Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206

International Journal of Current Research and Academic Review

ISSN: 2347-3215 Special Issue-3 (August-2016) pp. 195-206 Journal home page: http://w ww.ijcrar.com

Screening of Novel MAPK Inhibitor from Ethanolic Extract of Stem of aspera (Thumbai) using GCMS and In Silico Analysis

P.M. Keerthana1*, G. Shoba1,2 and R. Padmini1,2

1 Department of Biochemistry and Bioinformatics & Clinical Trial Management, Dr. MGR Janaki College of Arts and Science, Chennai, Tamil Nadu, 2 Department of Biotechnology, Mother Teresa Women‟s University, Kodaikanal, Tamil Nadu, India

*Corresponding author

KEYWORDS ABST RACT

Inflammation, Leucasaspera (Family: ) commonly known as “Thumbai” is Inflammatory bowel generally used as an insecticide and whole decoctions are traditionally disease, taken for cough and cold and to treat rheumatism, inflammation and bacterial MAP kinase, infections. The extracts of whole have been reported to have antiulcer,

Leucasaspera, antimicrobial, hepatoprotective, antinociceptive, antioxidant, cytotoxic and autodock. prostaglandin inhibitory activities. The mitogen activated protein kinases (MAPK) have been implicated in an ever-increasingly diverse array of pathways, including inflammatory signalling cascades. Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn‟s disease, are characterized by the perpetual production of inflammatory mediators. The

transduction pathway behind this over-production has highlighted the

potential mediating role for the MAPKs and their related signaling

components. The ethanolic extracts of Leucasaspera stems were investigated for their action in significant anti-inflammatory action of acute and chronic inflammation. The bioactive compounds of Leucasaspera includes 13, Docenyloxy, 2-(9-octadecenyloxy), pentadecanoicacid, cyclopropaneoctanic acid, 5,19-Cyclo-5a-androst-6-ene-3,17-dione. The emergence of bioinformatics has provided a platform to explore diseases at the molecular level using computational tech niques. In the present study, MAPK (Mitogen Activated Protein Kinase) drug targets were docked against bioactive compounds using AutoDock software. The highest binding energy was obtained using docking analysis and the protein–ligand interaction was studied and possible binding sites were predicted. The results suggested that the molecular target modulated by the bioactive constituents were useful indicators and may act as potent drugs for inflammatory bowel disease (IBD).

195

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 Introduction causes to the development of these conditions. Currently, three broad areas are Leucasaspera (Family: Lamiaceae) proposed to be involved in the development commonly known as „Thumbai is of IBD. distributed throughout India from the Himalayas down to Ceylon. Leucasasperais (i) genetic susceptibility including mutations also an antipyretic, it is a herb that has the of key proteins facilitating or enhancing the ability to help reduce fever. In some forms access of microbes to the epithelium of traditional medicine, the stem formed by and underlying layers. crushing the Samoolam, also known as the plant's flowers, seeds, roots, berries, bark or (ii) luminal antigenic drive (e.g. microbes leaves, can be inhaled to help treat nasal inhabiting the GI tract or food allergens) congestion, coughing, cold, headache and fever. In addition the juices of the flower (iii) environmental triggers, including stress can be extracted and used to help treat and smoking. Extensive genetic profiling sinusitis, as well as headaches. The plant is has succeeded in identifying several genes generally used as an insecticide, and whole or genetic loci, which have been associated plant decoctions are traditionally taken for with CD, UC or both conditions. Many of coughs and colds and to treat rheumatism, these genes or genetic loci have been found inflammation, and bacterial infections. A to be linked to the signalling pathways literature survey revealed that the extracts of regulating both the innate and adaptive whole plants have been reported to have immune system. antiulcer, antimicrobial, hepatoprotective, antinociceptive, antioxidant, cytotoxic, and The mitogen activated protein kinases prostaglandin inhibitory activities (MAPK) have been implicated in an ever- (Phytochemical and Biological Investigation increasingly diverse array of pathways, of two Bangladeshi Medicinal plants, including inflammatory signaling cascades. Leucasaspera and Curcuma zedoria A Inflammatory bowel diseases (IBD), such as dissertation submitted for the partial ulcerative colitis and Crohn‟s disease, are fulfillment of the course of Pharmaceutical characterized by the perpetual production of Research (PHRM 404) of the Department of inflammatory mediators. The transduction Pharmacy, East West University for the pathway behind this over-production has Degree of Bachelor of Pharmacy). highlighted the potential mediating role for the MAPKs and their related signaling Inflammatory Bowel Disease (IBD) components.

