In silico screening of 3,4 dihydropyrimidones as focal adhesion tyrosine kinase inhibitors

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1 Research Article ISS: Gopinathan arasimhan et al. / Journal of Pharmacy Research 2015,9(1), Available online through In silico screening of 3,4 dihydropyrimidones as focal adhesion tyrosine kinase inhibitors Gopinathan arasimhan*, Chitra Krishnan Department of Pharmaceutical Chemistry, Faculty of Pharmacy Sri Ramachandra University, Porur, Chennai-116, Tamilnadu, India. Received on: ; Revised on: ; Accepted on: ABSTRACT Credible role in metastasis and over expression in invasive tumors made Focal adhesions tyrosine kinase [FAK] a target for the development of anticancer drugs. Cancers of the thyroid, prostate, cervix, colon, rectum, oral epithelium and ovary have shown increased in FAK levels. In search for new drugs structure-based design is an important technique applied currently in order to speed up the lead finding and optimization In this study, Molecular modeling and docking analysis were used to predict and understand interactions between Focal Adhesion Kinase and twenty four compounds of 3,4 dihydropyrimidones. 3BZ3 atomic coordinates of Focal Adhesion Kinase was retrieved from protein data bank (PDB). All ligands were drawn by software chemsketch. Molegro virtual docker program that predicted interactions in terms of Dock score. The approach is applicable in engineering 3D structures of enzymatic models, and studying interactions of active site residues with ligands show that the three compounds: it is concluded that that 5-(1H-benzimidazol-2-yl)-4-(4-hydroxy-3-methoxyphenyl)-6- methyl-3,4-dihydropyrimidine-2(1h)-one, 5-(1H-benzimidazol-2-yl)-4-(4- and 5-(1Hbenzimidazol-2-yl)-4-(1H-indol-3-yl)-6-methyl-3,4- could be a potent anticancer target molecule against Focal adhesions tyrosine kinase which may be worth for further clinical trials. KEYWORDS: Anti-cancer, Focal adhesions tyrosine kinase, Molecular docking, Interactions, Molegro virtual docker. ITRODUCTIO The cell surface and the extracellular matrix are interceded by Focal adhesions. Intracellular signalling from focal adhesions alters cell adhesion, migration, proliferation, differentiation, and survival in response to signal in the extracellular milieu. Credible role in metastasis and over expression in invasive tumors made Focal adhesions tyrosine kinase [FAK] a target for the development of anticancer drugs 1. Increased FAK mra and protein level were observed in tumor tissue when compared to normal tissue which is revealed by real-time PCR analysis of colorectal carcinoma and liver metastases 2. Cancers of the thyroid, prostate, cervix,colon, rectum, oral epithelium and ovary have shown increased in FAK levels.in few cancer cell lines amplification of the FAK gene is reported. cells derived from head and neck cancer possessed gains in gene copy number 3. kinase or calcium-dependent protein tyrosine kinase. It is articulated in most tissues and cell types. It is evolutionarily potted in mammalian species as well as lower eukaryotic organisms. Increased cancer cell motility, invasiveness and proliferation is related with increased FAK expression in tumor cell. secretion and inhibition of cell migration, invasion and proliferation is reduced by inhibition of FAK function 4. Conventionally in drug research serendipity occupied a key role. ew receptor agonists and antagonists were designed by using neurotransmitters and hormones as templates. In search for new drugs structure-based design is an important technique applied currently in order to speed up the lead finding and optimization 5. METHODS AD MATERIALS The sole members of the family of non receptor protein tyrosine kinases are cell adhesion kinase, related adhesion focal tyrosine *Corresponding author.. Gopinathan Assistant professor, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sri Ramachandra University, porur, Chennai-116, Tamilnadu, India. Molecular Docking study To study protein-ligand interactions X-ray crystallographic 3-dimensional structure of human Focal adhesion kinase 1, 3bz3 atomic coordinates was retrieved from protein data bank (PDB). 3bz3 is a Focal adhesion tyrosine kinase enzyme with EC umber: classified under transferase class of enzymes, complexed with a selective inhibitor -methyl--{3-[({2-[(2-oxo-2,3-dihydro-1h-indol-5-yl)amino]- 5(trifluoromethyl)pyrimidin-4yl}amino)methyl]pyridin-2-yl}

2 Gopinathan arasimhan et al. / Journal of Pharmacy Research 2015,9(1), methanesulfonamide with A chain, with 2.20 A resolution and R-value respectively. Computational analysis was carried out on chain A of 3bz3. Twenty four molecules were selected to study the associated protein-ligands interactions. All ligands (Figure 1) were drawn by software chemsketch. -methyl--{3-[({2-[(2-oxo-2,3-dihydro-1h-indol-5-yl)amino] 5(trifluoromethyl) pyrimidin-4 yl}amino) methyl]pyridin-2-yl} methanesulfonamide ligand with surrounding active site residues within 3.31 A, hydrogen bonding interactions and the spatial orientation in binding pocket is given in Figure 3. The interacting residues surrounding the ligand within 3.5 A distance are Met 184, Gln 182 and Gln 161 respectively.all chemical structures were drawn using chem sketch software and all the files were converted to PDB file format. Protein target was downloaded from the PDB(Protein Data Bank). The protein PDB I.D is 3bz3.Active site in protein target were determined from the software. Docking of protein with dihydropyrimidine derivatives were carried out using Molegro software. H R H Figure 1. ligand bind with Focal adhesion tyrosine kinase enzyme Mol Dock Score scoring function was employed to predict the binding energy for active site residue-ligand interactions and docking studies computed for all ligands using Molegro virtual docker program that predicted interactions in terms of Dock score. All calculations were done on a Intel core I7 laptop with windows seven configuration. Docking was performed by using Molegro Virtual Docker (MVD) software package. MVD performs flexible ligand docking, so the optimal geometry of the ligand will be determined during the docking. To obtain better potential binding sites in the Focal adhesion tyrosine kinase enzyme (PDB ID: 3bz3), a maximum of five cavities was detected using default parameters. RESULTS AD DISCUSSIO Figure 2. Standard ligand bind with Focal adhesion tyrosine kinase enzyme. H 3 C Figure 3. General structure for ligand Virtual screening The twenty four ligand molecules having minimum energy were screened out as the possible inhibitors for COX-2 given in the (Table 1). 5-(1H-benzimidazol-2-yl)-4-(4-hydroxy-3-methoxyphenyl)-6-methyl- 3,4-dihydropyrimidine-2(1H)-one had highest moldoc score of kcal/mol. It had three hydrogen bonds. The Ile 108 of protein formed hydrogen bond with oxygen of hydroxyl group of ligand. Second hydrogen bond is formed between Pro 193 of protein with H of dihydropyrimidone of ligand. The bond lenths were found to be 2.78 A. and 2.85 A. respectively. The third hydrogen bond was formed between Cys 195 of protein and H of benzimidazole and bond length was found to be 2.88 A. Its thione derivative had mole dock score of kcal/mol. It had three hydrogen bonds as that of its urea derivative which owned the bond length were 2.66 A, 2.72 A and 2.85 A respectively. 5-(1H-benzimidazol-2-yl)-4-(1H-indol-3-yl)-6-methyl-3,4- had moldoc score of kcal/mol with one hydrogen bond formed between Ser216 of protein and H of indole of ligand claimed bond length of 2.97 A. Its thione derivative had moldoc score of kcal/mol. It had two hydrogen bonds. Hydrogen bond was formed between Ser 216 of protein with H of indole of ligand and the bond length was found to be 3.07A. The another hydrogen bond was formed between Pro 193 of X

3 Table 1. Gopinathan arasimhan et al. / Journal of Pharmacy Research 2015,9(1), S.o ame of the ligand3z3 Mol Dock Re-rank Hbond Hbond in A Residues Score score 1. 5-(1H-benzimidazol-2-yl)-4-(4- hydroxy-3-methoxyphenyl)-6-methyl- 3,4-dihydropyrimidine-2(1H)-one 2. 5-(1H-benzimidazol-2-yl)-4-(1H- indol-3-yl)-6-methyl-3, (1H-benzimidazol-2-yl)-4-(1H- indol-3-yl)-6-methyl-3, (1H-benzimidazol-2-yl)-4-(4- hydroxy-3-methoxyphenyl)-6-methyl- 3,4-dihydropyrimidine-2(1H)-thione 5. 5-(1H-benzimidazol-2-yl)-4-( (1H-benzimidazol-2-yl)-4-( (1H-benzimidazol-2-yl)-4-( (4-aminophenyl)-5-(1H benzimidazol-2-yl)-6-methyl-3, (1H-benzimidazol-2-yl)-4-( (1H-benzimidazol-2-yl)-4-( (4-aminophenyl)-5-(1H- benzimidazol-2-yl)-6-methyl-3,4- dihydropyrimidine-2(1h)-thione (1H-benzimidazol-2-yl)-4-( (1H-benzimidazol-2-yl)-4-(3- hydroxy phenyl)-6-methyl- 3, (1H-benzimidazol-2-yl)-4-(4- methoxyphenyl)-6-methyl-3, (1H-benzimidazol-2-yl)-4-(4- methoxyphenyl)-6-methyl-3, Three 2.78 Ile Pro Cys One 2.97 Ser Two 3.07 Ser Pro Three 2.66 Ile Cys Pro Three 2.87 Cys Pro Ile Three 2.91 Cys Pro Glu Two 2.59 Cys Tyr Three 2.73 Ile Pro Cys Two 2.89 Cys Pro Three 2.87 Cys Pro Ile Three 2.75 Ile Pro Cys Three 2.95 Glu Cys Pro Two 3.28 Cys Ser Two 2.99 Cys Pro Two 2.99 Cys Pro 193

4 Gopinathan arasimhan et al. / Journal of Pharmacy Research 2015,9(1), S.o ame of the ligand3z3 Mol Dock Re-rank Hbond Hbond in A Residues Score score (1H-benzimidazol-2-yl)-4-[4- (dimethylamino)phenyl]-6-methyl- 3,4-dihydropyrimidine-2(1H)-thione Two 3.77 Cys (1H-benzimidazol-2-yl)-4-[4 -(dimethylamino)phenyl]-6-methyl- 3,4-dihydropyrimidine-2(1H)-one (1H-benzimidazol-2-yl)-4-(furan-2- yl)-6-methyl-3,4-dihydropyrimidin- 2(1H)-one (1H-benzimidazol-2-yl)-4-(furan-2- yl)-6-methyl-3,4-dihydropyrimidin- 2(1H)-thione (1H-benzimidazol-2-yl)-4-( (1H-benzimidazol-2-yl)-4-( (1H-benzimidazol-2-yl)-4-( (1H-benzimidazol-2-yl)-6-methyl-4- phenyl-3,4-dihydropyrimidin-2(1h)- one (1H-benzimidazol-2-yl)-6-methyl- 4-phenyl-3,4-dihydropyrimidin-2(1H)- thione Three Ser Cys Arg Two 3.47 Arg Arg Three 3.29 Cys Glu Pro Two 2.9 Cys Pro Three 2.86 Ser Arg Pro protein and H of benzimidazole group of ligand and the bond length was found to be 3.08 A. 5-(1H-benzimidazol-2-yl)-4-(4- with three hydroen bond and possessed moldoc score of kcal/mol. The Cys 195 of protein formed hydrogen bond with H of benzimidazole group of ligand. Second hydrogen bond is formed between Pro 193 of protein with H of dihydropyrimidone group of ligand. The bond lenths were found to be 2.87 A. and 2.85 A. respectively. The third hydrogen bond was formed between Ile 108 of protein and hydroxyl of ligand.its bond length was found to be 2.73 A. Its thione derivative had moldoc score of kcal/mol and three hydrogen bond as that of its urea derivative which owned the bond length were 2.86 A, 2.71 A and 2.84 A respectively.5-(1h-benzimidazol-2-yl)-4-(3-hydroxyphenyl)-6- methyl-3,4- had moldoc score of kcal/mol and three hydrogen bond. The Cys 195 of protein formed hydrogen bond with H of benzimidazole group of ligand. Second hydrogen bond is formed between Pro 193 of protein with H of dihydropyrimidone group of ligand. The bond lenths were found to be 2.91 A. and 2.90 A. respectively. The third hydrogen bond was formed between Glu of protein and hydroxyl of ligand.its bond length was found to be 2.81 A. Its urea derivative had moldoc score of kcal/mol and had two hydrogen bonds. Hydrogen bond was formed between Ser 216 of protein with hydroxyl of ligand and the bond length was found to be 2.97A. The another hydrogen bond was formed between Cys 195 of protein and H of benzimidazole group of ligand and the bond length was found to be 3.28 A. 4-(4-aminophenyl)-5-(1Hbenzimidazol-2-yl)-6-methyl-3,4- with three hydroen bond and possessed moldoc score of kcal/mol. The Cys 195 of protein formed hydrogen bond with H of benzimidazole group of ligand. Second hydrogen bond is formed between Pro 193 of protein with H of dihydropyrimidone group near methyl substitution of ligand. The bond lenths were found to be 2.87 A. and 2.80 A. respectively. The third hydrogen bond was formed between Ile 108 of protein and amino group of ligand. Its bond length was found to be 2.73 A. Its thione derivative had moldoc score of and three hydrogen bond as that of its urea derivative which owned the bond length were 2.86 A,

