In Silico Screening Of Oncogene CDK2 Inhibitors And Validation As A Potential Anti-cancer Drugs

ObjectiveWith increased incidence of tumors, cancer has become one of the most serious chronic diseases that threaten human survivalin recent years. Cancer treatments include surgery, chemotherapy, radiation therapy, and biological therapy. Due to drug-resistant cancer patients on chemotherapy has increased in recent years, limit the existing chemotherapy in cancer patients. The development of new chemotherapy drug is extremely urgent. The activation and deactivation of cyclin-dependent kinase (cyclin-dependent kinases,CDKs)maintain cell cycle phase in an orderly manner. CDK2 phosphorylation of the Rb(retinoblastoma, Rb)is more important to switch G1 to s phasein cell cycle. Inhibition of CDK2 can inhibit proliferation of tumor cells. Hence, CDK2 is a more important target to anti-cancer drug. Computer-aided drug design is a computer simulation of molecular interaction, through the integrated use of bioinformatics tools, so as to achieve the objective selected, find the candidate drug. Protein-ligand docking using computer software (idock) predicts a small molecule of the best conformation and affinity strength when the non-Covalent binding to the target protein, through the strength of its affinity to rate high and low filter to target protein inhibitors. The experimental use of protein-ligand docking software from FDA registered drug targeted database screening CDK2 inhibitors can greatly save cost and time for new drugs. Candidates of CDK2 subsequentlywere validated in vivo and in vitro assay to verify its biological activity. The candidates of CDK2 inhibitors through protein-ligand docking software (idock) were validated in vivo and in vitro to find out new CDK2 inhibitor to treat colorectal cancer, liver cancer.Research methods:(1) It was verified by the Redocking to validate the accuracy and validity of idock software.(2) The candidates of target protein inhibitors was found out by the use of protein-ligand docking software (idock) CDK2.(3)The objective drug was found out by MTT inhibition rate of tumor cell.(4)The the objective to cell cycle was investigated to verify its role in causing cell cycle arrest.(5) The purposes of apoptosis was tested by flow cytometry.(6) It was tested by Western Blot to detect of cell cycle related protein expression of CDK2, cyclinDl, cyclinB1, cyclinE, and Rb, pho-CDk2, of pho-Rb.(7)The growth inhibitoray of cancer by treat tumor nude mice model was observed.(8) Structure analysis of the idock protein-ligand docking software illustrated molecular mechanism of the objective drugs inhibiting CDK2.Research results:(1) The result of redocking indicated that idock software in our assay was more accuracy, effectiveness improved 7%,8.69-37.51 times faster than that of the similar software.(2) 9candidates inhibitortargeted CDK2 were found and bought by protein-ligand docking software (idock) and then validated their bioavailability in vivo and in vitro.(3) We first evaluated the anti-cancer effect of the nine compounds by MTT assay. Among them, Adapalene had the lowest IC50:4.43μM for DLD1, and 7.135μM for LOVO. Adapalene exhibited the highest cytotoxicity compared to the control with statistical significance (p<0.05); Fluspirilene had the lowest IC50: 4.017μM for HepG2 and 3.468μM for Huh7. Fluspirilene exhibited the highest cytotoxicity compared to the control with statistical significance (p<0.05). Such inhibition effect was dose-and time-dependent.(4)Adapalene treatment significantly increased the percentage of cells in Gl phase compared to the control (p<0.05) in a dose- and time-dependent manner. At 30μM or 10μM concentration, Adapalene treatment continuously increased the percentage of G1 phase for 24 hours.Fluspirilene treatment significantly increased the percentage of cells in Gl phase compared to the control (p<0.05) in a dose-and time-dependent manner. At 30μM or 10μM concentration, Fluspirilene treatment continuously increased the percentage of G1 phase for 24 hours.(5) Adapalene treatment at 30μM or 10μM concentration significantly increased the percentage of apoptosis in DLD1and LOVO cell lines compared to the control in a dose-and time-dependent manner.Fluspirilene treatment at 30μM or 10μM concentration significantly increased the percentage of apoptosis in Huh7 and HepG2 cell lines compared to the control in a dose-and time-dependent manner(p<0.05).(6) Adapalene treatment DLDland LOVO colon cancer celllinesdemonstrated decreased theexpressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 onThr160 and Rb on Ser795, cyclinD1, cyclinB1 protein expression did not change. Fluspirilene treatment HepG2 and Huh7 liver cancer cell lines demonstrated decreased theexpressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 onThr160 and Rb on Ser795, cyclinD1, cyclinB1 protein expression did not change.(7) the Adapalene DLD1 subcutaneous tumor in BALB/c mice transplantation tumor model in nude mice in drug treatment, Adapalene (20 mg/kg) resulted in significant reduction of tumor weight and volume compared to the control (p<0.05); Fluspirilene Huh7 subcutaneous tumor in BALB/c mice transplantation tumor model in nude mice in drug treatment, Fluspirilene(20 mg/kg) resulted in significant reduction of tumor weight and volume compared to the control (p<0.05).(8)Though molecular docking and molecular dynamics study, Adapalene primarily bound with CDK2 by hydrophobic keys Phe82, Ile10, Leu134, Lys33, His84, competing with CDK2 binding site ATP Pocket. Fluspirilene mainly bound with CDK2 through hydrophobic keys Phe82, Ile10, Leu134, His84, competing with CDK2 binding site ATP pockets.Conclusion:This experiment using the computer software for protein-ligand idock screening candidates inhibitortargeted CDK2 and further validated their bioavailability in vivo and in vitro laboratory. We found out anti- Colorectal cancer drug Adapalene and anti-liver cancer drug Fluspirilene. Adapalene bound with CDK2 by hydrophobic keys Phe82, Ile10, Leu134, Lys33, His84, competing with CDK2 binding site ATP Pocket; Fluspirilene bound with CDK2 hydrophobic keys Phe82, Ile10, Leu134, His84, competing with CDK2 binding site ATP pockets, thus inhibiting CDK2 activity. By inhibiting CDK2 cell cycle pathway so that the cell blocked in the G1 phase, and further induced apoptosis and slowed the growth of tumor cells. Adapalene /Fluspirilene showed good and low toxicity of orally active agents, can be a potential anti-cancer and good CDK2 inhibitor of colon/liver cancer targeted drugs.