Inflammatory bowel disease (IBD) is an In the field of molecular modelling, docking umbrella term for several chronic is a method which predicts the preferred inflammatory conditions affecting the orientation of one molecule to a second gastrointestinal tract (GI), the two most when bound to each other to form a stable common entities being Crohn‟s disease complex. Docking is universally used to (CD) and ulcerative colitis (UC). The predict the binding orientation of small detailed aetiology for these enigmatic molecule drug candidates to their protein diseases is not currently fully understood, targets in order to predict the affinity and and while the histology and prognosis for activity of the ligands. Hence, docking plays the patients differ between CD and UC, they a significant role in the rational drug design. share similarities in the proposed underlying

196

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 Molecular docking studies indicate how two from genome sequencing projects. It or more molecular structures interact with contains a large amount of information each other for example, drug and enzyme or about the biological function of proteins receptor of protein, fit together. Molecular derived from the research literature. The docking software‟s are mainly used in drug UniProt/Swissprot Knowledgebase research. The most important application of UniProtKB is the central access point for docking software is virtual screening. extensive curated protein information, Virtual screening selects the most interesting including function, classification, and cross- and promising molecules from an existing reference (Uniprot C, "The Universal database for advanced research. This places Protein Resource UniProt in 2010". Nucleic demands on the used computational method Acids Res, 38 Database issue: D142–D148, which must be fast and reliable (Yang et al., (2009) (http://www.uniprot.org/) 2011; Ai et al., 2011; Amuthalakshmi, 2013). PDB

Materials and Methods The PDB is a repository for 3-D structural data of proteins and nucleic acids. These Collection of plant materials data, typically obtained by X-ray crystallography or NMR Spectroscopy, are The fresh stems of Leucasaspera plant were submitted by biologists and biochemists collected from the region of Chennai, from around the world, are released into the Tamilnadu. The collected plant was public domain, and can be accessed for free. carefully examined, identified, weighed, air The mission of the PDB is to maintain a dried in the shade and powdered. 20gms of single Protein Data Bank Archive of stem powder was extracted with 200ml of macromolecular structural data that is freely ethanol by using the above apparatus for and publicly available to the global 24hrs. The extract was then filtered and community (www.rcsb.org/pdb). stored in air tight containers. Pfam GC-MS Compound Identification The Pfam database contains information In the present study the analysis of Bio- about protein domains and families. Pfam-A active compounds from Leucasaspera stem is the manually curated portion of the extract was carried out through GC-MS database that contains over 10,000 entries. analysis. The Ethanolic extract of For each entry a protein sequence alignment Leucasaspera stem was employed for GC- and a hidden Markov model is stored. These MS analysis. In this investigation only five hidden Markov models can be used to chemical constituents have been identified. search sequence databases with the HMMER package written by Sean Eddy. Preparation of protein Because the entries in Pfam-A do not cover all known proteins, an automatically Uniprot generated supplement is provided called Pfam-B. Pfam-B contains a large number of UniProt is a comprehensive, high-quality small families derived from clusters and free online database of protein sequence produced by an algorithm called ADDA. and functional information, mainly derived (http://pfam.sanger.ac.uk)

197

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 http://www.modelling.leeds.ac.uk/qsitefinde structures. It uses Genetic Algorithms for r/ the conformational search and is a suitable method for the docking studies. The ACD Chem Sketch technique combines simulated annealing for conformation searching with a rapid grid Advanced chemistry development's based method of energy evaluation. Auto ACD/chem sketch is a chemically intelligent Dock tools are used to prepare, run and drawing interface that allows you to draw analyze the docking simulations, in addition almost any chemical structure including to modeling studies (http://autodock. organics, organometallics, polymers, and scripps.edu/resources/tools). markush structures. Use it to produce professional looking structures and diagrams PyMOL for reports and publications (www.acdlabs.com/../chemsketch/). PyMOL is an open-source tool to visualize molecules available from (z. It runs on OPEN BABEL Windows, Linux and MacOS equally well. PyMOL has excellent capabilities in Open Babel is a chemical toolbox designed creating high-quality images from 3D to speak the many languages of chemical structures, it has well developed functions data. It‟s an open, collaborative project for manipulating structures and some basic allowing anyone to search, convert, analyze, functions to analyze their chemical or store data from molecular modeling, properties. The possibilities to write scripts chemistry, solid-state materials, and plugins as well as to incorporate biochemistry, or related areas PyMOL in custom software are vast and (http://openbabel.org/). superior to most other programs. PyMOL has been written mostly in the Python Pass online language (www.python.org).

