CP-456773 (MCC950 (CP-456773) and CRID3) is an effective and specific cytokine release inhibitor and NLRP3 inflammasome inhibitor. CP-456773 inhibits IL-1β secretion and caspase 1 processing. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced IL-1β production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis.

HMDM:8.1 nM, BMDM:7.5 nM

MCC950以纳摩尔浓度阻断NLRP3的典型与非典型激活，特异性地抑制NLRP3而不影响AIM2、NLRC4或NLRP1的激活。在小鼠骨髓来源的巨噬细胞（BMDM）和人源单核细胞衍生的巨噬细胞（HMDM）中测试了MCC950对NLRP3炎症体激活的影响，BMDM中MCC950的半抑制浓度（IC50）约为7.5 nM，而在HMDM中具有相似的抑制能力（IC50=8.1 nM）。MCC950还呈剂量依赖性地抑制IL-1β的分泌，但不抑制TNF-α的分泌。在非典型途径激活下，MCC950特异性阻断由caspase-11直接引起的NLRP3激活和IL-1β的分泌。即使在10 μM的浓度下，MCC950也不抑制由沙门氏菌（Salmonella typhimurium）激活的NLRC4刺激的IL-1β和TNF-α的分泌。在响应S. typhimurium时，MCC950不抑制caspase-1的激活或IL-1β的处理。MCC950处理对细胞裂解物中的pro-caspase-1和pro-IL-1β的表达没有显著影响[1]。

MCC950通过减少白介素-1β (IL-1β) 的产生和减轻实验性自身免疫性脑炎症(EAE) 的严重程度，来延缓多发性硬化症模型的发展。MCC950预处理能降低IL-1β和IL-6的血清浓度，但对TNF-α的数量影响不大。在小鼠身上应用MCC950能延缓EAE的发病时间并减轻症状。通过对第22天牺牲小鼠的脑部单核细胞进行细胞内细胞因子染色和流式细胞术(FACS)分析显示，与PBS处理的小鼠相比，MCC950处理的小鼠中IL-17和IFN-γ产生的CD3+ T细胞的频率有轻微降低。具体来说，IFN-γ和特别是IL-17产生的细胞数量在CD3+ T细胞的CD4+和γδ+亚群中也有所减少。

Disk diffusion is conducted, except that 10 μg of each antibiotic compound is used per filter. Growth in liquid medium in the presence of CHIR-090 is evaluated as follows: cells from overnight cultures are inoculated into 50 mL portions of LB broth at an A600 of 0.02 and grown with shaking at 30°C. When the A600 reaches 0.15, parallel cultures are treated with either 6 μL of 500 μg/mL CHIR-090 in DMSO or 6 μL of DMSO. To assess cumulative growth, cultures are maintained in log phase growth by 10-fold dilution into pre-warmed medium, containing the same concentrations of DMSO or DMSO/CHIR-090, whenever the A600 reaches 0.4. The minimal inhibitory concentration is defined as the lowest antibiotic concentration at which no measurable bacterial growth is observed in LB medium containing 1% DMSO (v/v), when inoculated at a starting density of A600=0.01. Cultures are incubated with shaking for 24 h at 30°C in the presence of CHIR-090. Experiments are performed in triplicate[1].

MCC950 is dissolved in DMSO and stored, and then diluted with appropriate media before use[1]. BMDM are seeded at 5×105/mL or 1×106/mL, HMDM at 5×105/mL and PBMC at 2×106/mL or 5×106/mL in 96 well plates. The following day the overnight medium is replaced and cells are stimulated with 10 ng/mL LPS from Escherichia coli serotype EH100 (ra) TLRgrad for 3 h. Medium is removed and replaced with serum free medium (SFM) containing DMSO (1:1,000), MCC950 (0.001-10 μM), glyburide (200 μM), Parthenolide (10 μM) or Bayer cysteinyl leukotriene receptor antagonist 1-(5-carboxy-2{3-[4-(3-cyclohexylpropoxy)phenyl]propoxy}benzoyl)piperidine-4-carboxylic acid (40 μM) for 30 min. Cells are then stimulated with inflammasome activators: 5 mM adenosine 5'-triphosphate disodium salt hydrate (ATP) (1 h), 1 μg/mL Poly(deoxyadenylic-thymidylic) acid sodium salt (Poly dA:dT) transfected with Lipofectamine 200 (3-4 h), 200 μg/mL MSU (overnight) and 10 μM nigericin (1 h) or S. typhimurium UK-1 strain. Cells are also stimulated with 25 μg/mL Polyadenylic-polyuridylic acid (4 h). For non-canonical inflammasome activation cells are primed with 100 ng/mL Pam3CSK4 for 4 h, medium is removed and replaced with SFM containing DMSO or MCC950 and 2 μg/mL LPS is transfected using 0.25% FuGENE for 16 h. Supernatants are removed and analysed using ELISA kits. LDH release is measured using the CytoTox96 non-radioactive cytotoxicity assay[1].