Analysis of Therapeutic Potential and Computer Aided Virtual Screening of Elaeocarpus Ganitrus

IJSTE - International Journal of Science Technology & Engineering | Volume 4 | Issue 1 | July 2017 ISSN (online): 2349-784X

Analysis of Therapeutic Potential and Computer Aided Virtual Screening of Elaeocarpus Ganitrus Arpitha B M Assistant Professor Dayananda college of Engineering, Bengaluru

ShilpaShree N Assistant Professor Dayananda college of Engineering, Bengaluru

Naik Soumya Lakshmi P.G Student Dayananda college of Engineering, Bengaluru

Abstract Cancer accounts for 12% of the death worldwide. Most of the existing treatments for cancer have not revealed much successful results. Therefore, there is a requirement for a novel therapeutic approach. Cucurbitacins and ellagic acid are the two utmost important compounds present in Elaeocarpus ganitrus. Cucurbitacins belong to a group of tetracyclic triterpenoids and it is found to exhibit anti-tumor, anti-inflammatory, anti-microbial, anti-diabetic and hepatoprotective properties. Ellagic acid is found to be a polyphenol compound and it exhibits anticarcinogenic, antioxidant and antifibrosis properties. Present study, focuses on the inhibitory potential of cucurbitacins and ellagic acid against various cancers by computer aided virtual screening. 19 receptors were selected based on the literature study, depending on its functionality in various cancers. iGEMDOCK was used to carry out molecular docking and to study the interactions between cucurbitacins and ellagic acid against the selected target receptors. The docking studies revealed the anticancer properties of the ligands through the effective interaction pattern with cancer receptors. All the three ligands cucurbitacin D, cucurbitacin F and ellagic acid showed the best interaction with nitric acid synthase (NOS2) receptor with binding energy -153.27 kcal/mol, -154.09 kcal/mol and -134.22 kcal/mol respectively. This study would promote the development of novel drug candidates against various types of cancer when present therapies are not found to be very helpful.

Keywords: Anticancer, Cucurbitacins, Elaeocarpus ganitrus, Ellagic acid, iGEMDOCK ________________________________________________________________________________________________________ I.

INTRODUCTION

Elaeocarpus ganitrus is a large broad leaved evergreen tree which grows from the gangetic plain to the foothills of Himalayas. It is commonly known as Rudraksha . Elaeocarpus species belongs to the Elaeocarpaceae family (1). It has been treasured in all the ancient ayurvedic texts for its exceptional therapeutic properties. The active components presents in rudraksha are elaeocarpine, elaeocarpidine, rudrakine, isoelaeocarpine, epiisoelaeocarpiline, epielaeocarpiline, alloelaeocarpiline and pseudoelaeocarpiline (2).

The presence of ellagic acid and cucurbitacin was revealed from the phytochemical analysis of the bark of rudraksha which have shown cytotoxicity against tumour cells (3). Elaeocarpus ganitrus is believed to have several traditional uses such as blood purifier, rectifies chicken pox, cures piles, helps in blood pressure management etc. Elaeocarpus ganitrus is noted to have innumerable pharmacological activities that involve analgesic, hypoglycemic, anti-inflammatory, antiasthmatic, antidepressant, antihypertensive, antimicrobial, anticancer, antiulcerogenic, and anticonvulsant (2). Different extracts of Elaeocarpus ganitrus’s leaves, bark and fruits have been tested on human cancerous cell lines and on animal models (1). Cancer is identified as an uncontrolled growth and spread of abnormal cells. Cancer is the major cause of disease worldwide (4). To prevent this cancer burden, there is a significant need for a better anticancer therapeutics. Cucurbitacins are primarily known for their diverse biological and pharmacological properties, especially the anticancer activity (5) and found to act against various cancer (6). Ellagic acid is also found to have anticancer activity against various types of cancer (7). Ellagic acid, cucurbitacin D and cucurbitacin F have been identified and tested against the human oral epidermoid carcinoma cell line. These three compounds showed pronounced cytotoxic activity on the human cancerous cell lines (2).

