11. IN SILICO DOCKING ANALYSIS

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1 11. IN SILICO DOCKING ANALYSIS 11.1 Molecular docking studies of major active constituents of ethanolic extract of GP The major active constituents are identified from the ethanolic extract of Glycosmis pentaphylla which possess anti arthritic and inflammatory properties according to traditional claims and the 3D structures of the active constituents (Marmesin & (3ß)-Stigmast-5-en-3-yl (9Z)-) are drawn using ChemSketch software Materials and method Preparation of Ligand The major active constituents are identified from the selected medicinal plant Glycosmis pentaphylla which possess anti arthritic and inflammatory properties according to traditional claims and the 3D structures of the active constituents (Marmesin & (3ß)-Stigmast-5-en-3-yl (9Z)-) are drawn using chemsketch software and saved in mol format. The ligands are imported to the workspace and preparation of them is done. The docking scores of the active constituents are compared against the standard drugs methotrexate for anti arthritic activity & rofecoxib and valdecoxib for anti-inflammatory activity obtained from the drug bank in.mol format[wishart, D. S., et al.2008] Preparation of Enzyme The targets for docking studies are selected as human dihydrofolate reductase for anti arthritic activity and cyclooxygenase-2 for anti-inflammatory 58

2 activity. Docking analysis is done by initially selecting the target for the disease and followed by obtaining the 3D structure of human dihydrofolate reductase (4KAK) & Cyclooxygenase-2 (4COX) from protein data bank in.pdb format [Bernstein, F.C., et al.1977]. It is well known that PDB files often have poor or missing assignments of explicit hydrogen s, and the PDB file format cannot accommodate bond order information. Therefore, proper bonds, bond orders, hybridization and charges were assigned using the MVD. The potential binding sites of both the targets were calculated using the built-in cavity detection algorithm implemented in MVD. The search space of the simulation exploited in the docking studies was studied as a subset region of 25.0 Angstroms around the active side cleft. The water molecules are also taken in to consideration and the replaceable water molecules were given a score of Molegro Virtual Docker s docking search algorithms and scoring functions Ligand docking studies were performed by Molegro Virtual Docker (MVD), which has recently been introduced and gained attention among medicinal chemists. MVD is a fast and flexible docking program that gives the most likely conformation of ligand binding to a macromolecule. MolDock software is based on a new heuristic search algorithm that combines differential evolution with a cavity prediction algorithm [Thomson, R., et al.2006]. It has an interactive optimization technique inspired by Darwinian Evolution Theory (Evolutionary Algorithms - EA), in which a population of individuals is exposed to competitive selection that weeds out poor solutions. Recombination and mutation are used to generate new solutions. The scoring function of MolDock is based on the Piecewise Linear Potential (PLP), which is a simplified potential whose parameters are fit to protein-ligand structures and a binding data scoring function [Gehlhaar, D.K., et al.1998] that is further 59

3 extended in GEMDOCK (Generic Evolutionary Method for molecular DOCK) with a new hydrogen bonding term and charge schemes [Yang, J.M., et al.2004] MolDock Optimizer In MVD, selected parameters were used for the guided differential evolution algorithm: number of runs =5 by checking constrain poses to cavity option), population size=50, maximum interactions =2000, cross over rate=0.9, and scaling factor=0.5. A o variance-based termination scheme was selected rather than root mean square deviation (RMSD).To ensure the most suitable binding mode in the binding cavity, Pose clustering was employed, which lead to multiple binding modes Parameters for scoring functions MolDock score They ignore-distant-atoms option was used to ignore atoms far away from the binding site. Additionally, hydrogen bond directionality was said to check whether hydrogen bonding between potential donors and acceptors can occur. The binding site on the protein was defined as extending in X, Y & Z directions around the selected cavity with a radius of 25 Angstroms (142) Results and discussion The ability of the phytoconstituents to bind with the targets is given in terms of MolDock core & rerank score. The scores are used as the parameter for analysing the docking results. The phytoconstituents are ranked according to their rerank score. The ligand possessing the highest mol dock score shows a strong affinity towards its target. 60