PASS Online predicts over 3500 kinds of Docking studies biological activity, including pharmacological effects, mechanisms of Databases and tools action, toxic and adverse effects, interaction with metabolic enzymes and transporters, Bioinformatics is seen as an emerging field influence on gene expression, etc. with the potential to significantly improve Prediction is based on the analysis of how drugs are found, brought to the clinical structure activity-relationships for more than trials and eventually released to the 250,000 biologically active substances marketplace. Computer – Aided Drug including drugs, drug-candidates, leads and Design (CADD) is a specialized discipline toxic compounds. that uses computational methods to simulate drug receptor interactions. CADD methods Autodock are heavily dependent on bioinformatics tools, applications and data bases. The Auto Dock is a suite of automated docking structure of 2ojg was retrieved from PDB tools. The software is used for modelling database. Using Chemsketch the structures flexible small molecules such as drug of the compounds were generated by their molecules and its binding to receptor SMILES notation obtained from uniprot and proteins of known three dimensional

198

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 the structural analogues of these compound of UniProt (http://www.uniprot.org/ were sketched. The docking analysis was uniprot/). The source organism is Homo carried by AUTO DOCK docking software. sapiens. For our study we used bioinformatics tools, biological databases like PubMed, pubchem, Structure Retrieval uniprot, PDB (Protein Data Bank) and software‟s like auto dock and PYMOL. The three-dimensional structure of MAPK was available in the PDB database.The PDB Results and Discussion id is 2OJG. The 3D structure was visualized using the Rasmol Tool (shown in Fig. 4). Ethanolic extract of Leucasaspera

The Ethanolic extract of Leucasaspera stem MAPK have the specific domain region. was employed for GC-MS analysis. In this The protein kinase domain is a structurally investigation only six chemical constituents conservedprotein domain containing the have been identified (Table-1). They are 13- catalytic function of protein kinases. Docosenoic acid, 2-(9-octadecenyloxy), Cyclopropaneoctanoic acid, Pentadecanoic Preparation of Ligands acid, 5,19-Cyclo-5a-androst-6-ene-3. The peak levels of different chemical compounds GCMS analysis of Leucasaspera bioactive are shown in Fig 2. compounds are selected. The two- dimensional structures of the ligands were Autodock generated using the ACD/Chem Sketch tool.

Auto Dock is a suite of automated docking This software contains tools for 2D tools. The software is used for modelling cleaning, 3D optimization, and viewing. flexible small molecule such as drug These data are saved as a molecular format molecule binding to receptor proteins of file (MDL MOL format). The molecular known three dimensional structure. It uses format converter tool (Open Babel) is used Genetic Algorithms for the conformational to convert this file into the PDB format and search and is a suitable method for the is used during docking analysis. The docking studies. The technique combines structure and molecular formula of simulated annealing for conformation inhibitors were shown below. searching with a rapid grid based method of Docking Analysis of Mapk against energy evaluation. Auto Dock tools is used Bioactive Compounds to prepare, run and analyze the docking simulations, in addition to modeling studies. The inhibitors docked with MAPK receptor Auto Dock is the most cited docking using Autodock software (Version 4.2) . The software because it is very fast, it provides Graphical User Interface program "Auto- high quality predictions of ligand Dock Tools" was used to prepare, run, and conformations and good correlations analyze the docking simulations. Kollman between inhibition constants and united atom charges, solvation parameters experimental ones. and polar hydrogens were added into the Sequence Retrieval: Mapk receptor PDB file for the preparation of protein in docking simulation. The protein sequence for the P28482 was obtained from the protein sequence database

