Novel Noncatalytic Substrate-Selective p38α -Specific MAPK Inhibitors with Endothelial-Stabilizing and Anti-Inflammatory Activity This information is current as of September 26, 2021. Nirav G. Shah, Mohan E. Tulapurkar, Aparna Ramarathnam, Amanda Brophy, Ramon Martinez III, Kellie Hom, Theresa Hodges, Ramin Samadani, Ishwar S. Singh, Alexander D. MacKerell, Jr., Paul Shapiro and Jeffrey D. Hasday

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Novel Noncatalytic Substrate-Selective p38a-Specific MAPK Inhibitors with Endothelial-Stabilizing and Anti-Inflammatory Activity

Nirav G. Shah,*,1 Mohan E. Tulapurkar,*,1 Aparna Ramarathnam,* Amanda Brophy,† Ramon Martinez, III,† Kellie Hom,† Theresa Hodges,‡ Ramin Samadani,†,2 Ishwar S. Singh,* Alexander D. MacKerell, Jr.,†,x Paul Shapiro,†,2,3 and Jeffrey D. Hasday*,{,3

The p38 MAPK family is composed of four kinases of which p38a/MAPK14 is the major proinflammatory member. These kinases contribute to many inflammatory diseases, but the currently available p38 catalytic inhibitors (e.g., SB203580) are poorly effective and cause toxicity. We reasoned that the failure of catalytic p38 inhibitors may derive from their activity against noninflammatory p38 isoforms (e.g., p38b/MAPK11) and loss of all p38a-dependent responses, including anti-inflammatory, counterregulatory Downloaded from responses via mitogen- and stress-activated kinase (MSK) 1/2 and Smad3. We used computer-aided drug design to target small molecules to a pocket near the p38a glutamate–aspartate (ED) substrate-docking site rather than the catalytic site, the sequence of which had only modest homology among p38 isoforms. We identified a lead compound, UM101, that was at least as effective as SB203580 in stabilizing endothelial barrier function, reducing inflammation, and mitigating LPS-induced mouse lung injury. Differential scanning fluorimetry and saturation transfer difference–nuclear magnetic resonance demonstrated specific binding of UM101 to the computer-aided drug design–targeted pockets in p38a but not p38b. RNA sequencing analysis of TNF-a–stimulated http://www.jimmunol.org/ expression revealed that UM101 inhibited only 28 of 61 SB203580-inhibited and 7 of 15 SB203580-inhibited tran- scription factors, but spared the anti-inflammatory MSK1/2 pathway. We provide proof of principle that small molecules that target the ED substrate-docking site may exert anti-inflammatory effects similar to the catalytic p38 inhibitors, but their isoform specificity and substrate selectivity may confer inherent advantages over catalytic inhibitors for treating inflammatory diseases. The Journal of Immunology, 2017, 198: 3296–3306.

he p38 MAPK family of stress- and cytokine-activated toxicity and lack of efficacy. Of the 36 phase II clinical trials

kinases contribute to the pathogenesis of many human of p38 inhibitors listed on ClinicalTrials.gov (https://www. by guest on September 26, 2021 T diseases, including cancer (1), rheumatoid arthritis (2), clinicaltrials.gov), the results of only eight studies have been cardiovascular disease (3), multiple sclerosis (4), inflammatory published or listed on this site and showed little clinical benefit bowel disease (5), chronic obstructive pulmonary disease and (10–13) and/or moderate toxicity (12). asthma (6), and acute lung injury (ALI) (7). Among the many All available p38 inhibitors block catalytic activity either by important biological processes regulated by p38 MAPKs, regulation directly competing for ATP binding or by allosterically causing of endothelial and epithelial barrier function (8), leukocyte traf- conformational changes that preclude access of ATP to the catalytic ficking (9), and cytokine expression (2) are central to the patho- site (14). Davidson et al. (15) identified a purported p38a substrate- genesis of acute and chronic inflammatory disorders. Although selective inhibitor, CMPD1, which selectively inhibited MAPK- preclinical studies strongly support the pharmacologic targeting of activated kinase-2 (MK2) in in vitro p38 as treatment for inflammatory diseases, p38 inhibitors have had kinase assays, but CMPD1 bound near the p38a and very limited success in clinical testing because of dose-limiting was subsequently shown to lack substrate-selectivity when tested

*Division of Pulmonary and Critical Care Medicine, Department of Medicine, Uni- The sequences presented in this article have been deposited in the versity of Maryland School of Medicine, Baltimore, MD 21201; †Department of Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93330) Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, under accession number GSE93330. MD 21201; ‡University of Maryland Institute for Genome Science, Baltimore, MD x Address correspondence and reprint requests to Dr. Jeffrey D. Hasday, Division of 21201; Computer-Aided Drug Design Center, Department of Pharmaceutical Sci- Pulmonary and Critical Care Medicine, University of Maryland School of Medicine, ences, University of Maryland School of Pharmacy, Baltimore, MD 21201; and { 110 S. Paca Street, Room 2N149, Baltimore, MD 21201. E-mail address: Medicine and Research Services, Baltimore Veterans Administration Medical Cen- [email protected] ter, Baltimore, MD 21201 The online version of this article contains supplemental material. 1N.G.S. and M.E.T. contributed equally to this work. Abbreviations used in this article: ALI, acute lung injury; ATF, activating 2Current address: Medimmune, Gaithersburg, MD. factor; CADD, computer-aided drug design; CD, common docking; CRP, C-reactive 3P.S. and J.D.H. jointly supervised this study. protein; DSF, differential scanning fluorimetry; DUSP2, dual-specificity protein phosphatase-2; ED, glutamate–aspartate; HMVECL, human lung microvascular ORCIDs: 0000-0002-6030-4992 (K.H.); 0000-0003-4976-8793 (R.S.); 0000-0001- endothelial cell; i.t., intratracheal; MANOVA, multivariate ANOVA; MK2, 8287-6804 (A.D.M.). MAPK-activated protein kinase-2; MSK, mitogen- and stress-activated kinase; Received for publication December 8, 2016. Accepted for publication February 6, RNASeq, RNA sequencing; STD-NMR, saturation transfer difference–nuclear 2017. magnetic resonance; TEM, transendothelial migration; UM60, N2,N7-di(2-hydroxyethyl)- 9-oxo-9H-2,7-fluorenedisulfonamide; UM101, 4-chloro-N-(4-[(1,1-dioxo-1l∼6∼,4- This work was supported by National Heart, Lung, and Blood Institute Grant thiazinan-4-yl)methyl]phenyl)benzamide. R01HL69057 (to J.D.H. and P.S.) and U.S. Department of Veterans Affairs Grant BX002143 (to J.D.H.). Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602059 The Journal of Immunology 3297 in cells (16). Almost all available inhibitors are active against Fisher Scientific (PV3317), SignalChem (Richmond, BC, Canada) (S52- both p38a and p38b (17), and some are active against additional 50G), and Origene (Rockville, MD) (TP710017), respectively. p38 isoforms. Yet genetic and pharmacologic studies have CADD identification of lead compounds identified p38a as the proinflammatory isoform (18, 19), whereas other studies have demonstrated p38b signaling to be cytoprotective Based on the x-ray crystal structure of mouse p38a (PDB ID: 1P38), we used a step-wise iterative CADD process to screen an in silico database of (20, 21). Therefore, inhibition of p38b maycontributetobothlack small molecule compounds available from Maybridge Chemical Screening of efficacy and toxicity of non–isoform-selective p38 inhibitors. Collection for the potential to bind in a pocket near the ED substrate However, the extensive structural conservation of the catalytic module (Fig. 1A, 1B) using the same methods as were used to identify across most protein kinases presents a challenge to developing cata- novel ERK inhibitors (30). In silico preparation of p38a conformation was lytic inhibitors with high selectivity, especially for individual p38 performed using CHARMM36 protein and CHARMM general force field (31, 32) with the Nanoscale Molecular Dynamics program (33), to identify isoforms (17). local potential ligand-binding pockets (34). Protein structures were sub- Even if the catalytic inhibitors were absolutely selective for jected to clustering (35) to identify 20 representative protein conformations p38a, by design these compounds would block all p38a signaling to account for protein flexibility. Screening was performed in the following events, many of which are essential for reestablishing and main- stages: 1) potential inhibitor binding sites were identified (36); 2) com- pounds were ranked based on their van der Waals and electrostatic inter- taining homeostasis. For example, p38a not only activates expression action energies with the protein binding pockets using the program DOCK of proinflammatory cytokines, it also activates anti-inflammatory cy- (37–39) with size-based score normalization (40); 3) the top 50,000 tokines and counterregulatory dual-specificity protein phosphatase-2 compounds were subjected to a second in silico screen with additional (DUSP2) through the p38a substrate, mitogen- and stress-activated relaxation of the ligands during simulated binding and the top 1000 compounds were selected based on total interaction energy including score kinase (MSK) 1/2 (22, 23). The transient decrease and subsequent Downloaded from normalization based on ligand size; and 4) chemical fingerprint-based rebound of serum C-reactive protein (CRP) levels seen in clinical cluster analysis (41, 42) of the top scoring compounds using the pro- trials of p38 catalytic inhibitors (12, 13, 24) might be caused by the gram MOE (Chemical Computing Group) was performed to identify loss of the MSK1/2-dependent anti-inflammatory signaling. chemically diverse compounds and the final list of potential p38a-interacting As an alternative to the catalytic inhibitors, we targeted the compounds were selected based on a scalar bioavailability metric, 4DBA (43), which accounts for the physiochemical descriptors in Lipinski’s Rule of substrate binding groove of p38a, which stretches between two Five (44) (Fig. 1D). acidic patches, the common docking (CD) and glutamate–aspartate http://www.jimmunol.org/ (ED) domains (25, 26), and is distinct from the DEF substrate- Differential scanning fluorimetry binding pocket (27). Downstream substrates, upstream activating Binding of CADD-selected compounds to p38a and b isoforms was tested kinases, and possibly scaffolding molecules all interact with p38 experimentally using differential scanning fluorimetry (DSF), which through these sites (25). We used computer-aided drug design evaluates changes in the target protein melting temperature (ΔTm) due to (CADD) to target low m.w. compounds to a pocket near the p38a interactions with the test compound (45). SYPRO orange (Thermo Fisher Scientific) diluted 1:1000 in 10 mM HEPES, 150 mM NaCl (pH 7.5), and ED substrate binding site, which binds MK2 (28), a p38a substrate 1 mM unphosphorylated recombinant human p38a were added to 96-well known to mediate endothelial permeability and neutrophil trans- PCR plates. Then 50 nM to 200 mM test compound dissolved in 100% endothelial migration (TEM) in vitro and pulmonary edema in a DMSO (2% final DMSO concentration) was added, the plates were mixed, mouse lung injury model (7), whereas anti-inflammatory MSK1/2 sealed, centrifuged at 1000 rpm for 1 min, and melting curves were per- by guest on September 26, 2021 appears to bind to the CD site (26). Using this algorithm, we formed using an Applied Biosystems StepOne real-time PCR instrument. The melting point was determined from the first derivative curve. identified p38a-binding compounds with high efficiency, including a lead compound, 4-chloro-N-(4-[(1,1-dioxo-1l∼6∼,4-thiazinan-4-yl) Cell culture methyl]phenyl)benzamide (UM101), that selectively bound p38a HMVECLs were purchased from Promocell, Heidelberg, DE, maintained in and not p38b, stabilized endothelial barrier function in human Endothelial Cell Growth Medium MV2, used at passage 3–10, and studied lung microvascular endothelial cells (HMVECLs), inhibited LPS- at postconfluence according to the supplier’s protocol as we have previ- induced proinflammatory gene expression in THP1 cells, and was ously described (8, 9, 46, 47). The THP1 human monocyte cell line well tolerated and more potent than SB203580 in mitigating ex- (American Type Culture Collection no. TIB202) was maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium py- perimental ALI. ruvate, 10 mM HEPES buffer (pH 7.3), penicillin, streptomycin, 0.05 mM b-mercaptoethanol, and 10% defined FBS (Life Technologies, Grand Island, NY). HeLa cells (American Type Culture Collection no. CCL-2) Materials and Methods were cultured in DMEM with 4.5 g/l glucose, 1 mM sodium pyruvate, Chemicals, recombinant , and Abs 2mML-glutamine, penicillin, streptomycin, and 10% FBS. Prior to ex- Mouse anti-human p38a and rabbit anti-phospho-MK2 (T222) and perimental exposure, THP1 cells were differentiated by treating with 5 ng/ml phospho-STAT-1 (S727) were obtained from Cell Signaling Tehcnologies PMA (Sigma-Aldrich) for 24 h, washing with PBS, and culturing at 37˚C in (Danvers, MA). The coding sequences for human p38a variant 2 and p38b PMA-free media for an additional 24 h. (with N-terminal hemagglutinin tag) were amplified by PCR and cloned Endothelial permeability assay into pRSetA (Thermo Fisher Scientific, Waltham, MA). were introduced into p38a using Quikchange (Stratagene) and confirmed by Permeability of HMVECL monolayers was assessed by measuring bidirectional sequencing. To generate activated dual-phosphorylated p38a transendothelial flux of 10 kDa dextran conjugated to Cascade blue for in vitro kinase assays, p38a variant 2 was amplified by PCR and cloned fluorescent dye for 30 min at 37˚C in Matrigel-coated 3 mm pore size into the first multicloning site of pETDuet (EMD-Millipore) in-frame with Transwell plates as we have described (8). an N-terminal His tag sequence. A gene block containing the optimized sequence for the constitutively active human MKK6 S207G/T211G mutant TEM assay (29) was synthesized (Genscript, Piscataway, NJ) and cloned into the second multicloning site of pETDuet-p38a. Plasmids were transformed in Isolation of neutrophils from heparinized venous blood collected from BL21 and proteins were purified using cobalt columns healthy volunteers, calcein labeling, and measurement of IL-8–directed (TALON; Clontech Laboratories, Mountain View, CA), and confirmed by TEM through HMVECLs was measured as previously described (9, 48, SDS-PAGE and immunoblotting. The p38a protein expressed from the 49). The study was approved by the University of Maryland Baltimore pETDuet plasmid was confirmed to be .80% dual-phosphorylated by Institutional Review Board. MALDI-TOF in the University of Maryland School of Pharmacy Proteomics Mouse acute lung injury model Core. The compounds identified in the CADD screen were purchased from Maybridge Chemical (Belgium). Recombinant MK2, STAT-1a, and acti- Male CD-1 mice weighing 25–30 g were purchased from Charles River and vating (ATF) 2 protein were purchased from Thermo housed in the Baltimore Veterans Administration Medical Center Animal 3298 COMPUTER-AIDED DESIGN OF SUBSTRATE-SELECTIVE p38 INHIBITORS