5 Table 2 Ligand with different substitution X = O or S Gopinathan arasimhan et al. / Journal of Pharmacy Research 2015,9(1), its urea derivative which owned the bond length were 3.29 A, 2.86 A and 2.90 A respectively. H 3 C R O H 2 H H 3 C CH 3 H 3 C CH OH 3 O Cl OH H 3 C 2.84 A and2.75 A respectively. 5-(1H-benzimidazol-2-yl)-4-(2- with three hydroen bond and possessed moldoc score of kcal/mol. The Cys 195 of protein formed hydrogen bond with H of benzimidazole group of ligand. Second hydrogen bond is formed between Pro 193 of protein with H of dihydropyrimidone group of ligand. The bond lenths were found to be 3.35 A. and 2.91 A. respectively. The third hydrogen bond was formed between Glu 87 of protein and hydroxyl of ligand. Its bond length was found to be 2.95 A. Its thione derivative had moldoc score of and three hydrogen bond as that of O OH Cl OH 5-(1H-benzimidazol-2-yl)-4-(4-methoxyphenyl)-6-methyl-3,4- had moldoc score of kcal/mol. It had two hydrogen bonds. Hydrogen bond was formed between Cys 195 of protein with H of benzimidazole of ligand and the bond length was found to be 2.99 A. The another hydrogen bond was formed between Pro 193 of protein and H of dihydropyrimidone group near methyl substitution group of ligand and the bond length was found to be 2.61 A. Its thione derivative had moldoc score of and two hydrogen bond as that of its urea derivative which owned bond length of 2.99 A and 2.65 A respectively. 5-(1H- benzimidazol-2-yl)-4-(2-dihydropyrimidin- 2(1H)-one had moldoc score of kcal/mol. It had two hydrogen bonds. Hydrogen bond was formed between Cys 195 and Tyr 135 residues of protein with carbonyl oxygen of dihydropyrimidone of ligand and the bond length was found to be 2.59 A and 3.22 A. Thione derivative had moldoc score of kcal/mol and two hydrogen bond. Hydrogen bond was formed between Cys 195 of protein with H of benzimidazole of ligand and the bond length was found to be 2.89 A. The another hydrogen bond was formed between Pro 193 of protein and H of dihydropyrimidone group near methyl substitution group of ligand and the bond length was found to be 2.88 A. 5-(1H-benzimidazol-2-yl)-4-[4-(dimethylamino)phenyl]-6-methyl-3,4- dihydropyrimidine-2(1h)-thione had moldoc score of kcal/mol and one hydrogen bond.. Hydrogen bond was formed between Cys 195 residues of protein with H of benzimidazole of ligand and the bond length was found to be 3.77 A. Its urea derivative had mol doc score of kcal/mol and there was no hydrogen bond formation. 5-(1H-benzimidazol-2-yl)-4-(4-chlorophenyl)-6- methyl-3,4- had moldoc score of kcal/mol and two hydrogen bond. Hydrogen bond was formed between Cys 195 of protein with H of benzimidazole of ligand and the bond length was found to be 2.99 A. The another hydrogen bond was formed between Pro 193 of protein and H of dihydropyrimidone group near methyl substitution group of ligand and the bond length was found to be 2.71 A. Thione derivative had moldoc score of kcal/mol and there was no hydrogen bond. 5-(1H-benzimidazol- 2-yl)-4-(furan-2-yl)-6-methyl-3,4- had moldoc score of kcal/mol and three hydrogen bond. Two hydrogen bond was formed between Cys 195, Ser 216 residues of protein with oxygen of furan of ligand and the bond length was found to be A and A. The another hydrogen bond was formed between Arg 175 of protein of A ring and carbonyl oxygen of

6 Gopinathan arasimhan et al. / Journal of Pharmacy Research 2015,9(1), dihydropyrimidone group of ligand and the bond length was found to be 2.95 A. 5-(1H-benzimidazol-2-yl)-6-methyl-4-phenyl-3,4-dihydropyrimidin- 2(1H)-one had moldoc score of kcal/mol. It had three hydrogen bonds. The Arg 175 of protein of A ring formed hydrogen bond with oxygen of carbonyl of dihydropyrimidone group of ligand. Second hydrogen bond is formed between Ser216 of protein with H of dihydropyrimidone of ligand. The bond lenths were found to be 3.12 A. and 2.86 A. respectively. The third hydrogen bond was formed between Pro 193 of protein and H of benzimidazole and bond length was found to be 3.14 A. Thione derivative had no hydrogen bonds