Research On Activation Mechanism And Inhibitor Discovery Of NLRP3 Inflammasome

The NLRP3 inflammasome is a multimeric protein complex consisted of pattern recognition receptor NLRP3,adaptor protein ASC and effector protein pro-caspase-1.Upon activation,NLRP3 inflammasome promotes the maturation and secretion of IL1β and IL-18 and induces pyroptosis by mediating the autocleavage and activation of pro-caspase-1.Unlike other pattern recognition receptors,NLRP3 can be activated by various pattern molecules or danger signals with completely different morphological structures and molecular sequences,resulting in an extremely complex activation mechanism.In addition,because NLRP3 can be activated by various signals,NLRP3 inflammasome is involved in the onset and development of a wide range of human diseases,including non-alcoholic fatty liver disease,multiple sclerosis,atherosclerosis,tumors and others,but there are still no clinically available medications targeting the NLRP3 inflammasome.Therefore,our study aims to further reveal the activation mechanism of NLRP3 inflammasome and develop small molecule inhibitors targeting NLRP3,including two major parts:Ⅰ.The effects and mechanisms of S6Ks in NLRP3 inflammasome activationS6K proteins include S6K1 and S6K2,both mTOR pathway effectors with protein kinase activity,which can promote protein synthesis and cell growth.Dysregulation of the S6Ks has been implicated in the progression of several human diseases,including diabetes,cancer and obesity.However,the role of S6Ks in inflammation is still unclear.We investigated whether S6Ks is involved in the regulation of NLRP3 inflammasome activation.We found that:1.S6Ks inhibitor PF4708671 blocks NLRP3 inflammasome activation;2.S6Ks knockdown(siRNA)specifically inhibits NLRP3 inflammasome activation without affecting AIM2 or NLRC4 inflammasome activation;3.S6K2 knockdown does not affect the priming phase of NLRP3 inflammasome activation;4.mTOR does not affect NLRP3 inflammasome activation;5.S6Ks interact with NLRP3,and S6K2 binds the NACHT and LRR domain of NLRP3;6.S6Ks knockout does not affect NLRP3 inflammasome activation.Therefore,our study found that S6Ks may play a complex role in NLRP3 inflammasome activation,which can provide guidance for follow-up research.Ⅱ.The effects and mechanisms of an RRx-001 analogue in inhibition of NLRP3 inflammasome activationWe have previously identified RRx-001 as a potent inhibitor of NLRP3,and RRx001 effectively alleviate NLRP3-related inflammatory diseases in mice.However,RRx-001 contains unique high-energy nitro functional groups,so RRx-001 can decompose rapidly and generate highly toxic oxidant.Therefore,structural modification of RRX-001 is an important strategy to reduce the risk of toxicity and contribute to the search for safer NLRP3 inflammasome inhibitors.Through structural modification of RRX-001,we found that compound 149-01,an RRx-001 analogue without high-energy nitro functional groups,is a potent,specific and safe NLRP3 inhibitor.We found that:1.149-01 inhibits canonical,non-canonical and alternative NLRP3 inflammasome activation in mouse BMDM cells or human PBMC cells;2.149-01 does not affect the priming phase at concentrations that inhibit NLRP3 inflammasome activation;3.149-01 specifically inhibits NLRP3 inflammasome activation without affecting AIM2,NLRC4 or Pyrin inflammasome activation;4.14901 does not affect the upstream events required for NLRP3 activation,and 149-01 suppresses the assembly of NLRP3 inflammasome by preventing the NEK7-NLRP3 interaction;5.149-01 prevents the NEK7-NLRP3 interaction by covalently binding to cysteine 409 of NLRP3;6.149-01 treatment effectively attenuated several NLRP3related inflammatory diseases in mice,including systemic inflammation,peritonitis and experimental autoimmune encephalomyelitis,but does not affect the physiological parameters of naive mice.Therefore,our study suggested that 149-01 can inhibit NLRP3 inflammasome activation and alleviate NLRP3-related inflammatory diseases by targeting NLRP3 protein,while avoiding the therapeutic risks associated with the presence of highenergy nitro functional groups.Since RRx-001 has entered into phase III clinical trial of anti-tumor,149-01 and its analogues provide new options for the development of clinical drugs targeting NLRP3.