MOLECULAR DOCKING STUDIES OF CANTHIN-6-ONE FROM SIMAROUBA GLAUCA AGAINST EGFR TYROSINE KINASE

MOLECULAR DOCKING STUDIES OF CANTHIN-6-ONE FROM SIMAROUBA GLAUCA AGAINST EGFR TYROSINE KINASE

Chirag Prajapati^* and M. N. Reddy

Department of Biosciences, Veer Narmad South Gujarat University, Surat - 395007, Gujarat, India.

ABSTRACT: In recent years, in silico approaches have been predicting novel drug targets. The objective of this study was to analyze the inhibitory action of canthin-6-one alkaloid by computational docking studies. For this study, natural metabolite canthin-6-one isolated from Simarouba glauca was used as ligand for molecular interaction. The crystallographic structure of molecular target Epidermal Growth Factor Receptor (EGFR) tyrosine kinase was obtained from Protein Data Bank (PDB) database (PDB ID: 1M17). Gemcitabine, Cisplatin and Thiotepa were taken as the standard for comparative analysis. Computational docking analysis was performed by using online program PATCHDOCK. The canthin-6-one showed optimum binding affinity with a molecular target (EGFR - tyrosine kinase) with the binding energy of (-248.25) as compared to the standard drugs Gemcitabine (-188.48), Cisplatin (-45.26) and Thiotepa (-190.89). These results indicated that canthin-6-one could be one of the potential ligand to treat cancer. These potential drug candidates can further be validated in wet lab studies for its proper function.

EGFR - tyrosine kinase, Binding energy, Docking studies, Canthin-6-one

INTRODUCTION: Simarouba glauca belongs to family Simaroubaceae, commonly known as “The Paradise Tree” or “King Oil Seed Tree” or “Laxmitaru Tree”, is a versatile multipurpose evergreen tree having a height of 7-15 m with tap root system. It is a poly-gamo-dioecious tree and a potential source of biodiesel ¹. In India, it is mainly observed in Andhra Pradesh, Karnataka and Tamil Nadu etc. It can adapt a wide range of temperature, has the potentiality to produce 2000 - 2500 kg seed/ha/year ² can grow well in marginal lands / wastelands with degraded soils ³ and therefore considered as a major forest tree ⁴.

This plant is well known for its different types medicinal and pharmacological properties. The bark and leaf extract of S. glauca is well known for its different types of pharmacological properties such as haemostatic, antihelmentic, antiparasitic, antidysenteric, antipyretic and anticancerous ⁵. The leaf, fruit, pulp and seed of S. glauca are known to possess medicinal properties such as analgesic, antimicrobial, antiviral, astringent, emmenagogue, stomachic, tonic and vermifuge ⁶.

J. F. Rivero-Cruz et al., (2005) have demonstrated activity-guided fractionation of a chloroform-soluble extract of Simarouba glauca twigs collected from a plot in southern Florida afforded six canthin-6-one type alkaloid derivatives, canthin-6-one, 2- methoxycanthin- 6- one, 9-methoxycanthin-6-one, 2-hydroxycanthin-6-one, 4, 5- dimethoxycanthin- 6- one and 4, 5- dihydroxy-canthin-6-one ⁷.

Canthin-6-one was first isolated in 1952 by Haynes et al., from Australian Pentaceras australis (Rutaceae) ^{8, 9}. Since then, more than forty members of this class of alkaloids have been reported from several plants primarily of the Rutaceae ^{9, 10} and Simaroubaceae ^{9, 7} families, but also from Malvaceae, ⁹ Amaranthaceae, ⁹ Caryophyllaceae, ^{9, 11} Zygophyllaceae, ¹² and recently from fungi (Boletus curtisii Berk.) ¹³. Several of these alkaloids have been bioassayed and show interesting pharmacological activities ^{9, 14, 15}. Canthin-6-one and some derivatives isolated from Zanthoxylum chiloperone var. angustifolium displayed interesting antifungal activities ^{16, 17}.

Gemcitabine is a nucleoside analog used as chemotherapy. Gemcitabine is used in various carcinomas: non-small cell lung cancer, pancreatic cancer, bladder cancer and breast cancer ²⁰. Cisplatin is one of the most potent chemotherapy drugs widely used for cancer treatment ²¹. N, N'N'-triethylenethiophosphoramide (Thio TEPA) is a cancer chemotherapeutic member of the alkylating agent group, now in use for over 50 years. It is a stable derivative of N, N', N''- triethylene-phosphoramide (TEPA). It is mostly used to treat breast cancer, ovarian cancer and bladder cancer ²².