The main purpose of our study is to evaluate the therapeutic potential of ellagic acid, cucurbitacin D and cucurbitacin F against various cancer targets by computer aided virtual screening. Through the docking studies, the interactions were analyzed between the receptors of different types cancer and ellagic acid, cucurbitacin D and cucurbitacin F. The receptor of different types of cancer includes oral cancer, gastric cancer, pancreatic cancer, breast cancer, cervical cancer etc.

II. METHODOLOGY Selection of Receptors The receptors for our study were selected based on their functionality in different types of cancer pathways which were analyzed from Kyoto Encyclopedia of Genes and Genomes (8). The different types of cancers includes, Non-small cell lung cancer, Oral

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Analysis of Therapeutic Potential and Computer Aided Virtual Screening of Elaeocarpus Ganitrus (IJSTE/ Volume 4 / Issue 1 / 001)

cancer, Gastric cancer, pancreatic cancer, cervical cancer, penile cancer, breast cancer, hepatocellular carcinoma, small cell lung cancer, prostate cancer, endometrial cancer, glioma, esophageal cancer, bladder cancer, thyroid cancer, ovarian cancer, choriocarcinoma, colorectal cancer, cancer of anal canal, malignant pleural mesothelioma, synovial sarcoma, hairy-cell leukemia, osteosarcoma, alveolar rhabdomyosarcoma, multiple myeloma, laryngeal cancer, chronic myeloid leukemia (CML), burkitt lymphoma, adult T-cell leukemia, adrenal carcinoma, chronic lymphocytic leukemia (CLL), Hodgkin . The three dimensional structures of the cancer receptors were retrieved from PDB database.

Selection of Ligands The ligands were selected from extensive literature survey based on their cytotoxic activity against the human cancerous cell lines.

Structures of ellagic acid, cucurbitacin D and cucurbitacin F were obtained from NCBI PubChem database (9). The pharmacokinetics properties of ligand were evaluated using Pre-ADMET tool. Druglikeliness, ADME and toxicity properties were analyzed for all the three ligands. In druglikeliness, it predicts the suitability and violations of CMC like rule, lead like rule, MDDR like rule, rule of five and WDI like rule. In ADME, it predicts the blood brain barrier penetration, human intestinal absorption, Caco2 cell permeability and MDCK cell models for oral drug absorption prediction and skin permeability. And in toxicity, it predicts the toxicity based on AMES and rodent carcinogenicity assays.

Interaction Studies of Receptor and Ligand Docking is a process which is used to predict the orientation of the small molecules to the protein targets so as to determine the activity and affinity of the small molecule. Determination of the binding mode and affinity between the receptor and ligand is crucial in the understanding of the interaction mechanisms between them (10). Molecular docking was performed by using igemdock (11) . iGEMDOCK makes use of the genetic algorithm where, it selects the best solution from the population by calculating their fitness. Genetic algorithm is a general purpose optimization and search technique which is based on evolutionary process of natural selection. Genetic algorithm permits the individuals of a population to evolve under certain specific selection rules to a condition that maximizes the fitness function (12).

III. RESULT AND DISCUSSION Selection of Receptors The receptors for our study were selected based on their functionality in different types of cancer pathways. Nineteen cancer receptors were selected for the study. Phosphatase and tensin homolog (PTEN) is the second most mutated gene in human cancer and also a tumor suppressor gene which regulates negatively the PIK3/AKT pathway (13, 14). Matrix metallopeptidase 1(MMP1) are particularly associated with regulation of tumor cell growth, cell adhesion, apoptosis, cell signaling, metastasis, tumor cell invasion, immune survelliance amd angiogenesis. MMP1 gene when overexpressed leads to initiation of pathogenesis of tissue destruction in various diseases (15). Breakpoint cluster region (BCR) gene’s normal product is cytosolic phosphoprotein (16). It physiological importance is not well defined so the BCR-ABL fusion proteins have frequently been studied. BCR-ABL is involved in pathogenesis of chronic myeloid leukemia (17). Breast cancer 2 (BRCA2) is mainly involved in genome integrity control and in cell proliferation (18). Germline mutation in BRCA2 gene leads to susceptibility of the individual to breast cancer, ovarian and other epithelial cancer (19). Murine double minute 2 proto-oncogene (MDM2) is involved in negative regulation of the P53 tumor suppressor (20). Cyclin-dependent kinase 4 (CDK4) may be involved in tumorigenesis (21). It is involved in regulation of G1 to S phase transition and also has kinase activity which depends on one of the cyclins of D-type (22). Serine/threonine kinase 11 (STK11) also denoted by LKB1 gene, acts as a tumor suppressor gene. The cellular functions of LKB1 are carried out by interactions with number of proteins (23). Protein kinase B (AKT2) is evolving as gene involved in tumorigenesis and plays a major role in human malignancies (24). It is important in cellular processes such as cell metabolism, proliferation and survival (25). Nitric oxide synthase 2 (NOS2) drives metastasis and tumor growth and it has evolved as an oncogene in estrogene receptor-negative breast cancer.