199

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 Table.1 (Bio Activity Chemicals Identified in (Ethanol Extract) GC-MS -Peak Report –TIC

No R.Time Name of the chemical M.F M.W Peak Area compound Area %

1 22.62 13-Docosenoic acid, methyl C22H42O2 338.56768 5006992 18.9% ester (Z). g/mol 2 19.47 2-(9-Octadecenyloxy) C21H42O3 342.55638 3692544 7.3% g/mol

3 19.8 Cyclopropaneoctanoic C20H35O2 308.49864 3594464 6.7% acid,2-((2- g/mol pentylcyclopropyl)methyl)- methyl ester. 4 17.18 Pentadecanoic acid, 14- C15H30O2 242.3975 4181456 32.3% methyl, methyl ester. g/mol 5 17.83 15, 19-Cyclo-5a-androst-6- C18H22O3 284.40 g/mol 3429568 8.3% ene-3, 17-dione

Table.2 Preparation of Ligands

COMPOUND NAME 2D STRUCTURE 3D STRUCTURE H-Bond H-Bond Rotatable XLogP DONOR ACCEPTOR Bond 3 Count 13-docosenoic acid 1 2 19 8.7

2-(9- 2 3 19 6.7 Octadecenyloxy)

Cyclopropaneoctan 1 2 14 7.7 oic acid

Pentadecanoic acid, 1 2 13 5.8 14-methyl, methyl ester..

15, 19-Cyclo-5a- 2 0 0 2.01 androst-6-ene-3, 17- dione

200

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 Table.3 Molecular Interactions between 13-DOCOSENOIC ACID and the MAPK

MAPK 13-Docosenoic acid Distance Residue Atom (Ǻ) ARG65 NH2 O 2.4 THR188 OG1 H 4.0

GLN103 OE1 O 2.6

Table.4 Molecular Interaction between Cyclopropaneoctanoic acid and the MAPK

MAPK Cyclopropaneoctanoic Distance Residue Atom acid (Ǻ) ARG 65 NH1 O 3.2 ARG 65 CZ H 3.O

Table.5 Molecular Interaction between Pentadecanic Acid andtheMAPK

MAPK pentadecanic acid Distance (Ǻ) Residue Atom ARG65 NH O 2.5 TYR188 CE2 H 5.8 TYR34 OG1 O 4.4

Table.6 Molecular Interaction between 5,19-cyclo-5a-androst-6-ene-3, 17-dione and the MAPK

MAPK 5, 19-cyclo-5a-androst-6- Distance ene-3, 17-dione (Ǻ) Residue Atom GLN103 NH2 O 3.2 LEU154 CD2 O 3.6 ASP109 OD1 O 2.6

Fig.1 Leucasaspera

 Kingdom: Plantae  Class: Dicotyledonae  Subclass:  Order: Tubiflorae  Family: Labiatae  Genus: Leucas

201

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 Fig.2 Inflammatory Bowel Disease

Fig.3 Bio Activity Chemical Identified in Leucaaspera (Ethonol Extract) GC- MS-Peak Report-TIC

Fig.4 3D Visualization Using Rasmol Domain Analysis

202

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 Fig.5 13-DOCOSENOIC ACID AND MAPK (a)Binding energy score (b) Interaction between MAPK and13-DOCOSENOIC ACID was visualized using Auto dock (c) Hydrogen bond interaction between MAPK and13-DOCOSENOIC ACID was visualized using Pymol

(a) (b)

(c) Fig.6 CYCLOPROPANEOCTANOIC ACID AND MAPK (a)Binding energy score (b) Interaction between MAPK andCYCLOPROPANEOCTANOIC ACID was visualized using Auto dock (c) Hydrogen bond interaction between MAPK andCYCLOPROPANEOCTANOIC ACID was visualized using Pymol

(a) (b)

203

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206

(c)

Fig.7 PENTADECANIC ACID AND MAPK (a)Binding energy score (b) Interaction between MAPK andPENTADECANIC ACIDwas visualized using Auto dock (c) Hydrogen bond interaction between MAPK andPENTADECANIC ACIDwas visualized using Pymol

(a) (b)

(c)

