The Effect And Mechanism Of RRx-001 In Inhibition Of NLRP3 Inflammasome Activation And Alleviation Of NLRP3-driven Diseases

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 Ⅱ/Ⅲ clinical trials to treat brain cancer,colorectal cancer,small-cell lung cancer and non-small cell lung cancer.n addition to its cytotoxic antiproliferative effects on tumor cells,RRx-001 exerts anticancer effects by acting as a radiosensitizer,chemosensitizer,or immunosensitizer.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-1βexpression,but the concentrations at which RRx-001 inhibits the NLRP3 inflammasome are not sufficient to inhibit the LPS-induced priming process.These results suggested that RRx-001 specifically inhibits NLRP3 inflammasome activation.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.These results demonstrated that RRx-001 directly blocks the assembly of the NLRP3 inflammasome.3.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.Our results suggested that RRx-001 activity depends on both the twinNO2 and the bromoacetyl groups.4.RRx-001 covalently binds to the cysteine 409 of NACHT domain in NLRP3 via its bromoacetyl group and therefore blocks the interaction between NLRP3 and NEK7.The C409A mutation of NLRP3 resulted in the failure of RRx-001 to bind and inhibit NLRP3.These results suggested that RRx-001 inhibits NLRP3 inflammasome assembly by directly binding to NLRP3.5.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.