Epidermal growth factor receptors (EGFRs) are a large family of receptor tyrosine kinases (TK) expressed in several types of cancer, including breast, lung, esophageal, and head and neck. EGFR and its family members are the major contributors of a complex signalling cascade that modulates growth, signalling, differentiation, adhesion, migration and survival of cancer cells. Due to their multidimensional role in the progression of cancer, EGFR and its family members have emerged as attractive candidates for anti-cancer therapy ¹⁸. Specifically the aberrant activity of EGFR has shown to play a key role in the development and growth of tumour cells, where it is involved in numerous cellular responses including proliferation and apoptosis ¹⁹. In this study, docking of target protein (EGFR tyrosine kinase) was carry out with compound canthin-6-one and to evaluate the compound docking and active site binding.

MATERIALS AND METHODS:

Preparation of Macromolecule: The three dimensional structure of epidermal growth factor receptor (EGFR) tyrosine kinase (PDB ID: 1M17) was downloaded from the RCSB protein Data Bank ²³. The hydrogen atoms were added to the target protein molecule after removing the water molecules for docking.

Selection and Preparation of Ligand Structure: Ligand, which interacts with protein’s binding sites, is a small molecule. There are several possible mutual conformations in which binding may occur. These are commonly called binding modes ²⁴. The SDF file of canthin-6-one was obtained from PubChem database ²⁵ and then converted to PDB file with the help of Open Babel ²⁶ and the PDB file of standard drugs Gemcitabine, Cisplatin and Thiotepa was taken from DrugBank Database ³³.

Analysis of Target Active Binding Sites: The enzyme epidermal growth factor receptor (EGFR) tyrosine kinase was retrieved from the RCSB Protein Data Bank with PDB ID: 1M17. Active sites of the enzyme were identified by CASTp ³⁷ server. CASTp (Computed Atlas of Surface Topography of proteins) is used to verify the binding sites of a protein. It includes annotated functional information of specific residues on the protein structure ²⁷.

Molecular Docking Analysis: A computational ligand‑target docking approach was used to analyze structural complexes of the epidermal growth factor receptor (EGFR) tyrosine kinase with canthin-6-one and standard drugs in order to understand the structural basis of this protein target specificity. Finally, docking was carried out by online program PATCHDOCK ^{28, 34}. PATCHDOCK program serves to find different docking transformations which can yield good complementarity in its molecular shape based on molecular docking algorithm. The PDB format of both protein and alkaloid including standard drugs was sent to PATCHDOCK server for docking. The docked structure was further analyzed by using UCSF Chimera ³⁶.

RESULT AND DISCUSSION: In this present study, the interactions between the epidermal growth factor receptor (EGFR) tyrosine kinase (target) with canthin-6-one and standard drugs (ligands) was studied to explore their binding mode, docking study was performed using online program PATCHDOCK with UCSF Chimera. Epidermal growth factor receptor (EGFR) tyrosine kinase (PDB ID: 1M17) structure was derived from RCSB Protein Data Bank and used as a target for docking simulation. The details of canthin-6-one and standard drugs (Gemcitabine, Cisplatin and Thiotepa) are mentioned in Table 1. The 3D structure of protein and ligands was visualised by using Rasmol ³⁵ as show in Fig. 1 and Fig. 2 respectively.

TABLE 1: PROPERTIES OF CANTHIN-6-ONE COMPOUND AND REFERENCE DRUGS

FIG. 1: RASMOL VIEW OF 3D STRUCTURE OF EGFR - TYROSINE KINASE

Binding Site of the Protein: The detection of ligand ‐ binding sites is often the starting point for protein function identification and drug discovery. In our study, CASTp server predicted active site of our target protein - Epidermal growth factor receptor (EGFR) tyrosine kinase as shown in Fig. 3. The active sites of EGFR - tyrosine kinase protein comprises of amino acid residues are LEU694, GLY695, GLY697, ALA698, PHE699, VAL702, LYS704, ALA719, LYS721, GLU722, LEU723, LYS730, ALA731, LYS733, GLU734, ILE735, ASP737, GLU738, VAL741, MET742, CYS751, ARG752, LEU764, THR766, GLN767, LEU768, MET769, PRO770, GLY772, CYS773, ASP776, ARG812, ASP813, ARG817, LEU820, LYS828, THR830, ASP831, PHE832, GLY833, LEU834, ALA835, LYS836, ALA847, GLU848, GLY849, LYS851, VAL852.