Care Facility under American Association of Laboratory Animal Care–approved contained 150 mM NaCl, 50 mM phosphate (pH 7), 200 mM UM101, and conditions. All protocols were approved by the University of Maryland 5 mM p38 protein in D2O. Spectra were recorded on an Agilent DD2 Baltimore Institutional Animal Care and Use Committee. Inhibitors were 500-MHz spectrometer equipped with a 5-mm inverse proton–fluorine– tested in a mouse intratracheal (i.t.) LPS/febrile-range hyperthermia– carbon–nitrogen probe head at 27˚C. During each transient, the protein was induced ALI model as we have previously described (50). Mice were pre- saturated with a series of 58 GAUSSIAN-shaped pulses (50 ms with 1 ms treated with SB203580 or putative p38 inhibitors in #2%DMSO via 0.5 ml delay between pulses) using the vendor-supplied STD–excitation-sculpting i.p.injection1hpriortoi.t.instillationof50mg LPS and switch to a 37˚C pulse sequence, for a total saturation time of 3 s. The on-resonance irra- incubator, which increases core temperature to ∼39.5˚C. The dose of diation of protein was done at 0.5 ppm and off-resonance irradiation at SB203580 used, 1 mg (40 mg/kg body weight), was previously shown to be 30 ppm. The vendor-supplied WATERGATE pulse sequence was used to maximally effective in animal models of septic shock (51). Mice were eu- suppress the water signal in the STD spectrum. The on-resonance and off- thanized after 24 h, the lungs were lavaged with a total of 2 ml PBS, the cells resonance pulse sequences were subtracted internally. A total of 16,384 were counted, and the cell-free lavage fluid was analyzed for protein content transients were collected for each STD experiment with a 1 s delay be- using the Bradford method (Bio-Rad), as previously described (50). tween acquisitions, 6000 Hz spectral width, and 1.3 s acquisition time. Inhibition of substrate phosphorylation Statistical methods A functional analysis of UM101 to block p38-dependent phosphorylation of Data are presented as mean 6 SE. Differences among .2 groups were MK2 and STAT-1 was performed in HeLa cells. The cells were pretreated analyzed by applying a Tukey Honestly Significant Difference test to a with SB203580 or UM101 for 30 min and then activated with 25 mg/ml one-way ANOVA. Differences between dose-response curves was ana- anisomycin for 10–60 min. Cell were lysed in radioimmunoprecipitation lyzed by multivariate ANOVA (MANOVA). Differences with p , 0.05 buffer containing protease and phosphatase inhibitors and lysates were were considered significant. resolved by SDS-PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% nonfat dry milk, and probed with primary Abs against Results Downloaded from phosphorylated MK2 and STAT-1, and total p38a as a loading control. Bands were detected using secondary Abs conjugated to infrared fluorophores CADD modeling of p38 MAPK substrate-docking site and and infrared fluorescence imaging (Odyssey; LI-COR) and the data were compound identification quantified using the LI-COR image analysis software. The inhibitor effect on p38a phosphorylation of specific substrates was further analyzed by in vitro We used a CADD-based strategy to identify low m.w. compounds kinase assay in 20 ml reaction volume containing 75 ng of active p38a predicted to bind near the ED substrate-docking site of mouse (expressed from pETDuet), substrate proteins (100–150 ng), 5 mM SB203580 unphosphorylated p38a (MAPK14 variant-1; PDB:1P38), which 32 or 10–50 mMUM101,and5mCi of ATP [g- P] in kinase buffer [50 mM is .99% identical to human p38a (variant-2) (Fig. 1A). The ED http://www.jimmunol.org/ Tris-HCl, 10 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35, (pH and CD sites in p38a are located at either end of a substrate- 7.5)]. Reactions were performed at 37˚C for 10–120 min, then terminated by adding and equal volume of 23 SDS-PAGE sample buffer. The reaction binding cleft located on the opposite side of the protein from mixtures were separated by SDS-PAGE. The gels were dried and the the catalytic site (Fig. 1A). We identified a pocket near the ED radioactive phosphate incorporation into substrates was measured by binding site comprising 10 aa, only seven of which were identical phosphorimaging on a Typhoon FLA 7000 (GE Healthcare Life Sciences). in p38a and p38b (Fig. 1B). Overlay of structures of mouse The data were quantified using ImageQuant TL (GE Healthcare Life Sci- ences) and expressed as relative values. unphosphorylated (PDB:1P38) and dual-phosphorylated p38a (PDB:3PY3) revealed near-superposition of the targeted pocket in Cytotoxicity assay the two forms (Fig. 1C). by guest on September 26, 2021 Cytotoxicity was monitored in parallel HMVEC-L monolayers established An overview of the CADD screening and compound testing in 96-well culture plates using a colorimetric assay that measured the re- protocols is shown in Fig. 1D. The compounds in the Maybridge duction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- Screening Collection were analyzed for binding to the targeted (4-sulfophenyl)-2H-tetrazolium to a formazan dye (CellTiter 96; Promega, p38a pocket based on van der Waals and electrostatic interaction Madison, WI) according to the manufacturer’s protocol and quantifying product formation by measuring absorption at 490 nm. energies (40), chemical diversity by chemical fingerprint-based cluster analysis (41, 42), solubility, m.w., and number of hydrogen- Gene expression bonding functional groups that maximize bioavailability (43). A RNA integrity was confirmed by Agilent Bioanalyzer 2100 and all samples panel of 150 diverse compounds was selected for potential biological were confirmed to have RNA integrity scores of 10 prior to further analysis. testing; 20 of these structurally dissimilar compounds were obtained Poly(A)-enriched samples were reverse transcribed and sequenced using the for functional analysis (Supplemental Fig. 1, Table I). Illumina HiSeq platform to generate at least 90 million reads per sample. Intergenic sequence accounted for ,0.7% of all reads, indicating minimal Screening compounds for direct, selective interaction with genomic DNA contamination. Raw data were analyzed using the TopHat p38a read alignment tool and Homo sapiens genomic reference sequence (Ensembl version GRCh38.78). Differential gene expression was analyzed Test compounds at 10–100 mM were screened for binding to using the DESeq R package (Bioconductor) and the negative binomial recombinant p38a and ERK2 using DSF (45) (Fig. 1E, Table I). model. Criteria for significant differences in gene expression were: 1) false Five compounds caused concentration-dependent stabilization , . discovery rate 0.05; 2) expression level 10th percentile; and 3) $2-fold of p38a, indicating binding. Three of these also stabilized change. The differential gene expression patterns were further analyzed using the PathwayNet (Troyanskaya Lab, Princeton) and Ingenuity Path- ERK2 (3, 5, and 141, highlighted yellow in Fig. 1E) and two, N2,N7- way Analysis (Qiagen) tools. Cytokine gene expression in THP1 cells was di(2-hydroxyethyl)-9-oxo-9H-2,7-fluorenedisulfonamide (UM60) and analyzed by quantitative RT-PCR using primers in a commercially avail- UM101, highlighted blue, stabilized p38a but not ERK2. These two able PCR array (HCA-II array; Real Time Primers, Elkins Park, PA) and structurally dissimilar compounds (Fig. 1F), added at 100 mM, in- SYBR-green reaction mix (Bio-Rad), and a Bio-Rad iCycler IQ Optical ∼ Module according to the supplier’s protocol. Data were quantified using creased the melting temperature of p38a by 0.7˚C compared with a the gene expression Ct difference method (52) and standardized to the 6˚C increase with SB203580. levels of the housekeeping gene, GAPDH, using Ct values automatically determined by the thermocycler as we have previously described (53). The Effects of compounds on endothelial barrier functions RNA sequencing (RNASeq) data have been deposited in the Gene Ex- We tested the capacity of UM60 and UM101 to stabilize endo- pression Omnibus database (accession number GSE93330; https://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93330). thelial barriers to macromolecules and neutrophils in TNF-a– and hyperthermia-stressed HMVECL monolayers (8, 9) (Fig. 2). Combined Saturation transfer difference–nuclear magnetic resonance exposure to 1 ng/ml TNF-a and hyperthermia (39.5˚C) for 6 h in- A 40 mM stock solution of UM101 was made in d6-DMSO. Saturation creased HMVECL permeability for 10 kDa dextran 2.8-fold compared transfer difference–nuclear magnetic resonance (STD-NMR) samples with untreated 37˚C cells. Pretreating with 10 mM SB203580 for The Journal of Immunology 3299 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Design of substrate-selective p38 inhibitors. (A) Structure of p38a showing CD, ED, DEF, and activation site. (B) Comparison of p38a and b structure. CD and ED sites colored red and blue and the CADD target yellow. The sequence comprising the CADD target on p38a and the corresponding site on p38b differ in only 3 of 10 aa (highlighted yellow). (C) Overlap of CADD target structure in apo- (PDB:1P38; green) and dual-phosphorylated (PDB:3PY3; yellow) mouse p38a.(D) Overview of CADD screening strategy. (E) DSF screening of compounds added at 10, 25, 50, or 100 mMto recombinant p38a or ERK2 with binding indicated by increase in melting temperature. Compounds binding ERK2 and p38 a highlighted yellow. Those only binding to p38 a are highlighted in blue. (F) Chemical structure of UM60, UM101, and SB203580.

30 min reduced TNF-a–/hyperthermia-induced permeability by 50% 2-(4-sulfophenyl)-2H-tetrazolium assays when added to HMVECLs (Fig. 2A). Pretreatment with UM60 at 10 and 25 mM had no effect on at 100 mM for 48 h (data not shown). permeability, but 100 mMUM60reducedtheTNF-a–/hyperthermia- induced permeability increase by 71%, whereas UM101 at 10, 25, and Comparing effectiveness of SB203580 and UM101 in mouse 100 mM reduced the TNF-a–/hyperthermia-induced permeability in- ALI crease by 74, 89, and .100%, respectively. We compared the effectiveness of UM60, UM101, and SB203580 Preincubating HMVECLs at 39.5˚C for 6 h increased subsequent in mitigating transalveolar protein and neutrophil extravasation in a IL-8–directed neutrophil TEM from 22.8 6 0.45 3 103 to 31.8 6 0.54 mouse model of LPS-/hyperthermia-induced ALI (50) (Fig. 2C, 3 103 neutrophils (Fig. 2B). Pretreatment with 10 mM SB203580 2D). Mice received a single i.p. injection of 100, 300, 500, or 1000 mg reduced hyperthermia-augmented neutrophil TEM by 84%. UM60 at UM101, 1000 mg UM60, or 1000 mg SB203580 in 0.5 ml 2% DMSO 10 and 25 mM and UM101 at 10 mM reduced hyperthermia- 30 min prior to i.t. instillation of 50 mg LPS and transfer to hyper- augmented increase in TEM by 18, 89, and 95%. UM60 at 50 mM thermic chambers. Control mice received DMSO. Four of six UM60- and UM101 at 25 and 50 mM reduced TEM to less than baseline treated, one of six SB203580-treated, and 1 of 11 DMSO-treated control levels. Neither compound was toxic in lactate dehydrogenase mice died within 24 h. All 16 UM101-pretreated mice survived. Lung release and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- lavage from DMSO-pretreated, LPS-/hyperthermia-challenged mice 3300 COMPUTER-AIDED DESIGN OF SUBSTRATE-SELECTIVE p38 INHIBITORS

Table I. CADD-identified p38a ED site-binding candidates screened for p38a binding

