Necroptosis Small Molecule Inhibitors Screening And Anti-necroptosis Molecular Mechanism Investigation

Necroptosis has been demonstrated to be another kind of programed cell death(PCD),apart from apoptosis and autophage.The indentifications and function investigations of necroptotic signaling factors:RIPK1,RIPK3 and MLKL,have revealed the fine-tuning of necroptotic signaling.MLKL has been found to be the final executor of necroptosis,but the precise mechanisms of how oligomerized MLKL translocating to membrane fraction and provoking plasma membrane permeabilizatin still remain obscure.Necroptosis shows important roles in many diseases,and is going to be an important drug developing direction.For this screening and mechanism study for new anti-necroptosis small molecules,we have two objects.First,we hope to get more knowledge of the molecular mechanism of necroptosis signaling pathway,by identifying the targeting proteins of compound hits.Second,considering the different limitations of existing anti-necroptosis inhibitors as drug precursos,we try to get new structural necroptosis inhibitors as potential drug developing materials.Through a necroptosis cell death assay,with cotreatment of TNF-a,Smac mimetic and Z-VAD.fmk as necroptosis inducer,we finally got about 180 compound hits with anti-necroptosis activity,out of a pool with～310,000 different compouds.Acroding to the 3D structure alignment,different activities in other cell death assays and effects on key necroptosis biomarkers of these hits,they were classified into different groups,including Necrostatin-like RIPK1 ihibitors,NSA-like MLKL inhibitors,and so on.In oligomerization of RIPK3 inducing necroptosis assay and thermal shift assay screenings,we also got some inhibitors block necroptosis by inhibiting RIPK3’s kinase activity.According to the goals of compound screening,we finally chose compound C236N12 and S37J14 for further mechanism investigation.By using biochemical and chemicobiological methods,we have revealed that both of these two compounds target to MLKL and covalently bind at MLKL’s site Cys86.They block necroptosis by disrupting the formation of MLKL oligomers and preventing MLKL’s translocation to plasma membrane.In the process of compound targets investigation,we have developed a system that could activate necroptosis pathway by inducing RIPK3 or MLKL oligomerization,which dosen’t rely on the upstream signaling transduction.It would be useful in anti-necroptosis drug screening,necroptosis inhibitor target sites determing and necroptosis molecular mechanism research.Through SAR analysis and structure optimizatioin,C236N12’s anti-necroptosis bioactivity has been greatly improved,with EC50 equals to 2nM.It becomes a highly specific necroptosis inhibitor with great drugable potential.