAPK10 , PAK4 PAK4 PAK4 , , , , , , MAPKAPK2 GSK3B , PLK1 , PLK1 , , ADAM17 ESR2 , MAPKAPK2 MAPKAPK2 MAPKAPK2 , , , , ADORA2A FNTA ABL1 , MAPK9 MAPK9 MAPK9 PRKCI , , PDPK1 , , , VDR , , PRKCA , PAK4 PPP2R1A MAPK10 , , , , CDK6 MAPK1 MAPK1 MAPK1 LGALS1 TGFBR1 TGFBR1 TGFBR1 , , , , , FKBP1A , , , AURKA , , PRKACA AURKA APP MAPK8 MAPK8 , , MAP3K9 MAP3K9 MAP3K9 , , , XIAP PRKCI , , , MIF , MAPK14 , AURKA AURKA AURKA , , GRK6 GRK6 GRK6 MAPK9 , , , PIM1 RPS6KA1 , , , AKT1 , , RPS6KA1 , EGFR , GSK3B , , , CDK6 BRAF BRAF BRAF RAF1 , , , , PRKCA , , PRKCB PRKCB PRKCB F2 TRAF6 , , , PAK4 , , SRC , , CHEK1 TP53 PLK1 CSNK2A1 CASP3 , , , , , VCP ADRBK1 MAPK9 MAPK9 , , ADRBK1 , , AKT2 , IRAK4 MAPK3 MAPK3 MAPK3 YWHAZ , , ADRBK1 ADRBK1 ADRBK1 , , , ROCK1 PRKCQ , LCK CSNK2A1 CSNK2A1 CSNK2A1 PRKCI , , , PRKCQ , EGFR , , , , , PRKCI , , , , ROCK1 ROCK1 ROCK1 CDK2 CDK2 CDK2 TTK GLO1 , , , , , , , , ESR1 , AKT1 CHEK2 , , GRK6 GRK6 PDPK1 ATP7A , , PAK1 , , CSNK2A1 , CHEK1 , PIM1 CHEK1 , RPS6KA1 , PIM1 , , MAPK8 PDPK1 PDPK1 PDPK1 , NME1 CHEK1 CHEK1 CHEK1 , , , , BCL6 WEE1 WEE1 WEE1 DYRK1A DYRK1A DYRK1A , , , , MAPK1 , , , , , , , BTK MAPK1 , PIK3CG CDK5R1 SFN PIM1 , , , MAP2K1 , , , CSNK2A2 , , MAP2K1 MAP2K1 MAP2K1 , , , TTK AKT1 TTK , , NAE1 , TTK TTK TTK , , , , MAPK3 MAPK3 AKT1 AKT1 AKT1 PDPK1 , , , , , MAPKAPK3 , PRKACA , ROCK1 CDK5 CDK5 CDK5 PRKCQ , , , , , , NME2 TGM2 PTEN GSK3B GSK3B GSK3B MAPK9 , , , , , , TGFBR1 RXRA PRKCB , SGK1 NQO1 PRKCB , , TRAF2 , , SGK1 SGK1 SGK1 , , , , , , NR3C1 , CHEK2 , BRCA1 AKT1 IRAK4 IRAK4 IRAK4 , , , , , PAK4 NEK2 , , RAC1 KIT CD38 CSNK2A2 BRAF GRK6 , , DYRK1A DYRK1A , , , , MAPK1 CDK2 , , , CDK2 MAPKAPK3 , MAPKAPK3 MMP-9 MAPK10 MAPK10 MAPK10 MAPKAPK3 MAPKAPK3 MAPKAPK3 PRKCA MAP2K1 CASP1 , , GSTP1 , PRKCA PRKCA PRKCA , ROCK1 , , , , , , , , , MAPK14 MAPK14 MAPK14 , , , , PLG , , , , , CSNK2A2 AKT2 NEK2 CHEK2 AKT2 CDK5 GSK3B NEK2 CSNK2A2 AKT2 NEK2 CSNK2A2 AKT2 NEK2 CSNK2A2 CDK5R1 MAPKAPK2 MAP3K9 ROCK1 WEE1 GSK3B HRAS RARB BRAF MAPK1 CTSB BCL2L1 PPP2CA IGF1 Genes NEK2 CDK6 PLK1 CDK6 PLK1 CDK6 PLK1 SGK1 PRKCB MAPK3 -value 2.09 E -16 4.17 E -16 6.21 E -16 1.09 E -14 7.51 E -12 2.10 E -15 P 3.20 E -17 8.14 9.42 9.42 8.14 10.06 15.85 % 9.42 38 44 44 38 47 74 Count 44 S erine/threonine protein kinase S erine/threonine protein kinase, active site S erine/threonine protein kinase related S _TKc Protein serine/threonine kinase activity Enrichment score: 14.56 R egulation of programmed cell death Term Enrichment score: 14.64 S erine/threonine protein kinase ) Continued ( IN T ER P R O IN T ER P R O IN T ER P R O S M AR T G OT ER M_MF_F A T Annotation cluster 7 G OT ER M_BP_F A T Table 3 Category Annotation cluster 6 S P_P IR _K E YWO R D

4070 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 4.63 E -12 7.25 E -12 3.12 E -09 3.70 E -09 3.70 E -09 6.67 E -08 (Continued) 8.23 E -13 1.17 E -12 3.25 E -10 3.65 E -10 3.65 E -10 4.93 E -09 2.75 2.75 3.19 3.13 3.13 3.34

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, , SFN BIRC7 BIRC7 , LYN LYN LYN CASP8 CASP8 , , , , , , , PPP2CA PPP2CA PPP2CA EGFR , , , , IGF1 IGF1 BCL2 PTPN11 MAPK8 MAPK8 , , ALB ERBB2 ERBB2 , CDK5 CDK5 , , , , , , , , BIRC7 CXCR4 CXCR4 CXCR4 , , , , CDK5R1 CDK5R1 CDK5R1 PTK2B NUDT2 NUDT2 , , , SYK , F7 F7 , , , , , ALB CHEK1 TIAM1 TIAM1 , , IGF1 IL2 IL2 , , TGFBR1 TGFBR1 PTK2B PTK2B PTK2B , , , , , , , , BIRC7 BIRC7 PPP2R1A PPP2R1A PPP2R1A IGF1R , , PIM1 , , , , , PDPK1 PDPK1 PDPK1 INSR , , , ADAM17 ADAM17 ESR2 ESR2 , TRAF2 TRAF2 TRAF2 , , , , , , , ADORA2A ADORA2A CXCR4 IGF1R FNTA FNTA ABL1 ABL1 , , , PIN1 PIN1 PIN1 , , , , , PPARG MET MET , , , VDR BIRC7 , , , , PPP2R1A PPP2R1A , MET EGFR EGFR EGFR , , , , , , LGALS1 LGALS1 , , APP APP APP KIT KIT KIT , CASP3 CASP3 CASP3 IL2 , , , , , VDR TRAF6 XIAP XIAP PRKCI PRKCI , , , MIF , , , , , , , , MAPK9 MAPK9 MET , , , EGFR GSK3B GSK3B ERBB2 , , , ADAM17 ADAM17 ADAM17 , PRKCA , , , ELANE PRKCA PRKCA PRKCA TGFBR1 TGFBR1 , APP APP APP SFN SFN SFN , , , , , , F2 F2 TRAF6 TRAF6 MIF , , , , , , , , , , , SRC , TP53 TP53 PAK1 CASP3 CASP3 CASP3 , , , F2 F2 F2 , , , VCP VCP , , , , , SYK SYK SYK YWHAZ YWHAZ IL2 IL2 , , , , , LCK LCK ADAM17 SRC , , EGFR PRKCA TGFBR1 PLEK PLEK , , , , , , , , , GLO1 GLO1 , , ESR1 ESR1 CDC42 CDC42 CDC42 TGFBR1 FKBP1A FKBP1A FKBP1A , , , , , AKT1 AKT1 , , , , CHEK2 , , ADORA2A NCK2 NCK2 NCK2 , PDPK1 , , , , , ATP7A ATP7A INSR INSR INSR PRKACA , , , , , , TNK2 TNK2 , , FABP4 NME1 NME1 , BCL6 BCL6 , , CHEK2 , , IL2 BTK BTK PIK3CG PIK3CG , CDK5R1 CDK5R1 SFN PIM1 PIM1 , , , AKT1 AKT1 AKT1 , , , , , , , , , , MAP2K1 MAP2K1 MAP2K1 , , , CDK5 CDK5 CDK5 PAK1 PAK1 PAK1 , CHEK1 , CHEK1 , CHEK1 NAE1 NAE1 RAC1 , , , , , , , , , CDC42 MAP2K1 , HMGCR , SFN FABP4 FABP4 , , , , NCK2 , NME2 NME2 TGM2 TGM2 PTEN PTEN TNK2 , , , , , , RXRA RXRA PTEN PTEN PTEN , RB1 RB1 RB1 NQO1 NQO1 , , TRAF2 TRAF2 , , , , , , , , , , NR3C1 TRAF6 TRAF6 TRAF6 , , , , AKT1 ERBB2 ERBB2 ERBB2 BRCA1 BRCA1 , , , , , , PRKACA PRKACA PRKACA PPP2CA TTK TTK TTK , , , , ATP7A ATP7A , , , RAC1 RAC1 KIT NR3C1 , , CD38 CD38 , , , , , , PIM1 PIM1 PIM1 PPP2R1A FABP4 CDK5R1 , , , , , PTPN11 , MMP-9 MMP-9 CASP1 CASP1 , GSTP1 GSTP1 RAC1 RAC1 RAC1 , , ROCK1 ROCK1 , , , , PLG PLG , , , , , , , PTEN , HRAS RARB BRAF MAPK1 CTSB BCL2L1 PPP2CA IGF1 RARB BRAF MAPK1 CTSB BCL2L1 PPP2CA IGF1 MAP3K9 ELANE ADORA2A BCL2 ATP7A MAP3K9 ELANE ADORA2A BCL2 PTPN11 MAP3K9 ELANE ADORA2A BCL2 PTPN11 KIT MAP3K9 PRKCA CDK5 HRAS HMGCR HMGCR HMGCR TRAF2 2.52 E -15 4.05 E -15 1.72 E -12 2.04 E -12 2.04 E -12 3.67 E -11 15.85 15.63 10.49 10.71 10.71 8.78 74 73 49 50 50 41 R egulation of cell death R egulation of apoptosis Enrichment score: 10.94 R egulation of phosphorylation R egulation of phosphorus metabolic process R egulation of phosphate metabolic process R egulation of transferase activity G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 8 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4071 Dovepress He et al Dovepress FDR 7.37 E -08 4.07 E -07 1.44 E -10 5.48 E -10 2.53 E -09 1.67 E -08 1.01 E -07 1.08 E -07 1.28 E -07 8.67 E -07 7.74 E -07 3.55 E -06 4.49 E -06 Benjamini 5.24 E -09 2.33 E -08 2.18 E -12 4.45 E -11 3.68 E -11 1.08 E -09 4.50 E -09 4.43 E -09 4.92 E -09 2.26 E -08 2.14 E -08 8.44 E -08 1.02 E -07 Fold enrichment 3.40 3.34 12.97 5.85 6.97 7.72 6.47 6.87 6.81 4.68 5.61 4.21 4.17

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, , PLAU , , , , , , , ST14 F11 , , PRKACA PPP2CA F7 , , , PLG PRSS3 , , TGFBR1 KIT KIT DPP4 DPP4 DPP4 F2 NAALAD2 NAALAD2 , PLG PLG PLG PLG PLG , , PTPN11 , , , , , , , , , , , , F7 CTSG , , FABP4 DPP4 LPA , PPP2R1A , F7 F7 F7 F7 F7 , , , , , , , SFN SFN PLG PLG PLG , , GZMB , , , PPP2CA , PLAU PLAU , , , TPSB2 TPSB2 TPSB2 CDK5 MAP3K9 , , , , , PLG F7 F7 F7 DPP4 , F9 , , , , MAP2K1 , GZMB , NCK2 , , TPSB2 , GZMB GZMB GZMB GZMB GZMB PRKCA , , , , , , F7 RB1 , CFD CFD CFD CTSG CTSG , , , , CHEK1 CHEK1 F9 , , , , , CFD , PLAU PLAU PLAU F9 F9 F9 F9 F9 PTK2B , , , , , , , , , MAP3K9 PLAU CFD GZMB GZMB GZMB , , , , , , ELANE , EGFR , CFD CFD , , PTPN11 PPP2R1A GZMB , BIRC7 , F9 F9 F9 , , , , , ERBB2 ERBB2 TPSB2 CTSG CTSG CTSG ELANE , , , , , , , CTSG ELANE ELANE ELANE ELANE ELANE SYK , F9 PRSS3 , , , , , , KLK5 PTK2B CXCR4 , SERPINE1 SERPINE1 SERPINE1 , , , , , , , TPSB2 TPSB2 PIM1 CDK5 , , , CFD CFD CFD , PRKCA , , , , ELANE ELANE ELANE KLK5 , , , , CMA1 INSR , KLK5 KLK5 KLK5 KLK5 KLK5 , CMA1 , , , , , BCHE , GZMA , GZMA PRSS3 PRSS3 PRSS3 , RB1 PDPK1 , , , , CXCR4 , MET , ADORA2A ADORA2A , , , , CMA1 CMA1 F2 EGFR , , , IL2 TPSB2 TPSB2 TPSB2 , KLK5 KLK5 KLK5 , , , , , , , PLAU PLAU , , GZMA F10 PRSS3 F10 , APP , , TRAF6 , , GZMA GZMA GZMA GZMA GZMA , PLAU , , , , , , HTRA1 BIRC7 SYK , , , HTRA1 HTRA1 HTRA1 F2 ST14 PDPK1 ELANE , , , , , , , PRSS3 PRSS3 , , CMA1 CMA1 CMA1 CTSG CTSG HMGCR HMGCR HPN HPN CES1 F10 F10 F10 F10 GZMA F10 GZMA GZMA , , , , , , , , , , , , , , , , , , PAK1 LPA , CASP3 , CTSG F2 F2 LPA LPA LPA ADAM17 , , APP , , , , , INSR PLAU , PIM1 , , , , ST14 , F10 F10 F10 F10 , , , , PREP HPN HPN HPN HPN HPN FOLH1 PREP , , , , , , , IL2 , PRSS3 PRSS3 PRSS3 CMA1 CMA1 TGFBR1 , , , , , , , ST14 ST14 MET , , CDK5R1 CDK5R1 , TPSB2 CTSG FABP4 , , , , PRKACA CDC42 CASP3 , TRAF6 F2 F2 F2 F2 F2 HMGCR MMP-8 MMP-8 MMP-8 , F11 HPN F11 F11 F11 F11 HPN F11 HPN HPN , , , F11 , , , , , , , , , , , , , , , , , , , , HTRA1 , Genes TRAF2 AKT1 RAC1 MAP2K1 NCK2 AKT1 PAK1 ELANE ADAM17 CMA1 PLAT NAALAD2 LPA ST14 HTRA1 ST14 ST14 PLAT CMA1 HTRA1 PLAT ST14 PLAT ST14 TRAF2 PLAT PREP PLAT PLAT PLAT PLAT PLAT PREP PLAT PREP PREP -value P 4.06 E -11 2.24 E -10 9.89 E -14 3.26 E -13 1.74 E -12 9.94 E -12 6.51 E -11 6.91 E -11 8.22 E -11 5.61 E -10 6.36 E -10 2.29 E -09 2.90 E -09 % 8.56531 8.145 3.64 6.00 4.93 4.28 4.50 4.28 4.28 5.35 4.50 5.57 5.57 Count 40 38 17 28 23 20 21 20 20 25 21 26 26 Term R egulation of kinase activity R egulation of protein kinase activity Enrichment score: 10.39 S erine proteinase C harge relay system S erine protease Peptidase S 1 Peptidase S 1 and 6, chymotrypsin/ H ap Peptidase S 1 A , chymotrypsin Peptidase S 1/ 6, chymotrypsin/ H ap, active site S erine-type endopeptidase activity Tryp_ S Pc S erine-type peptidase activity S erine hydrolase activity ) Continued ( Table 3 Category G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 9 S P_P IR _K E YWO R D UP_ SE Q_F EA TU RE S P_P IR _K E YWO R D UP_ SE Q_F EA TU RE IN T ER P R O IN T ER P R O IN T ER P R O G OT ER M_MF_F A T S M AR T G OT ER M_MF_F A T G OT ER M_MF_F A T

4072 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 1.26 E -06 1.36 E -06 3.11 E -06 1.54 E -06 1.90 E -06 2.18 E -06 3.45 E -07 1.55 E -06 1.34 E -05 7.65 E -10 (Continued) 6.76 E -08 7.10 E -08 1.31 E -07 7.82 E -08 8.87 E -08 9.91 E -08 2.04 E -08 7.64 E -08 5.08 E -07 5.74 E -11 3.21 3.20 3.17 2.96 2.94 2.93 4.40 4.27 4.31 23.39