CADD no. Compound IDa m.w. logPb p38aDTm (˚C) at 100 mMc ERK2 DTm (˚C) at 100 mMc 2 SEW 06373 417 3.19 20.05 0.412 3 HTS 02798 415 0.67 0.282 0.337 4 HTS 13333 312 21.10 0.065 0.452 5 SCR 00846 418 2.22 0.808 0.628 8 AW 00509 317 1.13 20.07 0.531 13 SEW 06264 309 0.28 0.005 0.390 16 SCR 00610 339 1.69 20.052 0.444 23 SCR 01200 378 2.79 20.488 20.598 29 BTB 05645 350 3.07 20.353 0.342 31 KM 04113 304 1.83 20.278 0.153 43 CD 11992 300 1.16 20.485 0.151 55 SP 01164 2.11 1.92 20.506 0.022 60 BTB 13869 426 0.28 0.735 0.195 63 PD 00612 294 0.61 20.287 0.075 69 KM 00081 345 1.68 20.233 0.361 101 HTS 05732 378 2.31 0.667 0.0175 115 NRB 03986 278 3.88 20.156 0.246 141 SEW 02182 318 2.46 0.554 0.238 146 KM 10445 313 2.55 21.084 21.632 Downloaded from 150 HTS 03239 341 1.68 20.171 0.133 aCompound ID from Maybridge portfolio. blogP is the logarithm of the estimated octanol/water partition coefficient, a measure of compound solubility. cChange in melting temperature relative to DMSO control in DSF assay.

contained 1.09 6 0.19 mg/ml protein, and 3.97 6 1.07 3 106 Effect of SB203580 and UM101 on LPS-induced gene http://www.jimmunol.org/ neutrophils. Compared with DMSO-treated controls, lavage protein expression in human THP1 promonocytes concentration and neutrophil content in mice pretreated with 1000 mg The effects of UM101 and SB203580 on inflammatory cytokine SB203580 were reduced by 42 and 46.8%, respectively. Lavage expression were compared by pretreating PMA-differentiated protein concentration in mice pretreated with 100, 300, 500, and THP1 cells with 25 mM SB203580 or 10, 25, or 100 mMUM101 1000 mg UM101 was reduced by 0, 44.1, 43.9, and 92.9%, re- for 30 min, then stimulating with 100 ng/ml LPS, and harvesting spectively, and lavage neutrophil content was reduced by 44.4, RNA 4 h later for analysis by PCR-based cytokine array. Of 16 LPS- 49.5, 55.3, and 54%, respectively. stimulated genes in the array, SB203580 inhibited expression of by guest on September 26, 2021

FIGURE 2. Biological effects of p38 inhibitors. (A and B)Effectof10mM SB203580 (SB), or indicated concen- tration of UM60 or 101 on HMVECL permeability (A) and capacity for IL-8– directed neutrophil TEM (B). Cells were pretreated with DMSO or compounds for 1 h, then incubated with 10 ng/ml TNF-a for 6 h at 39.5˚C prior to per- meability assay (A) or at 39.5˚C without additional stimulus for 6 h prior to TEM assay (B). Mean 6 SE. *p , 0.0001 versus DMSO, †p , 0.0001 versus SB, ‡p , 0.005 versus 37˚C. (C and D) Male CD1 mice were pretreated with1mgSBor0.1–1mgUM101 prior to i.t. instillation of 50 mgLPS and hyperthermia exposure. *p , 0.05 versus DMSO. The Journal of Immunology 3301 seven: IL-1a, IL-8, TNFSF8 (CD30 ligand), TNFSF9 (CD137 ligand), CXCL5, CCL7, and CCL17 (Table II). UM101 inhibited expression of all SB203580-inhibited genes except TNFSF9, and c NS NS NS versus LPS 0.033 0.0001 0.0001 0.003 0.006 0.0001 0.0001 0.0001 p , , , , inhibited four SB203580-insensitive genes, IL-1b,CXCL1, , TNFSF15, and CCL5. Comparing effects of SB203580 and UM101 on TNF-a– induced gene expression in HMVECLs 5.2 3.33 0.5 1.0 1.8 0.015 0.6 9.5 0.025 1.2 51 0.2 12.7 NS 0.4 6.4 0.33 14.5 NS 1.12 0.086 M m 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 We compared the effects of UM101 and SB203580 on TNF-a– 6 UM101 100 80

induced gene expression in HMVECLs using RNASeq. We pre- 3.9 217 treated HMVECLs for 1 h with 10 mM SB203580 or 100 mM 130 UM101, and then stimulated with 10 ng/ml TNF-a for 3 h. We used a UM101 concentration 10-fold higher than its biologically effective dose in HMVECL barrier assays to ensure identifying NS NS 42.7 NS versus any partial overlap with SB203580. The TNF-a concentration and LPS 0.204 51.6 0.005 5.2 0.053 2.9 0.008 2.6 0.0001 3.1 p duration of stimulation used were based on published studies (54, , 55) and confirmed by preliminary quantitative RT-PCR analysis of IL-8 and IL-1b mRNA expression (data not shown). After filtering the RNASeq results for genes having $10 reads in at least one Downloaded from 9.0 13.5 0.041 88 2.5 4.1 0.564 19.7 0.8 3.9 0.006 20.7 0.2 2.9 57 1.0 0.9 0.0036 4 22.9 NS 1.0 0.0004 2.5 21.2 NS 6.9 0.334 32 1.1

sample per experiment, we found 511 genes that were upregulated M m 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 and 520 downregulated by $2-fold by TNF-a treatment 6 25 UM101 88 (Supplemental Table I). SB203580 inhibited expression of 61 7.8 TNF-a–induced genes, 28 of which were also inhibited by UM101 (Supplemental Fig. 2, Supplemental Table I, Table III). SB203580

increased expression of 38 genes, 10 of which were also increased http://www.jimmunol.org/ by UM101. Of the 28 genes inhibited by both SB203580 and c NS 191 NS 63.4 NS versus UM101, 22 coded for known proteins, including IL-1b, CCL17, LPS p MMP9, IDO1, CXCL5, 10 and 11, hyaluronan synthase-3, MUC4, and PLA2 (Table III). Of the 33 genes inhibited by SB203580 but not UM101, 24 coded for known proteins, including GM- CSF, IL-1a,TNF-a, IL-12 -b1, and hyaluronan 21 0.382 22.6 0.74 339 1.8 0.36 12.8 0.7 0.40 26.7 0.9 0.544 5.4 8.3 0.37 10.5 0.5 0.018 2.7 3.7 0.0002 8.7 41 3.1 0.3 0.0036 6.2 20.0 NS 132 4.7 0.008 11 12.4 NS 95.9 2.1 0.99 38.2 1.7 M m 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 synthase-2 (Table III). Quantitative RT-PCR confirmed the 6 3 10 UM101 63 RNASeq results for a subset of these genes and showed that 87 8.9 174 128 UM101, but not SB203580, increased expression of DUSP2 and by guest on September 26, 2021 10 (Supplemental Fig. 3). The differentially expressed genes were further analyzed using PathwayNet and Ingenuity tools to identify the transcription factors c and biological pathways regulated by the two inhibitors. PathwayNet NS NS NS NS versus LPS 0.0001 424 0.988 104 0.577 19.8 0.998 7.6 0.26 0.0001 23.2 0.0001 7.7 p , , analysis suggested that UM101 inhibits some of the SB203580- , inhibited transcription factors (STAT-1, c-Fos, c-Jun, NF-kB, , PPARg, and Sp1), but not others (ATF1, ATF2, Elk1, c/EBPb, USF1, SMAD3, FOXO1, and CREB via MSK1/2). Ingenuity 9.2 7.4 1.9 0.1 0.002 35.6 1.1 8.8 0.024 23.5 0.8 1.0 16 15.1 NS 0.3 4.6 3.7 0.001 30.4 9.6 3.1 0.0054 48 analysis suggested that both SB203580 and UM101 inhibit dendritic 1.1 M 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

cell maturation, triggering receptors expressed on myeloid cells-1, m high mobility group box 1, and NF-kB pathways and both in- SB20358025 crease liver X-receptor/retinoid X-receptor activation, whereas only SB203580 inhibits IL-6, acute phase, and cholecystokinin/gastrin- mediated pathways (Fig. 3A). UM101 at 100 mM reduced expres- sion of 115 genes and increased expression of 119 genes that were not b modified by SB203580 (Supplemental Table I), which ingenuity 0.0001 141 0.0089 187 0.0001 28.2 0.0012 10 0.0045 3.6 0.0001 3.2 0.9519 188 0.0001 4.2 0.0001 21.5 0.1195 4.1 ANOVA , , , , pathway analysis suggested reduced TLR and Wnt/b-catenin signal- , ing and increased NO in cardiovascular disease pathways (Fig. 3B). Comparing effects of SB203580 and UM101 on p38 MAPK

substrate phosphorylation profile a 24 9.0 0.5 3.3 0.0026 9.6 1.1 5.5 0.0073 20.6 0.9 2.9 12 27.8 0.1189 106 1.2 11.0 0.9887 125 6.1 13/1 0.6112 66.4 6.1 0.0046 20.7 1.8 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 To assess whether UM101 selectively inhibits phosphorylation 6 DMSO 7.6 8.1 453 171 188 consistent with its target, HeLa cells were pretreated for 30 min 115 with 10 mM SB203580, 50 mM UM101, or 0.1% DMSO vehicle control, then with the p38 activator, anisomycin (25 mg/ml), and phosphorylated MK2, and STAT-1 were analyzed by immuno- blotting (Fig. 3C). Anisomycin-stimulated phosphorylation of

MK2 and STAT-1 were reduced by both 10 mM SB203580 and Gene values from one-way ANOVA. values from the Tukey Honestly Significant Difference post hoc test. Cells were preincubated withp 0.4% DMSO or inhibitors for 1 h, then stimulated with 100 ng/ml LPS for 2 h. p IL-1A IL-1B CXCL1 24.5 IL-8 56.5 TNFSF15 9.6 TNFSF8 60.5 CCL5 CXCL5 49.7 CCL4 CCL20 82.5 CCL7 12.8 CXCL2 122 CCL17 56.9 TNF TNFSF9 50.8 BMP6

50 mM UM1010, but to a greater extent with SB203580. The All values area fold-change mRNA levelsb versus unstimulated PMA-differentiated THP1c cells.

in vivo phosphorylation studies were complemented by in vitro Table II. Effects of SB203580 and UM101 on LPS-induced cytokine expression in THP1 cells 3302 COMPUTER-AIDED DESIGN OF SUBSTRATE-SELECTIVE p38 INHIBITORS

Table III. Effect of SB203580 and UM101 in HMVECLs on TNFa-induced genes

Log Fold-Change Versus DMSO

Gene Symbol Gene Name SB203580 UM101 Genes inhibited by both SB203580 and UM101 PRRG4 rich Gla 4 21.782580534 21.198488233 TSLP Thymic stromal lymphopoietin 21.651439594 21.336652511 CCL17 Chemokine (C-C motif) ligand 17 21.834143455 22.730309773 EXOC3L4 Exocyst complex component 3-like 4 21.479163179 21.160471021 MMP9 Matrix metallopeptidase 9 21.157091348 21.091179627 IDO1 Ido 1 23.510632932 23.567987354 CXCL10 Chemokine (C-X-C motif) ligand 10 23.100915562 24.369836708 CD200 Cd200 21.729285649 21.538155406 SLC15A3 15, member 3 21.00338842 21.73105887 VDR 21.16718631 21.19731694 IL1B Il-1b 21.401586926 21.172530543 GPR88 G protein-coupled receptor 88 21.397083754 22.150599176 CD207 CD207 (langerin) 21.547757288 23.382437255 TCHH Trichohyalin 21.504958085 21.547665316 HAS3 Hyaluronan synthase 3 21.43377734 21.124339564

GBP1P1 Guanylate binding protein 1 21.363287203 21.755706078 Downloaded from MUC4 Mucin-4 22.859491876 21.057315692 ELOVL7 ELOVL fatty acid elongase 7 21.340933369 21.381063226 CXCL11 Chemokine (C-X-C motif) ligand 11 21.377905942 24.136354868 GBP4 Guanylate binding protein 4 21.259283076 22.835947907 PLA1A Phospholipase A1 member A 21.27452433 21.500633356 CXCL5 Chemokine (C-X-C motif) ligand 5 21.017427849 21.468307731 Genes inhibited by SB203580 but not UM101 CSF2 GM-CSF 21.634082807 NS http://www.jimmunol.org/ RND1 Rho family gtpase 1 21.15027449 NS SLC26A9 Solute carrier family 26, member 9 22.092734866 NS HAS2 Hyaluronan synthase 2 22.646197932 NS CD69 Cd69 21.467068659 NS ANKRD55 Ankyrin repeat domain 55 21.204412851 NS NKX3-1 NK3 1 21.105417452 NS EBI3 EBV induced 3 21.393947741 NS NKD2 Naked cuticle homolog 2 21.418158287 NS F2RL3 Coagulation factor II receptor-like 3 22.642662346 NS 2