Nitric oxide regulates the positive and negative feedback of NOS2 (26). Adenomatous polyposis coli (APC) help in the regulation of apoptosis. It also is involved in shuttling of the β-catenin between the nucleus and cytoplasm (27). V-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2) belongs to an ErbB2 family which are memberane-bound receptor tyrosine kinase and plays a major role in development of diseases (28). Mut L homolog 1 (MLH1) is one of the DNA mismatch repair genes (MMR) (29) . It is involved in some of the principle cellular functions and also indicates any damages to the cellular functions (30). Mut S homolog 2 (MSH2) is also one of the essential DNA mismatch repair genes (31). It plays a crucial role in enabling genomic stability (32) . Fast cell surface death receptor (FAS) is a transmemberane molecule which belongs to a member of tumor necrosis factor receptor (TNFR) superfamily which triggers programmed cell death. It plays a major role in regulation of life span of lymphocyte, control of immune response and cell mediated cytotoxicity (33). Tumor protein p53 (TP53) plays a crucial role in maintaining cellular and genomic integrity (34). It is a tumor suppressor gene which is involved in the regulation of cellular apoptosis, DNA repair and cell cycle arrest. Therefore, it is called as guardian of genome. It is also involved in protection of the cellular genome from the UV exposure (35). Platelet-derived growth factor beta polypeptide (PDGFB) is a growth factor and cellular promoter that provides gene expression in neurons (36). It is involved in activation of the PDGF receptors (37). It helps in carrying out cellular responses including survival, migration and proliferation (38). Breast cancer type 1 (BRCA1) is a tumor suppressor gene which

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Analysis of Therapeutic Potential and Computer Aided Virtual Screening of Elaeocarpus Ganitrus (IJSTE/ Volume 4 / Issue 1 / 001)

plays an important role in maintaining genome stability, DNA repair, regulation of transcription (39), ubiquitination of protein, cell cycle checkpoint and apoptosis (40). Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) is a group of lipid kinases and its functionality involves controlling the signaling pathways involving cell adhesion, proliferation, motility and survival (41). PIK3CA’s mutation, upregulation and amplification has been reported in studies on ovarian cancer (42). Kirsten rat sarcoma viral oncogene homolog (KRAS) on activation encodes a protein that acts as stimuli for hormones, growth factors and cytokines via the cell surface receptors (43).

Selection of Ligands Ligands were selected from extensive literature survey. Cucurbitacin D, cucurbitacin F and ellagic acid were chosen based on their therapeutic potential to act against various types of cancer. Cucurbitacins belong to tetracyclic triterpenoids. Plants containing cucurbitacins have been identified for their analgesic, antimicrobial, antipyretic, antitumor and anti-inflammatory properties. There are 17 molecules of cucurbitacins ranging from cucurbitacin A to cucurbitacin T and cucurbitacin D is especially found to show important anticancer activity in cancer cell lines of humans (5). Cucurbitacins, generally are the inhibitors of JAK/STAT pathways.

Ellagic acid is a polyphenolic compound. It has antioxidant, antifibrosis and anticarcinogenic properties (7) . It plays an important role in preventing the formation of tumors.