but with moldoc score of kcal/mol. The active site is present in A ring of the protein. The residue bind with the ligand is same as that of the standard compounds docked. The residues found in the active sites are as followsile 108, Pro 193, Cys 195, Ser 216, Glu 87, Tyr 135, Arg 175, Arg 176, Arg 215. The ligands favourite attraction site as follows Ile 108, Pro 193,Cys 195 and Ser 216. COCLUSIO In this study, the molecular docking was applied to correlate its docking score with the activity of a 3,4 dihydro pyrimidone derivatives.the docking studies provided good insights into the binding of pyrimidine derivatives at the molecular level,with the aim of rendering the more selective human tyrosine kinase inhibitors. Thio analogues are more potent than the other analogues. pyrimidinyl carboxylates were found to interact with at least one of the amino acid residue of the active binding site.it was clearly understood from the hydrogen bonding mentioned. 5-(1H-benzimidazol-2-yl)-4-(4-hydroxy-3- methoxyphenyl)-6-methyl-3,4-dihydropyrimidine-2(1h)-one had highest moldoc score of kcal/mol against 3bz3 (PDB ID) in docking analysis. Docking studies confirm that the main interaction of Focal adhesion tyrosine kinase enzyme Hydrogen bond and Hydrophobic interactions with the binding pockets made by H of benzimidazole and H and Carbonyl dihydropyrimidones of the ligands. This information has potential implications to understand the mechanism of Focal adhesion tyrosine kinase enzyme related enzymatic inhibition reactions, and also applicable in the prediction of more effective inhibitors and engineering 3D structures of other enzymes as well. Hence, it is concluded that that 5-(1H-benzimidazol-2-yl)-4- (4-hydroxy-3-methoxyphenyl)-6-methyl-3,4-dihydropyrimidine-2(1H)- one, 5-(1H-benzimidazol-2-yl)-4-(4- and 5-(1H-benzimidazol-2-yl)-4-(1H-indol- 3-yl)-6-methyl-3,4- could be a potent anti-cancer target molecule against Focal adhesion tyrosine kinase enzyme which may be worth for further clinical trials. In this study, computations on the interactions at the active site of Focal adhesion tyrosine kinase enzyme were carried out for twenty four ligands. In future, it may be necessary to explore the development of potential new Focal adhesion tyrosine kinase enzyme inhibitor drugs for treating cancer. The present study shall help in rational drug design and synthesis of new selective Focal adhesion tyrosine kinase enzyme inhibitors with predetermined affinity and activity and provides valuable information for the understanding of interactions between Focal adhesion tyrosine kinase enzyme and the novel 3,4 dihydropyrimidones. Hence we concluded that the Pyrimidine derivative is one of the Tyrosine kinase inhibitor. By using computational approaches derivatives designed showed good interactions with 3bz3 protein. ACKOWLEDGMETS The authors would like to express thanks Molegro ApS for providing a fully functional version of Molegro Virtual Docker software for a period of 30 days during which all the in-silico docking work was carried out. REFERECES 1. Daniel Lietha, Xinming Cai, Derek F.J. Ceccarelli,Yiqun Li, Michael D. Schaller, and Michael J. Eck, Structural Basis for the Autoinhibition of Focal Adhesion Kinase,Cell 129, June 15, 2007, Vita M Golubovskaya, Baotran Ho, Min Zheng, Andrew Magis, David Ostrov, Carl Morrison and William G Cance,Disruption of focal adhesion kinase and p53 interaction with small molecule compound R2 reactivated p53 and blocked tumor growth, Bio Medical Central Cancer,2013,13:342, Gordon W. McLean, eil O. Carragher, Egle Avizienyte, Jeff Evans, Valerie G. Brunton and Margaret C. Frame,The Role Of Focaladhesion Kinase In Cancer A ew Therapeutic Opportunity,ature Reviews,Cancer,Volume 5, July 2005, J. Thomas Parsons, Focal adhesion kinase: the first ten years, Journal of Cell Science, 2003,116, 8, Hugo Kubinyi, Structure-based design of enzyme inhibitors and receptor ligands, Current Opinion in Drug Discovery and Development, 1998, Vol 1,o 1, Source of support: il, Conflict of interest: one Declared