IL1A IL-1alpha 2.152720278 NS by guest on September 26, 2021 TP63 Tumor protein 63 21.312154792 NS HAND1 Heart/neural crest derivatives expressed 1 21.617984328 NS IL12RB1 IL 12 receptor, b 1 21.409364824 NS ERVFRD-1 Endogenous group FRD, member 1 21.234925956 NS TMEM52B Transmembrane protein 52B 21.109879612 NS BPGM 2,3-bisphosphoglycerate mutase 21.204500786 NS TOX Thymocyte selection-associated HMGB 21.422546092 NS HPCA 22.219945733 NS TNF Tnf-a 21.478211598 NS FAM101A Family with sequence similarity 101, 21.712019627 NS member A OLR1 Oxidized low density lipoprotein receptor 1 21.013271327 NS HEPACAM Hepatic and glial cell adhesion molecule 21.211648309 NS P2RX2 Purinergic receptor P2X 22.244463614 NS HMVECL treated 1 h with 0.4% DMSO, 10 mM SB20350, or 100 mM UM101, then 4 h with 10 ng/ml TNF-a. kinase assays, comparing the effects of 5 mM SB203580 with We confirmed selective binding of UM101 to the CADD- 50 mM UM101 on phosphorylation of MK2, STAT-1a, and ATF2 targeted pocket in p38a using STD-NMR (Fig. 4C–I). A 1D by p38a (Fig. 3D, 3E). Whereas SB203580 abrogated phosphorylation spectrum of UM101 in the presence of p38a is shown in Fig. 4D of all three substrates, UM101 partially reduced phosphorylation of the and the STD spectrum of the same sample is shown in Fig. 4E. three substrates by varying degrees with the greatest effect on STAT-1a. The peaks in the 1D spectrum are labeled according to tentative peak assignments of UM101 in aqueous form, based on assign- a Analyzing specific binding of UM101 to p38 ments of UM101 in 2 mM d6-DMSO, which were obtained from We used DSF to analyze concentration-specific binding of UM101 the use of 1D proton and [13C] and two-dimensional hetero- to p38a and p38b. Whereas SB203580 stabilized both p38a and nuclear multiple bond correlation experiments (data not shown). p38b, UM101 only stabilized p38a (Fig. 4A). To confirm that The shifts of the peaks in the STD spectrum correspond well to UM101 bound the CADD-targeted pocket, we used DSF to those of the 1D spectrum, thus indicating that protons in both compare binding of UM101 and SB203580 to wild-type p38a and aromatic rings of UM101 interact with p38a. In contrast, al- a p38a mutant with substitutions of 4 of the 10 amino acids de- though the 1D spectra for UM101 with p38b and mutated p38a fining the CADD-targeted pocket (R49K/HL107-8TF/K165R) were similar to that of UM101/p38a (Fig. 4F, 4H), the interac- (Fig. 4B). The mutant exhibited SB203580-binding that was tion of UM101 with p38b and mutated p38a is much weaker, as identical to wild-type p38a, but no detectable UM101 binding. indicated by the barely discernible peaks of the aromatic protons The Journal of Immunology 3303 Downloaded from http://www.jimmunol.org/ FIGURE 3. Biochemical effects of substrate-selective p38 inhibitors. (A and B) Heat maps from RNASeq showing IPA pathways inhibited by SB203580 alone or SB203580 and UM101 or (A) and those only inhibited by UM101 (B). (C) HeLa cells were pretreated with 50 mM UM101 or 10 mM SB203580 (SB) for 30 min, then treated with anisomycin for 10–60 min, and immunoblotted for phospho-MK2, phospho-STAT-1, and total p38. The means of the band densities from two experiments expressed as percent of total density for all bands are shown below each corresponding band. (D and E) In vitro kinase reaction containing activated p38a, ATP [g-32P], 5 mM SB203580, or 50 mM UM101, and the indicated substrate were incubated at 37˚C for indicated time, separated by SDS-PAGE, and analyzed by phosphorimaging. A representative of three gels (D) and means 6 SE of the 120-min band densities expressed as fold-change versus the 5-min control reaction band are shown. *p , 0.0001, †p , 0.01 versus control.

in the STD spectrum of UM101 with p38b (Fig. 4G) and mutated We identified a lead compound that has favorable biological by guest on September 26, 2021 p38a (Fig. 4I). effects in human cell culture models and in a mouse model of inflammatory lung injury. The CADD-targeted pocket in p38a Discussion differed from the corresponding pocket in p38b in 3 of 10 aa, In targeting a pocket near the ED substrate–docking site of which provided an opportunity for p38a selectivity. We confirmed p38a, we theoretically avoided interfering with CD-specific selective binding of UM101 to p38a using complementary tech- substrates, including MSK1/2 (25, 26), which limits inflam- nologies. DSF, which detects ligand-induced protein stabilization mation through expression of IL-10 and DUSP2 (22). Among (45), showed UM101 to cause a concentration-dependent increase the effects of MSK1/2 in mice is increased and pro- in melting temperature of p38a but not p38b (Fig. 3D). The longed LPS-induced expression of the CRP regulator, IL-6, smaller effect of UM101 compared with SB203580 on p38a suggesting a possible mechanism of the rebound in serum melting suggests lower p38a binding affinity of substrate-selective CRP observed in some clinical trials of catalytic p38 inhibitors versus catalytic inhibitors, which is similar to our prior experience (12, 13, 24). with substrate-selective ERK inhibitors (30). The smaller effect of Mouse unphosphorylated p38a/MAPK14 variant-1 differs from SB203580 on p38b than p38a is consistent with the known ∼10- its human variant-2 by only 2 aa, H48L and A263T, and from fold higher binding affinity of SB203580 for p38a (58). STD- mouse variant-2 and human variant-1 by only 14 aa between NMR, which measures low-affinity protein:ligand binding via residues 230 and 254. Neither these differences nor the nonscalar magnetization transfer from protein to ligand protons phosphorylation state of p38a (Fig. 1C) are predicted to significantly (59), confirmed specific UM101 binding to p38a and localized the alter the structure of the CD or ED sites of our CADD targeted interaction to its aromatic rings. We indirectly confirmed that pocket, thereby validating our use of mouse unphosphorylated p38a variant-1 for the CADD search and unphosphorylated recombinant UM101 bound to its CADD target by showing that mutating 4 of human p38a variant-2 protein for the DSF screen. Although DSF is 10 aa in the targeted pocket abrogated UM101 binding whereas less sensitive than other assays of ligand:protein binding (56), it is SB203580 binding was preserved. low cost and has relatively high throughput. DSF detected p38a In vitro kinase assays illustrate how UM101 and SB203580 differ binding by 25% of the CADD-identified compounds screened and in how they modify p38a activity. Whereas SB203580 abrogated selective p38a binding by 10%, demonstrating good efficiency of phosphorylation of all three substrates tested, MK2, ATF2, and STAT- boththeCADDandDSFscreeningstrategies. The 10% hit rate of 1a, UM101 only partially reduced substrate phosphorylation. It exerted our CADD search for substrate-selective p38a inhibitors was similar the greatest effect on STAT-1a and the least on MK2. Although x-ray to our prior experience with CADD-identified substrate-selective crystallographic data showing that MK2 binds the ED site of p38a led ERK inhibitors (30) and much greater than the usual 0.1–0.01% us to predict that UM101 would block its phosphorylation, MK2 si- hit rate using experimental screening alone (57). multaneously interacts with the CD site (28), which may explain why 3304 COMPUTER-AIDED DESIGN OF SUBSTRATE-SELECTIVE p38 INHIBITORS Downloaded from http://www.jimmunol.org/

FIGURE 4. Binding specificity of UM101 and SB203580. (A) DSF analysis of indicated concentration of UM101 and 10 mM SB203580 (SB) binding to ,†,x recombinant p38a and p38b. Mean 6 SE of four experiments. * p , 0.0001 versus p38a with DMSO, p38b with DMSO, and p38b with SB203580, by guest on September 26, 2021 respectively. p , 0.0001 for difference between UM101 binding to p38a and p38b by MANOVA. (B) DSF analysis of indicated concentration of UM101 and 10 mM SB203580 (SB) binding to recombinant wild-type p38a and a p38a mutant with four mutations in CADD-targeted pocket (highlighted and bolded). Mean 6 SE of four experiments. *,†p , 0.0001 versus wild-type with DMSO and mutant with DMSO, respectively. p , 0.0001 for difference between UM101 binding to wild-type and mutant p38a by MANOVA. (C–I) STD-NMR was performed with UM101 and p38a (D and E), p38b (F and G), and the p38a mutant (H and I). The 1D (D, F, and H) and STD spectra (E, G, and I) from the same sample are shown. The tentative peak assignments are indicated in (D). The structure of UM101 with the protons labeled is shown in (C).

UM101 exerted only modest effects on its phosphorylation in HeLa functions (Fig. 2A, 2B), UM101 modified expression of only 38 cells (Fig. 3C) and in in vitro kinase assays (Fig. 3D, 3E). of the 99 SB203580-modified genes. Both of the selective p38a binding compounds, UM60 and PathwayNet analysis showed UM101 blocked only seven of the UM101, exerted SB203580-like in vitro and in vivo endothelial- 15 SB203580-blocked transcription factors. MSK1/2 was among barrier–stabilizing and -cytokine–modifying effects, those spared by UM101, which is consistent with the targeting thereby validating our ED-targeting strategy. We note that the strategy for UM101 to the ED site. Because p38 MAPK exerts in vivo dose response for UM101 differs for transalveolar protein transcriptional and posttranscriptional control of gene expression leak and neutrophil migration, suggesting that different UM101- through phosphorylation of multiple protein substrates (60), inhibitible p38-dependent pathways may contribute to endothelial substrate-selective inhibitors like UM101 are likely to impact gene protein leak and neutrophil transmigration. Alternatively, UM101 expression and cell function differently than catalytic inhibitors. may exert its effects on neutrophil transmigration in vivo by af- For example, sparing of MSK1/2 phosphorylation by UM101 pre- fecting both endothelial cells and neutrophils as p38 activation in serves expression of counterregulatory genes, including DUSP2 and both cells contributes to neutrophil transendothelial transmigration 10 (22, 23), which were increased by treatment with UM101 but not (9). Because UM101 more effectively stabilized endothelial bar- by treatment with SB203580. riers than SB203580 (Fig. 2A, 2B) despite having less effect on The partial functional overlap of UM101 and SB203580 MK2 phosphorylation (Fig. 3C), we evaluated additional molec- revealed by RNASeq is consistent with the design of UM101 as a ular actions by comparing the effects of UM101 and SB203580 on noncatalytic substrate-selective inhibitor, but might also be the result global gene expression using RNASeq in TNF-a–treated HMVECLs. of off-target effects of SB203580, which include receptor-interacting TNF-a increased expression of 511 genes by $2-fold, of which 61 protein kinase-2, cyclin G-associated kinase, and casein kinase-1d were reduced and 38 increased by pretreatment with 10 mM (58). However, none of the SB203580-inhibited transcription fac- SB203580. Despite using a concentration of UM101 that was tors identified by the PathwayNet analysis are known substrates for .10-fold higher than required to stabilize HMVECL barrier these kinases as analyzed with PhosphoNetworks (61). The Journal of Immunology 3305

Although the high concentration of UM101 used in this initial pulmonary disease: a randomised, double-blind, placebo-controlled trial. Lancet analysis may have caused some p38-independent actions, we be- Respir. Med. 2: 63–72. 12. MacNee, W., R. J. Allan, I. Jones, M. C. De Salvo, and L. F. Tan. 2013. Efficacy lieve the following data support a conclusion that UM101 exerts its and safety of the oral p38 inhibitor PH-797804 in chronic obstructive pulmonary biological effects predominantly by modifying p38a: 1) DSF and disease: a randomised clinical trial. Thorax 68: 738–745. STD-NMR show p38a-specific binding of UM101; 2) p38a bind- 13. Schreiber, S., B. Feagan, G. D’Haens, J. F. Colombel, K. Geboes, M. Yurcov, V. Isakov, O. Golovenko, C. N. Bernstein, D. Ludwig, et al; BIRB 796 Study ing of UM101 was abrogated by mutating four of 10 target pocket Group. 2006. Oral p38 mitogen-activated protein kinase inhibition with BIRB aminoacids;3)UM60and101bothbindp38a and exert effects on 796 for active Crohn’s disease: a randomized, double-blind, placebo-controlled endothelial function similar to those of SB203580; 4) UM101 trial. Clin. Gastroenterol. Hepatol. 4: 325–334. 14. Pargellis, C., L. Tong, L. Churchill, P. F. Cirillo, T. Gilmore, A. G. Graham, partially blocked phosphorylation of the p38 substrates MK2 and P. M. Grob, E. R. Hickey, N. Moss, S. Pav, and J. Regan. 2002. Inhibition of p38 STAT-1 in TNF-a–stimulated HeLa cells; and 5) UM101 inhibited MAP kinase by utilizing a novel allosteric binding site. Nat. Struct. Biol. 9: 268–272. the expression of about half the genes inhibited by SB203580. 15. Davidson, W., L. Frego, G. W. Peet, R. R. Kroe, M. E. Labadia, S. M. Lukas, R. J. Snow, S. Jakes, C. A. Grygon, C. Pargellis, and B. G. Werneburg. 2004. UM101 may be more effective than SB203580 in stabilizing the Discovery and characterization of a substrate selective p38alpha inhibitor. Bio- endothelial barrier because of its selective sparing of potential chemistry 43: 11658–11671. counter-regulatory genes, such as GM-CSF (62), MSK1/2-dependent 16. Hendriks, B. S., K. M. Seidl, and J. R. Chabot. 2010. Two additive mechanisms impair the differentiation of ‘substrate-selective’ p38 inhibitors from classical anti-inflammatory genes (22, 23), and p38b-dependent prosurvival p38 inhibitors in vitro. BMC Syst. Biol. 4: 23. genes (20, 21). Although the apparent affinity of UM101 for p38a is 17. Marber, M. S., J. D. Molkentin, and T. Force. 2010. Developing small molecules much greater than for p38b, additional work is required to defini- to inhibit kinases unkind to the heart: p38 MAPK as a case in point. Drug Discov. Today Dis. Mech. 7: e123–e127. tively address whether it is functionally specific for the p38a isoform. 18. Beardmore, V. A., H. J. Hinton, C. Eftychi, M. Apostolaki, M. Armaka,

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Figure S1. Chemical structures of 20 compounds screened by DSF from Maybridge catalogue.