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, , , PLG

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, , , , , , PLG PLG PLG , , , ELANE AKT1 , MIF MIF , CXCR4 RAC1 EGFR , , , , , TRAF6 TRAF6 TP53 TP53 ESR1 NR3C1 NR3C1 NR3C1 , , , , , , , , PTEN BCL2 BCL2 BCL2 , GSTP1 PTK2B , , , PRKCA PRKCA PRKCA , TGFBR1 , , , , GSTT1 MIF , , , MAPK8 MAPK8 TEK , , , BRCA1 BRCA1 BRCA1 PTEN PTEN , , , SFN SFN SFN , , , , , BCL6 BCL6 PTK2B TRAF6 TP53 , , , , , PRKCI PRKCI ACE MAP2K1 , , , CDK5 , , KIT KIT PTEN NQO1 NQO1 NQO1 PPP2CA PPP2CA PPP2CA , , , MAPK8 BCL2 , , , , , , , , GSTZ1 , ACE CDK5 CDK5 CDK5 F7 , , , , MAP2K1 , F10 BCL6 , GLO1 GLO1 , , , , PRKCI HDAC7 HDAC7 CHEK2 CHEK2 CHEK2 , ADAM17 ADAM17 IGF1R , , BIRC7 BIRC7 , , , , , , , , KIT , TRAF6 TRAF6 TRAF6 LYN TIAM1 TIAM1 TIAM1 TP53 TP53 TP53 PTK2B , , , , BCL2L1 BCL2L1 BCL2L1 , , , , , , , IGF1R GSTA4 , , , EGFR , , GLO1 , , TGM2 TGM2 ADAM17 PIM1 PIM1 BIRC7 AKT1 , , , , , , , ABL1 ABL1 ABL1 IGF1 GSK3B GSK3B F10 , , , BCL2L1 BCL2L1 BCL2L1 , , , , , , , ADAM17 ADAM17 , , RXRA RXRA RXRA CASP1 CASP1 CASP1 AKT1 , , , CASP3 CASP3 CASP3 , , , , , , , ERBB2 ERBB2 ERBB2 CXCR4 GSTA3 , , , BCL2 BCL2 PRKCA , TGM2 PIM1 , , , , , PTEN , , GSK3B EGFR , , TGFBR1 TGFBR1 NME1 NME1 APP APP APP , , , , MAPK8 MAPK8 MAPK8 MMP-9 , , , , , , MMP-9 MMP-9 MMP-9 , RARB RARB RARB ALB ALB , , , ACE PTEN KIT , , , , , , MAPK1 MAPK1 , , INSR , , BCL2 , , TGFBR1 TGFBR1 TGFBR1 GSTA2 , , , GSTP1 , INSR , , VDR VDR VDR BRAF BRAF NME1 TGFBR1 , , , KIT , , , , , ADORA2A ADORA2A ADORA2A NME2 NME2 , , , BCL6 BCL6 BCL6 , , KDR KDR ALB FNTA FNTA , , , , , IGF1R , , , MAPK9 MAPK9 MAPK9 , , , , RAC1 , MAP2K1 MMP-9 RARG RARG RARG , , , , , BCL6 , ADORA2A ADORA2A ADORA2A AKT1 AKT1 AKT1 BRAF , , , , , , , GSTM2 PLG PLG HPGDS HDAC7 XIAP XIAP XIAP , , , , , ESR2 ESR2 NME2 , , , LCK LCK LCK , , , ROCK1 ROCK1 , , , TEK FNTA , , MMP-9 TGM2 TGM2 TGM2 , , , CASP3 CASP3 , , , , , MAPK1 F7 F7 BTK BTK BTK , , , RAC1 ERBB4 , , , , , INSR NUDT2 NUDT2 NUDT2 , HRAS , , , HRAS HRAS ESR2 CDK5 CDK5 , , , , , , EGFR EGFR ROCK1 GSTA1 CD38 CD38 CD38 BCL2 GSTO1 , , , , , , , , ADORA2A IL2 IL2 , RAC1 RAC1 RAC1 CDK5R1 CDK5R1 CDK5R1 , , , CASP3 , , , , , , ERBB4 SRC SRC SRC IGF1R IGF1R , F10 IGF1 IGF1 TEK , , , , , , , , , F7 F7 F7 , AKT1 , , , , YWHAZ AKT1 PIK3CG ESR1 GSTP1 AKT1 PIK3CG ESR1 GSTP1 IGF1R PIK3CG IGF1 PTEN CASP8 PPP2R1A MAPK1 PTEN CASP8 PPP2R1A MAPK1 PTEN CASP8 PPP2R1A MAPK1 BCL2 IGF1 CXCR4 IGF1 KDR ADAM17 GSTO2 YWHAZ YWHAZ TRAF2 TRAF2 TRAF2 NRP1 ERBB4 EGFR GSTM1 6.92 E -10 7.49 E -10 1.71 E -09 8.49 E -10 1.05 E -09 1.20 E -09 1.90 E -10 8.53 E -10 7.40 E -09 4.56 E -13 8.4 8.14 7.92 8.99 8.99 8.99 6.00 5.78 5.14 2.57 38 38 37 42 42 42 28 27 24 12 Enrichment score: 9.02 N egative regulation of programmed cell death N egative regulation of cell death N egative regulation of apoptosis Enrichment score: 8.99 Positive regulation of apoptosis Positive regulation of programmed cell death Positive regulation of cell death Enrichment score: 8.97 R egulation of cell motion R egulation of locomotion R egulation of cell migration Enrichment score: 8.68 GS T N -terminal Annotation cluster 10 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 11 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 12 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 13 UP_ SE Q_F EA TU RE

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4073 Dovepress He et al Dovepress FDR 5.94 E -09 1.47 E -08 3.22 E -07 3.24 E -06 5.06 E -06 7.40 E -06 0.018816 0.804529 4.77 E -06 2.07 E -04 3.36 E -04 1.56 E -05 1.17 E -04 5.49 E -04 1.61 E -05 1.81 E -04 Benjamini 2.85 E -10 4.47 E -10 1.96 E -08 1.17 E -07 1.71 E -07 2.37 E -07 9.77 E -05 0.01217529 1.97 E -07 5.58 E -06 8.90 E -06 1.89 E -07 3.06 E -06 1.26 E -05 5.84 E -07 4.95 E -06 Fold enrichment 19.50 17.31 14.88 17.02 13.56 13.11 4.18 3.90 5.88 5.26 5.45 6.31 5.48 4.75 3.41 3.06

,

, , , , ,

, , ,

, KDR

, , , , , , , , , G6PD , , FYN FYN FYN , GAB2 GAB2 , , , , , ROCK1 ROCK1 , , F7 , KDR , IGF1R PTK2 PTK2 GSTT1 GSTT1 GSTT1 GSTT1 GSTT1 GSTT1 GSTZ1 RET RET , KIT , , , , , , , , , , , , DAPP1 DAPP1 DAPP1 GSTM2 , , , KIT , ADAM17 ADAM17 , , , SRC SRC KDR HDAC7 F7 , , , IGF1 , PLAT PLAT , , , , FYN FYN GSTZ1 GSTK1 GSTZ1 GSTZ1 GSTZ1 GSTZ1 GSTZ1 , , NCK2 NCK2 NCK2 , , , , , , , , , , KIT GSTM1 , , HDAC7 , IGF1 INSR PTEN PTEN , , , , F7 PRKCA PRKCA , , , TYK2 TYK2 TYK2 NCK2 NCK2 TGFBR1 , , , , , , GSTA4 GSTA4 GSTA4 GSTA4 GSTA4 GSTA4 GSTA4 KIT KIT , , , , , , , INSR , , , BCL6 HDAC7 GSTT1 , , , SYK SYK IGF1 , , , MMP-9 BTK BTK BTK , KDR KDR , , , , , PEX5 PEX5 MMP-9 , , , GSTA3 GSTA3 GSTA3 GSTA3 GSTA3 GSTA3 GSTA3 INSR GSTP1 , , , , , , , , , SYK SYK SYK GSTA4 , , , , SFTPD SFTPD ADAM17 , , CDK5 CDK5 , MMP-9 ADAM17 , , , , PTPN11 PTPN11 PTPN11 MAP2K1 , , , , GSTA2 GSTA2 GSTA2 GSTA2 GSTA2 GSTA2 GSTA2 GSTP1 GSTP1 ABL1 ABL1 ABL1 , , , , , , , , , HMGCR HMGCR GSTP1 , , , GSTO1 MAP2K1 , , , , RAC1 RAC1 , BCL2 GSTA3 , , ESR2 ESR2 BCL2 , ADAM17 , SRC SRC SRC , , , , , , , ERBB4 MAP2K1 JAK3 JAK3 JAK3 LYN , , , , , , NRP1 NRP1 , , HCK HCK HCK GSTM2 GSTM2 GSTM2 GSTM2 GSTM2 GSTM2 GSTM2 HPGDS HPGDS GSTP1 GSTP1 GSTP1 BCL2 PRSS3 PRSS3 , , , GSTO1 GSTO2 , , , , , , , , , , , , ODC1 , PTK2B , , , , PTK2B , , ELANE ELANE , , , F10 , JAK1 JAK1 JAK1 , , , ERBB4 ERBB4 , , GRB2 GRB2 GRB2 , , , MET MET GSTA1 GSTA1 GSTA1 GSTA1 GSTA1 GSTA1 GSTA1 , , PTK2B PTK2B PTK2B GSTO1 GSTO1 GSTP1 GSTO1 GSTO1 GSTO1 , , , , , , , GSTZ1 , PLAU PLAU EGFR GSTA2 CDK5R1 CDK5R1 , , GSTO2 , , , , , , , , , , , , , , MAPK1 MAPK1 , F10 , F10 , , ZAP70 ZAP70 ZAP70 LYN LYN LYN , , , , , , Genes GSTM1 GSTO2 GSTT1 GSTO2 GSTO1 GSTO2 GSTO2 GSTK1 GSTO2 IGF1R MAPK1 LCK LCK LCK CXCR4 TGFBR1 SDCBP CXCR4 TGFBR1 SDCBP GSTM1 GSTM1 GSTM1 GSTM1 GSTM1 GSTA1 GSTM1 EGFR IGF1R PRKCA EGFR ITK ITK ITK PPARD PPARD -value P 3.81 E -12 9.51 E -12 1.92 E -10 2.08 E -09 3.25 E -09 4.75 E -09 1.57 E -05 5.18 E -04 2.63 E -09 1.14 E -07 1.85 E -07 1.08 E -08 7.50 E -08 4.51 E -07 8.87 E -09 9.98 E -08 % 2.57 2.57 2.57 2.14 2.36 2.36 3.00 2.356 4.07 3.64 3.43 3.64 3.64 3.64 6.64 6.64 Count 12 12 12 10 11 11 14 11 19 17 16 17 17 17 31 31 -transferase, -transferase, -transferase, -transferase/ S S S S Term G lutathione N -terminal G lutathione transferase activity GS T C -terminal G lutathione C -terminal G lutathione C -terminal-like G lutathione chloride channel, C -terminal G lutathione metabolism Thioredoxin fold Enrichment score: 7.42 Positive regulation of cell motion Positive regulation of locomotion Positive regulation of cell migration Enrichment score: 7.15 SH 2 domain SH 2 domain SH 2 domain Enrichment score: 7.12 C ell migration C ell motility ) Continued ( Table 3 Category IN T ER P R O G OT ER M_MF_F A T UP_ SE Q_F EA TU RE IN T ER P R O IN T ER P R O IN T ER P R O K EGG _P A T H W Y IN T ER P R O Annotation cluster 14 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 15 S P_P IR _K E YWO R D IN T ER P R O S M AR T Annotation cluster 16 G OT ER M_BP_F A T G OT ER M_BP_F A T

4074 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 1.81 E -04 6.90 E -04 7.69 E -06 9.97 E -05 0.004202 7.90 E -05 1.72 E -04 6.05 E -04 1.04 E -04 1.63 E -04 (Continued) 4.95 E -06 1.68 E -05 4.41 E -07 2.74 E -06 7.42 E -05 2.46 E -06 4.77 E -06 1.49 E -05 3.07 E -06 4.61 E -06 3.06 2.49 11.42 5.96 4.44 3.55 3.41 3.40 2.27 2.31

, , , ,

,

, , , , ,

,

,

,

,

, ,

, , ,

,

, , IGF1 ,

,

GAB2 , IL2 IL2 , , APP , , , TGFBR1 IGF1 ROCK1

CXCR4 CXCR4 MMP-13 , , , , , ,

, TGFBR1 , NCOA1 MMP-1 , PTEN , PRKCQ RXRA SFTPD HSP90AA1 MAPK8 , MET FNTA , , , , , , , , TNFRSF1A , MAP2K1 MAP2K1 PTK2 , , RET IGF1R , ADAMTS4 , AKT1 , , , KIT MMP-3 IL2 , , APP ADAM17 EPAS1 , PTK2B PTK2B ESRRG IL2 , , , , , , CXCR4 , RAC1 MMP-3 , ADAM17 ADAM17 RARG , , , , , MAPK1 SRC SDCBP TNKS2 ELANE IGF1R , , , , , EGFR EGFR , MAP2K1 PLAT , , MMP-13 , , , ADAMTS5 FYN , JAK3 AR , PEX5 , PPARG , , AKT1 HSP90AA1 , NR1H2 , , , PDPK1 PDPK1 SYK SYK PRSS3 PTK2B , , MMP-16 , , , , PTEN TGFBR1 , ADAMTS5 CDK2 , , BCKDHA , EGFR , , , ADAM17 TNKS2 PTPN11 PTPN11 MMP-16 , , , , PRKCA , NR1H2 TGFBR1 EGFR , , NCK2 , , MMP-3 LTA4H , ADAM17 THRB , IL2 RARG , , , ERBB2 ARNT , , KIT , , PPARG , INSR INSR , ADAMTS4 PAK4 SYK , , MAPK14 , , , , CDC42 CDC42 PDPK1 SYK EPAS1 SYK , , , AR , , , , CDK2 MME CDK5 CDK5 KDR , , , , , ARNT NR1H3 JAK3 ADAM17 , , , , ADAM17 PTPN11 THRA ROCK1 , , , PEX5 , THRB , INSR PPARGC1A , , , TRAF6 TRAF6 MMP-16 , , MAPK14 PTK2B , HMGCR ADAM17 AKT1 AKT1 AKT1 , , MMP-1 , , EGFR AKT2 , , , , HDAC2 PIM1 PIM1 , , SFTPD , , , , ADORA2A CDK5 , , MMP-7 ECE1 MAP2K1 , HPRT1 HRAS , CDK5 , , , , NR1H4 MMP-7 KIT KIT KIT ESR2 , FYN THRA , , , , F2 , , , PAK1 PAK1 TRAF6 MME MAPK14 , , , , PPARGC1A , ECE1 , NRP1 INSR INSR HMGCR , , MAP2K1 , , , , , RAC1 CXCR4 , ESR2 BIRC7 BIRC7 , , , , HDAC4 RB1 MMP-12 PIM1 , , , , COMT MMP-8 RB1 HRAS NCBP1 , TRAF6 , NCK2 , PAK1 RET , , ERBB2 ERBB2 ERBB2 , , , , MMP-8 MMP-20 , , , , , NRP1 BCKDHA NCOA2 , , MMP-7 , HDAC2 PRKACA PRKACA GAB2 , PRSS3 , , , , DDAH1 DDAH1 , MMP-20 , , ELANE ELANE ELANE CDK5R1 TP53 IDE , , , , , , , BIRC7 BRCA1 KDR , ESR2 , , , PPARA PPARA PTK2B , , PRKCA , , PTK2 RAC1 RAC1 PRKACA ADAMTS5 ACE MMP-9 , , , , , , , MET MET MET NR1H3 ADORA2A , , , NCOA1 MMP-8 MMP-12 PTK2B , , , HDAC4 IGF1 , , PLAU CDK5R1 , ADORA2A ADORA2A ADORA2A , , , , PPARD , , , ELANE KDM5A KDM5A , MMP-9 , CDK5 CD40 , , ELANE , , , , APP PLAT , , PPARD CXCR4 TGFBR1 SDCBP SRC SYK ESR2 PLAU MMP-13 MMP-12 ADAMTS4 MMP-1 MAP3K9 TGFBR1 MAP3K9 TGFBR1 MAP3K9 MET RARB RXRA MAPK1 TNFRSF1A ESRRG RARB TP53 NCOA2 NR1H4 BRCA1 ITGAL MMP-20 MMP-9 SPG7 TRAF2 TRAF2 TRAF2 HSP90AB1 HSP90AB1 9.98 E -08 3.80 E -07 4.58 E -09 6.40 E -08 2.72 E -06 4.35 E -08 9.47 E -08 3.34 E -07 5.71 E -08 9.00 E -08 6.64 8.35 2.57 3.43 3.43 5.78 5.78 5.35 11.13 10.49 31 39 12 16 16 27 27 25 52 49 L ocalization of cell C ell motion Enrichment score: 7.03 S hort sequence motif: cysteine switch Peptidase M, neutral zinc metallopeptidases, zinc- binding site Metalloendopeptidase activity Enrichment score: 6.95 Positive regulation of kinase activity Positive regulation of transferase activity Positive regulation of protein kinase activity Enrichment score: 6.74 Positive regulation of biosynthetic process Positive regulation of nitrogen compound metabolic process G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 17 UP_ SE Q_F EA TU RE IN T ER P R O G OT ER M_MF_F A T Annotation cluster 18 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 19 G OT ER M_BP_F A T G OT ER M_BP_F A T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4075 Dovepress He et al Dovepress FDR 4.24 E -04 0.001641 1.23 E -04 4.38 E -04 0.001302 0.018952 8.00 E -05 2.68 E -04 4.00 E -04 4.00 E -04 5.95 E -04 0.001342 0.001915 12.27639 7.69 E -06 Benjamini 1.11 E -05 3.73 E -05 5.99 E -06 1.05 E -05 2.25 E -05 3.31 E -04 2.30 E -06 6.73 E -06 1.86 E -05 1.86 E -05 1.17 E -05 2.53 E -05 3.49 E -05 0.08777958 4.41 E -07 Fold enrichment 2.21 2.18 8.93 7.80 6.76 8.04 15.32 13.41 13.16 13.16 11.62 10.82 10.39 3.09 11.42