4 In silico docking analysis of phytoconstituents from Glycosmis pentaphylla on human dihydrofolate reductase (PDB ID: 4KAK) ranking based on MolDock, Re rank score and H Bond interactions are represented in table 11.1, 11.2 and 11.3 respectively. In-silico docking analysis of phytoconstituents from Glycosmis pentaphylla on cyclooxygenase 2 (PDB ID: 4COX) ranking based on MolDock score, rerank score and H Bond interactions are represented in table 11.4, 11.5 and 11.6 respectively. The figure 11.1 to 11.7 corresponds to the docking pose evaluated and captured by the ligand energy inspector tool in the Molegro virtual docker. Table 11.1 In silico docking analysis of phytoconstituents from Glycosmis pentaphylla on human dihydrofolate reductase (PDB ID: 4KAK) ranking based on MolDock Name Ligand [00](3β-Stigmast-5- enoctadec- 3-ylß(9Z)-octadec-9- MolDock ß Rerank ß H Bond [00]Methotrexate Methotrexate [01]Methotrexate Methotrexate [01] enoctadec- 3-ylß(9Z)-octadec [02]Methotrexate Methotrexate [03]Methotrexate Methotrexate [04]Methotrexate Methotrexate [03] octadec- [02] octadec- [04] octadec- en- 3-ylß(9Z)-octadec-9- en- 3-ylß(9Z)-octadec-9- en- 3-ylß(9Z)-octadec [00]Marmesin Marmesin [01]Marmesin Marmesin [02]Marmesin Marmesin [04]Marmesin Marmesin [03]Marmesin Marmesin

5 Table 11.2 In silico docking analysis of phytoconstituents from Glycosmis pentaphylla on human dihydrofolate reductase (PDB ID: 4KAK) ranking based on Rerank Name Ligand MolDock ß Rerank ß H Bond [00]Methotrexate Methotrexate [03]Methotrexate Methotrexate [01]Methotrexate Methotrexate [04]Methotrexate Methotrexate [00]Marmesin Marmesin [01]Marmesin Marmesin [02]Marmesin Marmesin [03]Marmesin Marmesin [02]Methotrexate Methotrexate [04]Marmesin Marmesin [04] octadec- [00] octadec- [01] octadec- [02] octadec- [03] octadec- (3β-Stigmast-5-en- 3-ylß(9Z)-octadec

6 Table 11.3 In silico docking analysis of phytoconstituents from Glycosmis pentaphylla on human dihydrofolate reductase (PDB ID: 4KAK) ranking based on H Bond Name Ligand MolDock ß Rerank ß H Bond [04]Marmesin Marmesin [01]Methotrexate Methotrexate [02]Methotrexate Methotrexate [04]Methotrexate Methotrexate [00]Marmesin Marmesin [02]Marmesin Marmesin [01]Marmesin Marmesin [00]Methotrexate Methotrexate [02](3 β -Stigmast [03]Marmesin Marmesin [03]Methotrexate Methotrexate [00](3 β -Stigmast- [01](3 β -Stigmast- [03](3 β -Stigmast- [04](3 β -Stigmast

7 Table 11.4 In-silico docking analysis of phytoconstituents from Glycosmis pentaphylla on cyclooxygenase 2 (PDB ID: 4COX) ranking based on MolDock Name Ligand MolDock ß Rerank ß HBond [00]Celecoxib Celecoxib [01]Celecoxib Celecoxib [00]Marmesin Marmesin [00]Rofecoxib Rofecoxib [02]Celecoxib Celecoxib [03]Celecoxib Celecoxib [01]Marmesin Marmesin [03]Marmesin Marmesin [01]Rofecoxib Rofecoxib [02]Marmesin Marmesin [02]Rofecoxib Rofecoxib [04]Celecoxib Celecoxib [03]Rofecoxib Rofecoxib [00](3 β -Stigmast- [01](3 β -Stigmast- [02](3 β -Stigmast- [04](3 β -Stigmast- [03](3 β -Stigmast- en-3- ylß(9z)-octadec-9- en-3- ylß(9z)-octadec-9- en-3- ylß(9z)-octadec-9- en-3- ylß(9z)-octadec-9- en-3- ylß(9z)-octadec

