Molecular Modelling of 1H-Benzo [b] [1,5] Diazepine- 2(3H)-one Derivatives and Docking Studies Against Receptor Associated Protein

In the present investigation, some N1-benzoyl/ N1-(1,3,4-thiadiazol-2-yl amino acetyl) -7substituted4-methyl-1,5-benzodiazepine-2-one were designed and docked at active site of cavity 1# of GABA-A receptor associated protein (1KJT) to distinguish from their hypothetical binding mode. The X-ray crystal structure of mammalian GABA-A receptor associated protein (1KJT) obtained from protein data bank was used as target protein. In this investigation the comparative analysis of the docking experiments of modelled compounds with known GABA agonist, Lofendazam was carried out. The dock scores calculated for Lofendazam was -4.7373. Among the modelled compounds, following conformation were found to have lower dock scores as indicated in bracket in comparison to other confermers; N1-benzoyl-7bromo4-methyl-1,5-benzodiazepine-2one, conformer_C3 (-5.0915), N1-(1,3,4-thiadiazol-2-yl amino acetyl) -7-chloro-4-methyl-1,5benzodiazepine-2-one, Conformer_C2 (-4.6532). These conformers have more affinity for active site of GABA-A receptor associated protein than other molecules.


INTRODUCTION
1, 5 benziodiazepines have wide spectrum of biological activities including anticonvulsant activity [1][2]. In addition to currently known anticonvulsant agents, there is a need to develop such new heterocycles with the hope of having greater anticonvulsant potential. For the treatment of epileptic seizures, there is an everincreasing demand for research into novel compounds with fewer toxicities and side effects.There are various reports on docking studies of benzodiazepine containing heterocycles viz. traizole, pyrimidine, quinazoline [3][4]. The objective of the present investigation is to identify new active compounds for the target protein, GABARAP using the structure-based virtual screening.
The docking process involves two main steps: predicting the ligand conformation as well as its positioning within the active site and assessment of the binding affinity. Both of these procedures are concerned with sample methods and scoring schemes, respectively. Molecular docking helps by identifying potential active sites in proteins, determining the optimum shape of the ligand receptor complex, and estimating the binding energy for different ligands to create more effective ligands. The interaction energy is calculated in terms of dock score. The strength of the noncovalent interaction between two molecules after they docked is thus predited from the score. Most scoring functions are physicsbased molecular mechanics, with force fields that estimate the energy of the low_(negative) energy indicates a stable system thus, alikely binding interaction.The options of docking are; rigid docking which obatins suitable position for the ligand in receptor environment, flexible docking obtains a favored geometry for receptor-ligand interactions is obtained, full flexible docking obtains the ligand's torsion angles as well as the side chains of active site residues. Thus Molecular Docking is an effective and competent tool for in silico screening. It is playing an important and ever increasing role in rational drug design [5][6][7].

Hardware and Software
All Docking studies and conformational analysis were performed using the Molecular Design Suite (VLife MDS software package, version 4.3; from VLife Sciences, Pune, India) on Lenovo computer, i3 processor with XP operating system.

Structure Conformation Generation
The 2D structure draw application of Vlife2Ddraw was used to sketch compound structures, which were then converted to 3D structures. The AMBER approach was used to reduce and optimise all of the structures, with a root mean square gradient (RMS) of 0.01 kcal/mol and a 10,000 iteration limit. Monte Carlo was used to construct conformers for each structure using the AMBER force field approach. The drug -protein complex with lowest dock score was chosen for further investigation of the types of interactions. (Fig. 1 shows the conformers for which the lowest dock score was obtained).

Preparation of protein
The protein, Crystal Structure of the GABA(A) Receptor Associated Protein, GABARAP [1KJT] (PDB DOI: 10.2210/pdb1KJT/pdb) was downloaded from www.rcsb.org and energy minimization of the protein was done. During preprocessing all the bound water molecules, ligands, and cofactors were removed from the protein which was saved in .pdb format. The side chains that were not close to the binding cavity and did not participate in salt bridges were neutralized. This step was then followed by restrained minimization of co-crystallized complex, which reoriented side-chain hydroxyl groups and alleviated potential steric clashes. The complex obtained was minimized using AMBER force field. The minimization was terminated after completion of 5,000 steps or when the energy gradient dropped below 0.05 kcal/mol.

Preparation of ligands
Structures of the 1,5 benzodiazepaines derivatives ligands were sketched using builtin Vlife 2D draw taken in .mol2 format; converted into 3D structure and performed a geometry minimization of the ligands. AMBER Force Fields with default settings were used for the ligand minimization.
VlifeMDS software was used to prepare the ligand for docking. This procedure is outlined as follows.
 2D structures of ligands were drawn in Chemdraw.  2D Structures were converted to 3D.

 Conformers
were generated and optimized.  Docking was done by GA based docking.  Cavity # 1 was selected for docking and dock score was calculated.  Docked Complex was optimized.  Lowest dock score complex was further studied. (Fig. 1 shows the conformers for which the lowest dock score was obtained) During the docking procedure, the system searches the docked ligand's conformational, orientational, and positional space, eliminating undesirable conformations using scoring, and then optimising the structure using the AMBER force field. GABA-A receptor associated protein is used to do batch docking of proposed ligands in MDS.

Docking Results
The molecular docking studies of all possible three dimensional confirmations of N 1 -benzoyl/ N 1 -(1,3,4-thiadiazol-2-yl amino acetyl) -7substituted-4-methyl-1,5-benzodiazepine-2-one were done using Vlife MDS Biopredicta module using cavity#1 against GABA-A receptor associated protein (1KJT) obtained from Protein Data Bank as target protein. The intermolecular interactions between the ligand and the protein (receptor) were investigated. It was processed by deleting the solvent molecule as well as correcting the structure with respect to bonds and the Hatoms. Table 1 shows dock scores and binding energies of conformations of N 1 -benzoyl-7-substituted-4methyl-1,5 -benzodiazepine-2-ones. Table 2 shows dock scores and binding energies of conformations of N 1 -(1,3,4-thiadiazol-2-yl amino acetyl)-7-substituted-1,5-benzodiazepin-2-ones. Some of the molecules for which the confirmations shows lower dock scores were selected to study their binding interaction with the cavity#1 of the receptor. The binding patterns of the docked molecules were compared with standard ligand, Lofendazam. Fig. 2 also depicts its intercations.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable