Research On Action And Mechanism Of GZD824 As A Pan-FGFR Inhibitor Against FGFR1 Mutation Resistance In Vitro And In Vivo

Background and objective:Fibroblast growth factors receptors（FGFRs）are a subfamily of receptor tyrosine kinases（RTKs）,which mainly include FGFR1,2,3,4 subtypes.FGFR1-4 shows abnormal activation in different solid tumors and hematomas,and activates downstream signals to promote the malignant proliferation,migration and invasion of cells,which leads to the occurrence and development of tumors.Although a variety of FGFR inhibitors have shown benefits in many clinical patients with FGFR1 aberrations,FGFR1 mutations resistance has become a new unresolved clinical problem,especially the gatekeeper site V561-based mutation resistance.So far,second-generation inhibitors that are effective against FGFR1 mutations are still in the early stage of development,and there are no drugs approved for marketing.This thesis intends to systematically study the effect and mechanism of GZD824,a class 1.1 new drug developed by the team early and currently in the NDA stage,as a pan-FGFR inhibitor in overcoming FGFR1 mutation resistance in vivo and in vitro,in order to bring new treatment options for such patients.Methods:Firstly,we studied the kinase inhibitory effect of GZD824 by Z’-LYTE^TMkinase assay in vitro,and then constructed a series of stable expression FGFR1 wild-type and mutant Ba/F3 tool cells by electroporation transfection,using TEL-FGFR1 plasmid.Then investigate the anti-proliferative activity of GZD824 on those Ba/F3 model cell lines in vitro.Then the mechanism of GZD824 mediated anti-proliferation and overcoming resistance was tested by molecular docking,CETSA,Western Blot,immunoprecipitation,flow cytometry etc.Furthermore,a mouse xenograft model was constructed to investigate the anti-cancer effect of GZD824 in vivo,Ki-67 and TUNEL staining were used to study the effect of GZD824 on tumor tissue cell proliferation and apoptosis.Results:The results demonstrated that GZD824 shows kinase inhibitory effect of FGFR1/2/3/4,with IC₅₀of 14±0.96,2.77±0.082,8.10±0.15 and 30.27±21.66 n M respectively;In vitro proliferation experiments showed that GZD824 is effective for FGFR aberrant cells with different activation mechanisms,and can effectively overcome FGFR1 V561M/F,K656N/E mutation resistance,with IC₅₀ of 55±3.7,12±0.5 n M to Ba/F3-FGFR1-V561M,Ba/F3-FGFR1-V561F gatekeeper mutant resistant cells.Through the investigation of its mechanism of action,it was found that GZD824binds to the FGFR1 protein binding pocket stably in the binding mode of a type II inhibitor,and can inhibit the activation of FGFR and its downstream key signaling pathways in a dose-dependent manner,can inhibit cell proliferation and promote cell apoptosis.GZD824 can also degrade FGFR1 protein in Ba/F3 tool cells through the ubiquitin-proteasome system.It also showed that GZD824 can inhibit the growth of mice xenograft,and inhibit the proliferation of tumor tissue cells and induce apoptosis in vivo.Conclusions:This study showed that GZD824 as a pan-FGFR inhibitor can overcome FGFR1-V561M and other mutation resistance in vivo and in vitro,which provides an important basis for broadening the indications of GZD824 in clinical trials.