Pharmacological Study Of Cyclobentinib (CB1107) With Dual Inhibition On Drug-resistant Bcr-Abl And C-Kit Protein Tyrosine Kinases

At present,the first-line drug Imatinib for the treatment of chronic myeloid leukemia?CML?and gastrointestinal stromal tumors?GIST?has serious drug resistance problems,and the therapeutic effect of Imatinib on drug-resistant CML patients and GIST patients is weakened or ineffective.This has made the development of new small molecule targeted drugs to solve the problem of Imatinib-resistance become a research hotspot.The small molecule compound cyclobutinib?CB1107?studied in the subject is a novel compound entity obtained by optimizing and reforming the structure of Imatinib,through the rational drug design theory and skeleton transition method.The purpose of this study is to investigate the anti-tumor activity and mechanism of CB1107 on Imatinib-resistant chronic myeloid leukemia K562/G01 cell line and gastrointestinal stromal tumor GIST-1210 cell line.Combined with computer-aided drug design software,the molecular docking method was used to simulate the binding mode of receptor protein to small molecule compounds.The simulation results indicate that CB1107 forms three hydrogen bonds with Glu-305,Asp-400 and Met-337 to enter the catalytic domain of"DFG-out"state Abl^T315I kinase.Moreover,the six-ring structure extended to the hydrophobic regions?Leu-317,Val-398 and Ile-312?,and the affinity of CB1107 for the"DFG-out"conformation of Abl^T315I kinase was higher than Imatinib.The inhibitory effect of Imatinib and CB1107 on K562 cell line,K562/G01cell line,GIST-882 cell line and GIST-1210 cell line was detected by MTT assay.The IC₅₀ values of Imatinib to K562 cells and K562/G01 cells were0.33±0.13?mol/L and 6.56±0.28?mol/L,respectively.The results showed that the K562/G01 cells was 19.87-folds resistance to Imatinib.The IC₅₀ value of CB1107to K562/G01 cells was 0.43±0.08?mol/L,and this inhibitory effect was 15-folds more than Imatinib.The IC₅₀ values of Imatinib to GIST-882 cells and GIST-1210cells were 1.89±0.87?mol/L and 14.5±0.11?mol/L,respectively.The results showed that the GIST-1210 cells was 7.32-folds resistance to Imatinib.The IC₅₀ value of CB1107 to GIST-1210 cells was 10.11±0.41?mol/L.It was found that CB1107 has high selectivity to K562/G01 cells by detecting the inhibitory effect of CB1107 on different cell lines.CB1107 inhibited the two drug-resistant tumor cells in a time-dependent and concentration-dependent manner.Elisa results showed that CB1107 significantly inhibited the drug-resistant Bcr-Abl kinase and c-Kit kinase.The results of soft agar colony formation assay showed that CB1107 significantly inhibited the colony formation of two Imatinib-resistant tumor cells,indicating that the two drug-resistant tumor cells have high sensitivity to CB1107.Thepharmacodynamic study of CB1107 in mice demonstrated that CB1107 significantly inhibited the growth of solid tumors in drug-resistant human chronic myeloid leukemia mice,and the effect was significantly better than Imatinib.The mice administered did not show death or a significant decrease in body weight,indicating the in vivo safety of CB1107.The change of cell cycle kinetics after 48 hours of administration was detected by FCM method.The results showed that the number of cells in G1 phase increased significantly and the number of cells in S phase decreased after administration.And the number of cells in G1 phase was positively correlated with the concentration of drug,indicating that the drug blocked the cell cycle in G1 phase,and the blocking effect of CB1107 was stronger than that of Imatinib.Flow cytometry results indicated that CB1107 can induce apoptosis in both drug-resistant tumor cells.At the same time,this study examined the effects of CB1107 on the relative expression of two pathogenic genes and their encoded pathogenic proteins at the gene level and protein level,respectively.The results showed that CB1107 significantly down-regulated the Bcr-Abl and c-Kit protein encoded by the two genes,respectively,and the down-regulation of CB1107 was significantly stronger than Imatinib.The results of RT-PCR demonstrated that CB1107 significantly up-regulated the expression of Caspase-3 and Caspase-8 genes,and down-regulated the expression of MDR gene in K562/G01 cells.In situ analysis of the pathogenic protein expression of GIST-1210 cells by cellular immunofluorescence experiments was also consistent with the results of Western Blot.The results of the scratch test showed that CB1107 can inhibit the metastasis of tumor cells by inhibiting the migration ability of GIST-1210 cells.Through the above experimental studies,the following conclusions were obtained:Cyclobutinib?CB1107?has a good inhibitory effect on Imatinib-resistant K562/G01 and GIST-1210 cells and drug-resistant kinase expression,and is resistant to chronic myeloid leukemia NOD/SCID mice.Has very good in vivo efficacy and safety.CB1107 blocks the cycle of two drug-resistant tumor cells in G1phase,and inhibits cell proliferation by down-regulating the expression levels of pathogenic genes and pathogenic proteins of both cells,by up-regulating the expression of apoptosis-related genes and inhibiting phosphorylation of p-STAT5and p-Crk1 proteins.At present,most of the inhibitors of anti-T315I mutations are still in clinical trials.All of the above results provide a theoretical basis for the development of safer and more potent novel tyrosine kinase inhibitors against Imatinib resistance problem.