A Novel NLRP3 Inflammasome Inhibitor: Role And Mechanism Of Pyrazolol [4,3-d] Pyrimidine Derivatives In Inflammation

Activation of NLRP3 inflammasome plays a key role in the inflammatory process.When the innate immune system recognizes foreign pathogens such as bacteria and viruses,it will stimulate the activation of NLRP3 inflammasome,thus inducing macrophages to abnormally secrete IL-1β and promote the occurrence of inflammation.In the past decade,many NLRP3 inflammasome inhibitors have been synthesized by studying the structure and mechanism of NLRP3 inflammasome,but most of them have not been used in clinic due to some side effects.For example,although it has good anti-inflammatory activity,its bioavailability and solubility are poor.MCC950 has achieved good results in some preclinical disease models,but it can only be used as an instrumental drug due to its severe liver toxicity.Therefore,we need to synthesize and screen new low-toxicity NLRP3 inhibitors.Pyrazolol [4,3-d] pyrimidine is an active skeleton widely used in chemical synthesis,and allyl is considered to be an important group of NLRP3 inflammasome inhibitors.In this study,we obtained a variety of novel candidate compounds by introducing allyl and sulfoxyl active groups at site 5 and site 7,respectively.Further,bacterial endotoxins(lipopolysaccharides)and Nigericin were used to build an in vitro NLRP3 cell model,and these compounds were screened for their activity.At the same time,the rat model of adjuvant arthritis(AA)was constructed by Fredrin’s complete adjuvant and the mouse model of ulcerative colitis was induced by sodium glucan sulfate(DSS)for pharmacodynamics observation,hoping to find the compound that can effectively inhibit the NLRP3 inflammasome.Objective: The aim of this study was to explore novel compounds that effectively inhibit the activation of NLRP3 inflammasome and to explore their molecular mechanism of action.Methods:1.Synthesis and spectrum identification of target compoundsCandidate new compounds 1,2,3,4 were synthesized and identified by carbon spectrum and hydrogen spectrum to determine whether they were the target compounds.2.In vitro(1)Cytotoxicity evaluation of compoundsCytotoxicity was evaluated.Primary mouse bone marrow macrophages BMDMs were extracted,THP-1 cells were induced into macrophages by PMA,and the cytotoxicity of the compound was detected by CCK-8.(2)Pharmacological activities of the compounds on IL-1β release and pyroptosisBMDMs were treated with LPS and Nigericin at appropriate concentrations to activate the inflammasome.After treatment with LPS and Nigericin,ELISA and LDH were used to detect the expression of IL-1β and LDH in the cell supernatant,and computer high-content analysis was used to detect pyroptosis.(3)Mechanism of action of compound 2a.BMDMs cells were stimulated with LPS and Nigericin to activate NLRP3 inflammasome.b.BMDMs were simultaneously stimulated with NLRP3 agonists ATP and MSU.c.ASC cross-linking experiments were performed using cross-linking agents.d.Flow cytometry was used to detect the expression of ROS and immunofluorescence was used to detect the change of mitochondrial membrane potential after the compound intervention.3.In vivo(1)Animal safety evaluation of compound 2The animal toxicity of the compound was evaluated.Wistar rats were divided into treatment group and normal group.The drug administration group was given a maximum dose of 2000 mg/kg at one time,and the physiological morphology of the animals within 24 h and 14 d were observed.After 14 d,the main organs were sacrificed for pathological staining and blood routine and biochemical indexes were detected.(2)In vivo efficacy experiment of compound 2 in rat model of AAA rat model of adjuvant arthritis type AA was established.After the model was successfully administered,the changes in body weight and foot swelling of rats were detected,and joint inflammation of AA rats was scored using the arthritis scoring rules.HE staining was used to observe the pathological changes of the ankle joint after drug administration.(3)In vivo efficacy of compound 2 in mice model of ulcerative colitis induced by DSSA mouse model of ulcerative colitis was established using 3.5 % DSS induction.The mice were simultaneously administered for 7 consecutive days,and the body weight,DAI score and colon length of the mice were recorded.The pathological changes of colon were observed by HE staining.Results:1.By spectrogram analysis,the synthesized compound was the target compound.2.In vitro(1)Compound 1,2 and 3 showed no obvious toxicity in BMDMs and THP-1 cells,while compound 4 showed strong cytotoxicity.(2)In the NLRP3 inflammasome model stimulated by LPS and Nigericin,compound 2 had a good effect on inhibiting IL-1β and pyrosis.(3)a.Compound 2 after the activation of the NLRP3 inflammasome by Nigericin inhibited its induction of IL-1β and caspase-1 from BMDMs,but did not affect the expression of NLRP3 complex related proteins.b.Compound 2 inhibited the release of IL-1β and caspase-1 induced by the NLRP3 inflammasome activated by multiple agonists,but did not affect the expression of NLRP3 inflammasome complex related proteins.c.Compound 2 did not affect the expression of total ASC protein,but the polymerization of ASC decreased with the increase of the concentration of compound 2,which was also demonstrated by the ASC spot experiment.d.Compound 2 did not inhibit ROS production and mitochondrial damage after activation of inflammasome.3.In vivo(1)After the maximum dose of compound 2 of 2000 mg/kg,the pathological morphology of major organs and blood routine blood biochemical indexes of rats were normal.(2)Compound 2 effectively alleviated joint inflammation and foot swelling in AA rats.(3)Compound 2 effectively alleviates DAI score and intestinal inflammation in mice with ulcerative colitis.Conclusions:1.Compound 2 inhibits NLRP3 inflammasome activation in vitro.2.Compound 2 effectively improves inflammation in AA rats and ulcerative colitis mice.