8 Table 11.5 In silico docking analysis of phytoconstituents from Glycosmis pentaphylla on cyclooxygenase 2 (PDB ID: 4COX) ranking based on Rerank Name Ligand MolDock ß Rerank ß H Bond [00]Marmesin Marmesin [00]Celecoxib Celecoxib [01]Marmesin Marmesin [02]Celecoxib Celecoxib [03]Marmesin Marmesin [01]Celecoxib Celecoxib [02]Marmesin Marmesin [02]Rofecoxib Rofecoxib [04]Celecoxib Celecoxib [01]Rofecoxib Rofecoxib [03]Rofecoxib Rofecoxib [00](3 β -Stigmast [00]Rofecoxib Rofecoxib [03](3 β -Stigmast [03]Celecoxib Celecoxib [02](3 β -Stigmast- [04](3 β -Stigmast- [01](3 β -Stigmast

9 Table 11.6 In silico docking analysis of phytoconstituents from Glycosmis pentaphylla on cyclooxygenase 2 (PDB ID: 4COX) ranking based on H Bond Name Ligand MolDock ß Rerank ß H Bond [00]Marmesin Marmesin [01]Marmesin Marmesin [00]Rofecoxib Rofecoxib [03]Rofecoxib Rofecoxib [02]Celecoxib Celecoxib [00]Celecoxib Celecoxib [01]Rofecoxib Rofecoxib [02]Rofecoxib Rofecoxib [01]Celecoxib Celecoxib [02]Marmesin Marmesin [03]Celecoxib Celecoxib [01](3 β -Stigmast- [02](3 β -Stigmast- [04](3 β -Stigmast- [03](3 β -Stigmast- [00](3 β -Stigmast [03]Marmesin Marmesin [04]Celecoxib Celecoxib

10 Figure 11.1 In silico docking results of Celecoxib.4COX target Figure 11.2 In silico docking results of Rofecoxib.4COX targets 67

11 Figure 11.3 In silico docking results of Marmesin.4COX target Figure 11.4 In silico docking results of 3β-Stigmast-5-en-3-yl (9Z)-octadec-9-.4COX target 68

12 Figure 11.5 In silico docking results of Methotrexate.4KAK targets Figure 11.6 In silico docking results of Marmesin.4KAK targets 69

13 Figure 11.7 In silico docking results of 3β-Stigmast-5-en-3-yl (9Z)-octadec-9-.4KAK target 70

14 11.4 References Bolton, E. E., Wang, Y., Thiessen, P. A., & Bryant, S. H. (2008). PubChem: integrated platform of small molecules and biological activities. Annual reports in computational chemistry, 4, ACD ChemSketch Freeware, version 10.00(2012) Advanced Chemistry Development, Inc., Toronto, ON, Canada, Wishart, D. S., Knox, C., Guo, A. C., Cheng, D., Shrivastava, S., Tzur, D.,... & Hassanali, M. (2008). DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic acids research, 36(suppl 1), D901- D906. PMID: Bauman, J.D., Patel D., Das, K., A rnold E. (2013) Crystal structure of HIV-1 reverse transcriptase (RT) in complex with Rilpivirine (TMC278, Edurant), a non-nucleoside rt-inhibiting drug. Nat Chem 5: PDB ID: 4G1Q. Bernstein, F. C., Koetzle, T. F., Williams, G. J., Meyer, E. F., Brice, M. D., Rodgers, J. R.,... & Tasumi, M. (1978). The Protein Data Bank: a computer-based archival file for macromolecular structures. Archives of biochemistry and biophysics, 185(2), Thomsen, R., & Christensen, M. H. (2006). MolDock: a new technique for high-accuracy molecular docking. Journal of medicinal chemistry, 49(11), Gehlhaar, D.K., Verkhivker, G., Rejto, P.A., Fogel, D.B., Fogel, L.J., Freer, S.T.,(1995) Docking conformationally flexible small molecules into a protein binding site through evolutionary programming. In Proceedings of the Fourth International Conference on Evolutionary 71

15 Programming: 1-3 March 1995; San Diego Edited by: John R McDonnell, Robert G Reynolds, David B Fogel. MIT Press Gehlhaar, D.K., Bouzida, D., Rejto, P.A.(1998) Fully automated and rapid flexible docking of inhibitors covalently bound to serine proteases. In Proceedings of the Seventh International Conference on Evolutionary Programming: March 1998; San Diego Edited by: William, P. V., Saravanan, N., Donald, E. W., Eiben, A.E. Springer Yang, J.M., Chen, C.C.(2004) GEMDOCK: A generic evolutionary method for molecular docking. Proteins.55:

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