Screening Of Survivin Inhibitors Based On Computer Simulation Of Molecular Docking Technology And Study On Its Anti-gastric Cancer Activities

Objectives:Survivin is a tumor-specific apoptosis inhibitory protein that plays an important role in tumor formation and maintenance,and is considered to be an ideal anticancer target.However,the number of inhibitors of Survivin developed in recent years is limited,and most of these inhibitors reduce the level of Survivin by interacting with other biomolecules rather than directly interacting with Survivin proteins.Despite these challenges,the development of more effective and selective Survivin inhibitors is significant for better exploring anticancer drugs to inhibite the function of Survivin proteins.Therefore,in this study,we targeted Survivin protein and screened its inhibitors via computer simulation of molecular docking technology from the Novell Natural and Semi-Natural Diversity Small Molecular Database.The high-scoring small molecule Survivin inhibitors were found and its anti-gastric cancer effects were verified by in vitro and in vivo experiments.This study laid the theoretical foundation for the development of a new anti-gastric cancer drug targeting Survivin.Methods:1.Virtual screening:The three-dimensional structure of Survivin protein was determined in the PDB database.The Schrodinger docking method was used to predict the Survivin binding site and molecular docking,and the small molecule compounds with higher docking scores were screened.2.Verification of the actual binding ability:Survivin protein was immobilized on the surface of CM5 chip,and SPR technology was used to observe the actual binding ability between small molecule compound and Survivin protein,which was detected by Biacore X100 Protein Interaction System.3.In vitro experiment:Immunofluorescence was used to detect the expression of Survivin protein in gastric cancer cell lines:SGC 7901 and MKN 45 cells.Human normal gastric mucosal epithelial cell:GES 1 was used as normal control.MTT assay was used to detect the effect of small molecule inhibitors on the proliferation of gastric cancer cell line(SGC 7901 and MKN 45)and human normal gastric epithelial cell GES 1.TUNEL staining was employed to detect each group of cells'apoptosis.Cell cycle was also investigated by flow cytometry.Scratch test and Transwell chamber test were used to investigate the migration and invasive abilities of each group of cells.The expressions of Survivin,Bax,Bcl-2,Caspase-3,Caspase-9,Cyclin B1 and CDK1 proteins in each group of cells were detected by Western blot.4.In vivo experiment:Human gastric cancer cell SGC-7901 nude mouse xenograft model was established and randomly divided into control group,vehicle control group,UP group and BRU group.The effect of UP and BRU on the growth of xenografted tumor in nude mice was observed and the tumor inhibition rate were calculated.HE staining was used to observe the pathological changes of transplanted tumors and all the mouse organs in each group.The expression of Survivin,Bax,Bcl-2,Caspase-3 and Caspase-9 proteins in tumor tissues was detected by immunohistochemical staining.Results:1.Survivin protein(PDB ID 4A0I)inhibitors were screened from the Novell Natural and Semi-Natural Diversity Small Molecular Database(http://jkchemical.com/)by the Virtual Screening Workflow tool.50 small molecular compounds were screened out by 3 rounds of screening.Two small molecule compounds were selected from the 50 compounds by re-induced inductive docking.The two small molecule compounds were:Undecyl erythromycin STOCK1N-52340(Undecylprodigiosin,UP)and strychnine N17-X9570-357(Brucine,BRU).2.The actual binding ability of the above two small molecules to Survivin protein was verified by SPR technology.The results showed that the small molecule inhibitors(UP and BRU)had strong binding ability with Survivin protein,which was consistent with the computer simulation results.3.Immunofluorescence assay showed that survivin protein was overexpressed in gastric cancer cell:SGC7901 and MKN45,but in normal GES1 cell,survivin protein was not found expression.4.The effects of UP and BRU on SGC 7901 cells,MKN 45 cells and GES 1 cells were detected by MTT assay.The results showed that UP and BRU could inhibit the proliferation of SGC 7901 cells and MKN 45 cells,and presented concentration and time dependence(P<0.05).UP and BRU had no obvious effects on GES 1 cells.5.TUNEL assay showed that the number of apoptotic cells(SGC 7901 cells and MKN 45 cells)was significantly increased after UP and BRU treatment for 48 h,except for GES 1 cells,indicating that both UP and BRU could promote cell apoptosis,the difference was statistically significant(P<0.01).6.The results of flow cytometry showed that UP and BRU could block the cell cycle in G2/M phase of SGC 7901 cells and MKN 45 cells,but had no obvious effect on GES 1 cells.7.Scratch test showed that UP and BRU can inhibit the migration ability of SGC 7901 cells and MKN 45 cells,but no effect on GES 1 cells.Transwell chamber experiments also showed that UP and BRU could inhibit the invasion ability of SGC 7901 cells and MKN 45 cells,but no obvious effect on GES 1 cells.8.The expression of Survivin,Bax,Bcl-2,Caspase-3,Caspase-9,Cyclin B1 and CDK1 proteins were respectively detected Western blot.Results showed that the expression of Survivin,Bcl-2,CDK1 and Cyclin B1 proteins were significantly decreased,but Caspase-3(P<0.01).Caspase-9 and Bax were increased(P<0.01).9.The nude mouse xenograft model of human gastric cancer SGC 7901 cells was treated by intraperitoneal injection of UP and BRU.The growth of transplanted tumors was observed daily.The results showed that UP and BRU could significantly inhibit tumor growth in nude mice,andtumor inhibition rates were28.09%and 18.47%,respectively(P<0.01).10.Pathological anatomy and HE staining of transplanted tumor tissues and major organs in nude mice showed that the tumor tissues in UP and BRU treatment groups grew slowly as compared with the blank and vehicle control groups.There were no obvious pathological changes were found in the main organs of nude mice,such as heart,liver,spleen,lung and kidney.11.The expression of Survivin,Bax,Bcl-2,Caspase-3 and Caspase-9 protein in tumor tissues was detected by immunohistochemical staining.The expression of Survivin and Bcl-2 protein in UP and BRU groups was significantly decreased(P<0.01),while the expression of Caspase-3,Caspase-9 and Bax protein were significantly increased(P<0.01).Conclusions:1.Two potentially bioactive natural small molecule compounds were screened out by computer high-throughput screening and molecular simulation docking technology:STOCK1N-52340(UP)and N17-X9570-357(BRU).The actual binding capacity of UP and BRU to Survivin protein is consistent with the results of computer simulation.2.UP and BRU have significant proliferation inhibition effects on both human gastric cancer cell lines,which is consistent with the results predicted by computer simulation.UP and BRU could significantly inhibit the growth of human gastric cancer xenograft tumors in nude mice,and all the organs of nude mice did not have any pathological abnormal changes.3.In vitro and in vivo experiments confirmed that UP and BRU have anti-gastric cancer activity,and its possible mechanism is related to the induction of tumor cell apoptosis and cell cycle arrest.As have the advantages of high selectivity,affinity and effectivity,UP and BRU have the potential development values.