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ThesisItem Open Access Virtual high throughput screening of sodium channel blockers as potential insecticide leads(CCSHAU, 2018) Naina Kumari; Sudhir KumarSodium channels are integral transmembrane proteins responsible for the initiation and propagation of action potential. Sodium channel consists of α and β subunits. α subunit is responsible for voltage dependent ion conductance whereas β subunit is responsible for membrane localization. By the use of virtual high throughput screening (molecular docking), potential inhibitors of voltage gated sodium channel were identified. vHTS is a computational method for screening in silico collection of compound libraries. Using vHTS, the binding affinity of the compounds from in silico library and target receptor was predicted. The crystal structure of insect sodium channel (5X0M) consisting of 1553 amino acids was retrieved from RCSB PDB and the active site and binding pocket were identified by CastP tool.. On the basis of substructure similarity of well-known inhibitors of sodium channel, 2,06,404 small molecules/ligands were obtained from ZINC15 database. Out of 2, 06,404 ligands, 1,65,437 were selected on the basis of Lipinski’s rule of 5. Molecular docking of the ligands with the sodium channel protein was performed using USCF DOCK6. 4,854 Docked ligands were obtained on the basis of acceptable binding energy score. The ligands were further filtered by their ADMET properties prediction through vNN-ADMET and TEST. Out of 2,06,404 ligands, 47 ligands were identified as non-toxicant and potential insect sodium channel blockers. The interaction study of the 47 accepted leads with the sodium channel protein revealed that ARG 1138, ARG 1120, GLU 1435, LEU 1224, ILE 1152, ASP 1412, GLU 1123, LEU 1224 and TYR 1430 are the key residues involved in interaction through hydrogen bonding, electrostatic interactions or vander waals forces.ThesisItem Open Access Computational modelling and molecular dynamic simulation of glutamate decarboxylase of wheat (Triticum aestivum L.),(CCSHAU, 2018) Jakhar, Ritu; Sudhir kumarGlutamate decarboxylase (GAD) catalyses the decarboxylation of glutamate amino acid to γ – amino butyrate (GABA) in presence of Pyridoxal phosphate cofactor. GABA build up happens after GAD activation in plants in response to various biotic and abiotic stress such as hypoxia, temperature shock, water stress, salinity stress, acidosis, virus infection and mechanical manipulation. GAD assembled into dimer and subsequently in hexamer for activation. In plants, GAD dimerisation and activation is associated with binding of CaM and C-terminal of GAD. The N-terminal residues are required for the assembly and stabilization of hexameric state of GAD. Modelling and dynamics study can uncover the interaction forces involved in GAD activity. A 500 AA long wheat GAD sequence was retrieved from UniProtKB and further aligned using BLAST program to identify templates for comparative structure prediction. Modelling of GAD peptide (monomer subunit) by Modeller9.19 and Phyre2 server provided Model1 and Model2, respectively. Model1 was generated in two fragments for N- and C- terminal with Modeller9.19 and joined using Chimera visualization tool. Both models were subjected to energy minimisation using GROMOS force field and structure assessment by GROMOS, QMEAN and ANOLEA. Both models were further verified, validated and evaluated using WHATIF and SAVES server. The RMSD of models on superimposition with the template was found to be less than 2.0 Å. Models were further refined using NAMD, a molecular dynamics (MD) code designed for high-performance simulation of large biomolecular systems. Out of two models, Model1 was predicted better model than Model2 on the basis of RMSD between initial and simulated model.ThesisItem Open Access Computational modeling and molecular dynamic simulation of pyrophosphatase of rice (Oryza sativa L.)(CCSHAU, 2018) Manisha; Sudhir KumarInorganic pyrophosphatase plays a significant role in various processes in plants. It causes chalkiness and hydrolytic breakdown of ADP-glucose in plastidal compartment. It has significance in lipid metabolism, calcium absorption, DNA synthesis and biochemical transformations. The sequence of inorganic pyrophosphatse was retrieved from NCBI and template was identified using BLASTP. With 84% query coverage and 71% identity 4LUG was selected as template. Modeller 9.19 and RaptorX were used for computational modeling. Predicted models were refined by energy minimization with GROMOS force field from Swiss-pdb Viewer. Minimum energy calculated for Modeller 9.19 and RaptorX predicted models were -2394.489KJ/mol and -7365.312KJ/mol respectively. The structures were assessed by GROMOS, ANOLEA and QMEAN graphs. More favourable region was shown by GROMOS and ANOLEA as compare to QMEAN. WHATIF server programs were used for structures optimization and validation. Bond length Z-score, bond angle Z-score, coarse packing quality and Ramachandran Z-score, were approximately 0.4, 1.2, -0.9 and 0.1 respectively. SAVES server programs score for PROVE, VERIFY3D and ERRAT were approximately 4.2%, 81% and 91% respectively. Ramachandran plot calculated by PROCHECK showed approximately 94% amino acid in core and 6% in allowed region. The models visualization showed coils were dominantly present in both the structures. RMSD for the structures was less than 0.5. Explicit solvent molecular dynamic simulation was done by VMD and NAMD software. The total energy and RMSD graphs calculated after simulation were stable for the structures. Structure superimposition with template showed significant conserved region between template and predicted structures. RMSD calculated after simulation was less than 0.5 Å against for both models template. The model predicted by RaptorX was found better as compared to Modeller 9.19 predicted model.