Molecular Docking Studies of Bioactive Compounds from Allium sativum Against EML4-ALK Receptor

 

Padmini R^{1, 2}, Sitrarasi R¹, Razia M¹*

¹Department of Biotechnology, Mother Teresa Women’s University, Kodaikanal, Tamil Nadu

²Department of Biochemistry & Bioinformatics, Dr. MGR Janaki College of Arts and Science, Chennai, Tamil Nadu, India

 

ABSTRACT:

Lung cancer is the common cancer which leads to death in all developed countries and it is a malignant lung tumor characterized by uncontrolled cell growth in the tissues of the lung. Non-Small Cell Lung Cancer (NSCLC) which is a form of lung cancer, accounts for approximately 80% of all lung cancer cases in India. EML4-ALK, a fusion protein plays a major role in provoking lung cancer. Knowledge of the three-dimensional structure paves the way to understand the mechanism of protein and conduct structure based drug design. In order to develop 3D structure of EML4-ALK protein, homology modelling approach using the tool Modeller 9v14 was utilized. Garlic (Allium sativum) is a vegetable that belongs to allium family, is capable of allowing cancer cell death normally, the process called as apoptosis. The medicinal effects of garlic can be utilized for the prevention and cure of cancer. Molecular docking studies of the modelled EML4-ALK target protein with bioactive compounds of Allium sativum using AutoDock was performed. These computational studies will provide an insight of potential inhibitors against lung cancer.

 

 

INTRODUCTION:

Cancer is a collection of heterogeneous genetic diseases which is associated with abnormal cell growth. It is one of the big threat to human beings globally and currently,  there is considerable scientific and commercial interest has been developed to produce effective cancer medications¹. Lung cancer is the second most common cancer and  has the highest mortality rate worldwide. It is caused by the cells in the lung which becomes abnormal and multiplies uncontrollably to form a tumor².

 

It is a multifactorial disease which is mainly due to smoking and other factors are exposure to indoor and outdoor air pollution, exposure to radiation, and exposure to agents such as asbestos, nickel, chromium, and arsenic³. Lung carcinomas can be categorized based on its cell size as nonsmall cell lung carcinoma (NSCLC), including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma, which represents 80% of all lung cancer cases, and the remaining cases are small cell lung carcinoma (SCLC)^4,5.

 

A subset of non-small cell lung cancer harbors the EML4-ALK fusion gene. A small inversion within short arm of chromosome 2 results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Natural products, particularly dietary sources are important sources of potential chemotherapeutic agents and it is more economical and beneficial in identifying potential anticancer drugs⁵.

 

Garlic (Allium sativum) belongs to the Alliacae family, has been used has a traditional medicine, which has potent biological activity and has the ability to stimulate immunological response⁶.Garlic enhances immune system and exhibits various activities such as anticancer, hepatoprotective, antioxidant, hypoglycemic activity and it also reduces the blood cholesterol level which prevents cardiovascular disease. The chemopreventive properties of garlic is due to the high content of bioactive constituents⁷ that target several key events involved in the progress of various diseases, including cancer. Phytochemicals derived from garlic have been shown to suppress carcinogenesis⁸.  Excess garlic intake is associated with decreased risk of various types of cancer. The health benefits of garlic extracts could be attributed to phytochemicals such as Ajoene⁹, (S)-allylcysteine¹⁰, Alliin¹¹, Alicin¹², Diallyl disulfide¹³, Allylmethyl disulfide^{14, 15}.

 

Molecular docking approach is a new strategy in drug designing, which can be used to model the interaction between a small molecule and a protein at the atomic level, which allow us to characterize the behavior of small molecules in the binding site of target proteins as well as to elucidate fundamental biochemical processes¹⁶.This approach was utilized to prove that bioactive constituents can bind to receptor protein to find the potential inhibitors against lung cancer.

