Design,Synthesis,and Activity Study Of Novel NLRP3 Inhibitors As Oridonin Analogs

NLRP3 is an important pattern recognition receptor in the cytoplasm and a member of the NOD-like receptor family.The NLRP3 inflammasome consists of NLRP3,Pro-caspase-1and ASC,which is closely related to various inflammatory diseases.It is a promising drug development target for treating inflammation-related diseases.Inflammatory bowel disease（IBD）is a chronic immune system-mediated disease,characterized by a high incidence and recurrence rate of gastrointestinal,which seriously affects the quality of life of patients and significantly increases the risk of colon cancer.The occurrence of IBD is closely related to NLRP3 inflammasome,and targeted inhibition of NLRP3 inflammasome activation may be an effective strategy for treating IBD.Acute lung injury（ALI）is a serious respiratory disease with high incidence rate and mortality.It is characterized by alveolar injury,severe hypoxemia,severe inflammatory reaction and cytokine accumulation.Neutrophil extracellular traps（NETs）play a crucial role in the development of acute lung injury（ALI）,and the activation of NLRP3 inflammasome can boost the formation of NETs.Therefore,targeting the inhibition of NLRP3 inflammasome activation to reduce the formation of NETs may serve as a promising therapeutic strategy for ALI.Oridonin（Ori）is an enantio kaurane type tetracyclic diterpene compound isolated from Rabdosia rubescens.It is reported that Ori is a specific and covalent inhibitor for NLRP3 inflammasome.Ori forms a covalent bond with the cysteine 279of NLRP3 in NACHT domain to block the interaction between NLRP3 and NEK7,thereby inhibiting NLRP3 inflammasome assembly and activation.Importantly,Ori has both preventive or therapeutic effects on mouse models of peritonitis,gouty arthritis and type 2diabetes,via inhibition of NLRP3 inflammasome activation.Ori has the potential to serve as a lead for developing new therapeutics against NLRP3-driven diseases.However,current studies on the structure-activity relationship of Ori are mostly focused on its anti-tumor activity,and its structure optimization as an NLRP3 inhibitor has not yet been reported.Therefore,this project aims to use Ori as a lead compound to design and synthesize a series of Ori derivatives and conduct the first activity study on their potential as NLRP3 inhibitors.The ultimate goal is to obtain structurally novel,highly efficient,and low-toxicity Ori derivatives that can serve as potential lead compounds for the effective treatment of NLRP3inflammasome-related diseases,including IBD and ALI.Methods:1.Design,synthesis,and preliminary structure-activity relationship study of oridonin derivativesBased on the molecular hybridization principle in new drug design,target compounds were synthesized by introducing different substitutedα,β-unsaturated ester or aminoformate active fragments into Ori’s 14-OH.The structures of all compounds were determined by means of nuclear magnetic resonance hydrogen spectroscopy（¹H NMR）,nuclear magnetic resonance carbon spectroscopy(¹³C NMR),and high-resolution mass spectrometry（HRMS）.THP-1 cells were differentiated into macrophages（THP-M）upon PMA stimulation.THP-M cells were first stimulated with LPS for 4.5 hours,followed by 30 minutes of ATP stimulation（LPS+ATP）,leading to activation of the NLRP3 inflammasome and a substantial production of IL-1β.The cytotoxicity of the compounds on THP-M cells was analyzed through the CCK-8 assay.The inhibitory activity of the compounds on the activation of the NLRP3inflammasome was evaluated by ELISA,which detected the amount of IL-1βsecreted by THP-M cells after LPS+ATP stimulation.Based on the toxicity and activity data of the compounds,a preliminary structure-activity relationship evaluation was conducted to obtain a target compound（compound E6）with further exploration value.The purity of compound E6was analyzed using liquid chromatography-mass spectrometry.2.Inhibitory effect of compound E6 on NLRP3 inflammasome and its mechanism of actionFurther analysis of the cytotoxicity of