Design,Synthesis And Biological Evaluation Of Novel NLRP3 Inflammasome Inhibitors From Three Scaffolds

Inflammasomes are large,cytosolic andmultimeric protein complexes which respond to a diverse set of pathogen-associated molecular patterns(PAMPs)and danger-associated molecular patterns(DAMPs).Among the inflammasomes that have been identified,NLRP3 is the best characterized and the most elusive one.The aberrant activation of NLRP3 inflammasome has been involved in a subset of human hereditary or acquired inflammatory diseases,which makes NLRP3 inflammasome as an attractive target for drug discovery.In this thesis,we designed and synthesized small molecule inhibitors derived from three bioactive scaffolds for targeting NLRP3 inflammasome,and systematically explored the structure and activity relationship(SAR)of these compounds.In the first chapter,through high-throughput screening(HTS)campaign on an in-house bioactive compound library,CFTRinh-172(1-1)was identified as a potent ’hit’for blocking NLRP3 inflammasome activation.Preliminary cell-based SAR study led to the discovery of a potent and selective NLRP3 inhibitor,CY-09(Ⅰ-9).Molecular mechanism study revealed that Ⅰ-9 directly targeted NLRP3 protein for blocking NLRP3 inflammasome activation,but not inhibited AIM2 and NLRC4 inflammasomes.Ⅰ-9 exhibited in vivo efficacy in NLRP3-related mouse disease models and ex vivo effect in monocytes derived from patients with gout.Ⅰ-9 would serve as a good starting point for the development of new therapeutics against NLRP3 inflammasome related diseases.Further medicinal chemistry efforts were focused on chemical modification of thiazolidinone scaffold and a total of 38 derivatives were designed and synthesized.Systematic structure and activity relationship(SAR)study led to the identification of two sulfonamide derivatives Ⅰ-36 and Ⅰ-43b as new potent NLRP3 inhibitors with biochemical IC50s of 0.42 and 0.68 μM against IL-1β secretion.Ⅰ-36 and Ⅰ-43b were confirmed to block the activation of NLRP3 inflammasome by directly targeting NLRP3 protein.In vivo efficacy study revealed that Ⅰ-36 and Ⅰ-43b also showed remarkable therapeutic effects on mouse models of MSU-induced peritonitis.Another round of medicinal chemistry optimization based on Ⅰ-36 and Ⅰ-43b was performed with the synthesis of 61 derivatives having hydrophobic fragments.1-89 was identified having good inhibitory activity against NLRP3 with inhibition rate of 55.6%at 1 μM.Systematic SAR exploration of 2-thioxothiazolidin-4-one scaffold revealed that 3-trifluoromethylphenyl.2-naphthyl and cyclohexyl groups(Ring A),one carbon spacer(n=1)between Ring A and Ring B,the thioxo group(X=S)(Ring B)and ortho-methylsulfonyl and meta-sulfamoyl moiety(Ring B)were key structural features for achieving both potent inhibitory activity and selectivity against NLRP3.The representative compounds were exemplified by Ⅰ-36,Ⅰ-43b,Ⅰ-89.In the second part,23 derivatives were designed and synthesized based on the acrylate scaffold using pharmacophore-hybrid strategy.Through SAR study,we obtained two best compounds sulfonamide derivatives Ⅱ-36 and Ⅱ-37 as new NLRP3 inhibitors with inhibition rate of 95%and 92%at 1 μM,respectively.Ⅱ-36 and Ⅱ-37 specifically inhibited the NLRP3 inflammasome but not the NLRC4 and AIM2 inflammasomes nor TLR-mediated priming signals.In the third part,we have discovered that oridonin exhibited therapeutic effects on mouse models of peritonitis,gouty arthritis and type 2 diabetes through inhibition of NLRP3 activation as a covalent inhibitor.Subsequent medicinal chemistry efforts were carried out to reduce the cytotoxicity of oridonin.A total of 40 oridonin analogs were designed and synthesized,which led to the identification of.Ⅲ-21 and Ⅲ-42 with improved biochemical IC50s of 0.17 and 0.21 μM against NLRP3,respectively.But the selectivity window exemplified by the ratio of EC50 against cancer cells to NLRP3 biochemical IC50 needs to be further optimized.In summary,through rationally design and systematic SAR exploration of compounds derived from three novel chemotypes of thiazolidinone,acrylate and kaurene-type diterpenoid.we have discovered a series of potent and highly selective NLRP3 inhibitors,which would serve as good leads for the developments of targeted therapeutics against NLRP3-mediated diseases.Our results suggest that pharmacological targeting NLRP3 is a viable approach for treating NLRP3 related diseases.