Canthin-6-one                          Gemcitabine

Cisplatin                                 Thiotepa

FIG. 2: RASMOL VIEW OF 3D STRUCTURE OF LIGANDS

FIG. 3: ACTIVE SITES CAVITY OF EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) TYROSINE KINASE (1M17) FROM CASTP ONLINE SERVER

Interaction Studies: The goal of ligand-protein docking is to predict the predominant binding model(s) of a ligand with a protein of known three dimensional structure ²⁹. To study the binding mode of canthin-6-one in the binding site of epidermal growth factor receptor (EGFR) tyrosine kinase (target enzyme), docking studies was performed and energy values were calculated from the docked conformations of the protein-inhibitor complexes. The target protein - EGFR tyrosine kinase with canthin-6-one and standard drugs was docked using an automated molecular docking server PATCHDOCK in order to find out how the chemical compounds inhibit the target protein structure EGFR - tyrosine kinase based on the negative binding affinities values ³⁸ as shown in Fig. 4, 5, 6 and 7.

FIG. 4: DOCKING SCORE OF CANTHIN-6-ONE WITH EGFR-TYROSINE KINASE IS (-248.25)

FIG. 5: DOCKING SCORE OF GEMCITABINE WITH EGFR-TYROSINE KINASE IS (-188.48)

FIG. 6: DOCKING SCORE OF CISPLATIN WITH EGFR-TYROSINE KINASE IS (-45.26)

FIG. 7: DOCKING SCORE OF THIOTEPA WITH EGFR-TYROSINE KINASE IS (-190.89)

Docking studies yielded crucial information concerning the orientation of the inhibitors in the binding pocket of the target protein. The minimum binding energy indicated that the epidermal growth factor receptor (EGFR) tyrosine kinase (target enzyme) was successfully docked with canthin-6-one as showed in the Table 2 and Fig. 8.

TABLE 2: DOCKING SCORE OF CANTHIN-6-ONE AND REFERENCE DRUGS WITH EGFR-TYROSINE KINASE

The molecular docking results clearly show that the binding values of Canthin-6-one with EGFR tyrosine kinase are (-248.25). Whereas the binding value of the existing drugs Gemcitabine, Cisplatin and Thiotepa with EGFR tyrosine kinase is (-188.48), (-45.26) and (-190.89) respectively as in Table 2. Similar type of studies with Quercetin and Resveratrol compound was performed by Muthukala et al., ³⁰ and Manimaran et al., ³¹ respectively. The protein-ligand interaction plays an important role in structural based designing ³². Epidermal growth factor receptor (EGFR) tyrosine kinase protein residues Thr 766, Thr 830 was formed H-bond with canthin-6-one molecule. Canthin-6-one showed relatively good binding affinity (-248.25) as compared to Gemcitabine, Cisplatin and Thiotepa as standard which showed minimum binding energy of (-188.48), (-45.26) and (-190.89) respectively. Finally, Canthin-6-one with EGFR tyrosine kinase showed negatively high binding values when compared to the existing drugs, Gemcitabine, Cisplatin, Thiotepa. Thus, we can say that Canthin-6-one is a potential anticancer agent for EGFR tyrosine kinase protein structure which is clearly shown in Fig. 8.

Canthin-6-one                               Gemcitabine

Cisplatin                                   Thiotepa

FIG. 8: HYDROGEN BONDING INTERACTIONS BETWEEN LIGANDS WITH EGFR-TYROSINE KINASE

In the present in silico investigation, we elucidated one finding that we used the alkaloid compound (Canthin-6-one) derived from plant (Simarouba glauca) and found that this bioactive compound acted as potential anticancer agent for the target protein, namely, EGFR tyrosine kinase. Analysis of ligand binding interaction with the Epidermal growth factor receptor (EGFR) tyrosine kinase protein can be useful for new preventive and therapeutic drug for cancer. Based on the molecular drugs docking and binding affinities of the target protein EGFR tyrosine kinase with the ligands, it was found that the alkaloid Canthin-6-one has high binding values than the existing drugs Gemcitabine, Cisplatin, Thiotepa. The results obtained from this study would be useful in both understanding the inhibitory mode as well as in rapidly and accurately predicting the activities of new inhibitors on the basis of docking scores.