, , , ,

,

,

,

,

,

,

,

,

, PDE8A PDE8A PDE8A PDE8A PDE8A PDE8A PDE8A PDE8A

, , , , , , , , , AKT2 , PRKACA PRKACA PRKACA , , , NR1H4 TNFRSF1A PRKCQ MAPK14 , ESRRG , RARB , CDK2 , , , , IGF1R RXRA , , PDE4D PDE4D PDE4D PDE4D PDE4D PDE4D PDE4D PDE4D PRKCB , , , , , , , , MMP-3 PRKCI PRKCI PRKCI , , APP , , , INSR , , HSP90AA1 IGF1R , , TNKS2 BRCA1 , NCOA2 , , TGFBR1 , NR1H2 , TGFBR1 , GRK6 GRK6 GRK6 PRKCI AKT1 PDE9A PDE9A PDE9A PDE9A PDE9A PDE9A PDE9A PDE9A , , , , , , , , , , , , , IL2 TRAF6 , RARG , , MMP-16 , CDK2 NR1H2 , , AKT1 EPAS1 , , HDAC2 AR , , EPAS1 JAK3 , , PPARG PDE3B PDE3B PDE3B PDE3B PDE3B PDE3B PDE3B PDE3B , PPARGC1A ADAMTS4 , , , , , , , , , , RPS6KA1 RPS6KA1 RPS6KA1 RPS6KA1 KDM5A , , , , ARNT , , ARNT EGFR , , ADAM17 , RARG , NCOA1 THRB ESR2 , PPARGC1A BCKDHA , , , , PDE5A PDE5A PDE5A PDE5A PDE5A PDE5A PDE5A PDE5A , , , , , , , , MAPK14 MMP-1 RARB SYK , AR , , , , ROCK1 ROCK1 ROCK1 ROCK1 , , , , PPARG RB1 , , MMP-7 ESR2 THRA , ADORA2A , , PRKCQ , , AKT2 NR1H3 PDE4B PDE4B PDE4B PDE4B PDE4B PDE4B PDE4B PDE4B , , , , , , , , , , PTK2B SGK1 SGK1 SGK1 SGK1 , HDAC2 IGF1 , , , , , , THRB RB1 MMP-12 , , , BCKDHA , HRAS , MMP-8 INSR , , AKT2 AKT2 AKT2 NCOA2 MAPK1 , , , , FNTA , PDE4A PDE4A PDE4A PDE4A PDE4A PDE4A PDE4A PDE4A TP53 , , , , , , , , , , NR1H4 TP53 , PRKCQ PRKCQ PRKCQ PRKCQ THRA , , , , , , HDAC4 , PPARA NR1H3 APP , , , ADAMTS5 MMP-9 PRKCB PRKCB PRKCB IL2 , , , , , DDAH1 , NCOA1 , HDAC4 PDE7A PDE7A PDE7A PDE7A PDE7A PDE7A PDE7A PDE7A , ESRRG AKT1 AKT1 AKT1 AKT1 TRAF6 , HRAS , , , , , , , , , , , , , , , ELANE SYK , , BRCA1 , JAK3 , Genes HSP90AB1 KDM5A RXRA MAPK1 PTK2B IGF1 TNFRSF1A AKT2 ELANE TNKS2 F2 ADRBK1 ADRBK1 ADRBK1 MMP-13 PPARA PRKCA PRKCA PRKCA PRKCA PDE2A PDE2A PDE2A PDE2A PDE2A PDE2A PDE2A PDE2A MMP-20 -value P 2.34 E -07 9.04 E -07 7.32 E -08 2.81 E -07 1.07 E -06 1.22 E -05 5.13 E -08 1.72 E -07 2.38 E -07 2.38 E -07 4.89 E -07 8.68 E -07 1.24 E -06 0.008435 4.58 E -09 % 10.71 10.06 2.57 2.57 2.57 1.93 1.93 1.93 1.93 1.93 1.93 1.93 1.93 1.93 2.57 Count 50 47 12 12 12 9 9 9 9 9 9 9 9 9 12 - C yclic nucleotide - C yclic-nucleotide ′ ′ ,5 ,5 ′ ′ Term Positive regulation of cellular biosynthetic process Positive regulation of macromolecule biosynthetic process Enrichment score: 6.14 AGC -kinase C -terminal AGC -kinase, C -terminal S _TK_X Protein kinase, C -terminal Enrichment score: 5.96 3 phosphodiesterase Metal-dependent phosphohydrolase, H D region Metal ion-binding site: divalent metal cation 2 Metal ion-binding site: divalent metal cation 1 H Dc 3 phosphodiesterase activity C yclic-nucleotide phosphodiesterase activity Phosphoric diester hydrolase activity Enrichment score: 5.80 S hort sequence motif: cysteine switch ) Continued ( Table 3 Category G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 20 UP_ SE Q_F EA TU RE IN T ER P R O S M AR T IN T ER P R O Annotation cluster 21 IN T ER P R O IN T ER P R O UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE S M AR T G OT ER M_MF_F A T G OT ER M_MF_F A T G OT ER M_MF_F A T Annotation cluster 22 UP_ SE Q_F EA TU RE

4076 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 5.07 E -05 0.001021 0.001021 0.001437 0.001437 0.001385 0.003271 0.003271 0.003271 0.003597 0.003017 0.006349 0.009958 0.144244 0.388252 7.11 E -04 0.001923 0.006252 0.011118 2.43 E -05 5.07 E -05 (Continued) 1.54 E -06 4.15 E -05 4.15 E -05 5.39 E -05 5.39 E -05 3.07 E -05 6.73 E -05 6.73 E -05 6.73 E -05 7.15 E -05 4.63 E -05 8.76 E -05 1.85 E -04 0.00874563 0.01875986 3.01 E -05 6.70 E -05 1.90 E -04 3.19 E -04 1.32 E -06 1.54 E -06 16.09 14.88 14.88 14.24 14.24 11.09 12.43 12.43 12.43 8.31 9.62 10.78 13.90 8.41 9.27 15.59 9.09 9.44 7.44 23.39 16.09

,

, ,

, , , , , , , ,

, , , , , MMP-1 , , MMP-1 , MMP-12 MMP-12 , , MMP-1 MMP-1 , , MMP-3 MMP-12 MMP-12 MMP-12 MMP-12 MMP-12 MMP-12 MMP-12 MMP-12 , , , , , , , , , MMP-13 MMP-13 MMP-13 MMP-13 , , , , MMP-1 , MMP-12 , MMP-12 , MMP-13 MMP-13 , , MMP-12 MMP-12 MMP-1 , , , MMP-16 MMP-3 MMP-13 MMP-13 MMP-13 MMP-13 MMP-3 MMP-13 MMP-13 MMP-13 MMP-3 MMP-13 MMP-3 , , , , , , , , , , , , , MMP-12 , MMP-13 , MMP-3 MMP-3 MMP-13 , , , MMP-3 MMP-3 MMP-3 MMP-3 MMP-3 MMP-3 MMP-3 MMP-3 MMP-13 MMP-13 MMP-13 , , , , , , , , , , , MMP-16 MMP-16 ADAM17 MMP-16 MMP-16 , , , , , MMP-13 MMP-3 , , MMP-7 MMP-7 MMP-3 , , , MMP-3 MMP-3 MMP-3 , , , MMP-7 MMP-16 MMP-16 MMP-16 MMP-16 MMP-7 MMP-16 MMP-16 MMP-16 MMP-7 MMP-7 MMP-16 MMP-7 , , , , , , , , , , , , , MMP-3 MMP-7 , , MMP-8 MMP-8 MMP-7 , , , MMP-1 , MMP-8 MMP-8 MMP-8 MMP-8 MMP-8 MMP-8 MMP-8 MMP-8 MMP-8 MMP-8 MMP-8 MMP-8 MMP-16 MMP-16 MMP-16 MMP-8 , , , , , , , , , , , , , , , , MMP-8 MMP-8 , , MMP-9 MMP-9 MMP-8 , , , MMP-9 MMP-1 MMP-1 MMP-12 MMP-1 MMP-1 MMP-9 MMP-9 MMP-9 MMP-9 MMP-9 MMP-9 MMP-9 MMP-9 MMP-9 MMP-9 MMP-9 MMP-8 MMP-8 MMP-8 MMP-9 , , , , , , , , , , , , , , , , , , , , , PRKCB PRKCB , , MMP-9 APCS MMP-9 APCS , , , , MMP-9 , MMP-20 MMP-12 MMP-1 MMP-1 MMP-1 MMP-1 MMP-12 MMP-1 MMP-1 MMP-1 MMP-13 MMP-12 MMP-1 PRKCB MMP-1 PRKCB MMP-1 MMP-12 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 MMP-20 PRKCA PRKCA PRKCA PRKCA ACE MMP-20 3.26 E -08 6.08 E -07 6.08 E -07 8.56 E -07 8.56 E -07 8.89 E -07 2.10 E -06 2.10 E -06 2.10 E -06 2.31 E -06 2.48 E -06 5.21 E -06 6.39 E -06 1.06 E -04 2.87 E -04 4.23 E -07 1.15 E -06 3.72 E -06 6.62 E -06 1.45 E -08 3.26 E -08 1.93 1.71 1.71 1.71 1.71 1.93 1.71 1.71 1.71 2.14 1.93 1.71 1.50 1.50 1.28 1.71 2.14 1.93 2.14 1.71 1.93 9 8 8 8 8 9 8 8 8 10 9 8 7 7 6 8 10 9 10 8 9 Peptidoglycan binding like H emopexin-like 4 domain H emopexin-like 3 domain H emopexin-like 1 domain H emopexin-like 2 domain Peptidase, metallopeptidases H emopexin/matrixin, repeat H emopexin/matrixin, conserved site H emopexin/matrixin Peptidase M10 A and M12B, matrixin and adamalysin ZnMc H X Peptidase M10 A , matrix metallopeotidase Peptidase_M10 A _matrix Matrix metalloproteinase, stromelysin type Enrichment score: 5.73 Metal ion-binding site: calcium 3; via carbonyl oxygen Metal ion-binding site: calcium 1 Metal ion-binding site: calcium 2; via carbonyl oxygen Metal ion-binding site: calcium 2 Enrichment score: 5.69 Metal ion-binding site: zinc 2; catalytic Peptidoglycan binding like IN T ER P R O UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE IN T ER P R O IN T ER P R O IN T ER P R O IN T ER P R O IN T ER P R O S M AR T S M AR T IN T ER P R O P IR _ S UP ER F A M IL Y P IR _ S UP ER F A M IL Y Annotation cluster 23 UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE Annotation cluster 24 UP_ SE Q_F EA TU RE IN T ER P R O

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4077 Dovepress He et al Dovepress FDR 6.60 E -05 8.48 E -05 0.001385 0.002803 0.003017 0.027575 0.058172 0.095563 0.315166 0.451323 0.006014 0.00851 0.009499 0.003772 0.009255 Benjamini 3.39 E -06 9.65 E -07 3.07 E -05 2.43 E -05 4.63 E -05 4.29 E -04 7.95 E -04 0.00113118 0.0030583 0.00404851 1.21 E -04 1.59 E -04 1.74 E -04 7.88 E -05 1.71 E -04 Fold enrichment 28.65 15.45 11.09 16.96 9.62 9.33 10.62 7.83 6.56 7.58 3.08 2.86 2.92 2.18 2.02

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, , , PTEN TRAF6 BCL6 , , , VCP , SFN TIAM1 , , CASP7 KDR , CDK5 , MMP-1 MMP-1 MMP-1 , KDR , , , KIT , , ATP7A CASP1 PDCD6IP , , , BCL6 ZAP70 , MMP-12 , ROCK1 , NAE1 , ZAP70 , RB1 , KIT , MMP-1 MMP-13 MMP-1 MMP-13 MMP-1 MMP-1 , SFN , , , , , , , PTK2B CXCR4 BCL2L1 KDR , , , GZMB , , PLG TRAF6 , , MDM4 MMP-13 MMP-13 MMP-13 SGK1 RB1 , , , , CASP8 ZAP70 , , , , CDK6 CASP8 RB1 , , , GJA1 MMP-13 ECE1 , , RAF1 MMP-3 MMP-3 , TIAM1 BCL2L1 , , , , , MMP-12 MMP-13 MMP-13 MMP-13 PAK1 LCK , , , , , , BRCA1 , CDK6 CASP1 , TP53 CASP8 , BCL2 MAPK8 MMP-3 MMP-3 MMP-3 , , , , , , , CDK6 , IL2 GJA1 , , CASP3 BIRC7 MMP-3 , , BCL2 , , CXCR4 TGM2 , CDK5 , ACE MMP-16 MMP-3 MMP-16 MMP-3 MMP-3 TP53 , , ADORA2A , , , , , , BCL2 , MMP-13 , TRAF6 CTSD , , , TP53 APP , , TGFBR1 TP53 , IL2 , MME MMP-16 MMP-16 MMP-16 , , , , , F2 G6PD ECE1 , , , ACE XIAP , SYK ADAM17 CASP8 , GZMB , , , ADORA2A PPARG PAK1 , , CDC42 , , MMP-7 MMP-7 , , , MMP-3 MMP-16 MMP-16 MMP-16 LCK , , , , ACE , IL2 , EPAS1 TGFBR1 , , , LCK LGALS1 , CASP3 KIT SPG7 MMP-7 MMP-7 MMP-7 MMP-7 , , XIAP , , RAF1 , , , , RAC1 PDCD6IP TNFRSF1A , , , , , CDC42 ADAM17 , , TGM2 VCP , , MMP-9 LYN , , MMP-8 MMP-8 APP , , , MMP-7 MMP-7 MMP-7 MMP-7 TRAF6 EPAS1 , , , , CDC42 G6PD KIT , , , , , LCK HRAS BCL2 HRAS GZMA , , AKT1 , BIRC7 PPARG , , , , , MMP-8 MMP-8 MMP-8 MMP-8 MDM4 , , , , NAE1 , CASP8 PLEK , , , EPAS1 LYN , , BCL6 MMP-1 MMP-1 MMP-9 MMP-9 , , , , , G6PD TP53 MMP-8 MMP-8 MMP-8 MMP-8 UNG PPARG , PTEN , PLG , , , , , , CASP7 HDAC4 HDAC4 SYK PPARD , PPARD , , , , , , , ROCK1 TNFRSF1A RAC1 , , , PLEK MMP-9 MMP-9 MMP-9 MMP-9 PLEK MAPK8 , , , , , , , Genes MMP-9 MMP-1 MMP-12 MMP-12 ATP7A ADAM17 TRAF6 ATP7A SYK HDAC4 AKT1 BCL2 LGALS1 LCK HPRT1 PTK2B SGK1 BRCA1 ACE MMP-20 MMP-9 MMP-20 ACE MMP-9 ACE ACE MMP-9 LYN MMP-9 MMP-9 TRAF2 TRAF2 -value P 3.93 E -08 5.85 E -08 8.89 E -07 1.93 E -06 2.48 E -06 1.52 E -05 3.21 E -05 5.27 E -05 1.74 E -04 2.49 E -04 3.31 E -06 4.69 E -06 5.23 E -06 2.08 E -06 5.10 E -06 % 1.50 1.93 1.93 1.50 1.93 1.71 1.50 1.71 1.71 1.50 5.14 5.57 5.35 9.42 10.28 Count 7 9 9 7 9 8 7 8 8 7 24 26 25 44 48 Term 2; zinc site: ion-binding Metal in inhibited form Metalloproteinase Peptidase, metallopeptidases C ollagen degradation ZnMc Multicellular organismal catabolic process C ollagen catabolic process Multicellular organismal macromolecule metabolic process Multicellular organismal metabolic process C ollagen metabolic process Enrichment score: 5.36 H emopoiesis I mmune system development H emopoietic or lymphoid organ development Enrichment score: 5.32 Programmed cell death C ell death ) Continued ( Table 3 Category UP_ SE Q_F EA TU RE S P_P IR _K E YWO R D IN T ER P R O S P_P IR _K E YWO R D S M AR T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 25 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 26 G OT ER M_BP_F A T G OT ER M_BP_F A T