 

MATERIALS AND METHODS:

Sequence Retrieval:

UniProt¹⁷ is the major resource for storing and interconnecting information from various sources, and the most comprehensive catalog of protein sequence and functional annotation. The protein sequence for EML4-ALK gene of Homo sapiens was obtained from UniProt and its UniProt Id is J7MA22.

 

Struture Retrieval :

Comparative modeling depends on the knowledge of three-dimensional structure of homologous proteins¹⁸. The Template for EML4-ALK fusion protein for homology modeling was identified using BLASTP¹⁹ program against Protein Data Bank²⁰ (PDB) to identify the structural homologs. From the results, Crystal Structure of Human Anaplastic Lymphoma Kinase in Complex with Acyliminobenzimidazole Inhibitor (4FOB.PDB) is identified as a template.

 

Comparative Modeling of EML4 ALK Protein and Structure Validation :

Sequence alignment between the target and its respective template was used as input for model building process in MODELLER9v7²¹.The downloaded structure of the template was used to generate the 3D structure of EML4-ALK protein using the tool MODELLER9v.14. In order to identify the best model of respective drug targets, the models with low MOLPDF and DOPE score were assessed using PROCHECK²² validation package. From using PROCHECK statistics, structure verification was done to predict the quality of the 3D structure of the protein. Ramachandran plot helps to refine the disallowed residues using loop refinement and thereby the protein is refined. The refined protien is further used as a target for molecular docking studies.

 

Molecular Docking Studies:

Preparation of Ligand :

The major bioactive constituents of the Allium sativum which possess anti-cancer activity based on literature were retrieved from PubChem²³ database (Refer Table 1). Molinspiration²⁴, an online tool is used to analyze the molecular properties of the compounds. The ligands are imported to the workspace and preparation of them is done.  The 3Dstructure of the compounds was drawn using Chemsketch²⁵ and it is saved in Mdlmol format. These Mdlmol formats were converted into PDB format using OpenBabel²⁶, which were given as an input to AutoDock²⁷ tool. The PDB format of the ligands   was visualized using Discovery Studio Visualizer²⁸.

 

Table 1: Bioactive constituents of the Allium sativum

 

 

 

 

Molecular Docking:

Docking is a method, which predicts the preferred orientation of one molecule to another when bound to each other to form a stable complex. In silico molecular docking studies were carried out by AutoDock program which is recently introduced by Scripps Research Institute²⁹. The generated 3D structure of EML4-ALK protein was used as receptor and bioactive constituents of Allium sativum were used as ligands for docking studies.

 

RESULTS AND DISCUSSION:

Comparitive Modelling Studies:

Knowledge of the three-dimensional structure is a pre requiste for the studying the protein interactions, functions and their localization and it gives great importance for the design of drugs. To perform the homology modeling, the basic step is to find best matching template using similarity searching program like BLASTP against PDB database. Templates are selected on the basis of their sequence similarity with query sequence. From the results, Crystal Structure of Human Anaplastic Lymphoma Kinase in complex with Acyliminobenzimidazole Inhibitor (4FOB.PDB) was identified as a template for homology modelling (Figure1). The query sequence and template was then given as input to the MODELLER tool. Quality and reliability of structure was checked by PROCHECK, which analyze residue-by-residue geometry and overall structure geometry.

 

Ramachandran plot helps to refine the disallowed residues using loop refinement by MODELLER program and thereby the protein were refined (Figure2).The result of the Ramachandran plot after loop refinement showed 87.8% of residues in favorable region representing that it is a reliable and good quality model (Figure3). The generated 3d structure of EML4-ALK protein was used as a receptor for molecular docking studies.