CONCLUSION: In this study, the molecular docking was applied to explore the binding mechanism and to correlate its docking score with the activity of Canthin-6-one compound. Docking studies of the Canthin-6-one with epidermal growth factor receptor (EGFR) tyrosine kinase showed that this ligand is good molecule which docks well with EGFR - tyrosine kinase target. The results are helpful for the design and development of novel drug having better inhibitory activity against various types of cancer. From this study, we conclude that Canthin-6-one is one of the best anticancer phytochemical agents. These potential drug candidates can further be validated in wet lab studies for its proper function.

ACKNOWLEDGEMENT: The authors are thankful to UGC for providing BSR fellowship as a part of financial support and the staff members of Department of Biosciences and authorities of Veer Narmad South Gujarat University for providing support to the study.

CONFLICT OF INTEREST: The authors report no conflict of interest.

REFERENCES:

1. Armour RP: Investigations on Simarouba glauca in EI Salvador. Economic Botany 1959; 13: 41-66.
2. Joshi S and Hiremath S: Simarouba - A potential oilseed tree. Current Science 2000; 78: 694-697.
3. Govindaraju K, Darukeshwara J and Srivastava AK: Studies on protein characteristics and toxic constituents of Simarouba glauca oil seed meal. Food Chem. Toxicol. 2009; 47: 1327-1332.
4. Duhan A, Suthar Y, Moudgil H and Duhan S: Effect of processing on seed oil of Simarouba glauca (DC): an underutilized plant. ARPN Journal of Agricultural and Biological Science 2011; 6: 16-20.
5. Patil MS and Gaikwad DK: A Critical Review on Medicinally Important Oil Yielding Plant Laxmitaru (Simarouba glauca ). India. J Pharm Sci and Res. 2011; 3: 1195-1213.
6. Joshi S and Joshi S: Oil Tree- Laxmitaru glauca. University of Agricultural sciences, Bangalore and Indian council of Agricultural Research, New Delhi, India 2002; 86.
7. Rivero-Cruz JF, Lezutekong  R, Lobo-Echeverri T, Ito  A, Mi  Q, Chai HB, Soejarto  DD, Cordell GA, Pezzuto  JM, Swanson  SM, et al.: Cytotoxic constituents of the twigs of Simarouba glauca collected from a plot in southern Florida. Phytother. Res. 2005; 19: 136–140.
8. Haynes HF, Nelson ER and Price JR: Alkaloids of the Australian Rutaceae:Pentaceras australis  F.I. Isolation of the Alkaloids and Identification of Canthin-6-one. Aust. J. Sci. Res. Ser. A 1952; 5: 387–400.
9. Ohmoto T and Koike K: Review: In the Alkaloids; Brossi, A., Ed.; Academic: New York 1989; 36: 135-170.
10. Rahman MM, Islam MA, Khondkar P and Gray AI: Alkaloids and lignans fromZanthoxylum budrunga (Rutaceae). Biochemical Systematics and Ecology 2005; 33: 91–96.
11. Hsieh PW, Chang FR, Lee KH, Hwang TL, Chang SM and Wu YC: A new anti-HIV alkaloid, drymaritin, and a new c-glycoside flavonoid, diandraflavone, from Drymaria diandra. Nat. Prod. 2004; 67: 1175–1177.
12. Ma ZZ, Hano Y, Nomura T and Chen YJ: Alkaloids and phenyl propanoids fromPeganum nigellastrum. Phyto-chemistry 2000; 53:1075–1078.
13. Brockelmann MG, Dasenbrock J, Steffan B, Steglich W, Wang Y, Raabe G and Fleischhauer J: An unusual series of thiomethylated canthin-6-ones from the North American mushroom Boletus curtisii. Eur. J. Org. Chem. 2004; 4856–4863
14. Murakami C, Fukamiya N, Tamura S, Okano M, Bastow KF, Tokuda H, Mukainaka T, Nishino H and Lee KH: Multidrug-resistant cancer cell susceptibility to cytotoxic quassinoids, and cancer chemopreventive effects of quassinoids and canthine alkaloids. Bioorg. Med. Chem. 2004; 12: 4963–4968.
15. Ferreira ME, de Arias AR, de Ortiz ST, Inchausti A, Nakayama H, Thouvenel C, Hocquemiller R and Fournet A: Leishmanicidal activity of two canthin-6-one alkaloids, two major constituents of Zanthoxylum chiloperone angustifolium. J. Ethnopharmacol 2002; 80: 199–202.