compound E6 on THP-M cells and its inhibitory activity on NLRP3 inflammasome was conducted through the CCK-8 assay and enzyme linked immunosorbent assay to obtain the IC50 values for cytotoxicity and inhibition of IL-1βsecretion,and to calculate the compound E6 selectivity index.The impact of compound E6on the priming and activation stages was explored by measuring the expression levels of proteins associated with NLRP3 inflammasome activation using Western blot and enzyme linked immunosorbent assay.Specificity of compound E6 on inhibiting NLRP3inflammasome activation was evaluated by detecting its effects on NLRP3 inflammasome activation induced by different stimuli（ATP,Nigericin or MSU crystals）and on the activation of different inflammasomes（NLRP3,AIM2 or NLRC4 inflammasome）.The binding efficiency of compound E6 with NLRP3 was assessed by cell thermal shift assay and its binding mode with NLRP3 was explored by molecular docking.The inhibitory effect of compound E6 on pyroptosis was investigated by detecting the amount of LDH released,pyroptosis rate and PI positive staining rate of THP-M cells stimulated by LPS+ATP.The mechanism of compound E6 inhibition on pyroptosis was investigated by measuring the expression levels of related proteins during pyroptosis using Western blot analysis.3.The therapeutic effect of compound E6 on DSS-induced colitis in miceDSS was added to the drinking water to induce acute colitis in mice,which were treated through intraperitoneal injection.The severity of colitis was evaluated based on changes in body weight,fecal consistency,rectal bleeding,and colon length.The histopathological changes in the colon tissue were analyzed using H&E and AB-PAS staining.The expression levels of NLRP3 inflammasome activation related proteins in the colon tissue were detected using immunohistochemistry and western blot analysis to investigate the molecular mechanism of compound E6 in alleviating colitis in mice.4.Preliminary study on the inhibitory effect of oridonin derivative F1 on neutrophil extracellular trapsFollowing the molecular hybridization principle in the new drug design,compound F1was obtained by introducing cyclohexyl isocyanate into Ori’s 14-OH.The structure of compound F1 was determined using ¹H NMR,¹³C NMR and HRMS.Neutrophils were extracted from mouse bone marrow and their purity was identified using flow cytometry.The impact of compound F1 on the secretion of IL-1βfrom neutrophils stimulated with nigericin was investigated by enzyme linked immunosorbent assay.The inhibitory effect of compound F1 on nigericin induced NETs was investigated using immunofluorescence.After 30 minutes of intraperitoneal injection,LPS was used to induce acute lung injury in mice through nasal drip;after 12 hours of LPS stimulation,the mice were euthanized and lung tissue was removed for H&E staining.Results:1.Twenty-nine structurally novel and unreported Ori derivatives were designed and synthesized,and the structures of all compounds were verified by ¹H NMR,¹³C NMR and HRMS.Among them,compound E6 exhibited strong inhibitory activity against IL-1βwith a higher selectivity index（IC50=0.45±0.02μM and SI=36.49）.Compared with Ori（IC50=5.18±0.10μM and SI=5.04）,the inhibitory activity and selectivity index of compound E6were increased by 11.5-fold and 7.2-fold,respectively.2.Compound E6 did not affect the priming stage of NLRP3 inflammasome activation,but significantly inhibited the activation stage.Compound E6 significantly inhibited the activation of NLRP3 inflammasome induced by different stimuli（ATP,Nigericin or MSU crystals）,but had no effect on the activation of AIM2 and NLRC4 inflammasomes.Compound E6 could bind to NLRP3,and its binding efficiency and affinity were higher than those of Ori.Compound E6 significantly inhibited the release of LDH from THP-M cells stimulated by LPS+ATP,and reduced pyroptosis rate and the proportion of PI positive staining.Compound E6 effectively reversed the upregulation of GSDMD,GSDMD-NT,and Caspase-1 expression in THP-M cells induced by LPS+ATP stimulation.Meanwhile,compound E6 also