Figure S2. Quadrant map of RNASeq analysis. Genes with at least 10 reads in one sample per set and at least 2-fold increase with TNFα are shown. The key refers to the direction of change in UM101-treated cells/SB203580-treated cells vs. DMSO-treated cells.

Figure S3. qRT-PCR confirmation of RNASeq results for subset of genes. HMVECLs were preincubated with either 0.4% DMSO, 10 µM SB20350, or 100 µM UM101 for 1h, then stimulated with 10 ng/ml TNFα for 4h and qRT-PCR performed. Results were expressed as fold- change vs. unstimulated controls for GM-CSF (A), TNFα (B), CXCL5 (C), CXCL10 (D), CSCL11 (E), IL-1β (F), IL-1α (G), DUSP2 (H), and DUSP 11 (I). Mean±SE of 4 independent experiments. The significant differences are indicated on each panel.

Table S1: Comparison of effects of UM101 (101) and SB203580 (SB) on TNF-induced gene expression in HMVECLs log(2) vs. untreated control Enembl Gene number 101 vs. cont SB vs. cont bin* Gene Symbol Gene Name ENSG00000006468 1.14697638 1.421076261 1 ETV1 ets variant 1 [Source:HGNC Symbol;Acc:HGNC:3490] ENSG00000128917 1.25167179 1.332013518 1 DLL4 delta-like 4 (Drosophila) [Source:HGNC Symbol;Acc:HGNC:2910] ENSG00000196872 1.10366409 1.625147781 1 KIAA1211L KIAA1211-like [Source:HGNC Symbol;Acc:HGNC:33454] mitogen-activated protein kinase kinase 6 [Source:HGNC ENSG00000108984 1.34524718 1.277526381 1 MAP2K6 Symbol;Acc:HGNC:6846] ENSG00000229953 1.63323083 1.701151748 1 RP11-284F21.7 TLR4 interactor with leucine-rich repeats [Source:HGNC ENSG00000255690 1.81451563 1.593283603 1 TRIL Symbol;Acc:HGNC:22200] BMP and activin membrane-bound inhibitor [Source:HGNC ENSG00000095739 1.11321658 1.122810577 1 BAMBI Symbol;Acc:HGNC:30251] sema domain, transmembrane domain (TM), and cytoplasmic domain, ENSG00000137872 2.48510888 1.904493008 1 SEMA6D (semaphorin) 6D [Source:HGNC Symbol;Acc:HGNC:16770] potassium channel, inwardly rectifying subfamily J, member 12 [Source:HGNC ENSG00000184185 1.18112212 1.550553136 1 KCNJ12 Symbol;Acc:HGNC:6258] fibronectin leucine rich transmembrane protein 3 [Source:HGNC ENSG00000125848 1.25784422 1.46432561 1 FLRT3 Symbol;Acc:HGNC:3762] ENSG00000196664 -1.55359362 1.217944135 2 TLR7 toll-like receptor 7 [Source:HGNC Symbol;Acc:HGNC:15631] ENSG00000119714 -1.30406162 1.226150879 2 GPR68 G protein-coupled receptor 68 [Source:HGNC Symbol;Acc:HGNC:4519] leucine rich repeat and fibronectin type III domain containing 5 [Source:HGNC ENSG00000165379 -1.44393526 2.061452912 2 LRFN5 Symbol;Acc:HGNC:20360] proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) [Source:HGNC ENSG00000135378 -1.19848823 -1.78258053 3 PRRG4 Symbol;Acc:HGNC:30799] ENSG00000145777 -1.33665251 -1.65143959 3 TSLP thymic stromal lymphopoietin [Source:HGNC Symbol;Acc:HGNC:30743] ENSG00000102970 -2.73030977 -1.83414345 3 CCL17 chemokine (C-C motif) ligand 17 [Source:HGNC Symbol;Acc:HGNC:10615] long intergenic non-protein coding RNA 677 [Source:HGNC ENSG00000259717 -1.49522538 -1.6963101 3 LINC00677 Symbol;Acc:HGNC:20121] ENSG00000205436 -1.16047102 -1.47916318 3 EXOC3L4 exocyst complex component 3-like 4 [Source:HGNC Symbol;Acc:HGNC:20120] matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV ENSG00000100985 -1.09117963 -1.15709135 3 ido1 collagenase) [Source:HGNC Symbol;Acc:HGNC:7176] ENSG00000131203 -3.56798735 -3.51063293 3 IDO1 indoleamine 2,3-dioxygenase 1 [Source:HGNC Symbol;Acc:HGNC:6059] ENSG00000276408 -3.02136041 -2.36477309 3 RP11-490B18.5 ENSG00000169245 -4.36983671 -3.10091556 3 CXCL10 chemokine (C-X-C motif) ligand 10 [Source:HGNC Symbol;Acc:HGNC:10637] ENSG00000091972 -1.53815541 -1.72928565 3 CD200 CD200 molecule [Source:HGNC Symbol;Acc:HGNC:7203] solute carrier family 15 (oligopeptide transporter), member 3 [Source:HGNC ENSG00000110446 -1.73105887 -1.00338842 3 SLC15A3 Symbol;Acc:HGNC:18068] vitamin D (1,25- dihydroxyvitamin D3) receptor [Source:HGNC ENSG00000111424 -1.19731694 -1.16718631 3 VDR Symbol;Acc:HGNC:12679] ENSG00000125538 -1.17253054 -1.40158693 3 IL1B interleukin 1, beta [Source:HGNC Symbol;Acc:HGNC:5992] ENSG00000279805 -1.45388548 -1.28507313 3 CTA-212A2.1 ENSG00000202533 -2.81570772 -1.96733528 3 Y_RNA Y RNA [Source:RFAM;Acc:RF00019] ENSG00000181656 -2.15059918 -1.39708375 3 GPR88 G protein-coupled receptor 88 [Source:HGNC Symbol;Acc:HGNC:4539] ENSG00000116031 -3.38243726 -1.54775729 3 CD207 CD207 molecule, langerin [Source:HGNC Symbol;Acc:HGNC:17935] ENSG00000159450 -1.54766532 -1.50495809 3 TCHH trichohyalin [Source:HGNC Symbol;Acc:HGNC:11791] ENSG00000103044 -1.12433956 -1.43377734 3 HAS3 hyaluronan synthase 3 [Source:HGNC Symbol;Acc:HGNC:4820] guanylate binding protein 1, interferon-inducible pseudogene 1 [Source:HGNC ENSG00000225492 -1.75570608 -1.3632872 3 GBP1P1 Symbol;Acc:HGNC:39561] ENSG00000145113 -1.05731569 -2.85949188 3 MUC4 mucin 4, cell surface associated [Source:HGNC Symbol;Acc:HGNC:7514] ENSG00000164181 -1.38106323 -1.34093337 3 ELOVL7 ELOVL fatty acid elongase 7 [Source:HGNC Symbol;Acc:HGNC:26292] ENSG00000169248 -4.13635487 -1.37790594 3 CXCL11 chemokine (C-X-C motif) ligand 11 [Source:HGNC Symbol;Acc:HGNC:10638] ENSG00000162654 -2.83594791 -1.25928308 3 GBP4 guanylate binding protein 4 [Source:HGNC Symbol;Acc:HGNC:20480] ENSG00000144837 -1.50063336 -1.27452433 3 PLA1A phospholipase A1 member A [Source:HGNC Symbol;Acc:HGNC:17661] ENSG00000222365 -1.74625516 -2.69898993 3 SNORD12B small nucleolar RNA, C/D box 12B [Source:HGNC Symbol;Acc:HGNC:33573] olfactory receptor, family 2, subfamily I, member 1 pseudogene ENSG00000237988 -3.7376706 -1.08751395 3 OR2I1P [Source:HGNC Symbol;Acc:HGNC:8258] ENSG00000163735 -1.46830773 -1.01742785 3 CXCL5 chemokine (C-X-C motif) ligand 5 [Source:HGNC Symbol;Acc:HGNC:10642] ENSG00000277105 1.8720451 -1.66179692 4 FP236383.10 ENSG00000259498 1.51730088 0 5 RP11-244F12.3 regulating synaptic membrane exocytosis 1 [Source:HGNC ENSG00000079841 1.1627696 0 5 RIMS1 Symbol;Acc:HGNC:17282] ENSG00000104081 1.87839947 0 5 BMF Bcl2 modifying factor [Source:HGNC Symbol;Acc:HGNC:24132] leucine rich repeat and fibronectin type III domain containing 1 [Source:HGNC ENSG00000128011 1.21234677 0 5 LRFN1 Symbol;Acc:HGNC:29290] ENSG00000102760 2.39873086 0 5 RGCC regulator of cell cycle [Source:HGNC Symbol;Acc:HGNC:20369] ENSG00000272918 2.02570082 0 5 CTB-152G17.6 ENSG00000158715 1.09721113 0 5 SLC45A3 solute carrier family 45, member 3 [Source:HGNC Symbol;Acc:HGNC:8642] SH3 domain and tetratricopeptide repeats 2 [Source:HGNC ENSG00000169247 1.22087519 0 5 SH3TC2 Symbol;Acc:HGNC:29427] ENSG00000163235 1.00798575 0 5 TGFA transforming growth factor, alpha [Source:HGNC Symbol;Acc:HGNC:11765] ENSG00000138311 1.68539518 0 5 ZNF365 protein 365 [Source:HGNC Symbol;Acc:HGNC:18194] RNA, 7SL, cytoplasmic 471, pseudogene [Source:HGNC ENSG00000263426 1.90751532 0 5 RN7SL471P Symbol;Acc:HGNC:46487] SRY (sex determining region Y)-box 18 [Source:HGNC ENSG00000203883 3.1401464 0 5 SOX18 Symbol;Acc:HGNC:11194] ENSG00000152213 2.11880858 0 5 ARL11 ADP-ribosylation factor-like 11 [Source:HGNC Symbol;Acc:HGNC:24046] ENSG00000115641 1.20543525 0 5 FHL2 four and a half LIM domains 2 [Source:HGNC Symbol;Acc:HGNC:3703] ENSG00000163884 1.31377126 0 5 KLF15 Kruppel-like factor 15 [Source:HGNC Symbol;Acc:HGNC:14536] ENSG00000171223 1.07792014 0 5 JUNB jun B proto-oncogene [Source:HGNC Symbol;Acc:HGNC:6205] ENSG00000137875 1.24981 0 5 BCL2L10 BCL2-like 10 (apoptosis facilitator) [Source:HGNC Symbol;Acc:HGNC:993] ENSG00000119630 1.02473901 0 5 PGF placental growth factor [Source:HGNC Symbol;Acc:HGNC:8893] v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog ENSG00000157404 1.98981566 0 5 KIT [Source:HGNC Symbol;Acc:HGNC:6342] pyruvate dehydrogenase kinase, isozyme 4 [Source:HGNC ENSG00000004799 2.74875752 0 5 PDK4 Symbol;Acc:HGNC:8812] lymphoblastic leukemia associated hematopoiesis regulator 1 [Source:HGNC ENSG00000104903 1.17436711 0 5 LYL1 Symbol;Acc:HGNC:6734] hes-related family bHLH transcription factor with YRPW motif 1 [Source:HGNC ENSG00000164683 2.56462289 0 5 HEY1 Symbol;Acc:HGNC:4880] ENSG00000229436 2.97587733 0 5 AC073850.6 ENSG00000074590 1.86606392 0 5 NUAK1 NUAK family, SNF1-like kinase, 1 [Source:HGNC Symbol;Acc:HGNC:14311] neuralized E3 protein 3 [Source:HGNC ENSG00000163121 1.17777496 0 5 NEURL3 Symbol;Acc:HGNC:25162] ENSG00000171435 2.0275154 0 5 KSR2 kinase suppressor of ras 2 [Source:HGNC Symbol;Acc:HGNC:18610] ENSG00000225213 2.78814593 0 5 RP11-197M22.2 LON peptidase N-terminal domain and ring finger 3 [Source:HGNC ENSG00000175556 2.1400816 0 5 LONRF3 Symbol;Acc:HGNC:21152] ENSG00000172031 1.46275504 0 5 EPHX4 epoxide 4 [Source:HGNC Symbol;Acc:HGNC:23758] ENSG00000164284 1.60799521 