 

Figure 1 Template Indication Using BLAST P against PDB

 (A)

Figure 2: A) Ramachandran Plot Before Loop Refinement

 (B)

Figure 2: B) Ramachandran Plot After Loop Refinement

Figure 3: PROCHECK statistics for EML4-ALK protein

 

Figure 4: 3D structure of EML4-ALK

 

Molecular Docking:

Structure-based drug discovery (SBDD) is the most preferred technique to identify novel potential inhibitors against the protein of interest. The goal of ligand-protein docking is to predict the predominant binding mode(s) of a ligand with a protein of known three-dimensional structure. In AutoDock, crystallographic water molecules and non polar hydrogen atoms were removed from 3D structure of EML4-ALK protein and were used as receptor. The receptor was first modified by adding polar hydrogen atoms and kollman charges using AutoDock tools (ADT).^. The torsional bonds of ligand were set free by ligand module in AutoDock tools- ADT.

 

AutoDock saved the prepared file in PDBQT format. AutoGrid was used for the preparation of the grid map using a grid box. Grid point spacing was calculated around the docking area for all theligand atom types using AutoDock parameters. AutoDock was employed for docking using protein and ligand information along with grid box properties in the configuration file. Then Lamarckian Genetic Algorithm (LGA) was chosen to search for the best conformations. AutoDock employs iterated local search global optimizer.

 

Docking results from each calculation were clustered on the basis of root mean square deviation (RMSD) between the Cartesian coordinates of ligands and were ranked according to binding energy. The conformer of each ligand with lowest binding free energy was chosen for docking. Docked pose of the different compounds with protein, Hydrogen bond interactions and its 2D representation were visualized using DS visualizer (Table 2) .The information about the Binding energies, Number of hydrogen bonds formed and the distance between them was given in the Table 3. From the analysis, (S)-Allylcysteine with least binding energy showed the best interaction with the active site receptors.

 

 

 

 

 

 

 

Table 2 Hydrogen bond interactions and 2D diagram between EML4-ALK and bioactive constituents of Allium sativum is visualized

using DS visualizer

Compound	Hydrogen bond interactions  and 2D diagram between EML4 ALK and bioactive constituents of Allium sativum is visualized using DS visualizer
AJOENE
ALLICIN
ALLIIN
S - ALLYL CYSTEINE
ALLYL METHYL DISULFIDE
DIALLYL DISULFIDE

 

Table 3 Binding energy and binding information between EML4-ALK and bioactive constituents of Allium sativum

S.NO	COMPOUND	STRUCTURE	BINDING ENERGY	BINDING INFORMATION
				HYDROGEN BOND INTERACTION	DISTANCE (Å)
1.	AJOENE		-3.77	GLU 240 (N….O)	3.11
2.	ALLICIN		-4.19	PHE 447 (N….O) ASP 446  (OD2….O)	2.8 3.29
3.	ALLIN		-4.28	ASP 525(N….O) ASP 525(OD1….H)	2.78 1.8
4.	(S)-ALLYLCYSTEINE		-4.54	LYS 528(N….O) LYS 528(NZ….O) LYS 528(NZ….O) PRO 526(H….O) PRO 526 (O….H)	2.68 2.8 2.6 2.38 2.03
5.	ALLYL METHYL DISULFIDE		-2.88	GLU417(N….S) SER 484(N….S)	3.39 2.85
6.	DIALLYL DISULFIDE		-3.23	GLU343(OE1….S) LYS 326(NZ….S)	2.895 3.73

 

CONCLUSION:

Molecular docking application lies in the concept of structure-based virtual screening, for identifying new active compounds towards a particular target protein, which provides effective way to develop novel, specific and safe drugs. The present study indicates that the bioactive compounds from Allium sativum which shows a strong binding affinity towards the modelled EML4-ALK protein, helps to understand the underlying mechanisms which leads to lung cancer. In docking study, six bioactive constituents of Allium sativum is subjected for docking against EML4-ALK protein. Out of six compounds(S)-Allylcysteine had maximum hydrogen bond interaction and mimimum binding energy value. Further experimental studies are required to elucidate the molecular mechanisms of this compound to find the potential drug with better therapeutic effects against lung cancer.

 

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Accepted on 11.07.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(11): 3741-3747.