4078 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 0.011004 0.014551 0.001176 0.009641 0.081769 0.009734 0.017402 0.11556 0.01085 0.01085 0.021303 0.410281 0.002605 0.005799 0.05845 1.260168 (Continued) 1.95 E -04 2.46 E -04 2.75 E -05 1.75 E -04 9.94 E -04 2.88 E -04 3.13 E -04 0.00154179 1.95 E -04 1.95 E -04 3.47 E -04 0.00375601 8.76 E -05 1.82 E -04 3.49 E -04 0.00613378 2.01 2.12 3.96 3.32 2.87 20.46 17.87 12.36 4.46 4.46 4.21 3.23 10.52 8.02 4.80 3.39

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SFN , TIAM1 , , , , , , MAPK14 PTEN PYGL PYGL , , CDK5 , , , PPP1CC PPP1CC PPP1CC , , , CASP1 PDCD6IP GZMA GNMT EHMT2 , , , , COMT , LPA LPA LPA LPA NAE1 , , , , , EHMT2 , , SFN , PTK2B PYGL , , G6PD G6PD CXCR4 BCL2L1 DLAT DLAT DLAT , , , , GZMB PLG PLG PLG PLG PLG , , , , , , , , , TRAF6 COMT , GAMT GNMT , MDM4 ROCK1 , , , , GNMT F7 F7 F7 F7 , , , , , TPI1 TPI1 G6PD , , GJA1 , FBP1 FBP1 FBP1 TIAM1 BCL2L1 ECE1 , , , , , , RAF1 , , MAPK10 MAPK10 MAPK10 , , , PAK1 BRCA1 , , SGK1 GAMT , , COMT SETD8 , TPI1 , CD40 CD40 CD40 CD40 MAPK8 , , , , , COMT , GJA1 , , PDK4 PDK4 PDK4 , , , PPP1CA PPP1CA CASP3 BIRC7 , , , , CXCR4 F9 F9 F9 F9 , , , , , CDK5 TGM2 , MAPK8 MAPK8 MAPK8 , , , , ADORA2A TRAF6 SETD8 , , GAMT CTSD , , F8 F8 F8 F8 , APP , , , , GAMT , PPP1CA KHK KHK PDK3 PDK3 PDK3 , METTL1 , , , , , , TP53 , , ECE1 F2 , , XIAP GZMB ADORA2A CASP8 , , , , CASP1 MAPK9 MAPK9 MAPK9 , , , , PLAU PLAU PLAU PLAU SETD8 PDCD6IP , , , , , AKT1 AKT1 AKT1 , LCK , , , SETD8 METTL1 , PNMT , , NNMT NNMT , , , CASP3 LGALS1 , SPG7 XIAP RAF1 , ADORA2A ADORA2A ADORA2A ADORA2A , , , RAC1 , , , , TNFRSF1A , ALDH5A1 ALDH5A1 ALDH5A1 , PAK1 , , , , VCP , PCK1 PCK1 PCK1 MAPK3 MAPK3 MAPK3 , , , APP PDHA1 PDHA1 , , , , MDM4 , , PNMT PNMT TPMT , TPMT , , METTL1 , , HNMT PLEK PLEK PLEK PLEK , HRAS HRAS , , , , , BCL2 GZMA BIRC7 , , AKT1 SERPINC1 SERPINC1 SERPINC1 SERPINC1 , , , , , , , , PPARD PPARD PPARD CASP8 NAE1 , , , , , PDHB PDHB PDHB , , , F2 F2 F2 F2 F10 F10 F10 F10 , , , , , , , , GSK3B GSK3B , , PNMT HNMT HNMT AMD1 MAPK8 TP53 MAPK14 MAPK14 MAPK14 , , , , , , PTEN PDHA1 , , , PLG EHMT2 CASP7 PPARD PPARD , , , , , NNMT , , EHMT1 RBP4 RBP4 RBP4 ROCK1 TNFRSF1A , , RAC1 , , , , F11 F11 F11 F11 , , , , , , LCK , TRAF2 HPRT1 PTK2B SGK1 BRCA1 AKT1 BCL2 LGALS1 F2 PPARGC1A GSK3B PPARGC1A MAPK14 PPARGC1A MAPK14 SERPINE1 SERPINE1 SERPINE1 SERPINE1 NNMT TPMT EHMT2 GNMT TRAF2 PDK1 PDK1 PDK1 MAPK1 MAPK1 MAPK1 PLAT PLAT PLAT PLAT EHMT1 EHMT1 EHMT1 TYMS 6.06 E -06 8.02 E -06 6.48 E -07 5.31 E -06 4.51 E -05 5.80 E -06 1.12 E -05 7.48 E -05 5.98 E -06 5.98 E -06 1.17 E -05 2.26 E -04 1.55 E -06 3.45 E -06 4.03 E -05 8.74 E -04 10.28 8.99 4.28 4.50 4.50 1.28 1.28 1.28 3.21 3.21 3.21 3.21 1.93 2.14 2.57 2.57 48 42 20 21 21 6 6 6 15 15 15 15 9 10 12 12 - l -adenosyl- -methionine S -methionine l l -adenosyl- - A denosyl- Death A poptosis Enrichment score: 5.27 G lucose metabolic process H exose metabolic process Monosaccharide metabolic process Enrichment score: 4.77 S hort sequence motif: TXY M A P kinase, conserved site M A P kinase activity Enrichment score: 4.76 Blood coagulation C oagulation H emostasis Regulation of body fluid levels Enrichment score: 4.68 R egion of interest: S binding Binding site: methionine S Methyltransferase G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 27 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 28 UP_ SE Q_F EA TU RE IN T ER P R O G OT ER M_MF_F A T Annotation cluster 29 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 30 UP_ SE Q_F EA TU RE UP_ SE Q_F EA TU RE S P_P IR _K E YWO R D S P_P IR _K E YWO R D

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4079 Dovepress He et al Dovepress 3.04 E -06 3.062766 FDR 0.006647 0.047923 0.21798 4.10 E -08 0.02584329 Benjamini 1.31 E -04 6.86 E -04 0.00225032 Fold enrichment 1.84 1.27 7.78 7.18 6.93

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ALB , , , RARB , FNTA VDR HGS , , , ADH1B ABO SIRT5 ARNT , ADH1B , , , ITK NR3C1 , MDM2 ADAM17 LTA4H , , , , AKT2 , MMP-20 ADH5 , EHMT1 RXRA , , , , PPP1CC ECE1 , , MMP-16 ASPA MMP-13 AIRE , , ASPA , , , HDAC4 CPB2 ADAMTS5 , , , MMP-20 BIRC7 CPA4 EEA1 , , NR1H2 , AKT2 , QPCT , , ADH1C , NR1H4 ARNT MMP-7 BRAF CA13 CPA4 , ACE , , , , , APP , , APP , UBR2 , , TP53 ESR2 , ADAM17 , , NAALAD2 ABL1 , S100B , , ZCWPW1 ITK ATP7A , , , RAF1 MME PYGO1 VDR ADH7 ARNT XIAP , , , , , , , NR1H3 , CA4 MMP-7 RXRA ROCK1 , ADH1C , L3MBTL2 , , PRKCI , , ZCWPW1 , , HMHA1 HMHA1 , PARP1 , FOLH1 GART NR1I2 PPARGC1A PPARGC1A , , , , , , MMP-16 PPARG ARG1 SIRT5 , TPH1 , , , , TRAF2 ADH5 NR1H3 PDE5A , , , , BRAF BCL6 RARG , ESR1 , , , NR1H4 , MMP-8 FNTB MMP-8 FNTB PPARG , , , INSR INSR PPARGC1A , , CA1 , , , , ESR1 PTPN1 NAALAD2 NR1I3 , BRCA1 AR , PRKCQ , , , , BIRC7 CA13 , KDM4A , , , NR1H2 IDE , METAP2 IDE , NR1H4 PGR PPP1CA , , PGR , , NQO2 , , ROCK1 , , TRAF6 PPP2CA INSR , , , HGS ADAMTS4 PPARD , , , PRKCI NR3C1 NR3C1 XIAP MDM4 , , , , , BRCA1 FOLH1 PRKCA PRKCB BTK POR , BTK , , , MME , , , EHMT2 LTA4H , , , TRAF6 RARG MMP-9 , MMP-9 AIRE , GLO1 , L3MBTL2 , , ADH4 , , CA1 METAP1 SPG7 CA2 , DDAH1 IGF1 IGF1 , , NR3C1 , , TP53 , , , , , FYN , PRKCQ TRAF2 MMP-1 , , MMP-1 AR EHMT2 KDM5A ADAMTS5 ACACB CDA PPP1CC GLO1 , , , , , CA2 , , , , , BCL6 MMP-12 , , , NQO2 SPG7 PLEK PLEK IGF1 S100B , RARB , , , , , , S100A7 PPARA S100A7 NR3C1 PDE9A , , , , , QPCT , ALB XDH , CDA , , UBR2 PRKCB , , PRKCA ESRRG PDE8A ESRRG PYGO1 MMP-3 MMP-3 CPA1 , , , , , , , MDM4 , NR1I2 CPB2 PPARD , PPP2CA THRB , , THRB , , PIR HDAC4 HDAC4 HDAC4 DDAH1 ADH7 , , F8 MMP-13 PCK1 , , , , , , , , , ECE1 MDM2 , , PDE9A PPP1CA ACE EHMT1 KDM5A MMP-12 ADH4 KDM4A THRA ADH1A MAT1A PIM1 ADAMTS4 ARG2 TGFBR1 NR1I3 PPARA HIF1AN ESR2 CA4 EEA1 RAF1 Genes AKT1 AKT2 PARP1 AKT1 AKT1 THRA CPA1 ADH1A -value 0.00201 P 3.66 E -06 2.64 E -05 1.20 E -04 2.10 E -09 26.34 % 2.14 1.93 1.71 20.99 123 Count 10 9 8 98 Transition metal ion binding Term Enrichment score: 4.64 R egulation of carbohydrate metabolic process R egulation of cellular carbohydrate metabolic process R egulation of glucose metabolic process Enrichment score: 4.32 Zinc ) Continued ( G OT ER M_MF_F A T Table 3 Category Annotation cluster 31 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 32 S P_P IR _K E YWO R D

4080 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 34.1204 0.025154 0.179443 0.179443 0.179443 0.002238 0.016313 0.028912 2.111955 5.571159 0.047358 0.047358 (Continued) 0.19883278 3.95 E -04 0.00196673 0.00196673 0.00196673 1.82 E -04 4.54 E -04 4.90 E -04 0.01896331 0.04321176 6.89 E -04 6.89 E -04 1.21 7.80 7.14 7.14 7.14 15.14 10.23 9.23 4.71 3.98 2.16 2.16

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MMP- HGS , , , , , , VDR ADH1B COMT , , , , ADH4 MMP-13 HPRT1 HPRT1 MDM2 , EHMT1 , , , , , HPRT1 HPRT1 HPRT1 , , , UBR2 TGM2 TGM2 , , , ASPA , AIRE MDM2 MDM2 , ITK , , , ESR2 , MAP2K1 MAP2K1 NR1H2 ADH1C , , NR3C1 , , SULT1E1 APP KDM4A SULT1C2 SULT1C2 SULT1C2 SULT1C2 , , , FKBP1A FKBP1A , , , , , COMT COMT COMT SULT1A2 ZCWPW1 , , RXRA , , , , VCP VCP , , ADAM17 , , CASP8 CASP8 , , , CPA4 , S100B , APCS APCS , , MME EEA1 , , CA4 PEX5 PEX5 BRAF MMP-7 MMP-16 , , , , LTA4H , , , NCK2 NCK2 PPARG RAC1 RAC1 , , , , , HMHA1 , SULT1C2 SULT1A2 SULT1A2 SULT1A2 SULT1A2 , NR1I2 , , , , SULT1A2 SULT1A2 SULT1A2 SULT1A1 , NR1H3 , , , , , TRAF2 ADH7 GJA1 GJA1 , , , , ESR1 , QPCT , ROCK1 MMP-8 FNTB , PPARD , PTK2B PTK2B , , HSP90AA1 HSP90AA1 , , , , NAALAD2 IDE IDE NR1I3 , , , MAOA , , NR1H4 PPARGC1A PPARGC1A ADH5 IDE , SULT1A2 SULT2B1 SULT2B1 SULT2B1 SULT2B1 RAF1 , , PRKCI , , PGR , , , , , , SULT1A1 SULT1A1 SULT1A1 , , , , , RARG SPG7 PTK2 PTK2 , , , , ADAMTS4 GRB2 GRB2 , , , FOLH1 BTK , , PPP2R1A PPP2R1A CA13 HDAC4 TP53 CDK5 CDK5 , , , , TRAF6 , SIRT5 , , , , MMP-9 SULT1B1 MAOA MAOA MAOA , , , , , NQO2 IGF1R IGF1R PPARA , , , , SULT1A1 SULT1A1 SULT1A1 SULT1A1 SULT1A1 AR , , , , , , HRAS HRAS SYK SYK , , , , XIAP , PRKCQ CD40 CD40 GLO1 MMP-1 , BIRC7 , , BRCA1 , , , , ADAMTS5 , CPB2 EPAS1 EPAS1 EPAS1 EPAS1 , , , , , S100A7 CA1 INSR INSR , , , , DDAH1 PRKCA TRAF2 TRAF2 , , , , CDA , TP53 TP53 , , SULT1B1 SULT1B1 SULT1B1 SULT1B1 SULT1B1 SULT1C3 SULT1C3 SULT1C3 SULT1C3 TNFRSF1A TNFRSF1A , , , , , , , , , PRKCB , , , MMP-3 CA2 , PYGO1 EHMT2 PARP1 , PNMT PNMT PNMT PNMT , , , , , , , THRB CPA1 , L3MBTL2 , , GNMT GNMT ECE1 MIF MIF , , , , , KDM5A , THRA ADH1A RARB PDE5A ACE PTPN1 ESRRG 20 MMP-12 MDM4 BCL6 ATP7A HPRT1 ATP7A ATP7A ATP7A SULT2A1 SULT1C3 SULT2A1 SULT1E1 SULT2A1 SULT1E1 SULT2A1 SULT1E1 SULT2A1 SULT1E1 HSD17B10 SRC CDA ALDH5A1 HSD17B10 SRC CDA ALDH5A1 0.026629 1.39 E -05 9.89 E -05 9.89 E -05 9.89 E -05 1.65 E -06 9.72 E -06 1.86 E -05 0.00138 0.0037 2.61 E -05 2.61 E -05 20.77 1.93 1.71 1.71 1.71 1.50 1.71 1.71 1.71 1.71 7.71 7.71 97 9 8 8 8 7 8 8 8 8 36 36 Zinc ion binding Enrichment score: 4.22 Phenol metabolic process Diol metabolic process process metabolic C atechol C atecholamine metabolic process Enrichment score: 4.16 A lcohol sulfotransferase N ucleotide phosphate- binding region: P A S S ulfotransferase S ulfotransferase activity Transferase activity, transferring sulfur- containing groups Enrichment score: 4.11 Protein complex assembly Protein complex biogenesis G OT ER M_MF_F A T Annotation cluster 33 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 34 P IR _ S UP ER F A M IL Y UP_ SE Q_F EA TU RE IN T ER P R O G OT ER M_MF_F A T G OT ER M_MF_F A T Annotation cluster 35 G OT ER M_BP_F A T G OT ER M_BP_F A T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4081 Dovepress He et al Dovepress FDR 0.298718 0.578406 0.118797 0.196338 0.212381 0.221543 0.142086 0.191517 0.191517 0.282881 0.078194 0.087828 2.369144 Benjamini 0.00291459 0.0048243 0.00136967 0.00208753 0.00220533 0.0022739 0.00161717 0.00206093 0.00206093 0.00277529 9.64 E -04 0.00105367 0.0157027 Fold enrichment 1.87 1.79 3.12 3.28 3.11 3.63 4.07 3.94 3.94 4.09 3.76 3.52 3.19

