Discovery And Mechanistic Study Of Novel Class Of Small Molecule Kinase Inhibitors Based On High Throughput Screening With The Functional Isogenic BaF3 Cell Library

Gain of function mutations or overexpression of protein kinases play a critical role in cancerogenesis. Small-molecule inhibitors targeted for protein kinases, accordingly, have been developed as an important part of the oncology armamentarium. The first small-molecule kinase inhibitor approved by FDA was Imatinib, which achieved great success in clinical trials in 2001 and makes kinase inhibitors, as a kind of target-specific and adverse effects limited agents, edged into the first-line therapy for cancer treatment. Nevertheless the increasing common use of kinase inhibitors has revealed their own resctrictions, such as the toxicity resulted from off-target effects or the drug resistance developed during tumor treatment. Therefore, there are utmet clinical needs for developing novel kinase inhibitors which bear higher selectivity or have the efficacy to overcome more drug resistance. In addition, despite there are almost 530 kinds of protein kinases being discoverd, the biological or pathological roles of many kinases are not well understood yet. Thus, the development of selective inhibitor targeted these kinases is of vital importance for investigating the functional mechanism of specific protein kinases and finding out new therapeutic targets.Based on our functional isogenic BaF3 cell library and high-throughput screening facility, we dicoverd 5 selective small-molecule kinase inhibitors: CHMFL-110, CHMFL-074, CHMFL-155, CHMFL-159 and STK16-IN-1. After evaluating the in-vitro and in vivo activity of these compounds, we found:(1) CHMFL-110 is a selective c-KIT inhibitor, which completely abolished ABL and FLT3 kinase activity compared to the two clinically used c-KIT kinase inhibitor, Imatinib and Sunitinib. CHMFL-110 displayed great antiproliferative efficacy against gastrointestinal stromal tumors (GISTs) cell lines GIST-T1 and GIST-882 (GI50:0.021 and 0.043μM, respectively). In addition, CHMFL-110 possessed acceptable bioavailability and effectively suppressed the tumor growth in inoculated xenograft model and might be a potential drug candidate for GISTs therapy. (2) CHMFL-074 is a potent Bcr-Ab1 inhibitor (IC50:25nM), which not only exhibited stronger inhibitory activity against native Abl kinase than Imatinib but also inhibited a number of clinical relavant ABL mutations such as E255K, F317I, F317L, M351T, Q252H, Y253F and F369P. In the Bcr-Abl positive chronic myeloid leukemia (CML) patient primary cells and CML cell inoculated xenograft mouse model, CHMFL-074 achieved dose-dependent antiproliferative and tumor progression suppression efficacy. CHMFL-074 might be a prospective drug candidate for fighting CML. (3) CHMFL-155, which bears similar structure of CHMFL-074, has both Bcr-Abl (IC50: 46 nM) and c-KIT (IC50:75nM) kinase inhibitory activity. As CHMFL-155 possessed a good oral PK property, it is under preclinical evaluation for both CML and GISTs therapy. (4) CHMFL-159 is a highly selective PDGFRa inhibitor, which exhibited strong potency against PDGFRa kinase but not structurally similar kinases, such as Abl, c-KIT, FLT3, VEGFR. For PDGFRp, CHMFL-159 also exhibited 44-fold selectivity difference. In the PDGFRa-dependent cancer cell line EOL-1, CHMFL-159 displayed strong anti-proliferative efficacy and strongly blockage of the PDGFRa mediated signaling pathway at low concentration. Furthermore, CHFML-159 displayed great anti-tumor efficacy in EOL-1 cell inoculated xenograft mouse model, in which 100% TGI (tumor growth inhibition) was obtained with dosage of 100 mg/kg/day. The high selectivity and potency of CHMFL-159 suggested that it may be a useful pharmacological tool to further elucidate the pathology of PDGFRa in the related tumors. (5) STK16-IN-1 is a highly selective inhibitor against STK16 kinase (1C50:0.295 μM). In MCF-7 cells, treatment of STK16-IN-1 results in accumulation of binucleated cells and G2/M phase cell cycle arrest, which can be recapitulated by RNAi-mediated STK16 knockdown. STK16-IN-1 provides a useful pharmacological tool for further elucidating the biological function of STK16.