0 5 GRPEL2 GrpE-like 2, mitochondrial (E. coli) [Source:HGNC Symbol;Acc:HGNC:21060] Ras association (RalGDS/AF-6) domain family (N-terminal) member 9 ENSG00000198774 1.92290695 0 5 RASSF9 [Source:HGNC Symbol;Acc:HGNC:15739] inositol polyphosphate-4-phosphatase, type II, 105kDa [Source:HGNC ENSG00000109452 1.25820227 0 5 INPP4B Symbol;Acc:HGNC:6075] ENSG00000071282 1.2458539 0 5 LMCD1 LIM and cysteine-rich domains 1 [Source:HGNC Symbol;Acc:HGNC:6633] ENSG00000163545 1.15652631 0 5 NUAK2 NUAK family, SNF1-like kinase, 2 [Source:HGNC Symbol;Acc:HGNC:29558] inhibitor of DNA binding 1, dominant negative helix-loop-helix protein ENSG00000125968 2.63377664 0 5 ID1 [Source:HGNC Symbol;Acc:HGNC:5360] ENSG00000099260 1.50498454 0 5 PALMD palmdelphin [Source:HGNC Symbol;Acc:HGNC:15846] ENSG00000176641 1.2877484 0 5 RNF152 ring finger protein 152 [Source:HGNC Symbol;Acc:HGNC:26811] ENSG00000139874 1.37617951 0 5 SSTR1 somatostatin receptor 1 [Source:HGNC Symbol;Acc:HGNC:11330] ENSG00000137834 2.51438228 0 5 SMAD6 SMAD family member 6 [Source:HGNC Symbol;Acc:HGNC:6772] ENSG00000259721 1.0709029 0 5 RP11-758N13.1 ENSG00000181800 2.42803687 0 5 CELF2-AS1 CELF2 antisense RNA 1 [Source:HGNC Symbol;Acc:HGNC:23515] prostaglandin E receptor 4 (subtype EP4) pseudogene 2 [Source:HGNC ENSG00000184523 2.14599043 0 5 PTGER4P2 Symbol;Acc:HGNC:9598] solute carrier organic anion transporter family, member 4A1 [Source:HGNC ENSG00000101187 1.12788457 0 5 SLCO4A1 Symbol;Acc:HGNC:10953] ENSG00000237512 1.72226772 0 5 UNC5B-AS1 UNC5B antisense RNA 1 [Source:HGNC Symbol;Acc:HGNC:45096] ENSG00000156463 1.43711148 0 5 SH3RF2 SH3 domain containing ring finger 2 [Source:HGNC Symbol;Acc:HGNC:26299] transient receptor potential cation channel, subfamily C, member 6 ENSG00000137672 1.41739616 0 5 TRPC6 [Source:HGNC Symbol;Acc:HGNC:12338] ENSG00000138135 2.4356406 0 5 CH25H cholesterol 25-hydroxylase [Source:HGNC Symbol;Acc:HGNC:1907] ENSG00000183691 1.18113108 0 5 NOG noggin [Source:HGNC Symbol;Acc:HGNC:7866] ENSG00000139174 2.23155754 0 5 PRICKLE1 prickle homolog 1 (Drosophila) [Source:HGNC Symbol;Acc:HGNC:17019] 19 open reading frame 35 [Source:HGNC ENSG00000188305 1.46592995 0 5 C19orf35 Symbol;Acc:HGNC:24793] ENSG00000082497 3.01359039 0 5 SERTAD4 SERTA domain containing 4 [Source:HGNC Symbol;Acc:HGNC:25236] vav 3 guanine nucleotide exchange factor [Source:HGNC ENSG00000134215 1.69084 0 5 VAV3 Symbol;Acc:HGNC:12659] ENSG00000242902 1.87508823 0 5 RP11-309L24.2 ENSG00000027075 1.03323842 0 5 PRKCH protein kinase C, eta [Source:HGNC Symbol;Acc:HGNC:9403] ENSG00000203280 1.22563855 0 5 CTA-221G9.12 ENSG00000006459 1.00029861 0 5 KDM7A (K)-specific demethylase 7A [Source:HGNC Symbol;Acc:HGNC:22224] ENSG00000171408 3.15161753 0 5 PDE7B phosphodiesterase 7B [Source:HGNC Symbol;Acc:HGNC:8792] family with sequence similarity 84, member A [Source:HGNC ENSG00000162981 1.53241836 0 5 FAM84A Symbol;Acc:HGNC:20743] ENSG00000118946 1.93243268 0 5 PCDH17 protocadherin 17 [Source:HGNC Symbol;Acc:HGNC:14267] ENSG00000146376 1.27126839 0 5 ARHGAP18 Rho GTPase activating protein 18 [Source:HGNC Symbol;Acc:HGNC:21035] ENSG00000204086 2.05144911 0 5 RPA4 replication protein A4, 30kDa [Source:HGNC Symbol;Acc:HGNC:30305] histocompatibility (minor) serpin domain containing [Source:HGNC ENSG00000221887 1.05893107 0 5 HMSD Symbol;Acc:HGNC:23037] ENSG00000196196 1.26354255 0 5 HRCT1 histidine rich carboxyl terminus 1 [Source:HGNC Symbol;Acc:HGNC:33872] ENSG00000172548 2.84971177 0 5 NIPAL4 NIPA-like domain containing 4 [Source:HGNC Symbol;Acc:HGNC:28018] ENSG00000156804 2.04515428 0 5 FBXO32 F-box protein 32 [Source:HGNC Symbol;Acc:HGNC:16731] IBA57 antisense RNA 1 (head to head) [Source:HGNC ENSG00000203684 1.5984677 0 5 IBA57-AS1 Symbol;Acc:HGNC:32062] C2 calcium-dependent domain containing 4B [Source:HGNC ENSG00000205502 1.63750711 0 5 C2CD4B Symbol;Acc:HGNC:33628] ENSG00000163734 1.26141193 0 5 CXCL3 chemokine (C-X-C motif) ligand 3 [Source:HGNC Symbol;Acc:HGNC:4604] ENSG00000181444 1.48713949 0 5 ZNF467 zinc finger protein 467 [Source:HGNC Symbol;Acc:HGNC:23154] ENSG00000275342 1.45192287 0 5 SGK223 Tyrosine-protein kinase SgK223 [Source:UniProtKB/Swiss-Prot;Acc:Q86YV5] Rho guanine nucleotide exchange factor (GEF) 28 [Source:HGNC ENSG00000214944 1.43116765 0 5 ARHGEF28 Symbol;Acc:HGNC:30322] ENSG00000198795 1.37664308 0 5 ZNF521 zinc finger protein 521 [Source:HGNC Symbol;Acc:HGNC:24605] ENSG00000108932 1.95156031 0 5 SLC16A6 solute carrier family 16, member 6 [Source:HGNC Symbol;Acc:HGNC:10927] glucose-fructose domain containing 1 [Source:HGNC ENSG00000145990 1.18764084 0 5 GFOD1 Symbol;Acc:HGNC:21096] 5-hydroxytryptamine (serotonin) receptor 1D, G protein-coupled ENSG00000179546 1.7287987 0 5 HTR1D [Source:HGNC Symbol;Acc:HGNC:5289] piccolo presynaptic cytomatrix protein [Source:HGNC ENSG00000186472 1.73331066 0 5 PCLO Symbol;Acc:HGNC:13406] 1-acylglycerol-3-phosphate O-acyltransferase 9 [Source:HGNC ENSG00000138678 1.55650245 0 5 AGPAT9 Symbol;Acc:HGNC:28157] GrpE-like 2, mitochondrial (E. coli) pseudogene 2 [Source:HGNC ENSG00000225814 1.57236046 0 5 GRPEL2P2 Symbol;Acc:HGNC:41970] phosphodiesterase 3A, cGMP-inhibited [Source:HGNC ENSG00000172572 1.01708765 0 5 PDE3A Symbol;Acc:HGNC:8778] amyloid beta (A4) precursor protein-binding, family A, member 1 ENSG00000107282 1.0986938 0 5 APBA1 [Source:HGNC Symbol;Acc:HGNC:578] ENSG00000171877 1.0590391 0 5 FRMD5 FERM domain containing 5 [Source:HGNC Symbol;Acc:HGNC:28214] subfamily 3, group C, member 2 [Source:HGNC ENSG00000151623 1.83557493 0 5 NR3C2 Symbol;Acc:HGNC:7979] ENSG00000189184 1.39874706 0 5 PCDH18 protocadherin 18 [Source:HGNC Symbol;Acc:HGNC:14268] chromosome 11 open reading frame 96 [Source:HGNC ENSG00000187479 1.56424224 0 5 C11orf96 Symbol;Acc:HGNC:38675] ENSG00000178726 1.31386114 0 5 THBD thrombomodulin [Source:HGNC Symbol;Acc:HGNC:11784] Pim-1 proto-oncogene, serine/threonine kinase [Source:HGNC ENSG00000137193 2.05607477 0 5 PIM1 Symbol;Acc:HGNC:8986] ADAM metallopeptidase with thrombospondin type 1 motif, 1 [Source:HGNC ENSG00000154734 1.08386589 0 5 ADAMTS1 Symbol;Acc:HGNC:217] ENSG00000143772 1.05692317 0 5 ITPKB inositol-trisphosphate 3-kinase B [Source:HGNC Symbol;Acc:HGNC:6179] ENSG00000140022 1.42852098 0 5 STON2 stonin 2 [Source:HGNC Symbol;Acc:HGNC:30652] zinc finger and BTB domain containing 20 [Source:HGNC ENSG00000181722 1.75771309 0 5 ZBTB20 Symbol;Acc:HGNC:13503] ENSG00000184058 2.32518737 0 5 TBX1 T-box 1 [Source:HGNC Symbol;Acc:HGNC:11592] ENSG00000043591 1.38727807 0 5 ADRB1 adrenoceptor beta 1 [Source:HGNC Symbol;Acc:HGNC:285] ENSG00000126550 2.94676056 0 5 HTN1 histatin 1 [Source:HGNC Symbol;Acc:HGNC:5283] odd-skipped related transciption factor 1 [Source:HGNC ENSG00000143867 1.2361215 0 5 OSR1 Symbol;Acc:HGNC:8111] nuclear receptor subfamily 5, group A, member 2 [Source:HGNC ENSG00000116833 1.34604824 0 5 NR5A2 Symbol;Acc:HGNC:7984] ENSG00000166292 2.00173044 0 5 TMEM100 transmembrane protein 100 [Source:HGNC Symbol;Acc:HGNC:25607] ENSG00000188487 1.04413181 0 5 INSC inscuteable homolog (Drosophila) [Source:HGNC Symbol;Acc:HGNC:33116] ENSG00000176697 1.76616859 0 5 BDNF brain-derived neurotrophic factor [Source:HGNC Symbol;Acc:HGNC:1033] runt-related transcription factor 1; translocated to, 1 (cyclin D-related) ENSG00000079102 1.5770273 0 5 RUNX1T1 [Source:HGNC Symbol;Acc:HGNC:1535] ENSG00000162599 1.02633907 0 5 NFIA /A [Source:HGNC Symbol;Acc:HGNC:7784] ENSG00000188763 1.52543754 0 5 FZD9 frizzled class receptor 9 [Source:HGNC Symbol;Acc:HGNC:4047] coxsackie and adenovirus receptor [Source:HGNC ENSG00000154639 1.36734426 0 5 CXADR Symbol;Acc:HGNC:2559] ENSG00000227946 1.24976159 0 5 AC007383.3 ENSG00000143341 1.16130281 0 5 HMCN1 hemicentin 1 [Source:HGNC Symbol;Acc:HGNC:19194] KLF7 intronic transcript 1 (non-protein coding) [Source:HGNC ENSG00000237892 1.07996952 0 5 KLF7-IT1 Symbol;Acc:HGNC:41355] ENSG00000103522 1.30364536 0 5 IL21R interleukin 21 receptor [Source:HGNC Symbol;Acc:HGNC:6006] UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 2 ENSG00000162630 1.457488 0 5 B3GALT2 [Source:HGNC Symbol;Acc:HGNC:917] ENSG00000106069 1.15244395 0 5 CHN2 2 [Source:HGNC Symbol;Acc:HGNC:1944] ENSG00000169047 1.03338349 0 5 IRS1 insulin receptor substrate 1 [Source:HGNC Symbol;Acc:HGNC:6125] ENSG00000226476 1.25685284 0 5 RP11-776H12.1 leucine-rich single-pass 1 [Source:HGNC ENSG00000181016 1.30372056 0 5 LSMEM1 Symbol;Acc:HGNC:22036] ENSG00000121966 3.89708969 0 5 CXCR4 chemokine (C-X-C motif) receptor 4 [Source:HGNC Symbol;Acc:HGNC:2561] ENSG00000189143 1.4778446 0 5 CLDN4 claudin 4 [Source:HGNC Symbol;Acc:HGNC:2046] ENSG00000257642 2.54048396 