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, , , , , , MDM4 CASP3 , PEX5 PEX5 , INSR CD40 CD40 , , , TNK2 TNK2 TNK2 SFTPD , , HIST1H3G , , , , CD38 , TRAF6 , , AKT1 AKT1 PRKCQ , , , IL2 LCK GJA1 GJA1 , IL2 , CD38 , , PPP2R1A PPP2R1A TP53 TP53 , , , , TNK2 , , MDM2 , , HIST1H4A HIST1H4A CD40 NCK2 , , , , MDM4 , CD38 IDE IDE ADAM17 ADAM17 ADAM17 , , , , , , SYK SYK SYK , IL2 , , TTK , PTPN11 PTPN11 , , , CASP1 CASP3 IL2 NCK2 , , , , F2 F2 F2 PTK2 PTK2 , , , SNUPN SNUPN , , MIF , , , PRKCQ , GRB2 GRB2 INSR INSR MDM2 PLA2G2A PRKACA , , IGF1 , , , , , SYK , TRAF6 , , CASP1 CDK5 CDK5 , , , NCK2 BCL2 BCL2 BCL2 PNP TRAF6 IL2 , , , , , , , PRKCQ MIF , , HRAS HRAS , , BCL6 GNMT GNMT , TTK TTK TTK , , TRAF6 SYK ALDH5A1 ALDH5A1 TNFRSF1A TNFRSF1A , , , , , CD40 CD40 TGFBR1 , , , , PNP PNP TRAF6 , , , , , , CD40 ADAM17 , PRKCQ , , HIST1H3A HIST1H3A , , PRKACA , CDA CDA , , IGF1 IGF1 IGF1 TRAF2 F2 , , , NCBP1 , BCL6 , BRAF BRAF , , , , ZAP70 IGF1R IGF1R , TRAF6 IL2 PRKCI PRKCI CD40 CD40 PNP , , , , , , , , , VCP VCP GSK3B , , , ERBB2 , PRKACA BCL2 , , MIF MIF TGM2 TGM2 SYK , , , , , TRAF2 NCBP1 BCL6 ZAP70 , , , SFTPD , TGFBR1 TGFBR1 TGFBR1 TGFBR1 , , , , , PTPN11 NCK2 NCK2 ERBB2 ERBB2 ERBB2 , , , , , , MAP2K1 MAP2K1 , , TGFBR1 TGFBR1 , , SRC SRC , , GSK3B GSK3B , , LCK IL2 ADORA2A CASP8 CASP8 LYN LYN LYN , TRAF6 , , , , , , , ADORA2A , , BCL2 SFTPD ZAP70 , , , APCS APCS , , IL2 IL2 IL2 , , , HPRT1 HPRT1 , , PDPK1 PDPK1 RBP4 HSD17B10 HSD17B10 LYN BCL6 RAC1 RAC1 , , , LCK CDK5 , , PLEK ADORA2A ADORA2A ADORA2A , , , , CASP3 PPARGC1A PPARGC1A , , , , , , , , , PRKCA PRKCA PRKCA ADORA2A ADORA2A SYK SYK SYK , , , , , , , , SFTPD , Genes NCBP2 FKBP1A PTK2B HSP90AA1 CDK5 FKBP1A PTK2B HSP90AA1 CDK5 HIST1H3G NCK2 LCK CASP3 ZAP70 INSR INSR INSR SYK PRKCQ PRKCQ CASP1 NCBP2 TRAF2 TRAF2 TRAF2 TRAF2 EGFR EGFR EGFR PRKCA RBP4 RBP4 PRKCQ -value P 1.65 E -04 3.20 E -04 6.55 E -05 1.08 E -04 1.17 E -04 1.22 E -04 7.83 E -05 1.06 E -04 1.06 E -04 1.56 E -04 4.31 E -05 4.84 E -05 0.00132 % 8.78 9.00 3.85 3.43 3.64 3.00 2.78 2.78 2.78 2.57 3.21 3.43 2.57 Count 41 42 18 16 17 14 13 13 13 12 15 16 12 Term Macromolecular complex assembly Macromolecular complex subunit organization Enrichment score: 4.00 R egulation of cell activation R egulation of lymphocyte activation R egulation of leukocyte activation R egulation of T-cell activation Enrichment score: 3.97 Positive regulation of phosphorylation Positive regulation of phosphate metabolic process Positive regulation of phosphorus metabolic process Positive regulation of protein amino acid phosphorylation Enrichment score: 3.85 R egulation of establishment of protein localization R egulation of protein localization R egulation of protein transport Enrichment score: 3.73 ) Continued ( Table 3 Category G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 36 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 37 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 38 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 39

4082 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 0.030568 0.030568 2.072751 2.698772 0.022889 0.070998 0.144174 0.26354 0.50945 0.557892 0.873863 1.488171 2.636383 0.13268 (Continued) 5.03 E -04 5.03 E -04 0.02767984 0.03373603 3.66 E -04 9.07 E -04 0.00163049 0.00262906 0.00445775 0.00471944 0.00684232 0.01076448 0.01716579 0.00321839 25.53 25.53 9.93 8.61 2.47 2.73 2.85 2.33 2.08 2.22 2.48 2.37 1.99 1.50

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NCK2 , , , , ATP7A PPP1CC , , PDHA1 PRKCA PRKCA , , , PTK2 PDHB RXRA , SIRT5 , , ERBB2 ERBB2 ERBB2 HIST1H4A MDM2 , , , , , , PTEN PTEN RB1 CDC42 CDC42 PARP2 , , , S100B , , , , CDH23 CDH23 HPRT1 BCKDHA S100B S100B PRKCA ERBB2IP ERBB2IP ATP7A , , , , , , , , , , EGFR EGFR , , RPS6KA1 CASP8 IGF1 PTPN11 , , , , IGF1R C1QC , , RXRA RXRA RXRA APP , , , , APP APP TLE1 , , CHEK2 , MAPK3 , , IGF1R IGF1R PARP1 RAC1 RAC1 HPRT1 HPRT1 PEX5 , , EGFR PDK1 , , , , , , , , ADORA2A S100B S100B S100B , , , , F8 , PTPN11 CDK5 CASP7 CDH23 CDH23 , , AGXT , , , PTK2 HDAC2 ATP7A , , PTK2 PTK2 , , , , BCKDHB GJA1 GJA1 GJA1 ABL1 BCL2 BCL2 , , , , , , , NR3C1 HPRT1 HPRT1 PPP1CA , , , , ADORA2A ADORA2A TP53 ADORA2A CDH23 , , , ATP7A ATP7A ATP7A , , , , , CDK5 DLG1 DLG1 PRKCI , MAP2K1 , , , IGF1R , HSD17B4 , IGF1R IGF1R KIT , XRCC1 , , , DUSP3 , PDK4 , , PTPN11 GJA1 GJA1 ERBB2 ERBB2 ERBB2 , PTK2B PTK2B , , CSNK2A1 PNKP , , , , , , , KDM4A RAC1 , GATM , , BCL6 BCL6 RXRA , , , NRP1 , MAP2K1 MAP2K1 PTPN11 PTPN11 PTPN11 MAP2K1 , , , , , ATP7A , PEX5 ATP7A ATP7A RAC1 , TGFBR1 , , , , PDK3 , INSR INSR INSR INSR PTEN , CHEK1 , , , , , , HDAC4 LYN ERBB2 ERBB2 NUDT21 , , BCL2 CASP3 , , , , , CXCR4 CXCR4 MAPK1 , , PLEK RAC1 RAC1 , ALDH2 , IDE , , , , NRP1 NRP1 ADORA2A ADORA2A ADORA2A NRP1 CDK5 CDK5 CDK5 BCL2 , , , , , , , , , , RXRA APP , EPAS1 CDK5 CDK5R1 , PCK1 FARS2 , , ERBB2 ERBB2 ERBB2 ERBB2 , RARG PTPN11 PTPN11 PTPN11 , , , , , , , , , , PTK2 PTK2 , , BCL2 BCL2 GSTK1 CDK2 , , , CXCR4 , , ROCK1 UBE2E1 NRP1 NRP1 NRP1 , , PRKCI PRKCI PRKCI , , , ADORA2A ADORA2A TSG101 , , , PTK2B DYRK1A , , , PARN CES1 , , APP APP , , , , RAC1 RAC1 RAC1 CDK5R1 CDK5R1 NEK2 FGFR1 CDK5R1 , , , CDK5 CDK5 CDK5 , , , HPRT1 , , BRCA1 , ERBB4 ERBB4 ERBB4 ERBB4 , , , , MAP2K1 , , , , , KDM5A MDC1 WEE1 , , PTK2B CXCR4 CXCR4 , , , , RXRA RXRA RXRA NRP1 NRP1 , , , , , HDAC7 ME2 BCL2 BCL2 BCL2 IGF1R IGF1R S100B , NCBP1 EGFR EGFR , MAP2K1 EGFR RET EGFR , , , , , GJA1 KAT2B SDCBP CXCR4 , , CDK5R1 CDK5R1 CDK5R1 , , , , , , , , , , , , , ALB CFD KDM1A NXF1 IGF1R IGF1R IGF1R IGF1R , , MAPK14 HPRT1 HPRT1 , HPRT1 , , , , , , , , , PTK2 PTK2 PTK2 , , , EGFR EGFR EGFR EGFR FGFR1 CDC42 PRKCA S100B ERBB2 S100B GJA1 CXCR4 APP MAP2K1 CDC42 PRKCA PRKCQ APP MAP2K1 GJA1 CXCR4 GJA1 CXCR4 APP MAP2K1 AKT1 BRD4 PPARGC1A MAPK8 BCHE DHX9 DLAT PLK1 HDAC8 PRKCA PRKCA FGFR1 FGFR1 CDK5R1 PRKCA PRKCA CDK5R1 NCBP2 1.96 E -05 1.96 E -05 0.001343 0.002245 1.26 E -05 3.91 E -05 7.95 E -05 1.45 E -04 2.81 E -04 3.08 E -04 4.83 E -04 8.26 E -04 0.001471 9.64 E -05 1.07 1.07 1.07 1.07 6.42 4.93 4.28 5.57 6.42 5.57 4.28 4.28 5.57 17.77 5 5 5 5 30 23 20 26 30 26 20 20 26 83 domain l EG F receptor, Furin-like cysteine-rich region Furin-like repeat FU Enrichment score: 3.73 C ell projection organization N euron projection development N euron projection morphogenesis N euron development N euron differentiation C ell morphogenesis C ell projection morphogenesis C ell part morphogenesis C ellular component morphogenesis Enrichment score: 3.64 Membrane-enclosed lumen IN T ER P R O IN T ER P R O IN T ER P R O S M AR T Annotation cluster 40 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 41 G OT ER M_ CC _F A T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4083 Dovepress He et al Dovepress 1.189132 0.238856 0.240933 0.276544 0.388594 FDR 0.199949 0.01759311 0.00243752 0.00244468 0.00272817 0.00359443 Benjamini 0.00410352 1.44 2.00 2.05 1.96 1.95 Fold enrichment 1.50

, , , ,

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,

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RXRA , , ,

VDR VDR

,

ME2 , WEE1 , , , , , , , , ALB , VDR , KDM1A PDHB , , RXRA NXF1 MAPK14 KAT2B NR1I2 , , , , , , PDHA1 HDAC8 , , PDHB , ME2 BCHE PDHA1 PLEK PPARGC1A NR1I2 , , , , PLK1 , , , HDAC7 NXF1 NCOA2 , , , NR1I2 BCHE DHX9 EHMT1 EHMT1 MDM4 MDM4 , , , , HDAC8 , PLK1 MAPK8 , , KDM1A KDM1A , , DLAT , C1QC , , , , DHX9 KDM1A NR1H2 CASP8 , , , HDAC4 , EHMT1 PARP2 C1QC VCP , , , , , PPARGC1A , CASP8 DLAT VCP , MAPK8 , , HDAC2 PPP1CC EHMT1 NR1I2 RARG RARG , , , , , , , HDAC8 SETD8 SETD8 , HDAC4 SIRT5 AGXT , , MDM4 , MDM2 , , , , HIST1H4A NR1H2 NR1H2 RARG CASP7 , , , BCKDHA AGXT , , , BRCA1 BCL2L1 NR1H2 PARP1 , HIST1H4A , , , RB1 , , BCKDHA , SIRT5 , , CASP7 RARG PPP1CC , , RPS6KA1 , PRKCA PRKCA MDM2 , NCOA2 , PARP2 , HDAC4 , FABP4 FABP4 , , , RPS6KA1 , , RB1 BRCA1 , SETD8 , , HDAC8 XRCC1 , PPARG PPARG MAPK3 , PDK1 ABL1 TLE1 , , , , , , IGF1 CHEK2 , PPARG PPARG NR1H4 , XRCC1 BCKDHB PDK1 , RB1 , , , , , , , CHEK2 , CSNK2A1 PRKCA RB1 F8 , , , , TLE1 , PARP1 BRCA1 , KIT HDAC2 NR1H4 NR1H4 MAPK3 HDAC4 MDM2 , MDM2 , , , , FABP4 , , PEX5 , , HDAC2 CSNK2A1 , INSR , , , MDM4 , PEX5 TLE1 , PDK4 PPP1CA , , TSG101 TSG101 , , KDM4A , , TSG101 TSG101 , TLE1 TP53 , , , NR3C1 , BCKDHB RB1 CASP3 SIRT5 ABL1 , , , HSD17B4 , IDE , , , RARB , NR1H4 RARB HSD17B4 , IDE , , , UBA3 UBA3 , , , RARB BRCA1 DUSP3 , NR3C1 TP53 , , MDM2 CASP3 , DUSP3 PPP1CA PNKP , KIT , PDK3 , SETD8 , , , APP , THRB THRB , , , THRB THRB , , RARG TLE1 , , SIRT5 TP53 , PDK4 , , , ALDH2 , BCL6 BCL6 , RARB , , RB1 , , BCL6 KDM4A , BACE2 , BACE2 TSG101 , , UBA3 RARG , TSG101 NUDT21 PARN PNKP , , , , NUDT21 , PARN , THRA THRA FABP4 THRA THRA , CHEK1 , HDAC4 EPAS1 TP53 , , , , , MAPK1 , CHEK1 , HDAC4 , , SIRT5 , , , PDK3 , , TLE1 ELANE BCL6 EED , , , KDM5A SFTPD , SFTPD , , , DYRK1A , ALDH2 UBE2E1 , BACE2 HDAC2 HDAC2 NCBP1 , PCK1 CES1 , NCBP1 , , TP53 GSTK1 PCK1 , GSTK1 , , , PPARD PPARD CDK2 PPARD PPARD , , , MAPK1 FARS2 , KDM5A FARS2 , , ELANE , MDM2 , , CFD KDM1A , , EED KDM1A , EED , EPAS1 , , SIRT5 , , RXRA , , , , , , SND1 SFTPD , , PPARA FNTA RXRA NCOA2 HDAC8 PPARA VDR TP53 UBA3 PPARA FNTA PLEK NCOA2 HDAC8 PPARA EED ELANE HDAC2 MAPK14 HDAC7 Genes NCBP2 AKT1 BRD4 WEE1 NEK2 MDC1 CES1 BRCA1 DYRK1A UBE2E1 KAT2B BRCA1 NCBP2 AKT1 BRD4 CDK2 NEK2 MDC1 -value 1.32 E -04 1.33 E -04 1.53 E -04 2.14 E -04 P 1.45 E -04 8.68 E -04 7.71 7.28 7.92 7.71 % 17.34 16.27 36 34 37 36 Count 81 76 Enrichment score: 3.60 N egative regulation of macromolecule biosynthetic process N egative regulation of gene expression N egative regulation of biosynthetic process N egative regulation of cellular biosynthetic process Term Organelle lumen I ntracellular organelle lumen ) Continued ( Annotation cluster 42 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Table 3 Category G OT ER M_ CC _F A T G OT ER M_ CC _F A T

4084 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 0.500666 0.88072 1.465914 0.034005 0.360451 0.437076 0.519307 1.785517 2.001185 (Continued) 0.00440251 0.00683604 0.0106896 5.12 E -04 0.00342307 0.00396061 0.00447801 0.01261501 0.0137637 2.05 1.93 1.90 2.04 1.93 2.21 2.02 1.83 1.91

