Discovery,Pharmacological Mechanism And Disease Intervention Strategy Of The NLRP3 Inflammasome Inhibitors

The NLRP3 inflammasome plays a crucial role in innate immune-mediated inflammation.It has been reported that aberrant activation of the NLRP3 inflammasome leads to a variety of human inflammatory disorders,such as gout,type 2 diabetes(T2D),neurodegenerative diseases,atherosclerosis and inflammatory bowel disease(IBD).To provide the strategies for treating NLRP3-driven diseases,some NLRP3-targeted inhibitors with potential beneficial effects have been identified.Although several NLRP3 inhibitors have reached clinical trials,whether these agents can be used to treat NLRP3-related diseases in humans remains to be determined.Therefore,there is still a need for NLRP3 inhibitors that are closer to clinical application,more active,and safer.RRx-001 is an anticancer agent currently in phase ?/? clinical trials to treat brain cancer,colorectal cancer,small-cell lung cancer and non-small cell lung cancer.RRx-001 is a well-tolerated agent without clinically significant toxic effects.Although RRx-001 has shown promising anticancer activities,its effects and target on inflammatory diseases have not been reported.In this study,we showed that RRx-001 ameliorates inflammatory diseases by acting as a potent covalent NLRP3 inhibitor.We found that:1.RRx-001 inhibits the activation of classical and nonclassical NLRP3 inflammasome,but not AIM2,NLRC4 or pyrin inflammasome.RRx-001 blocks NLRP3 inflammasome activation in both murine and human cells.RRx-001 also inhibits potassium efflux-independent NLRP3 inflammasome activation.Treatment with higher concentrations of RRx-001 before LPS stimulation decreases pro-IL-1?expression,but the concentrations at which RRx-001 inhibits the NLRP3 inflammasome are not sufficient to inhibit the LPS-induced priming process.2.Inhibition of G6PD is not essential for the inhibition of the NLRP3 inflammasome by RRx-001.RRx-001 has no effects on the upstream signaling events of NLRP3 activation,including potassium efflux,chloride efflux,mitochondrial damage and production of mitochondrial ROS.RRx-001 inhibits NLRP3 inflammasome activation by directly blocking the interaction between NLRP3 and NEK7,which is critical for the assembly of the NLRP3 inflammasome.3.RRx-001 activity depends on both the twin-NO2 and the bromoacetyl groups.The twin-NO2 groups in RRx-001 improve the inhibitory activity of RRx-001.The bromoacetyl group is essential for RRx-001-mediated inhibition of the NLRP3 inflammasome.4.RRx-001 directly binds to NLRP3 and therefore blocks the interaction between NLRP3 and NEK7.RRx-001 specifically interacted with NLRP3 but not AIM2,NLRC4 or NLRP1b.RRx-001 covalently binds to the cysteine 409 of NACHT domain in NLRP3 via its bromoacetyl group.5.RRx-001 treatment attenuates the symptom of LPS(lipopolysaccharide)-induced systemic inflammation,DSS(dextran sulfate sodium)-induced colitis,EAE(experimental autoimmune encephalomyelitis)and metabolic disorders in HFD(high fat diet)-induced diabetic mice.RRx-001 ameliorates inflammatory diseases by acting as an NLRP3 inhibitor.In this study,we found that RRx-001 is a potent,covalent NLRP3-targeted inhibitor that has strong beneficial effects on NLRP3-driven inflammatory diseases.Since RRx-001 has been proven to have low toxicity and safety in clinical trials,our study identifies RRx-001 as a new potential therapeutic for NLRP3-driven diseases.