MCC950 sodium (CP-456773 sodium) is a potent and selective inhibitor of the inflammatory vesicle NLRP3 (IC50=7.5 nM in BMDMs; IC50=8.1 nM in HMDMs). MCC950 sodium has no effect on other inflammatory vesicles such as AIM2, NLRC4 or NLRP1.

NLRP3:7.5nM

方法：小鼠骨髓衍生的巨噬细胞 BMDM 用 LPS (10 ng/mL) 刺激 3 h，用 MCC950 sodium (1-1000 nM) 刺激 30 min，再用 ATP (5 mM) 处理 1 h，使用 ELISA 方法检测 IL-1β 和 TNF-α 水平。
结果：MCC950 处理细胞可剂量依赖性地抑制 BMDM 中 IL-1β 的释放。LPS 依赖性 TNF-α 分泌未被 MCC950 损害。[1]
方法：小鼠巨噬细胞、人冠状动脉内皮细胞和平滑肌细胞用 MCC950 sodium (0.02-20 μM) 处理 3 天，使用 Alamar Blue assay 检测细胞活力。
结果：MCC950 sodium 对三种细胞无毒性作用。[2]

方法：为检测体内抗 NLRP3 活性，将 MCC950 sodium (20 mg/kg) 腹腔注射给人 CAPS 疾病 MWS 的小鼠模型，每天一次，持续四周。
结果：MCC950 拯救 CAPS 小鼠模型并抑制人 MWS 细胞中的 NLRP3。[1]
方法：为研究对体内实验性脊髓损伤模型和体外神经元损伤的药理作用，将 MCC950 sodium (10-50 mg/kg) 腹腔注射给脊髓损伤 (SCI) 的 C57BL/6 小鼠。
结果：MCC950 改善了 SCI 小鼠的握力、后肢运动、脊髓水肿和病理损伤。它通过阻断 NLRP3 炎症小体组装以及促炎细胞因子 TNF-α、IL-1β 和 IL-18 的释放来发挥这种作用。[3]

kinase activity assays: All assays are carried out in 384-well white microtiter plates. Compounds are 4-fold serially diluted in 8 steps, starting from 10 μM. The reaction mixture consisted of 25 μL assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 5 mM β-glycerol phosphate). For FLT3 assays, the reaction contains 2.0 μg/mL FLT3 enzyme, 5 μM of poly(Glu,Tyr) substrate and 4 μM of ATP. For JAK1 assays, the reaction contains 2.5 μg/mL of JAK1 enzyme, 10 μM of poly(Glu,Ala,Tyr) substrate and 1.0 μM of ATP. For JAK2 assays, the reaction contained 0.35 μg/mL of JAK2 enzyme, 10 μM of poly (Glu,Ala,Tyr) substrate and 0.15 μM of ATP. For JAK3 assays, the reaction contained 3.5 μg/mL of JAK3 enzyme, 10 μM of poly (Glu,Ala,Tyr) substrate and 6.0 μM of ATP. For TYK2 assays, the reaction contained 2.5 μg/mL of TYK2 enzyme, 10 μM of poly (Glu,Ala,Tyr) substrate and 0.15 μM of ATP. The reaction is incubated at room temperature for 2 h prior to addition of 13 μL PKLight? detection reagent. After 10 min incubation luminescent signals are read on a multi-label plate reader.

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].

C57BL/6 mice were injected intraperitoneally with 50 mg/kg MCC950.