Interaction Studies of Receptors and Ligands Docking was performed to analyze the interaction between the receptor and ligand and to predict the binding mode between them.

The docking was performed between the different cancer receptors and the ligands Cucurbitacin D, Cucurbitacin F and Ellagic acid. The cancer receptors act a drug target and the ligands as an inhibitor. The output given is the fitness table and the interaction table. The best docked complex was selected based on the lowest binding energy of docked complex, interactions such as vander waal’s, hydrogen bonding and electrostatic, and number of interacting residues. Our study showed that all three ligands cucurbitacin D, cucurbitacin F and ellagic acid showed effective interaction with NOS2 receptors with binding energy -153.27 kcal/mol, -154.09 kcal/mol and -134.22 kcal/mol respectively. (Figure 1) The interaction studies of all the three ligands with various receptors are tabulated. (Table 1)

(A) Docked complex of Cucurbitacin D with NOS2 and with its interacting residues.

(B) Docked complex of Cucurbitacin F with NOS2 and with its interacting residues.

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Analysis of Therapeutic Potential and Computer Aided Virtual Screening of Elaeocarpus Ganitrus (IJSTE/ Volume 4 / Issue 1 / 001)

(C) Docked complex of ELLAGIC ACID with NOS2 and with its interacting residues.

Fig. 1: Docked complex structures with interacting residues

RECEPTORS PTEN MMP1 BCR BRCA2 MDM2 CDK4 STK11 AKT2 AKT2 NOS2 APC ERBB2 MLH1 MSH2 FAS TP53 PDGFB BRCA1 PIK3CA KRAS

Table - 1 Binding Energy of the ligands and receptors CURARBITACIN D CURARBITACIN F ELLAGIC ACID Energy (kcal/mol) Energy (kcal/mol) Energy (kcal/mol) -79.7613 -120.352 -116.502 -126.103 -126.668 -111.416 -89.21 -96.986 -79.3421 -94.4869 -94.9928 -79.0727 -103.24 -97.4172 -89.5172 -111.709 -109.853 -88.958 -128.681 -130.162 -112.617 -119.906 -111.219 -88.6915 -120.435 -124.784 -120.312 -153.271 -154.094 -134.223 -93.8057 -96.1389 -77.9788 -106.166 -113.291 -95.5251 -112.608 -111.19 -86.6296 -112.577 -113.46 -121.215 -103.151 -102.64 -88.1525 -134.033 -123.531 -110.356 -108.616 -106.917 -86.4984 -119.122 -118.129 -97.4853 -113.04 -100.689 -99.8376 -146.935 -141.806 -120.256

IV. CONCLUSION Cancer is one of the second leading causes of death and is responsible for 12% of death worldwide. Currently available treatments for cancer causes drug resistance and adverse side effects. Hence, there is a need for an alternative promising therapy with potent anticancer properties. Cucurbitacin D and F possess excellent anti-tumor, anti-inflammatory, anti-microbial, anti-diabetic and hepato-protective properties. Ellagic acid exhibits anti-carcinogenic, antioxidant and anti-fibrosis properties. Present study revealed the inhibitory properties of cucurbitacins and ellagic acid against various cancer drug targets by bioinformatics approach.

The Insilico studies revealed that cucurbitacins and ellagic acid has the potential to act as a potent anticancer drug candidate. In this study, cucurbitacins showed better inhibitory activities against nitric acid synthase (NOS2), Kirsten rat sarcoma viral oncogene homolog (KRAS), tumor protein p53 (TP53) and mut L homolog 1 and ellagic acid showed better inhibition towards nitric oxide synthase (NOS2), mutS homolog 2 (MSH2), RAC serine/threonine-protein kinase (AKT2). Our study demonstrated the inhibitory properties of cucurbitacins and ellagic acid that would pave way for novel drug candidates against various types of cancer when

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Analysis of Therapeutic Potential and Computer Aided Virtual Screening of Elaeocarpus Ganitrus (IJSTE/ Volume 4 / Issue 1 / 001)

present therapies fail to overcome cancer. Hence, our study will help in the development of better therapeutic remedies against cancer.

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