Design,synthesis And Anti-NLRP3 Inflammasome Activity Study Of Chalcone Analogues

Background and purposeNLRP3 inflammasome is a multi-protein complex.Inhibiting NLRP3inflammasome can mediate inflammation and related diseases.In recent years,anti-inflammatory drug development targeting NLRP3 inflammasome has become a hot research field,but there is currently no drug on the market for this target.Although Several NLRP3 inflammasome inhibitors have been successively promoted to clinical phase I or II studies,their safety issues have not yet been identified.Our research group screened drugs that have been used clinically or have been in late-stage clinical trials,and identified GFT505 with low inhibitory activity(IC₅₀=39.6μM)against NLRP3inflammasome-mediated IL-1βrelease.As a drug candidate for the treatment of NASH,GFT505 is a PPAR-α/δdual agonist with chalcone scaffold.In May 2020,it was disclosed to fail in clinical Phase III trials.Despite this,the chalcone scaffold was at least proved to possess good safety.Here,based on the chalcone scaffold of GFT505,we synthesized a series of analogues and studied the anti-NLRP3 inflammasome activity,drug-like properties,biological mechanism,and in vivo efficacy of related animal models to obtain more advantageous lead compound,providing new ideas and direction for the development of novel NLRP3 inflammasome inhibitors.Research methodPart I:Based on the chalcone scaffold of GFT505,39 analogues were designed and synthesized,and the analogues were tested for their inhibitory activity on IL-1βrelease mediated by activation of NLRP3 inflammasome.Further,according to the inhibitory activity,Michael receptor reactivity assay and cytotoxicity test were carried out to comprehensively evaluate the drug-like properties of representative compounds.Part II:The effects of compound 40 on the expression of NLRP3,ASC,Pro-IL-1β,Pro-caspase-1 and the release of IL-1βand Caspase-1 were verified,as well as the effect of compound 40 on pyroptosis.Meanwhile,the selective effect of compound 40 on NLRP3 inflammasome was verified,and the effect of compound 40 on production of intracellular ROS and mitochondrial ROS was also detected.Part III:the efficacy of compound 40 in vivo was verified through the mice model of LPS-induced sepsis and DSS-induced colitis.ResultPart I:Among the analogues of GFT505,compound 40 shows a good inhibitory activity on the release of IL-1βmediated by NLRP3 inflammasome in vitro with IC₅₀of 1.3μM.Further studies have shown that compound 40 has no obvious cytotoxicity,and its therapeutic index is as high as 119.7.At the same time,the Michael acceptor reactivity experiment also confirmed that compound 40 is a safe covalent compound with weak electrophilicity.Part II:Compound 40 did not affect the expression of NLRP3,ASC,Pro-IL-1βand Pro-caspase-1,but inhibited the release of Caspase-1 and IL-1β.Compound 40could inhibit LDH activity,indicating that 40 could inhibit pyroptosis.Further experiments confirmed that compound 40 selectively inhibited NLRP3 inflammasomes,but not AIM2 or NLRC4 inflammation.Compound 40 could also inhibit production of intracellular ROS and mitochondrial ROS.Part III:In the mice model of LPS-induced sepsis,compound 40 can significantly reduce the level of IL-1βin serum and peritoneal fluid.In the mice model of DSS-induced colitis,compound 40 can dose-dependently attenuate the decrease in colon length and fecal occult blood,the increments level of IL-1βin colon was also prevented by compound 40.Conclusion1)Based on the chalcone scaffold of GFT505,39 analogues were designed and synthesized.Among them,compound 40 exhibited a 30-fold increase in inhibitory activity against NLRP3 inflammasome mediated IL-1βrelease in vitro(IC₅₀=1.3μM)compared with GFT505.2)The drug-like evaluation showed that 40 is a safe covalent compound with weak electrophilicity,and the therapeutic index is as high as 119.7.3)Further studies have shown that 40 can specifically inhibit the activation of NLRP3 inflammasomes,which may be mediated by inhibiting the production of mitochondrial ROS.4)Finally,compound 40 has a significant therapeutic effect on mice model of LPS-induced sepsis and DSS-induced colitis.All in all,compound 40 is a more advantageous lead compound,which can be further developed and studied as a NLRP3 inflammasome inhibitor.