Discovery,Target Identification And Structural Optimizations Of Necroptosis Inhibitor TAK-632

Necroptosis is a caspase-independent,cell death receptor-mediated programmed necrosis.It is mainly regulated by receptor interaction protein kinase 1?RIPK1?,receptor-interacting protein kinase 3?RIPK3?and mixed protein kinase domain-like protein?MLKL?.Many diseases,such as neurodegenerative diseases and ischemic diseases,viral infectious diseases are reported to have close relationship with necroptosis.Therefore,the discovery of necroptosis inhibitors is of great significance to the clinical treatment of related diseases.This study focuses on the discovery,target research and structural optimizations of novel necroptosis inhibitors.The main contents include three sections.1.Screening of necroptosis inhibitors,synthesis of biotin probes and target identificationUsing the in-house fluoride library?about 500 compounds?,we found that TAK-632 showed a promising protective effect on necroptosis models.TAK-632 almost completely protected HT-29 cells stimulated by TNF?,Smac analogs and z-VAD-FMK?TSZ?at 20?M with no significant toxicity and the EC₅₀ was 1.44±0.62?M.The mechanistic study showed that TAK-632 could specifically bind to RIPK1 and RIPK3.In order to further verify its targets,four biotin probes of TAK-632 were designed and synthesized.The bioactive results showed that the probe TAK-632-Biotin-4 had anti-necroptotic activity of HT-29 cells at high concentrations.Pull down assay confirmed the targets of TAK-632.In the TNF-?induced systemic inflammatory response syndrome?SIRS?mouse model,the survival rate was 70%when TAK-632 was administered at a dose of 100 mg/kg,and the body temperature of the mice increased significantly after 12 h.In this section,TAK-632 was identified as a novel anti-necroptosis lead compound for further structural optimization.2.Discovery and biological evaluation of dual-targeting TAK-632 analogues as necroptosis inhibitorsTAK-632 has a promising anti-necroptotic activity in vitro.However,it showed moderate in vivo efficacy at a high dose and the structure-activity relationship is still unclear.Through the Twenty-six target compounds were obtained by knowleage-based drug design and a preliminary SAR was concluded.Among them,compound B31j,a of noncyano-derivative of TAK-632,exhibited 8 times higher anti-necroptotic activity(EC₅₀=0.17±0.03?M)than that of TAK-632.The enzymatic activity aganist RIPK1?K_d=97 nM?and RIPK3?K_d=77 nM?were significantly better than TAK-632.The in vivo assay showed that B31j significantly increased mouse survival to 90%at the dose of 50 mg/kg compared to TAK-632 at a same dose.When the dose was 25 mg/kg,the survival rate was increased to 70%.The body temperature of the mice was quickly recovered at the two doses.Therefore,noncyano-TAK-632 analogue B31j could be used as a dual-targeting necroptosis inhibitor,providing an important lead compound for subsequent structural optimization.3.Discovery and biological evaluation of TAK-632 analogues selectively targeting RIPK3 as novel necroptosis inhibitorsOn the basis of the dual-targeting TAK-632 derivative B31j,fourteen analogues were designed and synthsized by two rounds of structural optimization.Most of the compounds showed nanomolar protective activity.Among them,the urea-containing compounds had high selectivity toward RIPK3.The enzymatic activity of compound C1m to RIPK3 was 61.7 times higher than that of RIPK1 with the anti-necroptotic activity of EC₅₀=0.44±0.1?M.It also protected the necroptosis of mouse-derived cells?L929?.The mechanistic study showed that compound C1m could inhibit the phosphorylation of RIPK3 and MLKL and directly block the formation of RIPK1 and RIPK3 necrosome.In the SIRS mouse model,the survival rate of mice was 90%when the oral dose of compound C1m was 5 mg/kg.At 25 mg/kg and 50 mg/kg,the survival rate was increased to 100%,which was much higher than that of TAK-632 and B31j.Collectively,a class of RIPK3 small molecule inhibitors were identified as anti-necroptosis inhibitor.