The Study Of TAK-117 Inhibiting Effect On Necroptosis

Background Necroptosis is an important form of cell death,which participates in the occurrence and development of many diseases.A variety of drugs that target and inhibit necroptosis are used in clinical research,as a new target for the treatment of diseases.Objective Necroptosis is a form of cell death independent of apoptosis and necrosis.TAK-117 can rescue TNF-α induced necroptosis.We want to clarify the mechanism of TAK-117 inhibiting necroptosis through related experiments.Methods(1)Observe the EC50 of TAK-117 to rescue necroptosis and screen out the IC50 of the drug through cytotoxicity test.(2)TAK-117 is a PI3 K inhibitor.Observe whether other PI3 K inhibitors can inhibit programmed necrosis of cells.(3)In vitro kinase experiment to detect the effect of TAK-117 on RIPK1 kinase activity.(4)Western blot(WB)is used to detect changes in cell protein expression and post-translational modification before and after drug rescue.(5)Immunoprecipitation(Immunoprecipitation,IP)was used to detect the effect of TAK-117 on complex’s stability.(6)Use Biotin-TAK-117 to determine the direct target of TAK-117.(7)TNF-α was injected into the tail vein to create a systemic inflammatory response syndrome(SIRS)disease model and observed the protective effect of TAK-117 on SIRS.Results(1)TAK-117 can rescue the necroptosis on the cells of mouse within a safe concentration range.(2)TAK-117 rescues the necroptosis of cells is a unique function of TAK-117,independent of the function of inhibiting PI3 K.(3)TAK-117 inhibits the occurrence of programmed necrosis but does not directly inhibit the kinase activity of RIPK1.(4)TAK-117 can inhibit phosphorylation of RIPK1 and MLKL.(5)The IP results showed that:TAK-117 destroyed the stability of the protein components of complex I and complex II and inhibited the ubiquitination of RIPK1.(6)Biotin-TAK-117 interacts with RIPK1 and RIPK3 to inhibit necroptosis.(7)TAK-117 can save the death of mice caused by TNF-α in vitro.Conclusion TAK-117 has the function of suppressing necroptosis both in vivo and in vitro,and this function is independent of inhibiting PI3 K.TAK-117 interacts with RIPK1 and RIPK3 to inhibit its ubiquitination and indirectly inhibits the kinase activity of RIPK1 to save.This research explain the mechanism of TAK-117 inhibiting necroptosis while expanding its functions.