, , ,

,

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, ,

,

,

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,

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,

, ,

APP TP53 , VDR VDR , , , , TLE1

VDR , , IL2 RXRA

, FABP4 SIRT5 TGFBR1 , , , , TP53 TGFBR1 , RARB RARB RARB , APP NR1H2 , , , , , TP53 , , MAPK9 , UBA3 NCOA1 ESR2 MDM2 , , , , , HDAC4 , HDAC4 , RXRA , BRCA1 JAK3 APP APP APP SIRT5 , , , , , , TP53 KDM1A KDM1A , RXRA RB1 EPAS1 NCOA2 , , , ARNT , , EPAS1 , NR1H2 , MDM2 ESRRG NR1H3 , , , UBA3 TNFRSF1A , , ESRRG , NR1I2 , , , HDAC4 CDK2 TNKS2 , , , MAPK14 IL2 IL2 TP53 , , , TNFRSF1A , IGF1 , EHMT1 RXRA , , , EPAS1 RARG UBA3 , PPARG MAPK14 , RXRA , NR1H2 COMT NR1H2 , , , RXRA NCOA1 , , , NR1I2 , , , PPARGC1A MAP2K1 TNFRSF1A TNFRSF1A TNFRSF1A NR1H4 JAK3 , , JAK3 , , , NR1H2 , CDK2 , , HDAC2 , , BRCA1 BRCA1 AR , RXRA , , , TP53 , , RARG HDAC2 , , NR1H2 RARG , , EHMT1 NR1I2 , , ESR2 INSR PPARG PPARG PPARG EPAS1 RARG , , , , , TGFBR1 HDAC2 , , , , ARNT HDAC4 HDAC2 , AR , , NR1H2 NR1H2 NR1H2 , BRCA1 RXRA ADORA2A , , , , , NCOA2 , MAPK14 , MAPK14 AR , , EHMT1 ARNT , , RB1 , IL2 BCKDHA NR1H4 NCOA2 , RARG , , , , , HDAC8 PPARGC1A PPARGC1A RARG , , , , HDAC2 EPAS1 , , PPARG KDM5A TSG101 TSG101 TSG101 NCOA2 KIT ARNT ARNT ARNT BRCA1 , THRB , , , , , , , , , , , EPAS1 NCOA2 , AR IGF1 , JAK3 RARG , , , , BCKDHA RARB HDAC4 , , HDAC2 HDAC2 , , , ESR2 ESR2 , , IL2 NR1H3 PPARGC1A NCOA1 BCKDHA NR1H4 , , , , MDM4 HDAC8 , , , , RARB THRB THRA , THRB THRB THRB , , NCOA2 , , , PPARG PPARG PPARG PPARG RARG , ESR1 , , , , , , MAP2K1 NCOA1 BCL6 , RB1 RB1 , , , , HDAC4 , JAK3 BRCA1 NR1H4 APP , , , NR1H3 , AR BCKDHA RARB MAPK14 CD40 PPARGC1A , NCOA2 NCOA2 , , , , , , , , HDAC8 MAPK1 , SETD8 THRA NR1H4 HRAS THRA THRA THRA MDM4 NR1H3 , , , , , TP53 , , , , , CDA THRB THRB THRB THRB , IGF1 IGF1 , , , , , RARG , , , RB1 HDAC4 , , RARB HDAC4 BRCA1 , , BCL6 ESR2 ESR2 , , , , AR , EED NR1H4 NR1H3 NR1H3 MAPK14 HDAC2 NCOA1 NCOA1 , , FABP4 , , , PPARA , MDM4 , , TP53 TP53 , , PPARD PPARD PPARD THRA THRA NCBP1 THRA THRA , SETD8 , , TNFRSF1A HDAC4 TLE1 , , , , , , , , ESRRG , , KDM5A NR1H4 , , , RB1 , , IL2 RB1 RB1 , BCL6 CDA , , , BRCA1 , , , PPARA EED RB1 SETD8 KDM1A SIRT5 MDM2 EED TLE1 FABP4 IGF1R BCKDHA IGF1 MAPK1 RARB RXRA CDK2 JAK3 NR1H4 PPARGC1A RARB ESRRG NCOA2 KDM5A ESRRG MAPK1 NR1H4 IGF1 HDAC2 PPARA PPARA NCBP1 PPARA PPARA PPARA PPARA PPARA 2.76 E -04 4.87 E -04 8.13 E -04 1.87 E -05 1.99 E -04 2.41 E -04 2.87 E -04 9.92 E -04 0.001113 6.64 7.07 6.85 8.99 7.92 5.78 6.85 7.28 6.42 31 33 32 42 37 27 32 34 30 N egative regulation of transcription N egative regulation of nitrogen compound metabolic process N egative regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolic process Enrichment score: 3.59 Positive regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolic process Positive regulation of gene expression Positive regulation of transcription from RNA polymerase II promoter Positive regulation of RNA metabolic process Positive regulation of transcription Positive regulation of transcription, D NA dependent G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 43 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4085 Dovepress He et al Dovepress FDR 0.21798 0.540924 0.70135 0.70135 0.25015 0.25015 3.129 0.149307 0.1668 0.1668 0.518482 0.607989 0.729273 0.826394 Benjamini 0.00225032 0.00461973 0.00566472 0.00566472 0.00252379 0.00252379 0.01988844 0.00166731 0.00183952 0.00183952 0.00449264 0.00504753 0.0058375 0.00655668 Fold enrichment 6.93 9.58 9.10 9.10 30.33 30.33 15.17 4.39 4.33 4.33 3.55 3.75 4.96 4.87

,

,

, , , PNP , BCL6 , CD40 CD40 CD40 , , , TRAF6 , AKT2 , ZAP70 BCL6 BCL6 BCL6 SYK SYK , , , , , , CD40 IL2 IL2 , LCK , , , ARNT , SFTPD SFTPD SFTPD , , , PNP PNP BCL6 , , , CD38 , ZAP70 ZAP70 ZAP70 AKT2 AKT2 AKT2 , , , , , , CD40 CD40 , , PPARGC1A , ZAP70 , NCK2 , ARNT ARNT ARNT BCL6 BCL6 ERBB2 ERBB2 ERBB2 LCK , , , , , INSR , , , , , PRKCQ , NCK2 CASP3 CASP3 CASP3 ZAP70 ZAP70 , , , , , , NR3C1 PNP , , ADAM17 ADAM17 ADAM17 , , , PPARGC1A PPARGC1A PPARGC1A , , , IGF1 NCK2 NCK2 NCK2 NCK2 NCK2 , , , , , , PRKCQ , CD40 , PIM1 PIM1 PIM1 IL2 , , , INSR INSR INSR , , , , SYK SYK SYK , , , SYK PLEK CD38 , , , PRKCQ PRKCQ PRKCQ PRKCQ PRKCQ HDAC4 IGF1 IGF1 IGF1 AKT1 AKT1 AKT1 , , , , , , , , , , , , IL2 IL2 IL2 , , , SYK , Genes AKT1 PNP PNP PNP TRAF6 IL2 AKT1 AKT1 AKT1 EGFR EGFR EGFR CD38 CD38 CD38 TRAF2 TRAF2 CD38 CD38 -value P 1.20 E -04 2.99 E -04 3.88 E -04 3.88 E -04 1.38 E -04 1.38 E -04 0.00175 8.23 E -05 9.20 E -05 9.20 E -05 2.86 E -04 3.36 E -04 4.03 E -04 4.57 E -04 % 1.71 1.28 1.28 1.28 0.86 0.86 0.86 2.57 2.57 2.57 2.78 2.57 1.93 1.93 Count 8 6 6 6 4 4 4 12 12 12 13 12 9 9

/ S 1 / S 1 Term Enrichment score: 3.57 R egulation of glucose metabolic process Positive regulation of glucose metabolic process Positive regulation of cellular carbohydrate metabolic process Positive regulation of carbohydrate metabolic process Enrichment score: 3.49 Positive regulation of cyclin-dependent prote in kinase activity during G R egulation of cyclin- dependent protein kinase activity during G Positive regulation of cyclin-dependent protein kinase activity Enrichment score: 3.44 R egulation of lymphocyte proliferation R egulation of mononuclear cell proliferation R egulation of leukocyte proliferation Positive regulation of cell activation Positive regulation of lymphocyte activation Positive regulation of lymphocyte proliferation Positive regulation of mononuclear cell proliferation ) Continued ( Table 3 Category Annotation cluster 44 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 45 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 46 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T

4086 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats 0.826394 1.300425 1.650107 6.043888 0.555258 1.006445 1.316686 0.280714 0.695681 1.506777 4.316077 0.959108 0.959108 4.185354 0.00655668 0.00971853 0.01184284 0.03453311 0.00471957 0.00764671 0.00979929 0.0015486 0.0157035 0.0206159 0.04574947 0.00731779 0.00731779 0.02523826 4.87 3.43 4.40 3.59 3.79 3.84 3.71 3.14 3.24 2.69 2.33 5.51 5.51 5.06

, , , ,

, , ,

, PNP , PPP2CA PPP2CA PPP2CA , , , TNK2 , MAP3K9 MAP3K9 MAP3K9 , , , TRAF6 SYK , , LCK , SYK SYK , FYN FYN FYN , , , , IL2 , ADORA2A ADORA2A ADORA2A CD40 , , , IL2 IL2 , , , HPRT1 HPRT1 , , TJP1 TJP1 TJP1 , , , PNP , MAP3K9 PNP PNP , , BCL6 , , CD40 CD40 HMGCR HMGCR HMGCR , , NCK2 NCK2 NCK2 , , , , , , SFTPD FYN , , CD40 TRAF6 ,

,

ZAP70 , , BTK BTK BTK , , , TJP1 CASP3 CASP3 CASP3 , , , , FKBP1A FKBP1A , , ZAP70 , BCL6 LCK , DLG1 DLG1 DLG1 , SRC SRC SRC , , , , , , ZAP70 BTK , , FKBP1A , TP53 TP53 , , PDPK1 PDPK1 PDPK1 , , , ERBB2 LCK NCK2 SRC NCF4 NCF4 NCF4 , , ZAP70 , , , , , , SFN TP53 , , BCL2 BCL2 ARHGAP9 ARHGAP9 ARHGAP9 , , , , , DLG1 , HCK HCK HCK , , , NCK2 NCF4 CASP3 , NCK2 , RB1 , , , PRKCQ PPP2R1A PPP2R1A PPP2R1A , , , , BCL2 FYN FYN , , , SFN SFN ABL1 ABL1 ABL1 , , , , , GRB2 HCK GRB2 GRB2 , , , , FYN NCK2 AKT1 AKT1 AKT1 , FABP4 , CD38 PRKCQ RB1 RB1 , , , , , , PRKCQ HPRT1 , , , , ARHGAP9 , CXCR4 CXCR4 TNK2 TNK2 TNK2 , , LYN LYN LYN LYN , , , SYK , , , , , CD38 IL2 PPARG FABP4 FABP4 LCK ABL1 LCK LCK TRAF2 PRKCQ TRAF2 PRKCA PRKCA PRKCA ITK ITK ITK ITK ACE ACE CXCR4 CD40 4.57 E -04 7.21 E -04 9.16 E -04 0.003429 3.07 E -04 5.57 E -04 7.30 E -04 1.94 E -04 4.16 E -04 9.74 E -04 0.003617 5.31 E -04 5.31 E -04 0.002353 1.93 2.57 1.93 1.93 2.57 2.36 2.36 3.43 3.00 3.43 3.43 1.71 1.71 1.50 9 12 9 9 12 11 11 16 14 16 16 8 8 7

Positive regulation of leukocyte proliferation Positive regulation of leukocyte activation R egulation of T-cell proliferation Positive regulation of T-cell activation Enrichment score: 3.30 N egative regulation of transferase activity N egative regulation of protein kinase activity N egative regulation of kinase activity Enrichment score: 3.14 SH 3 domain SH 3 domain S rc homology-3 domain SH 3 domain Enrichment score: 3.06 Mononuclear cell proliferation L eukocyte proliferation L ymphocyte proliferation D A V I D, Database for nnotation, Visualization, and ntegrated Discovery; U , ursolic acid; FD R false discovery rate. G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 47 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Annotation cluster 48 S P_P IR _K E YWO R D UP_ SE Q_F EA TU RE IN T ER P R O S M AR T Annotation cluster 49 G OT ER M_BP_F A T G OT ER M_BP_F A T G OT ER M_BP_F A T Abbreviations:

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4087 Dovepress He et al Dovepress 1.26 E -04 FDR 5.97 E -09 8.92 E -09 7.43 E -07 1.22 E -06 3.93 E -06 1.42 E -05 2.78 E -05 2.87 E -05 6.73 E -05 1.11 E -04 1.49 E -06 Benjamini 7.75 E -10 5.79 E -10 3.21 E -08 3.95 E -08 1.02 E -07 3.07 E -07 5.16 E -07 4.65 E -07 9.71 E -07 1.44 E -06 3.25 Fold enrichment 2.64 4.69 5.52 4.29 4.40 4.73 4.35 3.99 4.57 2.67

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4088 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats (Continued) 1.99 E -04 3.15 E -04 4.54 E -04 5.24 E -04 6.48 E -04 9.72 E -04 0.001345 0.001555 0.00221 0.002349 0.002583 0.006771 0.016317 0.018816 0.043123 0.119869 2.16 E -06 3.14 E -06 4.21 E -06 4.54 E -06 5.25 E -06 7.42 E -06 9.69 E -06 1.06 E -05 1.43 E -05 1.45 E -05 1.52 E -05 3.82 E -05 8.82 E -05 9.77 E -05 2.15 E -04 5.76 E -04 3.09 3.98 3.73 3.31 3.99 4.59 4.23 3.78 4.97 3.68 3.83 2.80 2.39 4.18 3.46 3.18

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Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4089 Dovepress He et al Dovepress FDR 0.124225 0.147114 0.173569 0.227192 0.265376 0.426361 0.44736 0.545693 0.592084 0.599243 0.808763 0.906871 1.483839 1.689783 1.886424 2.086826 2.51844 2.849446 5.017313 Benjamini 5.76 E -04 6.59 E -04 7.51 E -04 9.52 E -04 0.0010769 0.0016786 0.0017096 0.0020265 0.0021381 0.0021056 0.002769 0.0030264 0.0048377 0.0053789 0.0058663 0.0063436 0.0074937 0.0083009 0.0144151 Fold enrichment 3.34 3.29 3.24 2.19 2.74 3.34 2.84 4.63 3.23 3.73 3.13 1.84 3.64 2.51 2.85 3.83 2.20 2.30 3.36

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R -mediated Helicobacter pylori γ ) Term A dipocytokine signaling pathway E pithelial cell signaling in infection L ong-term depression E ndocytosis G n RH signaling pathway A cute myeloid leukemia S mall-cell lung cancer Thyroid cancer G lycolysis/ gluconeogenesis Tyrosine metabolism N OD-like receptor signaling pathway M A PK signaling pathway A ldosterone-regulated sodium reabsorption Fc phagocytosis p53 signaling pathway Prion diseases A xon guidance Oocyte meiosis Pyruvate metabolism Continued ( Table 4 Category pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG

4090 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats (Continued) 5.551863 5.747625 5.791613 6.46863 6.721689 7.833234 7.886251 7.890864 7.998282 8.257365 8.540799 8.91132 12.36319 14.40039 17.80881 24.69256 30.86838 37.46662 39.90887 44.44857 47.66781 53.06039 0.0156455 0.0158742 0.0156745 0.0172046 0.0175475 0.0201485 0.0199102 0.0195569 0.0194747 0.019771 0.020118 0.0206644 0.028606 0.0330669 0.0408596 0.0576238 0.0732134 0.090792 0.0966722 0.1091442 0.117864 0.1343859 2.40 2.15 2.63 2.59 2.35 3.12 2.87 2.21 2.52 2.13 3.41 2.81 3.60 1.85 1.89 4.39 2.90 1.79 4.97 2.61 2.89 2.25

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, HLA-E , IGF1 , PRKCB CDK2 GAMT CASP3 , , , RAMP1 , , AKT2 , FABP7 PTPN11 AKT1 , , , PDE5A PRKCB ADH7 MAPK3 , , , SRC , MAPK14 NME2 , CD40 , CASP1 , , , WEE1 MLLT4 MAPK3 , , TPMT , ALDH2 , , CALCRL , MLLT4 FABP4 , BRAF , RAF1 PRKCB , TNFRSF1A MMP-9 , , PDE4B GART , , , SFN , ADH1B HLA-B PPP1CC , , , , MAPK1 ALDH3A1 BRAF , RAC1 , , BCL2L1 , , AKT2 MAOB , HPRT1 , SRC , , GJA1 PNP ADK FABP3 , , , , NCF4 PRKACA IL2 BTK PPP2CA IRAK4 , , RAF1 , , , , , ADH5 HLA-A MAP2K1 , , TP53 IRAK4 CHEK2 , , , , CDA ALDH2 , ARG2 , TRAF6 PTEN , , , MAPK14 RPS6KA1 , , AGXT GNAI1 , PPARG AKT1 HPRT1 , PDE4A CD40 , , , , , GNAI1 HLA-G RB1 , , , RAC1 , AKT1 RAC1 , , ALDH2 PLA2G2A NAT2 , , , CSNK2A1 MAOB , MAOA PRKCI , ADORA2A , RPS6KA1 , , EIF4E , MAPK8 ABL1 JAK3 , GRB2 SULT1E1 , , AKT2 , , RXRA , CXCR4 , GNMT , , , HLA-E PDE7A BCL2 PDE4D , , BCL2L1 , , CASP8 ROCK1 CHEK1 , , , TJP1 , NAT1 , , ADH1C MAPK10 MAPK3 AIRE MAOA HRAS GATM , CTSG , , , , GNAI1 , , , PRKCB , ROCK1 PDPK1 , , CHKB , FYN MDM2 MAPK9 , GAMT PRKACA PTK2B , , CD40 TP53 , , , , , , NT5M PRKACA MAP2K1 ITGAL CDK6 PDE3B , , , SULT1A2 , CES1 , , , , PRKCQ MAPK14 , , CD40 CHKB , HRAS , ZAP70 CMA1 , HNMT , , PPP1CA , ITK PPP1CA XIAP PTK2 , , , TTK , , PPP1CC PPARD CASP3 MAPK1 DCK , CASP8 MAOB NR1H3 HLA-B , GSK3B , , , , , , , , , HRAS MAP2K1 MAPK3 ALDH7A1 CASP8 NME1 , , , , UMPS , , , CASP3 TAP1 ADH4 , CDC42 MME , , , , SULT2B1 , TP53 , PLK1 TRAF2 AKT1 MAP2K1 TJP1 RAC1 PCK1 ITGAL MAPK1 , , , , ATIC PDE8A , , , PIK3CG MAPK1 EGFR BRAF PPP2R1A CDC42 , , BCL2 PDPK1 MAOA , , GZMB , , , , , , ARG1 , , , , , TYMP NUDT2 PRKACA HLA-G ALDH7A1 LCK , , , , , , REN , PIK3CG CASP7 PRKCA PRKCB PRKCA RAF1 PPARA MMP-1 PRKCA MAPK1 XDH PIK3CG AKT2 PIK3CG MAPK3 EIF4G1 ABL1 PRKCA PRKCQ UNG ODC1 AMD1 ASPA XDH PDE2A PDE9A PRKCA CSNK2A2 ACE HLA-A YWHAZ HDAC2 SULT1A1 ALDH7A1 ADH1A GATM TNFRSF1A 0.00474 0.004911 0.00495 0.005547 0.005771 0.006762 0.006809 0.006813 0.00691 0.007143 0.007398 0.007733 0.010917 0.01285 0.01618 0.023312 0.030239 0.038296 0.041478 0.04772 0.052437 0.060969 3.00 3.64 2.57 2.57 3.00 1.93 2.14 3.21 2.57 3.43 1.71 2.14 1.50 4.07 3.64 1.07 1.50 3.21 0.86 1.50 1.28 1.71 14 17 12 12 14 9 10 15 12 16 8 10 7 19 17 5 7 15 4 7 6 8 A poptosis L eukocyte transendothelial migration L ong-term potentiation PP AR signaling pathway G ap junction Drug metabolism mTO R signaling pathway Toll-like receptor signaling pathway Viral myocarditis Vascular smooth muscle contraction Primary immunodeficiency A rginine and proline metabolism H istidine metabolism Purine metabolism Tight junction R enin–angiotensin system A llograft rejection C ell cycle S ulfur metabolism Fatty acid metabolism G lycine, serine, and threonine metabolism ALS pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4091 Dovepress He et al Dovepress

it shows that UA exerts an ameliorating effect on DMN- induced liver fibrosis in rats through reducing the accumula- tion of collagen and suppressing the expression of α-SMA FDR 59.39099 60.74629 61.67824 62.41638 63.75741 64.08485 66.4176 68.17856 in rat HSCs.

