MOLECULAR DOCKING ANALYSIS OF Aerva lanata PHYTO CONSTITUENTS AS LEAD FOR MICROBIAL INHIBITORS

ABSTRACT Molecular docking study was performed using Maestro Schrödinger suite 8.5 mainly on twenty nine phytoconstituents reported from the plant Aerva lanta for their antimicrobial potential. The crystal structure of protein data bank (PDB-ID: 3SRW) was obtained from RCSB (Research Collaboratory for Structural Bioinformatics) website. The ligands were obtained from the reported literature search of Aerva lanta plant. The top hits were analyzed for their binding affinity with the dihydrofolate reductase enzyme. The docking results revealed that rutin (Glide score: -11.75) exhibited better binding interaction to dihydrofolate reductase receptor.


INTRODUCTION
Natural goods have gained a lot of significance since ancient times due to their lesser side effects, higher safety and efficacy against health illness.In current era, plants have screened for their potential uses as an alternative medicines to allopathic medicines for the treatment of many diseases.The lesser side effect of natural products is due to their natural antioxidant properties. 1 As a result, it is essential to unlock their potential for the development of newer drugs against health risks.
Over the past several years, the increase of bacteria drug-resistance and the rapid emergence of new infections have intensely decreased the efficiency of the drugs against pathologies caused by certain microorganisms.This situation rises up the urgent need for the development of new antibacterial agents, preferentially, from natural sources. 2 The active principle of bioactive compounds has shown tremendous therapeutic applications either singly or in combination to inhibit the life processes of microbes.
Aerva lanata (Linn.)Juss.Ex Schult.belongs to the family Amaranthaceae, is one of the important plant grow in the warmer parts of India ascending to 1,000 m.In Sanskrit A. lanata is known as paashaanabheda, gorakshaganjaa, satkabhedi, aadaanpaak.It is commonly known as sirupeelar in Tamil or Siddha. 3The plant is extensively used in urinary disorders like Ashmari (Urinary calculi), Mootrakrichra (Dysuria), Mootravikara etc by most of the Ayurveda and Siddha practitioners in southern India, in the name of Pashanabheda.As the plant bears almost all the properties similar to that of the original source of Pashanabheda. 4Herbs are perennial, 5-50 cm tall.Stem branched from base; branches ascending or stoloniferous, white lanose.Leaves opposite or nearly whorled, sessile, grayish green, subulate, linear, 1-2.5 cm, abaxially white lanose, adaxially glabrous, base attenuate, sometimes vaginate. 5,6Flowers are small in size, sessile, greenish or dull white in colour, clustered with spikes.The phytoconstituents reported from Aerva lanata plant are flavanoids, tannins, anthra-quinons, alkaloid, phenol, proteins, amino acids and carbohydrates. 7wever, from literature reviewed till date, it is obvious that there is no information available about the in silico antimicrobial activity of phytoconstituents from Aerva lanata.The present docking studies was done to explore the lead molecules from Aerva lanata for antimicrobial activity.

MATERIALS AND METHOD
Molecular docking simulation was performed using Glide module in Maestro 8.5 version software (Schrodinger LLC suite).Schrodinger suite was installed in a system having configurations core TM processor with 2 GB RAM and 320 GB ROM with CentOS Linux as the operating system.

Protein Preparation
The X-ray crystal structure of antimicrobial target protein (PDB-ID: 3SRW) was accessed from RCSB protein data bank. 8The crystal structure of dihydrofolate reductase enzyme receptor was reported to complex with 7-(2-ethoxynaphthalen-1-yl)-6-methylquinazoline-2,4-diamine.Protein preparation was performed using Protein Preparation wizard in Maestro.In this step, water molecules were removed, bond orders were assigned, all hydrogen's in the structure were added, and bonds to metals were deleted and the formal charge in the metal & neighboring atoms were adjusted that more than the 5Ǻ specified distance.The next stage is to inspect and change the protonation state for the residue in the workspace for minimal structural errors.The final step in the protein preparation process was to refine the structure, with a restrained minimization.In order to determine the potential binding sites, a grid based cavity prediction algorithm has been used.Protein preparation method follows OPLS-2005 force field for energy minimization.

Ligand Preparation
The ligands were built using Chem Draw Ultra 10.0 converted to 3D structure from the 2D structure using the same software.Chem Draw Ultra 8.0 is a robust collection of tools designed to prepare high quality, all atom 3D structure for large numbers of drugs-like molecules, starting with the 2D or 3D structure in mol format. 9The resulting structures are saved in Mol format and imported to Maestro project file.The simplest use of Chem Draw Ultra 10.0 is to produce a single, low energy, 3D structure with correct chiralities for each successful proposed input structure.Further steps were performed using LigPrep module in Maestro.While performing ligand preparation step, chiralities were determined from 3D structure and original states of ionization were retained.Tautomers were generated discarding current conformers.The conformational space was searched using the Monte Carlo method.All rotable single bonds were included in the conformational search.Each search was continued until the global energy minima were found at least 10 times.The energy minimizations were carried out using the least square OPLS_2005 force field.The conformational searches were done for aqueous solution using the generalized Born/solvent accessible surface (GB/SA) continuum salvation model. 10

Grid generation & docking calculation
Glide searches for favorable interactions between ligand molecules and the receptor.Grids were generated using Receptor Grid Generation module in Glide following the standard procedure recommended.Grid generation defined the active sites of the protein and generated the electrostatics grid.Constraints were included in the grid files.The shape and the properties of the receptor were represented on a grid by several different sets of field that provide progressive more accurate scoring of the ligand poses.Ligand molecule was picked so that it can be excluded from the grid generation with the van der Waals radius scaling 1.00. 10The ligands were docked using docking functionality with extra precision (XP) mode.The most feasible orientation of the ligands in the binding pocket is predicted, and the strength of the interaction in the particular orientation was quantified from a scoring function.

RESULT & DISCUSSION
The hydrogen and nitrogen atoms of internal ligand were bound with the hydrophobic pocket of the receptor with residue MET 528 and CYS 530 by forming two H-bonds of length 3.091 Å and    3).shown good binding affinity with the protein (Figure 2 to Figure 5).This study throws a light on further experimentally validating these drug lead entities as microbial inhibitors.
3.091 Å having the docking score of -3.81.Ligands Rutin, Quercetin, Myrecetin exhibited better binding affinity towards the receptor as compared to other ligands.Rutin showed very good affinity towards the receptor with highest dock score of -11.75.It formed two hydrogen bonds of length 2.186 Å and 2.146 Å and oxygen and hydrogen atoms of ligands with ASP 351 residue of receptor.