204

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 Fig.8 5, 19-CYCLO-5A-ANDROST-6-ENE-3, 17-DIONE AND MAPK (a)Binding energy score (b) Interaction between MAPK and5, 19-CYCLO-5A-ANDROST-6-ENE-3, 17- DIONEwas visualized using Auto dock (c) Hydrogen bond interaction between MAPK and5, 19- CYCLO-5A-ANDROST-6-ENE-3, 17-DIONEwas visualized using Pymol

(a) (b)

(c) Autodock results were analyzed to study the active site is closer, then the interactions is interactions and the binding energy of the much higher than the other sites. docked structure. The best ligand-receptor structure from the docked structures was Conclusions chosen based on the lowest energy and minimal solvent accessibility of the ligand. Phytochemical analysis in ethonolic extract The docking results were visualized using of Leucasaspera stem by using GC-MS the Acceryls Visualizer discovery studio revealed presence of 13,docenyloxy, tool. pantadecanicacid, cyclopropaneoctanic acid, 5,19-cyclo-5a-androst-6-ene-3,17-dione. A bond is formed between two atoms by Bioactive compounds from Leucasaspera overlapping the atomic orbitals. This overlap stem has anti-inflammatory activity and of atomic orbitals to form molecular orbitals insilico docking analysis was carried out occurs only at certain distances between the against drug target MAPK using Autodock atom. When the amino acid residues of the 4.2 software. The result shows that

205

Int.J.Curr.Res.Aca.Rev.2016; Special Issue-3: 195-206 compounds from Leucasasperastem has the course of PharmaceuticalResearch good binding energy with least docking (PHRM 404) of the Department of score and the bioactive compound. Based on Pharmacy, East West University for Binding energy and Hydrogen bond formed the Degree of Bachelor of Pharmacy. and Inhibitory constantvalue the docking Broom, O.J., B. Widjaya, J. Troelsen, J. results were analyzed. Cyclopropaneoctanic Olsen and O.H. Nielsen. acid forms two hydrogen bond interaction in Mcfarland, L.V. 2008. State-of-the-art of the docking energy of 1.24 Kcal/Mol and irritable bowel syndrome and 13-Docenyloxy, pentadecanicacid, 5,19-and inflammatory bowel disease research Cyclo-5a-androst-6-ene-3,17-dione forms in 2008. World J. Gastroenterol., 14: three hydrogen bond interaction with the 2625–9. docking energy of -1.46, -1.02, and -7.69 Mitogen activated protein kinases: a role in Kcal/Mol, respectively. The work is inflammatory bowel disease.ei_403 significant in emphasizing the potent 272-280. inhibitory effects of bioactive compounds on Molecular docking studies of B-RAF MAPK activity and may offer therapeutic expression inhibitors identified from advantages in thetreatment and prevention Strychnospotatorum(Thethankottai) of Bowel‟s disease. Phytol - A chemical constituent of Leucasaspera through HPTLC, U.G. References Basarkar Assistant Professor, P.G. Dept. of Botany, G.E. Society‟s, HPT Ai, Y., S.T. Wang. 2011. Med. Chem. Res., Arts RYK Science College, Nasik- 20: 1298–1317. 422 005, Maharashtra, India. Chemical Amuthalakshmi, S. 2013. Adv. Biol. Res., compounds investigation of lucas 7(6): 248-252. aspera leaves – a potential folklore Anandan, R., Eswaran, A., Doss, G. medicinal plant. Sangeetha and S.P. Anand. Sasikala, R.P., Stela, P.G. and Meena, K.S. Phytochemical and Biological Bioinformatics Infrastructure Facility Investigation of two Bangladeshi Centre, Queen Mary‟s College, Medicinal plants, Leucasaspera and University of Madras, Chennai, India. Curcuma zedoria. A dissertation Yang, Y., J. Qin. 2011. J. Chem. Inf. Model, submitted for the partial fulfillment of 51: 680–692.

How to cite this article:

Keerthana, P.M., G. Shoba and Padmini, R. 2016. Screening of Novel MAPK Inhibitor from Ethanolic Extract of Stem of Leucas aspera (Thumbai) usingGCMS and In Silico Analysis. Int.J.Curr.Res.Aca.Rev. Special Issue-3: 195-206.

206