Study On The Anti-tumor Activity And Mechanism Of A New Generation Of Tyrosine Kinase Inhibitor PN17-1 On Drug-resistant Cells K562/G01 And GIST-1210

Imatinib is a protein tyrosinase inhibitor that is clinically used to treat chronic myeloid leukemia（CML）with severe mutations in the Bcr-abl fusion gene and stomachs with mutations in the c-Kit gene Intestinal stromal tumor（GIST）,the early stage of treatment has a better treatment effect for patients.However,with the increase in the amount of clinical drugs and in-depth testing studies,it has been found that some patients have serious imatinib resistance problems,which greatly weakens or even invalidates the therapeutic effect of the first-line treatment drug imatinib.The main causes of drug resistance are Bcr-abl^T315Imutations in chronic myeloid leukemia（CML）and secondary mutations in the c-Kit gene of gastrointestinal stromal tumors（GIST）,which makes imatinib resistant to drug-resistant individuals.The follow-up treatment did not achieve the desired effect.Based on the problem of imatinib resistance,the development of new small-molecule targeted drugs has become a research hotspot.The purpose of this research is to develop a safe and effective small molecule targeted tyrosine kinase inhibitor that can overcome imatinib resistance.The small molecule compound PN17-1 in the subject is a new compound obtained by computer-aided drug design methods,combined with computer scoring results based on the strength of the compound’s penetration into the molecular target cavity and the modification of the structure of imatinib.This article aims to study the in vitro anti-tumor activity and mechanism of compound PN17-1 in the K562/G01 cell line of chronic myelogenous leukemia and gastrointestinal stromal tumor GIST-1210 cell line resistant to imatinib.Computer-aided drug design simulation results show that compound PN17-1forms two hydrogen bonds with methionine at position 318 and aspartic acid at position 381 of the Bcr-abl^T315Imutation chamber（PDB ID:3IK3）,and has a tetralin structure It can form a rigid hydrophobic structure,and the acetylene bond in the structure can make the compound penetrate deep into the molecular target cavity and form a strong force with the target protein.The scoring results are consistent with the computer-aided drug design results（15.7757）;the computer-aided drug design simulation results show that the compound PN17-1passes through the 640 glutamic acid and 810 aspartic acid of the c-Kit mutant protein（PDB ID:1T46）,And Serine 673 form three hydrogen bonds,and the tetralin structure can form a rigid hydrophobic structure.The acetylenic bond in the structure can make the compound penetrate into the molecular target cavity and form a strong force with the target protein.The scoring results are consistent with the computer-aided drug design results（14.5017）.MTT colorimetry to study the antitumor activity of compound PN17-1 in vitro,the growth curves of imatinib-resistant tumor cells K562/G01 and GIST-1210.Then MTT colorimetry was used to detect the drug resistance index of two drug-resistant cells,K562/G01 and GIST-1210,and the results showed that the drug resistance index of the drug-resistant CML K562/G01 cells compared with the non-resistant K562 cells reached 10.519 Stomach.Compared with the non-drug resistant cell GIST-882 cell,the drug resistance index of intestinal stromal tumor drug-resistant cell GIST-1210 cell is 7.495.The compound PN17-1 and the positive control drug imatinib were further determined by MTT colorimetric method.In vitro proliferation inhibition of tumor cells,chronic myelogenous leukemia cells（K562 cells and K562/G01 cells）,gastrointestinal stromal tumor cells（GIST-882cells and GIST-1210 cells）,the results showed that compound PN17-1 is resistant to The IC50 value of drug cell K562/G01 cells is 0.286±0.07μmol/L.The IC₅₀value of compound PN17-1 on drug-resistant GIST-1210 cells was 2.325±0.16μmol/L.Based on the results of the MTT colorimetric method,the compound PN17-1 has a significantly better in vitro proliferation inhibitory effect on the drug-resistant tumor cells K562/G01 and GIST-1210 cells than the positive control drug imatinib.Furthermore,the inhibitory effect of PN17-1 on the proliferation of different kinds of common tumor cells was detected by MTT colorimetry assay,and the selectivity of PN17-1 to tumor cells was investigated.The experimental results show that the compound PN17-1 has a higher selectivity for drug-resistant K562/G01 cells,and its effect of inhibiting cell proliferation is significantly better than other types of tumor cells.The results of time-effect curve and dose-effect curve showed that the effect to the two drug-resistant tumor cells K562/G01 and GIST-1210 cells was time-and concentration-dependent.The soft agar clone formation experiment was conducted to investigate the sensitivity of compound PN17-1 to two drug-resistant tumor cells K562/G01 and GIST-1210 cells.The results showed that the inhibition on the colony formation of tumor cells was significantly better than that of the positive control drug imatinib.The inhibitory effect of compound PN17-1 and the positive control drug imatinib on normal cell human embryonic kidney cells（HEK-293）was further detected by MTT colorimetric experiment,and the toxic effect of the compound on normal cells was detected.Study the mechanism of the compound on the two drug-resistant cells K562/G01 cells and GIST-1210 cells.Flow cytometry was used to investigate the effect of compound PN17-1 on the cell cycle kinetics of tumor cells.The experimental showed that proportion of G0/G1 phase cells in the administration group increased significantly with the increasing concentration of compound PN17-1.The experimental results show that The compound blocks the normal cell cycle process by blocking the cell cycle in G0/G1 phase.In addition,the cell cycle G0/G1 phase arrest effect of compound PN17-1 was significantly better than the positive control drug imatinib at various concentrations.The effect of compound PN17-1 on cell apoptosis was investigated by flow cytometry experiments.The experimental results proportion of compound cell apoptosis in the administration group gradually increased with the increase of drug concentration,and at each concentration,compound PN17-1 had a significantly better pro-apoptotic effect on tumor cells.The positive control drug imatinib.At the same time,the effect of compound PN17-1 on the expression of pathogenic proteins bcr-abl and c-kit and the expression of related signal pathway proteins were investigated from the protein level.The experimental results showed that compound PN17-1 can down-regulating the mutant Bcr-abl and c-Kit protein significantly,and at each concentration,the effect of down-regulating the expression of the pathogenic protein was significantly better than that of the positive control drug imatinib.And compound PN17-1 can significantly down-regulate the protein phosphorylation of STAT5 and Crkl.The effects of compound PN17-1 on the migration and invasion of tumor cell line GIST-1210 were investigated by scratch test and Transwell test.The results showed that the compound PN17-1 could significantly inhibit the migration of adherent tumor cells GIST-1210.The results of Transwell chamber experiment showed that the effect of compound PN17-1 on the migration and invasion of imatinib-resistant tumor cells GIST-1210 was significantly better than that of the positive control imatinib.The inhibitory effects of PN17-1 and imatinib on normal human embryonic kidney cells（HEK-293）were further detected by MTT colorimetry assay,and the toxicity of PN17-1 to normal cells was detected.Conclusion:Compound PN17-1 has a good in vitro proliferation inhibitory activity against two kinds of imatinib-resistant cells K562/G01 cells and GIST-1210cells.Compound PN17-1 blocked the cell cycle of two drug-resistant tumor cells in G0/G1 phase,blocked the normal process of the cell cycle,and down-regulated the expression and down-regulation of the cytopathic protein Bcr-abl protein and c-Kit protein.Signal pathway protein expression,down-regulation of STAT5 and Crkl protein phosphorylation inhibit cell proliferation.The experimental results provide a theoretical basis for the development of safe and effective small molecule inhibitors of protein tyrosine that can overcome imatinib resistance.