UA restores serum SOD level and

Benjamini 0.155886 0.1591473 0.1607853 0.1616832 0.1650651 0.164358 0.172031 0.1775696 suppresses serum MDA in DMN-induced liver fibrotic rats Growing evidence shows that oxidative stress has been implicated in the pathogenesis of liver fibrosis.32 The antioxidative effect of UA was examined in rats treated with 1% DMN by determining Fold enrichment 3.11 6.39 1.56 1.58 1.46 2.98 3.73 2.22 the serum levels of SOD and MDA. SOD has a capability of

, detoxifying superoxide and protecting cells against oxidative

,

,

, stress, whereas MDA is a principal toxic product of lipid peroxi- MAPK8 , F2

, 33 ADAM17

, dation under oxidative stress. As shown in Figure 3A, DMN- MAP2K1 PRKCB , , TNFRSF1A ,

PAK4 alone treated group exhibited low serum level of SOD, and there , PRKACA , BRAF , BACE1 , was a 29.4% decrease (P,0.001), compared to the vehicle group; MAPK10 , MAPK1 TIAM1 , , whereas UA treatment remarkably increased the serum level of MAPK9 , CASP8 ROCK1 , , TP53 , SOD. In comparison to the DMN group, there was a 1.6- and NAE1 PTK2 , , RAC1 ,

HRAS 2.0-fold rise in the serum level of SOD (P,0.001). On the con- , MAPK3 , MMP-7 CDK5 , , trary, there was an 8.4-fold increase in the serum level of MDA PPP1CA MAPK3 , , GSK3B ITGAL , , NNMT CASP7

, in DMN-alone treated group, compared to the vehicle group , MME , ROCK1 , GRB2 , (Figure 3B, P,0.001). UA treatment suppressed DMN-induced PNP MAPK1 , , FGFR1 , IDE PPP2CA , , GSK3B GART ,

, elevation in the serum level of MDA. There was a 78.4% and PPARD , NT5M , PAK1 CDC42 MAP2K1 84.2% reduction in the serum level of MDA, compared to DMN , , , ATIC PIK3CG , , NAT2 , BACE2 CDK5R1 , ,

CSNK2A1 group (Figure 3B, P,0.001). Taken together, the data show , CD38 RAC1 , , PPP2R1A , DHFR FGFR2 MAPK1 , NAT1 PPP1CC that UA treatment can attenuate oxidative stress through the , , , , CASP3 , regulation of serum level of SOD and MDA in DMN-induced Genes NAMPT XDH HSD17B10 APP PRKCA CSNK2A2 EGFR RAF1 MAPK3 EGFR TYMS P I K3 CG , M A PK1, PK3, PK9, PK8, PK10, INSR liver fibrotic rats.

value UA inhibits proliferation in HSC-T6 cells

P- 0.072217 0.074832 0.076679 0.078172 0.080954 0.081647 0.086763 0.090845 Since we have observed the beneficial effects of UA in DMN- induced liver fibrotic rats, we further investigated the molecular

% 1.07 0.64 3.64 3.43 4.50 1.07 0.86 1.50 mechanisms that underlie the antifibrotic effect of UA in vitro. We first examined the effect of UA (50 μM), DPI (20 μM, an NOX inhibitor), AG490 (50 μM, a specific and potent inhibi-

Count 5 3 17 16 21 5 4 7 tor of JAK2), and PD98059 (30 μM, a potent and selective inhibitor of MAP kinase) on the proliferation of HSC-T6 cells using MTT assay (Figure 4). Treatment of HSC-T6 cells with leptin (100 ng/mL) markedly induced cell proliferation over 48 hours (P,0.001). Treatment of HSC-T6 cells with UA, AG490, DPI, or PD98059 prevented leptin-induced

) proliferation over 48 hours (Figure 4). DPI exhibited the most Term N icotinate and nicotinamide metabolism C affeine metabolism A lzheimer’s disease Wnt signaling pathway R egulation of actin cytoskeleton Dorso–ventral axis formation One carbon pool by folate Type II diabetes mellitus potent preventive effect, and the data showed that UA exerted K EGG , Kyoto E ncyclopedia of G enes and enomes; D A V I D, Database for nnotation, Visualization, ntegrated Discovery; U ursolic acid; FD R false discovery rate; ALS amyotrophic lateral sclerosis. a comparable inhibitory effect to DPI to the cell proliferation. Continued ( Taken together, the initial data suggest that ROS, JAK2, and MAPK/ERK signaling pathways are involved in the leptin-

Table 4 Category pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG pathway K EGG Abbreviations: promoted proliferation in HSC-T6 cells.

4092 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats

Figure 1 Effect of UA on DMN-induced hepatic fibrogenesis in rats. Notes: Rats were administered with 1% DMN dissolved in saline (1 mL/kg body weight) via intraperitoneal injection for 3 consecutive days per week for 4 weeks to induce liver fibrosis. Representative photomicrographs of the liver histology from groups (n=5) treated with vehicle control (A), DMN alone (B), DMN +20 mg/kg UA (C), and DMN +40 mg/kg UA (D). Magnification:× 100. Abbreviations: UA, ursolic acid; DMN, dimethylnitrosamine.

UA suppresses intracellular ROS cells, compared to basal level (P,0.001; Figure 5A). The generation in HSC-T6 cells leptin-induced effect on ROS generation was abolished We also examined the effect of UA on the ROS generation by pretreatment with UA (50 μM), NAC (10 mM, a ROS in the HSC-T6 cells using DCF-DA fluorescence (Figure 5A scavenger), DPI (20 μM), or AG490 (50 μM) in HSC-T6 and B). Within 1 hour, leptin-stimulated HSC-T6 cells cells (P,0.001; Figure 5A). Of note, UA suppressed showed a significant increase in ROS production compared leptin-induced ROS generation in a time-dependent man- to the vehicle control group. There was a 3.4-fold elevation ner over 24 hours, which was similar to DPI (Figure 5B). in the intracellular ROS level in leptin (100 ng/mL)-treated NAC potently scavenged intracellular ROS level within 1 hour; however, the ROS scavenging effect of NAC was gradually attenuated over 24 hours (Figure 5B). Taken Table 5 Effect of UA on rat liver collagen hyperplastic degree in together, the data suggest that UA reduces intracellular ROS experimental hepatic fibrotic rats (Mean± SD, n=8) level mainly through the inhibition of enzymatic source of Group Dose (mg kg-1) N - + ++ +++ ROS, rather than the scavenging of intracellular ROS. Normal wt 8 8 0 0 0 Model• wt 8 0 1 4 3 UA-2•, 20 8 1 4 2 1 UA inhibits leptin-induced NOX UA-3•,▲ 40 8 1 5 2 0 activation in HSC-T6 cells Notes: •P,0.01 vs normal; ▲P,0.01, P,0.05 vs model. An increased number of + Since we observed the inhibitory effect of UA on intracel- symbols represent an increase in collagen hyperplastic degree with treatment. - represents lular ROS generation via the regulation of enzymatic source, without treatment. Abbreviations: UA, ursolic acid; SD, standard deviation; wt, without treatment. we further examined the effect of UA on the activity of NOX

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Figure 2 Effect of UA on the expression of α-SMA in rat liver. Notes: Rats were administered with 1% DMN dissolved in saline (1 mL/kg body weight) via intraperitoneal injection for 3 consecutive days per week for 4 weeks to induce liver fibrosis. (A) Representative blot of α-SMA from groups treated with vehicle control, DMN alone, DMN +20 mg/kg UA, and DMN +40 mg/kg UA. (B) Bar graph showing the relative expression level of α-SMA in rat liver. β-Actin acts as an internal control. Data are expressed as mean ± SD (n=5). ***P,0.001. + and - represent with or without treatment, respectively. Abbreviations: UA, ursolic acid; α-SMA, α-smooth muscle actin; DMN, dimethylnitrosamine; SD, standard deviation. in HSC-T6 cells. As shown in Figure 6, the NOX activity UA inhibits leptin-induced p47phox remarkably increased in the HSC-T6 cells when treated membrane translocation with leptin (100 ng/mL) for 1 hour, 12 hours, or 24 hours, p47phox translocation from cytoplasm to cell membrane compared to the basal level (P,0.001). UA (50 μM) mark- occurs during NOX activation; thus, the translocation was edly inhibited leptin-induced NOX activation after 1 hour, examined through testing the content of the membrane- 12 hours, or 24 hours. UA’s inhibitory effect on NOX bound p47phox and the total cellular p47phox in HSC-T6 activity was similar to that of DPI (20 μM) and AG490 cells. There was a marked alteration in the leptin (100 ng/ (50 μM). However, NAC showed no effect on leptin-induced mL)-induced translocation of p47phox, whereas the total NOX activation. Taken together, the data further showed level of p47phox did not dramatically change in the pres- that UA suppresses intracellular ROS production via the ence of UA (50 μM), DPI (20 μM), or AG490 (50 μM) modulation of enzymatic source, NOX, in HSC-T6 cells. (Figure 7A and B). Stimulation of HSC-T6 cells with

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Figure 3 UA increased serum SOD level and decreased serum MDA level in liver fibrotic rats. Notes: Rats were administered with 1% DMN dissolved in saline (1 mL/kg body weight) by intraperitoneal injection for 3 consecutive days per week for 4 weeks to induce liver fibrosis. Bar graphs showing the serum level of SOD (A) and MDA (B) in rats received treatment of vehicle control, DMN alone, DMN +20 mg/kg UA, and DMN +40 mg/kg UA. Data are expressed as mean ± SD (n=5). ***P,0.001. Abbreviations: UA, ursolic acid; SOD, superoxide dismutase; MDA, maleic dialdehyde; DMN, dimethylnitrosamine; SD, standard deviation.

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8$ %DVDO /HSWLQ OHSWLQ 8$ '3,OHSWLQ $*OHSWLQ 3'OHSWLQ Figure 4 UA inhibits the proliferation of HSC-T6 cells. Notes: Leptin (100 ng/mL) stimulated HSC-T6 cells proliferation at 12 hours, 24 hours, and 48 hours. Leptin-induced HSC-T6 cell proliferation was significantly suppressed by the pretreatment with UA (50 μM), AG490 (50 μM), DPI (20 μM), or PD98059 (30 μM). Data are expressed as mean ± SD from six independent experiments. The symbol *indicates the comparison between leptin and other treatments, *P,0.001. Abbreviations: UA, ursolic acid; HSC, hepatic stellate cell; DPI, diphenyleneiodonium; SD, standard deviation. leptin for 30 minutes remarkably induced the translocation leptin-induced translocation of p47phox and normalized the of p47phox from cytoplasm to cell membrane. There was a membrane-bound p47phox. These effects were observed in 1.5-fold increase in the membrane- bound p47phox in leptin- DPI- and AG490-treated cells as well (P,0.05; Figure 7A stimulated cells, compared to the control cells (P,0.01; and B). Collectively, UA blocks the translocation of p47phox Figure 7A and B). Pretreatment of cells with UA blocked the from cytoplasm to cell membrane, contributing to the

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Figure 5 UA inhibits leptin-induced ROS production in HSC-T6 cells. Notes: HSC-T6 cells were seeded into six-well plates and pretreated with vehicle control, UA (50 μM), NAC (10 mM), DPI (20 μM), or AG490 (50 μM) for 30 minutes. Then, the cells were stimulated by leptin (100 ng/mL), and the intracellular ROS level was measured using DCF-DA fluorescence probes (10 μM). (A) HSC-T6 cells were stimulated by leptin for 1 hour showing a significant induction in ROS production that was blocked by the pretreatment with UA, NAC, DPI, or AG490. (B) UA suppressed the leptin-induced ROS generation in a time-dependent manner and exhibited a similar inhibitory effect on ROS generation to DPI over 24 hours. Data are expressed as mean ± SD from six independent experiments. ***P,0.001. Abbreviations: UA, ursolic acid; ROS, reactive oxygen species; HSC, hepatic stellate cell; NAC, N-acetyl-l-cysteine; DPI, diphenyleneiodonium; DCF-DA, 2′,7′- dichlorofluorescin diacetate; SD, standard deviation.

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com 4095 Dovepress He et al Dovepress inhibitory effect of NOX activity and ROS generation in As shown in Figure 9A and B, 30-minute stimulation of HSC-T6 cells. HSC-T6 cells with 100 ng/mL leptin remarkably increased the level of p-ERK1 and p-ERK2 by 1.6- and 1.4-fold, com- UA inhibits the leptin-induced expression pared to the control group, respectively (P,0.05 or 0.01). of gp91phox, p22phox, p67phox, and Rac1 Pretreatment of cells with UA (50 μM) completely blocked To further investigate the effect of UA on the activity of NOX the leptin-induced phosphorylation of ERK1/2 in HSC-T6 in HSC-T6 cells, we examined the effect of UA on the expres- cells (P,0.01; Figure 9A and B). Incubation of HSC-T6 cells sion level of NOX subunits, including gp91phox, p22phox, p67phox, with DPI (20 μM), AG490 (50 μM), or PD98059 (30 μM) and Rac1, which are important to NOX activity. As shown in markedly inhibited leptin-induced phosphorylation of ERK1 Figure 8A and B, after HSC-T6 cells were stimulated with lep- (P,0.05 or 0.01; Figure 9A and B). There was a decrease tin (100 ng/mL) for 12 hours, the expression level of gp91phox, in the level of p-ERK2 but without statistical significance p22phox, p67phox, and Rac1 was remarkably increased 1.9-, 1.7-, when treated with DPI, AG490, or PD98059 (P.0.05; 2.1-, and 1.9-fold compared to the control group, respectively Figure 9A and B). Furthermore, the effect of UA on PI3K/ (P,0.01 or ,0.001). Pretreatment of cells with UA (50 μM) Akt and p38 MAPK signaling pathways was examined in markedly decreased the leptin-induced protein expression of HSC-T6 cells. As shown in Figure 10A and B, stimulation gp91phox, p22phox, p67phox, and Rac1 by 71.9%, 62.5%, 61.0%, of HSC-T6 cells with leptin markedly increased the level of and 71.9%, compared to the leptin-treated cells, respectively p-p38 MAPK, PI3K, and p-Akt (P,0.05 or 0.001). However, (P,0.001). These results suggest that UA potently negatively pretreatment of cells with UA dramatically suppressed leptin- regulate the expression level of NOX4 subunits, contributing induced increase in the level of p-p38 MAPK, PI3K, and to the reducing effect of ROS generation. p-Akt (P,0.05 or 0.001; Figure 10A and B). Notably, DPI, SB203580, and LY294002 showed a similar effect to UA on UA inhibits ERK, PI3K/Akt, and p38 MAPK leptin-induced elevation in the level of p-p38 MAPK, PI3K, signaling pathways in HSC-T6 cells and p-Akt (P,0.001; Figure 10A and B). Taken together, NOX-derived ROS regulate signal transduction involved in UA negatively regulates ERK, PI3K/Akt, and p38 MAPK liver fibrosis in HSCs. We evaluated the effect of UA on the signaling pathways in HSC-T6 cells, which contributes, at modulation of PI3K/Akt, p38 MAPK, and ERK1/2 signaling least in part, to the underlying mechanism of the antifibrotic pathways in HSC-T6 cells using Western blotting analysis. effect of UA.



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Figure 6 UA suppressed leptin-induced NOX activation in HSC-T6 cells. Notes: Cells were pretreated for 30 minutes with UA (50 μM), NAC (10 mM), or AG490 (50 μM) and incubated for 1 hour, 12 hours, or 24 hours in the presence of leptin (100 ng/mL). Cells were detached by trypsinization and processed as described in the Materials and methods section. Data are expressed as mean ± SD from six independent experiments. ***P,0.01. Abbreviations: UA, ursolic acid; NOX, NADPH oxidase; HSC, hepatic stellate cell; NAC, N-acetyl-l-cysteine; SD, standard deviation.