0 5 RP11-474B16.1 ENSG00000250271 2.3827306 0 5 RP11-64D22.5 ENSG00000188483 1.76997593 0 5 IER5L immediate early response 5-like [Source:HGNC Symbol;Acc:HGNC:23679] potassium channel tetramerization domain containing 16 [Source:HGNC ENSG00000183775 1.04020782 0 5 KCTD16 Symbol;Acc:HGNC:29244] dickkopf WNT signaling pathway inhibitor 1 [Source:HGNC ENSG00000107984 1.55122779 0 5 DKK1 Symbol;Acc:HGNC:2891] major facilitator superfamily domain containing 4 [Source:HGNC ENSG00000174514 0 1.187467833 6 MFSD4 Symbol;Acc:HGNC:25433] ENSG00000270379 0 1.090459878 6 HEATR9 HEAT repeat containing 9 [Source:HGNC Symbol;Acc:HGNC:26548] ENSG00000240859 0 1.361880252 6 AC093627.10 ENSG00000236671 0 1.65509644 6 PRKG1-AS1 PRKG1 antisense RNA 1 [Source:HGNC Symbol;Acc:HGNC:45029] ENSG00000261707 0 1.157366293 6 RP11-264M12.2 ENSG00000273669 0 3.698760209 6 RP11-405M12.4 BEN domain containing 3 pseudogene 1 [Source:HGNC ENSG00000231345 0 1.358686175 6 BEND3P1 Symbol;Acc:HGNC:45014] ENSG00000134253 0 1.102241435 6 TRIM45 tripartite motif containing 45 [Source:HGNC Symbol;Acc:HGNC:19018] ENSG00000138336 0 1.764266324 6 TET1 tet methylcytosine dioxygenase 1 [Source:HGNC Symbol;Acc:HGNC:29484] ENSG00000120162 0 1.233788658 6 MOB3B MOB kinase activator 3B [Source:HGNC Symbol;Acc:HGNC:23825] complement component 3a receptor 1 [Source:HGNC ENSG00000171860 0 1.018148884 6 C3AR1 Symbol;Acc:HGNC:1319] ENSG00000167676 0 1.03160807 6 PLIN4 perilipin 4 [Source:HGNC Symbol;Acc:HGNC:29393] ENSG00000237234 0 1.660842435 6 RP1-142L7.5 ENSG00000164124 0 1.053810276 6 TMEM144 transmembrane protein 144 [Source:HGNC Symbol;Acc:HGNC:25633] v- avian myeloblastosis viral oncogene homolog [Source:HGNC ENSG00000118513 0 1.129823007 6 MYB Symbol;Acc:HGNC:7545] solute carrier family 26 (anion exchanger), member 4 [Source:HGNC ENSG00000091137 0 1.166998057 6 SLC26A4 Symbol;Acc:HGNC:8818] ENSG00000198483 0 1.435322281 6 ANKRD35 ankyrin repeat domain 35 [Source:HGNC Symbol;Acc:HGNC:26323] long intergenic non-protein coding RNA 1573 [Source:HGNC ENSG00000237886 0 1.572819969 6 LINC01573 Symbol;Acc:HGNC:51192] negative regulator of [Source:HGNC ENSG00000174004 0 2.276022317 6 NRROS Symbol;Acc:HGNC:24613] SHC (Src homology 2 domain containing) family, member 4 [Source:HGNC ENSG00000185634 0 1.664336782 6 SHC4 Symbol;Acc:HGNC:16743] ENSG00000259886 0 1.176664637 6 DNA-damage-inducible transcript 4-like [Source:HGNC ENSG00000145358 0 1.325502189 6 DDIT4L Symbol;Acc:HGNC:30555] ENSG00000269896 0 1.288382555 6 RP4-740C4.5 ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent) [Source:HGNC ENSG00000135828 0 1.083709403 6 RNASEL Symbol;Acc:HGNC:10050] ENSG00000259162 0 1.526671578 6 RP11-203M5.6 Uncharacterized protein {ECO:0000313|Ensembl:ENSP00000485568} ENSG00000279109 -3.34035348 0 7 AC008641.1 [Source:UniProtKB/TrEMBL;Acc:A0A096LPF4] interferon-induced protein with tetratricopeptide repeats 5 [Source:HGNC ENSG00000152778 -1.09722258 0 7 IFIT5 Symbol;Acc:HGNC:13328] ENSG00000128284 -1.30507158 0 7 APOL3 apolipoprotein L, 3 [Source:HGNC Symbol;Acc:HGNC:14868] ENSG00000213886 -4.03961136 0 7 UBD ubiquitin D [Source:HGNC Symbol;Acc:HGNC:18795] ENSG00000164116 -1.52230552 0 7 GUCY1A3 guanylate cyclase 1, soluble, alpha 3 [Source:HGNC Symbol;Acc:HGNC:4685] ENSG00000137462 -1.00204889 0 7 TLR2 toll-like receptor 2 [Source:HGNC Symbol;Acc:HGNC:11848] tumor necrosis factor receptor superfamily, member 9 [Source:HGNC ENSG00000049249 -2.1008906 0 7 TNFRSF9 Symbol;Acc:HGNC:11924] ENSG00000169181 -2.09559802 0 7 GSG1L GSG1-like [Source:HGNC Symbol;Acc:HGNC:28283] open reading frame 147 [Source:HGNC ENSG00000162888 -1.79122856 0 7 C1orf147 Symbol;Acc:HGNC:32061] DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 [Source:HGNC ENSG00000107201 -1.24135357 0 7 DDX58 Symbol;Acc:HGNC:19102] ENSG00000179826 -2.51338055 0 7 MRGPRX3 MAS-related GPR, member X3 [Source:HGNC Symbol;Acc:HGNC:17980] ENSG00000132109 -1.28670066 0 7 TRIM21 tripartite motif containing 21 [Source:HGNC Symbol;Acc:HGNC:11312] DnaJ (Hsp40) homolog, subfamily A, member 1 pseudogene 3 [Source:HGNC ENSG00000215007 -1.00787291 0 7 DNAJA1P3 Symbol;Acc:HGNC:39339] ENSG00000204682 -1.16284472 0 7 CASC10 cancer susceptibility candidate 10 [Source:HGNC Symbol;Acc:HGNC:31448] ENSG00000108688 -1.87034276 0 7 CCL7 chemokine (C-C motif) ligand 7 [Source:HGNC Symbol;Acc:HGNC:10634] superoxide dismutase 2, mitochondrial [Source:HGNC ENSG00000112096 -1.07302144 0 7 SOD2 Symbol;Acc:HGNC:11180] solute carrier family 6 (neurotransmitter transporter), member 13 ENSG00000010379 -2.69730597 0 7 SLC6A13 [Source:HGNC Symbol;Acc:HGNC:11046] ENSG00000169403 -1.57938798 0 7 PTAFR platelet-activating factor receptor [Source:HGNC Symbol;Acc:HGNC:9582] ENSG00000115604 -3.25905915 0 7 IL18R1 interleukin 18 receptor 1 [Source:HGNC Symbol;Acc:HGNC:5988] ENSG00000133401 -1.02465503 0 7 PDZD2 PDZ domain containing 2 [Source:HGNC Symbol;Acc:HGNC:18486] ENSG00000095587 -2.25101727 0 7 TLL2 tolloid-like 2 [Source:HGNC Symbol;Acc:HGNC:11844] ENSG00000134256 -1.35774774 0 7 CD101 CD101 molecule [Source:HGNC Symbol;Acc:HGNC:5949] ENSG00000272463 -1.14133941 0 7 RP11-532F6.3 immunoresponsive 1 homolog (mouse) [Source:HGNC ENSG00000102794 -1.59424636 0 7 IRG1 Symbol;Acc:HGNC:33904] IL10RB antisense RNA 1 (head to head) [Source:HGNC ENSG00000223799 -1.63414439 0 7 IL10RB-AS1 Symbol;Acc:HGNC:44303] CD74 molecule, major histocompatibility complex, class II invariant chain ENSG00000019582 -1.10558246 0 7 CD74 [Source:HGNC Symbol;Acc:HGNC:1697] ENSG00000121577 -1.23573024 0 7 POPDC2 popeye domain containing 2 [Source:HGNC Symbol;Acc:HGNC:17648] ENSG00000215268 -1.73731446 0 7 LA16c-60G3.8 transient receptor potential cation channel, subfamily M, member 6 ENSG00000119121 -1.47857665 0 7 TRPM6 [Source:HGNC Symbol;Acc:HGNC:17995] solute carrier family 6 (neurotransmitter transporter), member 4 ENSG00000108576 -1.22990555 0 7 SLC6A4 [Source:HGNC Symbol;Acc:HGNC:11050] ENSG00000274818 -1.88549909 0 7 RP1-292L20.3 ENSG00000198133 -1.93896982 0 7 TMEM229B transmembrane protein 229B [Source:HGNC Symbol;Acc:HGNC:20130] ENSG00000130477 -1.25410528 0 7 UNC13A unc-13 homolog A (C. elegans) [Source:HGNC Symbol;Acc:HGNC:23150] Ras association (RalGDS/AF-6) domain family member 5 [Source:HGNC ENSG00000266094 -1.05796373 0 7 RASSF5 Symbol;Acc:HGNC:17609] solute carrier organic anion transporter family, member 5A1 [Source:HGNC ENSG00000137571 -1.09049933 0 7 SLCO5A1 Symbol;Acc:HGNC:19046] ENSG00000272512 -1.59155142 0 7 RP11-54O7.17 ENSG00000124391 -1.72783256 0 7 IL17C interleukin 17C [Source:HGNC Symbol;Acc:HGNC:5983] solute carrier family 41 (magnesium transporter), member 2 [Source:HGNC ENSG00000136052 -1.32722235 0 7 SLC41A2 Symbol;Acc:HGNC:31045] glycoprotein Ib (platelet), alpha polypeptide [Source:HGNC ENSG00000185245 -1.94643325 0 7 GP1BA Symbol;Acc:HGNC:4439] chromosome 1 open reading frame 95 [Source:HGNC ENSG00000203685 -1.80611826 0 7 C1orf95 Symbol;Acc:HGNC:30491] ENSG00000149654 -1.33161296 0 7 CDH22 cadherin 22, type 2 [Source:HGNC Symbol;Acc:HGNC:13251] ENSG00000230943 -1.57412905 0 7 RP11-367G18.1 ENSG00000215277 -3.23339357 0 7 RNF212B ring finger protein 212B [Source:HGNC Symbol;Acc:HGNC:20438] MAM domain containing glycosylphosphatidylinositol anchor 1 [Source:HGNC ENSG00000112139 -1.28619609 0 7 MDGA1 Symbol;Acc:HGNC:19267] ENSG00000143494 -1.51352046 0 7 VASH2 vasohibin 2 [Source:HGNC Symbol;Acc:HGNC:25723] poly (ADP-ribose) polymerase family, member 8 [Source:HGNC ENSG00000151883 -1.17607508 0 7 PARP8 Symbol;Acc:HGNC:26124] receptor (chemosensory) transporter protein 4 [Source:HGNC ENSG00000136514 -1.97502424 0 7 RTP4 Symbol;Acc:HGNC:23992] cytochrome P450, family 3, subfamily A, polypeptide 5 [Source:HGNC ENSG00000106258 -1.01259197 0 7 CYP3A5 Symbol;Acc:HGNC:2638] ENSG00000243649 -2.55517135 0 7 CFB complement factor B [Source:HGNC Symbol;Acc:HGNC:1037] ENSG00000164342 -1.02909511 0 7 TLR3 toll-like receptor 3 [Source:HGNC Symbol;Acc:HGNC:11849] ENSG00000115956 -2.34795374 0 7 PLEK pleckstrin [Source:HGNC Symbol;Acc:HGNC:9070] ENSG00000144476 -1.71846582 0 7 ACKR3 atypical chemokine receptor 3 [Source:HGNC Symbol;Acc:HGNC:23692] ENSG00000157601 -1.41737641 0 7 MX1 MX dynamin-like GTPase 1 [Source:HGNC Symbol;Acc:HGNC:7532] sterile alpha motif domain containing 9-like [Source:HGNC ENSG00000177409 -1.14654994 0 7 SAMD9L Symbol;Acc:HGNC:1349] interferon-induced protein with tetratricopeptide repeats 3 [Source:HGNC ENSG00000119917 -1.85654737 0 7 IFIT3 Symbol;Acc:HGNC:5411] ENSG00000271503 -1.76834422 0 7 CCL5 chemokine (C-C motif) ligand 5 [Source:HGNC Symbol;Acc:HGNC:10632] ENSG00000117226 -1.1447048 0 