16. Thouvenel C, Gantier JC, Duret P, Fourneau C, Hocquemiller R, Ferreira ME, de Arias AR and Fournet A: Antifungal compounds from Zanthoxylum chiloperone angustifolium. Phytother. Res 2003; 17: 678–680.
17. Dai J, Li N, Wang J and Schneider U: Fruitful Decades for Canthin-6-ones from 1952 to 2015: Biosynthesis, Chemistry, and Biological Activities.Molecules. 2016; 21: 493.
18. Grandis JR and Sok JC: Signaling through the epidermal growth factor receptor during the development of malignancy. Pharmacol Ther. 2004; 102: 37-46.
19. Wells A: EGF receptor. Int J Biochem Cell Biol. 1999; 31: 637-43.
20. Available from: https://www.drugbank.ca/drugs/DB00441.
21. Ana-Maria F, Dietrich B: Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects. Cancers 2011; 3: 1351-1371.
22. Available from: https://www.drugbank.ca/drugs/DB04572.
23. Available from: http://www.rcsb.org/pdb/home/home.do.
24. Kitchen DB, Decornez H, Furr JR and Bajorath J: Docking and scoring in virtual screening for drug discovery: methods and applications. Nature reviews. Drug discovery 2004; 3: 935–49.
25. Wang Y, Xiao J, Suzek TO, Zhang J, Wang J and Bryant SH: PubChem: a public information system for analyzing bioactivities of small molecules. Asian J Biotech 2009; 4: 120-128.
26. O’Boyle N, Banck M, James CA, Morley C, Vandermeersch T and Hutchison GR: Open Babel: An open chemical toolbox. Journal of Cheminformatics 2011; 3: 33.
27. Liang J, Edelsbrunner H and Woodward C: Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design. Prot Sci 1998; 7: 1884-97.
28. Duhovny D, Nussinov R and Wolfson HJ: Efficient Unbound Docking of Rigid Molecules. In Gusfield et al., Proceedings of the 2'^nd Workshop on Algorithms in Bioinformatics(WABI) Rome, Italy, Lecture Notes in Computer Science 2452, Springer Verlag 2002; 185-200.
29. Mittal RR, McKinnon RA and Sorich MJ: Comparison data sets for benchmarking QSAR methodologies in lead optimization. J Chem. Inf. Model 2009; 49: 1810-1820.
30. Borappa M, Kanagarajan S and Kamalanathan A: In silico docking of Quercetin compound against the Hela cell line proteins. Int. J. Curr. Pharma. Res. 2015; 7: 13-16.
31. Manimaran M, Sivakumari K and Ashok K: Molecular Docking Studies of Resveratrol against the human oral cancer cell line proteins (KB Cells). Int. J. Curr. Adv. Res. 2015; 4: 415-420.
32. Heena VS, Sunil HG and Rama P: Molecular- Docking studies of potent Anticancer Agent. J. Comput. Sci. Syst. Biol 2012; 5: 012-015.
33. Available from: https://www.drugbank.ca/.
34. Schneidman-DD, Inbar Y, Nussinov R and Wolfson HJ: PatchDock and SymmDock: servers for rigid and symmetric docking. Nucl. Acids. Res. 2005; 33: W363-367.
35. Roger S and James MW: RasMol: Biomolecular graphics for all. Trends in Biochemical Sciences (TIBS). 1995; 20: 374.
36. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC and Ferrin TE: UCSF Chimera - A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004; 25: 1605–1612.
37. Joe D, Zheng O, Jeffery T, Andrew B, Yaron T and Jie L: CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated resiudes. Nucleic Acid Research 2006; 34: W116-W118.
38. Mashiach E, Schneidman DD, Peri A, Shavit Y, Nussinov R and Wolfson HJ: An integrated suite of fast docking algorithms. Proteins 2010; 78: 3197- 204.
    

    ---

    How to cite this article:

    Prajapati C and Reddy MN: Molecular docking studies of canthin-6-one from Simarouba glauca against EGFR tyrosine kinase. Int J Pharm Sci Res 2017; 8(12): 5130-36.doi: 10.13040/IJPSR.0975-8232.8(12).5130-36.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---