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Figure 7 UA suppressed leptin-induced translocation of the p47phox from cytoplasm to cell membrane in HSC-T6 cells. Notes: Cells were pretreated with UA (50 μM), DPI (20 μM), or AG490 (50 μM) for 30 minutes and then were stimulated by leptin (100 ng/mL) for another 30 minutes. The membrane bound and total level of p47phox were examined by Western blotting assay. (A) Representative blots of membrane (M-) p47phox and total (T-) p47phox in HSC-T6 cells. (B) Bar graph showing the relative level of M-p47phox in HSC-T6 cells. Data are expressed as mean ± SD from six independent experiments. β-Actin acts as an internal control. *P,0.05; **P,0.01. Abbreviations: UA, ursolic acid; HSC, hepatic stellate cell; DPI, diphenyleneiodonium; SD, standard deviation.

UA promotes the expression of MMP-1 plays a causal role in the pathogenesis of liver fibrosis11,35,36 but inhibits the expression of TIMP-1 and that antioxidant may be a promising agent to treat liver 13,37 and type I collagen in HSC-T6 cells fibrosis through the attenuation of oxidative stress. In the present study, we have predicted the molecular interactome of Finally, we examined the effect of UA on the protein expres- UA, and there are 611 molecular proteins possibly interact- sion of TIMP-1 and MMP-1 and mRNA expression of type I ing with UA. The subsequential functional and mechanistic collagen in HSC-T6 cells. HSC-T6 cells were treated with experiments show that UA reverses DMN-induced liver leptin at 100 ng/mL for 24 hours, the TIMP-1 protein fibrosis through the attenuation of oxidative stress with the expression level and the type I collagen mRNA expression involvement of ERK, PI3K/Akt, and p38 MAPK signaling level markedly increased, but the MMP-1 protein expres- pathways in vitro and in vivo. sion level significantly decreased (Figures 11A and B In recent years, there is a growing interest in the investiga- and 12A and B). Pretreatment of cells with UA (50 μM), tion of the beneficial effects and molecular mechanisms of DPI (20 μM), SB203580 (10 μM), or LY294002 (10 μM) UA that is a natural pentacyclic triterpenoid carboxylic acid inhibited leptin-promoted expression of TIMP-1 (P,0.001; and a major component of some traditional medicinal herbs.38 Figure 11A and B), while UA and DPI remarkably increased Increasing evidence shows that UA possesses a variety of leptin-suppressed expression level of MMP-1 (P,0.001; bioactivities, including antioxidative, anti-inflammation, and Figure 11A and B). SB203580 and LY294002 did not show anticancer activities, although the full spectrum of molecular significant regulatory effect on the expression of MMP-1 in targets has not yet been revealed.38,39 In the present study, the HSC-T6 cells (P.0.05; Figure 11A and B). Moreover, pre- computational experiment provided an interactome of UA, treatment of HSC-T6 cells with UA, DPI, AG490, or PD98059 with 611 potential molecular targets been predicted. The markedly blocked the leptin-induced mNRA expression of bioinformatic analysis showed various signaling pathways type I collagen (P,0.05 or ,0.01; Figure 12A and B). Col- that may be able to explain the beneficial effects of UA. These lectively, UA inhibits liver fibrosis with the involvement of results can provide a clue for us to investigate the beneficial ERK, PI3K/Akt, and p38 MAPK signaling pathways. effects and underlying mechanisms of UA. Discussion Employment of computational and bioinformatic Liver fibrosis represents a major challenge with considerable approaches have become a practical and valuable way to morbidity and mortality worldwide, due to the complexity of efficiently predict the molecular interactome of a chemical etiology.34 Compelling evidence indicates that oxidative stress molecule. The DDI-CPI tool has been used to predict the

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Figure 8 Effect of UA on the expression of NOX subunits of HSC-T6 cells. Notes: Cells were pretreated with UA (50 µM) for 30 minutes and then stimulated with leptin (100 ng/mL) for another 12 hours. The expression level of gp91phox, p22phox, p67phox, and Rac1 was examined using Western blotting assay. (A) Representative blots of gp91phox, p22phox, p67phox, and Rac1 in HSC-T6 cells. (B) Bar graphs showing the relative level of gp91phox, p22phox, p67phox, and Rac1 in HSC-T6 cells. Data are expressed as mean ± SD from six independent experiments. β-Actin acts as an internal control. **P,0.01; ***P,0.001. Abbreviations: UA, ursolic acid; NOX, NADPH oxidase; HSC, hepatic stellate cell; SD, standard deviation.

4098 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats

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Figure 9 UA inhibits ERK signaling pathway in HSC-T6 cells. Notes: Cells were pretreated with UA (50 µM), DPI (20 µM), AG490 (50 µM), or PD98059 (30 µM) for 30 minutes and then stimulated with leptin (100 ng/mL) for 30 minutes. The phosphorylation level of ERK1/2 was examined using Western blotting assay. (A) Representative blots of p-ERK1/2 in HSC-T6 cells. (B) Bar graphs showing the relative level of p-ERK1 and p-ERK2 in HSC-T6 cells. Data are expressed as mean ± SD from six independent experiments. β-Actin acts as an internal control. *P,0.05; **P,0.01. Abbreviations: UA, ursolic acid; ERK, extracellular signal-regulated kinase; HSC, hepatic stellate cell; DPI, diphenyleneiodonium; SD, standard deviation. potential targets, and DAVID has been employed to analyze resulting in an imbalance in the amounts of oxidants and anti- the molecular targets and related signaling pathways that are oxidants. NOXs are multicomponent enzymes mediating the regulated by UA. An approach using DDI-CPI server offers a transfer of electrons from cytosolic NADPH to O2 to produce - 43–45 46 fast, efficient, and inexpensive strategy to predict the potential O2 , and they are the primary enzymatic source of ROS. targets, identify drug repositioning potential, and evaluate NOXs are differentially expressed and distributed among the and determine adverse drug reactions of a chemical/drug tissues and subject to regulation by various factors. More- via molecular docking of small compound across human over, NOXs derived from ROS are also important signaling proteome,19–21,24,40,41 although this web-based program has molecules under pathophysiological conditions. It is known limitations that may affect the accuracy of the outcome.21 that ROS is involved in a wide range of cellular processes,46–49 Our findings showed that UA may modulate a number of including host defence, inflammation, cellular signaling, gene functional proteins and related signaling pathways. These expression, cellular death, cellular senescence, regulation of proteins and signaling pathways have important roles in the cell death, O2 sensing, biosynthesis, protein cross-linking, regulation of redox homeostasis, cell proliferation, apopto- regulation of cellular redox potential, reduction of metal ions, sis, energy metabolism, xenobiotics metabolism, lipid and regulation of matrix metalloproteinases, angiogenesis, and carbohydrate metabolism, and inflammatory response. cross-link with the nitric oxide system. This role suggests Oxidative stress plays a causal role in the development that NOXs should be considered a potential pharmacological of liver fibrosis.11,35,36,42 It is caused by an increase in the gen- target for antifibrotic therapies.26,50 eration of pro-oxidant molecules and/or a decrease in the To date, seven members of the NOX family have been antioxidants in a given cellular compartment, in which the distinguished on the basis of the membrane spanning cata- pro-oxidants overweigh the capability of antioxidant systems, lytic subunits of NOX or DUOX that they utilize to transfer

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Figure 10 UA inhibits p38 MAPK and PI3K/Akt signaling pathways in HSC-T6 cells. Notes: Cells were pretreated with UA (50 µM), DPI (20 µM), SB203580 (10 µM), or LY294002 (10 µM) for 30 minutes and then stimulated with leptin (100 ng/mL) for 30 minutes. The level of p-p38 MAPK, p38 MAPK, PI3K, p-Akt, and Akt was examined using Western blotting assay. (A) Representative blots of p-p38 MAPK, p38 MAPK, PI3K, p-Akt, and Akt in HSC-T6 cells. (B) Bar graphs showing the relative level of p-p38 MAPK, p38 MAPK, PI3K, p-Akt, and Akt in HSC-T6 cells. Data are expressed as mean ± SD from six independent experiments. β-Actin acts as an internal control. *P,0.05; ***P,0.001. + and - represent with or without treatment, respectively. Abbreviations: UA, ursolic acid; p38 MAPK, p38 mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; HSC, hepatic stellate cell; DPI, diphenyleneiodonium; SD, standard deviation.

4100 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats

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Figure 12 Effect of UA on the mRNA expression of collagen I in HSC-T6 cells. Notes: Cells were pretreated with UA (50 μM), DPI (20 μM), AG490 (50 μM), or PD98059 (30 μM) for 30 minutes and then stimulated with leptin (100 ng/mL) for another 24 hours. The mRNA expression level of type I collagen I was examined using PCR assays. (A) Representative blots of type I collagen I in HSC-T6 cells. (B) Bar graph showing the relative level of type I collagen I in HSC-T6 cells. Data are expressed as mean ± SD from six independent experiments. β-Actin acts as an internal control. *P,0.05; **P,0.01. + and - represent with or without treatment, respectively. Abbreviations: UA, ursolic acid; HSC, hepatic stellate cell; DPI, diphenyleneiodonium; PCR, polymerase chain reaction; SD, standard deviation.

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62 electrons from NADPH to O2 and produce ROS. They are the phagocytosis of apoptotic bodies by HSCs. The level NOX1, 2, 3, 4, 5 as well as DUOX1- and 2-containing of ROS in cells and tissues is controlled by the tissue- and oxidases.46,51–54 The core structure of all NOX isoforms stimulus-specific expressions of NOX protein and their regula- consists of six conserved transmembrane domains and a tory subunits and by the acute regulation via calcium, protein cytosolic C terminal. Increased activity and expression of phosphorylation, guanine nucleotide exchange on Rac, and the NOX isoforms has been demonstrated in a wide variety of assembly of regulatory subunits.54 Our results demonstrated diseases and/or disorders, which has been marked by the that leptin induced an increase in the expression level of the excessive NOX-generated ROS resulted from upregula- NOX4 regulatory subunits, including gp91phox, p22phox, p67phox, tion of activity and/or expression of various NOX family and Rac1 in HSC-T6 cells and that pretreatment with UA members.55–57 In the last decade, extensive attention has blocked the leptin-induced increase in the expression level of been focused on the redox control and the underlying gp91phox, p22phox, p67phox, and Rac1. Furthermore, pretreatment molecular mechanisms of NOX-dependent ROS generation. of UA inhibited the translocation of p47phox from cytoplasm to In addition, the regulation on NOX isoforms has also been cell membrane. The negative regulatory effect of UA on the studied.55–57 It has been shown that the regulation of NOX expression of NOX4 subunits explains the reduction of ROS isoforms varies from transcriptional level to posttranslational generation in HSC-T6 cells and rats. Consequently, the UA- level, although the underlying mechanisms of NOX regula- attenuated oxidative stress leads to a reversal of liver fibrosis tion have not been fully revealed.55–57 and protection of HSC-T6 cells, evident from the decrease in NOX4, like the other NOX isoforms, its structure consists the accumulation of type I collagen in HSCs and the expression mainly of a six-transmembrane domain known as the gp9phox of TIMP-1 and type I collagen at transcriptional or translational domain; NOX4 predominantly generates hydrogen peroxide rather level, but increase the level of MMP-1. than superoxide, although it is able to generate superoxide under Furthermore, increasing evidence shows that post- specific conditions.35,42,58 Despite their extensive similarity in struc- translational modification of NOX represents an important ture and enzymatic function, members of the NOX family differ in mechanism for NOX regulation. The phosphorylation of their mechanism of activation. In particular, NOX4 requires inter- NOX subunits and their assembly into active complexes are action with a second membrane-bound subunit, p22phox.54,59 NOX4 mediated by various mechanisms, of which many of them are is a multiprotein complex that generates ROS in both phagocytic redox sensitive. It has been demonstrated that a number of and nonphagocytic cells, regulating intracellular signaling.11,36 stimuli and pathways are involved in the activation of NOX In the liver, NOX4 plays a central role in fibrogenesis.11 by phosphorylation. They comprise phospholipases (PLC/γ, A functionally active form of NOX4 is expressed in HSCs, and PLD), arachidonic acid metabolites, GTP-binding proteins the ROS derived from NOX4 act as a second messenger for pro- (Ras, Rac1/2), PKC, PI3K, MAPK, and nonreceptor protein fibrogenic factor signal transduction in HSCs. HSCs activation tyrosine kinases.63,64 Previous studies showed that NOX- is a critical event in the occurrence and development of fibrosis, mediated ROS generation through the activation of Akt and because activated HSCs are the primary source of extracellular MAPK phosphorylation, leading to an increase in AP-1-DNA- matrix in liver upon injury.2 This role suggests that NOX4 may binding activity, promotion in the expression of type I col- act as a potential therapeutic target for antifibrotic therapies.26,50 lagen, TGF-β1, and other inflammatory cytokines26 and that The NOX subunits and regulatory protein are essential NOX-mediated ROS production interplays with p38 MAPK50 for the superoxide generating function and activity of NOX and JAK1/2-STAT3/ERK signaling pathways in HSCs.65 In isoforms. The subunits and regulatory proteins required for agreement with previous studies, the data clearly show that NOX4 activation include membrane-bound and cytosolic UA modulates the NOX activity and expression with the proteins – p22phox, which is a membrane-bound protein help- involvement of ERK, PI3K/Akt, and p38 MAPK signaling ing to stabilize the NOX proteins and dock cytosolic factors, pathways in HSCs via the employment of specific chemical and p47phox, p67phox, the small GTPase Rac, and the modula- inhibitors. In aggregate, it suggests that targeting NOX4 repre- tory p40phox, which are cytosolic proteins working together to sents a promising strategy for liver fibrosis treatment. Notably, activate the NOX enzymes.46,47,60 Rac1, a member of the Rho recent studies have indicated that the NOX1/4 dual inhibitor family of small GTPase proteins, regulates the activation of GKT137831 can significantly inhibit the activation of HSCs NOX4 and the production of ROS and plays an important and the development of liver fibrosis.12,66–68 This inhibitor is role in regulating NOX activity.59 Over-expression of Rac1 in currently being investigated in Phase II clinical trials. There- HSCs promotes liver injury and fibrosis in mice,61 and induces fore, NOX-targeting drugs with low toxicity that are effective

4102 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2015:9 Dovepress Dovepress UA reverses liver fibrosis in rats against fibrosis are expected to lead to a breakthrough in the 12. Cohen-Naftaly M, Friedman SL. Current status of novel antifibrotic therapies in patients with chronic liver disease. Therap Adv Gastroen- treatment of liver fibrosis. However, there are many factors terol. 2011;4(6):391–417. that may affect the therapeutic outcome of antifibrotic agents 13. Rosenbloom J, Mendoza FA, Jimenez SA. Strategies for anti-fibrotic in the treatment of liver fibrosis.69 Thus, more well-designed therapies. Biochim Biophys Acta. 2013;1832(7):1088–1103. 14. Dufour D, Pichette A, Mshvildadze V, et al. Antioxidant, anti- studies are needed to investigate the therapeutic effect and inflammatory and anticancer activities of methanolic extracts from underlying mechanism of the antifibrotic effect. Ledum groenlandicum Retzius. J Ethnopharmacol. 2007;111(1): 22–28. In summary, the present study has depicted a full spectrum 15. Saravanan R, Viswanathan P, Pugalendi KV. Protective effect of ursolic of molecular targets and related signaling pathways that pos- acid on ethanol-mediated experimental liver damage in rats. Life Sci. sibly respond to UA treatment. The benchmarking results have 2006;78(7):713–718. 16. Saraswat B, Visen PK, Agarwal DP. Ursolic acid isolated from clearly shown that UA exhibits a potent antifibrotic effect in Eucalyptus tereticornis protects against ethanol toxicity in isolated rat rats evident from the reduction in the accumulation of type I hepatocytes. Phytother Res. 2000;14(3):163–166. 17. Shen YM, Zhu X, Zhang KH, et al. [Effect of ursolic acid on prolifera- collagen in rat HSCs. The underlying molecular mechanism tion and apoptosis of hepatic stellate cells in vitro]. Zhonghua Gan Zang and beneficial effect of UA can be ascribed to the oxidative Bing Za Zhi. 2008;16(4):298–301. stress attenuating effect through negative regulation of NOX4 18. Steinkamp-Fenske K, Bollinger L, Völler N, et al. Ursolic acid from the Chinese herb danshen (Salvia miltiorrhiza L.) upregulates eNOS activity and expression with the involvement of ERK, PI3K/ and downregulates Nox4 expression in human endothelial cells. Akt, and p38 MAPK signaling pathways in HSCs. These Atherosclerosis. 2007;195(1):e104–e111. 19. Huang da W, Shermann BT, Lempicki RA. Systematic and integrative results render UA as a promising antifibrotic agent via target- analysis of large gene lists using DAVID bioinformatics resources. ing NOX4, and further studies are warranted to evaluate the Nat Protoc. 2009;4(1):44–57. safety and efficacy of UA in the treatment of liver fibrosis. 20. Luo H, Chen J, Shi L, et al. 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