Screening Of NLRP3 Inflammasome Inhibitors And Study On Its Anti-Inflammatory Activity

Background:NLRP3 is a NOD family of pattern recognition receptors that recognize molecules(PAMP,DAMP)released by pathogens or damaged cells to activate NLRP3 inflammasome and induce inflammation.Studies have shown that NLRP3 inflammasome activation is associated with a variety of inflammatory diseases.However,the inhibitors found so far have problems such as easy off-target,high liver toxicity and poor drug efficacy.Therefore,new inhibitors need to be further developed.MCC950 has been reported to be highly specific to NLRP3,and although MCC950 was discontinued in clinical trials for the treatment of rheumatoid arthritis due to elevated serum liver enzyme levels,this molecule is still considered to be a potent pharmacological tool in elucidating the mechanism of action of NLRP3 inflammasome.At present,two kinds of small molecular fluorescent probes targeting NLRP3 have been reported,which are mainly composed of MCC950 and its derivatives connected with linker and fluorophore,and have large molecular weight,resulting in poor permeability of cell membrane and weak binding ability to target protein.In addition,natural products are one of the sources of small molecule drug design.Natural product Celastrol has been reported to have NLRP3 inhibitory activity,but it has disadvantages such as poor water stability,low bioavailability and narrow therapeutic window.Objective:1.The laboratory modified and optimized the skeleton of MCC950,and we built a cell model for NLRP3 inflammasome activity screening,hoping to obtain small molecular compounds with simple and novel structure,small molecular weight,good membrane permeability,strong activity and good fluorescence characteristics.2.By screening the library of Celastrol derivatives,the novel structure,strong activity and reduced toxicity derivatives were found as NLRP3 inhibitors.Methods:First,THP-1 was induced to differentiate into macrophages by PMA,then stimulated with LPS,then treated with the compound,and finally activated with NLRP3 inflammasome by Nigericin.ELISA was used to detect IL-1β in the cell supernatant.The effect of candidate compounds on the expression of NLRP3 inflammasome-associated proteins,including Caspase-1 and IL-1β expression in supernatant,was detected by western blotting.Imaging power and fluorescence intensity of fluorescent probes in cells are detected by confocal immunofluorescence and flow cytometry.In vivo efficacy was evaluated using DSS-induced mouse enteritis model and CCl4-induced mouse liver fibrosis model.Results:1.Screening for NLRP3 inhibitor candidate compounds MCB-21-221 and SOMCL19-150.2.MCB-21-221 and SOMCL-19-150 inhibit the activation of NLRP3 inflammasome by inhibiting ASC oligomerization.3.Compound MCB-21-221 has better fluorescence characteristics for cellular fluorescence imaging of NLRP3 protein,and can be used as an effective tool for the study of NLRP3 biological function.4.The compound MCB-21-221 can improve DSS-induced enteritis in mice.5.The compound SOMCL-19-150 has lower in vivo toxicity than Celstrol.6.The compound SOMCL-19-150 can alleviate DSS-induced colon shortening and colon tissue damage in mice,and reduce inflammatory cell infiltration.7.SOMCL-19-150 alleviates CCl4-induced liver fibrosis in mice,inhibits the expression of α-SMA,COLIA1,and reduces Caspase-1 maturation and IL-1β secretion in liver tissue.Conclusion:1.The fluorescent inhibitor MCB-21-221 can inhibit NLRP3 inflammasome activation,improve mouse enteritis,and has the specificity of NLRP3 cell imaging,which provides a potential fluorescent probe molecule for the study of NLRP3 biological function.2.The natural product derivative SOMCL-19-150 was verified to have a protective effect on NLRP3 inflammasome-related inflammatory diseases in mouse colitis model and mouse liver fibrosis model,which provides a new idea for the prevention and treatment of inflammatory diseases.