effectively reversed the upregulation of GSDMD,Pro-caspase-4,GSDMD-NT,and Caspase-4 expression in THP-M cells induced by cytoplasmic LPS stimulation.3.After treatment with DSS,the mice exhibited symptoms such as weight loss,loose stools,and bloody diarrhea,with significantly elevated disease activity index.In addition,mice in the DSS group had reduced survival rates and a significantly shortened colon.Treatment with compound E6（10 mg/kg or 5 mg/kg）effectively alleviated the colitis symptoms in the mice,and the effect was superior to that of Ori（10 mg/kg）.Furthermore,the colonic tissue of the mice in the DSS group showed histopathological changes,including reduced goblet cell numbers,abnormal mucus secretion,crypt necrosis,and inflammatory cell infiltration,indicating damage to the intestinal barrier.However,treatment with compound E6（10 mg/kg or 5 mg/kg）markedly suppressed these histopathological changes in the colonic tissue of the mice,and the effect was superior to that of Ori（10 mg/kg）.The expression of IL-1β,Caspase-1,and TNF-αin the colonic tissue of the mice in the DSS group increased significantly,which was effectively suppressed by treatment with compound E6（10 mg/kg or5 mg/kg）.The elimination half-lives(T_1/2)of compound E6 in human and rat liver microsomes were 53.4 min and 31.8 min,respectively.The pharmacokinetic parameters of compound E6 in SD rats were as follows:elimination rate constant（Kel）was 0.107±0.0221h,half-life(T_1/2)was 6.64±1.15 h,clearance rate（CL）was 105±22.4 m L/kg/min,and volume of distribution at steady state(Vd_ss)was 23.1±4.00 L/kg.4.Compound F1 was designed and synthesized,and its structure was confirmed by ¹H NMR,¹³C NMR,and HRMS.After stimulation with nigericin,secretion of IL-1βby neutrophils was significantly increased,while compound F1 significantly inhibited the secretion of IL-1β,with a greater inhibitory effect than Ori.Treatment with nigericin resulted in neutrophils showing disappearance of nuclear lobulation,rounding of the nuclear region,chromatin decondensation,and formation of a reticular chromatin structure.Concurrently,co-localization of Cit-H3 with DNA was observed,and formation of NETs was significantly increased.Treatment with compound F1（0.5μM or 2μM）resulted in a significant reduction in the expansion of chromatin,reticular chromatin structure,co-localization of Cit-H3 with DNA,and formation of NETs.However,treatment with Ori（2μM）did not effectively reverse these effects induced by nigericin.Compound F1（20 mg/kg）can effectively reduce pulmonary inflammatory cell infiltration and alveolar hemorrhagic damage caused by LPS nasal drip stimulation in mice.Conclusions:1.The introduction ofα,β-unsaturated ester or aminoformate fragments at position 14 of Ori has significant effects on the inhibitory activity and cytotoxicity of the compounds.The inhibitory activity and cytotoxicity of the compounds obtained by introducingα,β-unsaturated ester or diphenyl substituted amino formate fragments at position 14 of Ori are synchronized,while compounds with benzene ring substituted amino formate fragments exhibit highly efficient and low toxicity properties.2.Compound E6 specifically inhibits the activation of NLRP3 inflammasome by targeting NLRP3,and can inhibit the secretion of IL-1βand cell pyroptosis by inhibiting the NLRP3/Caspase-1/GSDMD pyroptosis pathway and Caspase-4/GSDMD pyroptosis pathway.3.Compound E6 exhibits therapeutic effects on DSS-induced colitis in mice,and its mechanism of action is related to the inhibition of NLRP3 inflammasome activation.Meanwhile,compound E6 exhibits good metabolic stability in human liver microsomes and acceptable pharmacokinetic properties in SD rats.Therefore,compound E6 has the potential to become a lead compound for the treatment of NLRP3 inflammasome-related diseases,including IBD.4.Compound F1 effectively inhibits the formation of NETs induced by nigericin,and its mechanism of action may be related to the inhibition of NLRP3 inflammasome activation.Meanwhile,compound F1 may have therapeutic effect on LPS-induced acute lung injury in mice.