7 GBP3 guanylate binding protein 3 [Source:HGNC Symbol;Acc:HGNC:4184] ENSG00000163840 -1.33113792 0 7 DTX3L deltex 3 like, E3 ubiquitin ligase [Source:HGNC Symbol;Acc:HGNC:30323] ENSG00000010030 -1.22076729 0 7 ETV7 ets variant 7 [Source:HGNC Symbol;Acc:HGNC:18160] Uncharacterized protein {ECO:0000313|Ensembl:ENSP00000463376} ENSG00000261884 -1.33109855 0 7 CTC-479C5.12 [Source:UniProtKB/TrEMBL;Acc:J3QL48] proline-serine-threonine phosphatase interacting protein 2 [Source:HGNC ENSG00000152229 -1.01792906 0 7 PSTPIP2 Symbol;Acc:HGNC:9581] solute carrier family 8 (sodium/calcium exchanger), member 3 [Source:HGNC ENSG00000100678 -2.05140713 0 7 SLC8A3 Symbol;Acc:HGNC:11070] long intergenic non-protein coding RNA 92 [Source:HGNC ENSG00000225194 -2.42016879 0 7 LINC00092 Symbol;Acc:HGNC:31408] ENSG00000140968 -1.18984187 0 7 IRF8 interferon regulatory factor 8 [Source:HGNC Symbol;Acc:HGNC:5358] ENSG00000006210 -1.21827215 0 7 CX3CL1 chemokine (C-X3-C motif) ligand 1 [Source:HGNC Symbol;Acc:HGNC:10647] ENSG00000221963 -1.13921379 0 7 APOL6 apolipoprotein L, 6 [Source:HGNC Symbol;Acc:HGNC:14870] helicase with zinc finger 2, transcriptional coactivator [Source:HGNC ENSG00000130589 -1.06732828 0 7 HELZ2 Symbol;Acc:HGNC:30021] apolipoprotein B mRNA editing , catalytic polypeptide-like 3G ENSG00000239713 -1.6622438 0 7 APOBEC3G [Source:HGNC Symbol;Acc:HGNC:17357] enkurin, TRPC channel interacting protein [Source:HGNC ENSG00000151023 -1.01391885 0 7 ENKUR Symbol;Acc:HGNC:28388] ENSG00000187123 -1.31939789 0 7 LYPD6 LY6/PLAUR domain containing 6 [Source:HGNC Symbol;Acc:HGNC:28751] ENSG00000253831 -3.05071374 0 7 ETV3L ets variant 3-like [Source:HGNC Symbol;Acc:HGNC:33834] ENSG00000246130 -3.03517986 0 7 RP11-875O11.2 ENSG00000128335 -1.81220789 0 7 APOL2 apolipoprotein L, 2 [Source:HGNC Symbol;Acc:HGNC:619] ENSG00000108702 -4.94661977 0 7 CCL1 chemokine (C-C motif) ligand 1 [Source:HGNC Symbol;Acc:HGNC:10609] ENSG00000105963 -1.20498886 0 7 ADAP1 ArfGAP with dual PH domains 1 [Source:HGNC Symbol;Acc:HGNC:16486] ENSG00000170075 -1.44911565 0 7 GPR37L1 G protein-coupled receptor 37 like 1 [Source:HGNC Symbol;Acc:HGNC:14923] ENSG00000267607 -1.20212344 0 7 CTD-2369P2.8 ENSG00000142961 -1.12661434 0 7 MOB3C MOB kinase activator 3C [Source:HGNC Symbol;Acc:HGNC:29800] ENSG00000159200 -1.16291334 0 7 RCAN1 regulator of calcineurin 1 [Source:HGNC Symbol;Acc:HGNC:3040] interleukin 3 receptor, alpha (low affinity) [Source:HGNC ENSG00000185291 -1.50319193 0 7 IL3RA Symbol;Acc:HGNC:6012] solute carrier family 19 (thiamine transporter), member 3 [Source:HGNC ENSG00000135917 -1.31314337 0 7 SLC19A3 Symbol;Acc:HGNC:16266] ENSG00000179817 -1.72393982 0 7 MRGPRX4 MAS-related GPR, member X4 [Source:HGNC Symbol;Acc:HGNC:17617] C1q and tumor necrosis factor related protein 1 [Source:HGNC ENSG00000173918 -1.18090506 0 7 C1QTNF1 Symbol;Acc:HGNC:14324] ENSG00000198879 -1.35783919 0 7 SFMBT2 Scm-like with four mbt domains 2 [Source:HGNC Symbol;Acc:HGNC:20256] ENSG00000272078 -1.14951076 0 7 RP4-734G22.3 ENSG00000269794 -1.74769407 0 7 AC010642.2 ENSG00000115919 -1.21621287 0 7 KYNU kynureninase [Source:HGNC Symbol;Acc:HGNC:6469] ENSG00000255521 -1.8970116 0 7 RP4-607I7.1 poly (ADP-ribose) polymerase family, member 14 [Source:HGNC ENSG00000173193 -1.34012573 0 7 PARP14 Symbol;Acc:HGNC:29232] chromosome 11 open reading frame 88 [Source:HGNC ENSG00000183644 -1.60990165 0 7 C11orf88 Symbol;Acc:HGNC:25061] ENSG00000253522 -1.26587239 0 7 CTC-231O11.1 ENSG00000236453 -1.89698452 0 7 AC003092.1 ENSG00000131979 -1.57621384 0 7 GCH1 GTP cyclohydrolase 1 [Source:HGNC Symbol;Acc:HGNC:4193] C-type lectin domain family 2, member D [Source:HGNC ENSG00000069493 -2.17420622 0 7 CLEC2D Symbol;Acc:HGNC:14351] ENSG00000069696 -1.13235612 0 7 DRD4 dopamine receptor D4 [Source:HGNC Symbol;Acc:HGNC:3025] , CUB domain, EGF-like 2 [Source:HGNC ENSG00000175356 -1.39848028 0 7 SCUBE2 Symbol;Acc:HGNC:30425] ENSG00000128165 -1.1779326 0 7 ADM2 adrenomedullin 2 [Source:HGNC Symbol;Acc:HGNC:28898] ENSG00000166856 -1.19923371 0 7 GPR182 G protein-coupled receptor 182 [Source:HGNC Symbol;Acc:HGNC:13708] ENSG00000199161 -1.61684899 0 7 MIR126 microRNA 126 [Source:HGNC Symbol;Acc:HGNC:31508] ENSG00000050730 -1.98731652 0 7 TNIP3 TNFAIP3 interacting protein 3 [Source:HGNC Symbol;Acc:HGNC:19315] ENSG00000255750 -1.89097244 0 7 RP11-283G6.5 chromosome 6 open reading frame 58 [Source:HGNC ENSG00000184530 -2.35058533 0 7 C6orf58 Symbol;Acc:HGNC:20960] peroxisomal biogenesis factor 11 gamma [Source:HGNC ENSG00000104883 -1.38601473 0 7 PEX11G Symbol;Acc:HGNC:20208] egl-9 family hypoxia-inducible factor 3 [Source:HGNC ENSG00000129521 -2.70164981 0 7 EGLN3 Symbol;Acc:HGNC:14661] ENSG00000204482 -1.24757693 0 7 LST1 leukocyte specific transcript 1 [Source:HGNC Symbol;Acc:HGNC:14189] interferon induced with helicase C domain 1 [Source:HGNC ENSG00000115267 -1.3445539 0 7 IFIH1 Symbol;Acc:HGNC:18873] ENSG00000162692 -2.18018207 0 7 VCAM1 vascular cell adhesion molecule 1 [Source:HGNC Symbol;Acc:HGNC:12663] ENSG00000261618 -1.28643443 0 7 RP11-79H23.3 solute carrier family 52 (riboflavin transporter), member 3 [Source:HGNC ENSG00000101276 -1.17059156 0 7 SLC52A3 Symbol;Acc:HGNC:16187] cell adhesion associated, oncogene regulated [Source:HGNC ENSG00000064309 -1.46512339 0 7 CDON Symbol;Acc:HGNC:17104] proline-rich transmembrane protein 2 [Source:HGNC ENSG00000167371 -1.4282411 0 7 PRRT2 Symbol;Acc:HGNC:30500] CD40 molecule, TNF receptor superfamily member 5 [Source:HGNC ENSG00000101017 -1.55836634 0 7 CD40 Symbol;Acc:HGNC:11919] colony stimulating factor 2 (granulocyte-macrophage) [Source:HGNC ENSG00000164400 0 -1.63408281 8 CSF2 Symbol;Acc:HGNC:2434] ENSG00000172602 0 -1.15027449 8 RND1 Rho family GTPase 1 [Source:HGNC Symbol;Acc:HGNC:18314] solute carrier family 26 (anion exchanger), member 9 [Source:HGNC ENSG00000174502 0 -2.09273487 8 SLC26A9 Symbol;Acc:HGNC:14469] ENSG00000234290 0 -1.00067832 8 AC116366.6 ENSG00000170961 0 -2.64619793 8 HAS2 hyaluronan synthase 2 [Source:HGNC Symbol;Acc:HGNC:4819] ENSG00000110848 0 -1.46706866 8 CD69 CD69 molecule [Source:HGNC Symbol;Acc:HGNC:1694] ENSG00000164512 0 -1.20441285 8 ANKRD55 ankyrin repeat domain 55 [Source:HGNC Symbol;Acc:HGNC:25681] ENSG00000167034 0 -1.10541745 8 NKX3-1 NK3 homeobox 1 [Source:HGNC Symbol;Acc:HGNC:7838] ENSG00000105246 0 -1.39394774 8 EBI3 Epstein-Barr virus induced 3 [Source:HGNC Symbol;Acc:HGNC:3129] naked cuticle homolog 2 (Drosophila) [Source:HGNC ENSG00000145506 0 -1.41815829 8 NKD2 Symbol;Acc:HGNC:17046] coagulation factor II (thrombin) receptor-like 3 [Source:HGNC ENSG00000127533 0 -2.64266235 8 F2RL3 Symbol;Acc:HGNC:3540] ENSG00000115008 0 -2.15272028 8 IL1A interleukin 1, alpha [Source:HGNC Symbol;Acc:HGNC:5991] ENSG00000073282 0 -1.31215479 8 TP63 tumor protein p63 [Source:HGNC Symbol;Acc:HGNC:15979] heart and neural crest derivatives expressed 1 [Source:HGNC ENSG00000113196 0 -1.61798433 8 HAND1 Symbol;Acc:HGNC:4807] ENSG00000096996 0 -1.40936482 8 IL12RB1 interleukin 12 receptor, beta 1 [Source:HGNC Symbol;Acc:HGNC:5971] ENSG00000275582 0 -1.05575947 8 RP4-681N20.5 group FRD, member 1 [Source:HGNC ENSG00000244476 0 -1.23492596 8 ERVFRD-1 Symbol;Acc:HGNC:33823] ENSG00000165685 0 -1.10987961 8 TMEM52B transmembrane protein 52B [Source:HGNC Symbol;Acc:HGNC:26438] ENSG00000172331 0 -1.20450079 8 BPGM 2,3-bisphosphoglycerate mutase [Source:HGNC Symbol;Acc:HGNC:1093] thymocyte selection-associated high mobility group box [Source:HGNC ENSG00000198846 0 -1.42254609 8 TOX Symbol;Acc:HGNC:18988] ENSG00000258521 0 -1.0229865 8 RP11-638I2.9 ENSG00000279133 0 -1.46999903 8 RP11-342K2.1 ENSG00000121905 0 -2.21994573 8 HPCA hippocalcin [Source:HGNC Symbol;Acc:HGNC:5144] ENSG00000232810 0 -1.4782116 8 TNF tumor necrosis factor [Source:HGNC Symbol;Acc:HGNC:11892] family with sequence similarity 101, member A [Source:HGNC ENSG00000178882 0 -1.71201963 8 FAM101A Symbol;Acc:HGNC:27051] oxidized low density lipoprotein (lectin-like) receptor 1 [Source:HGNC ENSG00000173391 0 -1.01327133 8 OLR1 Symbol;Acc:HGNC:8133] ENSG00000257671 0 -1.03664909 8 RP3-416H24.1 ENSG00000269826 0 -1.64046441 8 RP11-158I3.3 ENSG00000176907 0 -1.2182476 8 C8orf4 open reading frame 4 [Source:HGNC Symbol;Acc:HGNC:1357] hepatic and glial cell adhesion molecule [Source:HGNC ENSG00000165478 0 -1.21164831 8 HEPACAM Symbol;Acc:HGNC:26361] chromosome 15 open reading frame 54 [Source:HGNC ENSG00000175746 0 -1.57742953 8 C15orf54 Symbol;Acc:HGNC:33797] purinergic receptor P2X, ligand gated ion channel, 2 [Source:HGNC ENSG00000187848 0 -2.24446361 8 P2RX2 Symbol;Acc:HGNC:15459]

* bin number refers to quadrant map in figure S2.