MCC950 is a potent and selective NLRP3 inhibitor with IC50s of 7.5 and 8.1 nM in BMDMs and HMDMs, respectively.
MCC950 blocks canonical and non-canonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibits NLRP3 but not AIM2, NLRC4 or NLRP1 activation. The effect of MCC950 on NLRP3 inflammasome activation is tested in mouse bone marrow derived macrophages (BMDM) and human monocyte derived macrophages (HMDM). The IC50 of MCC950 in BMDM is approximately 7.5 nM, while in HMDM it has a similar inhibitory capacity (IC50=8.1 nM). MCC950 also dose dependently inhibit IL-1β but not TNF-α secretion.MCC950 specifically blocks caspase-11-directed NLRP3 activation and IL-1β secretion upon stimulation of the non-canonical pathway. NLRC4-stimulated IL-1β and TNF-α secretion (as activated by Salmonella typhimurium) are not inhibited by MCC950 even at a concentration of 10 µM. MCC950 does not inhibit caspase-1 activation or IL-1β processing in response to S. typhimurium. The expression of pro-caspase-1 and pro-IL-1β in cell lysates is not substantially affected by MCC950 treatment[1].
MCC950 reduces Interleukin-1p (IL-1β) production and attenuates the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Pre-treatment with MCC950 reduces serum concentrations of IL-1β and IL-6 while it does not considerably decrease the amount of TNF-α. Treatment of mice with MCC950 delays the onset and reduced the severity of EAE. Intracellular cytokine staining and FACS analysis of brain mononuclear cells from mice sacrificed on day 22 shows modestly reduced frequencies of IL-17 and IFN-γ producing CD3+ T cells in MCC950 treated mice in comparison with PBS-treated mice. IFN-γ and particularly IL-17 producing cell numbers are also reduced in both the CD4+ and γδ+ sub-populations of CD3+ T cells[1].
MCC950是一种强效且选择性的NLRP3抑制剂，在BMDMs和HMDMs中的IC50分别为7.5和8.1纳摩尔。
MCC950可以在纳摩尔浓度下阻止NLRP3的典型和非典型激活。 MCC950特异性地抑制NLRP3而不是AIM2，NLRC4或NLRP1的激活。在小鼠骨髓源性巨噬细胞（BMDM）和人单核细胞衍生的巨噬细胞（HMDM）中测试了MCC950对NLRP3炎症小体激活的影响。在BMDM中，MCC950的IC50约为7.5 nM，在HMDM中具有类似的抑制能力（IC50 = 8.1 nM）。 MCC950还剂量依赖性地抑制IL-1β但不是TNF-α分泌。 MCC950特异性地阻断caspase-11定向的非经典途径刺激引起的NLRP3激活和IL-1β分泌。即使在10μm浓度下，也无法通过Salmonella typhimurium诱导NLRC4刺激引起IL-1β和TNF-α分泌来抑制MCC950。 MCC950不会通过S.typhimurium诱导抑制caspase-1活化或IL-1β加工反应。 经过McC95处理后，细胞裂解物中pro-caspase-1和pro-IL-1β的表达没有受到实质性影响[1]。
MCC950可以减少白细胞介素-1β（IL-1β）的产生，缓解实验性自身免疫脑脊髓炎（EAE）的严重程度，这是多发性硬化症的一种疾病模型。预先使用MCC950可降低血清中IL-1β和IL-6的浓度，但并不会显着降低TNF-α的数量。用MCC950治疗小鼠可以延迟EAE的发作时间，并减轻其严重程度。在第22天牺牲后对小鼠脑单个核细胞进行细胞因子染色和流式细胞分析显示，在与PBS处理组相比较下，接受MCC950治疗组CD3+ T 细胞中 IL-17 和 IFN-γ 产生频率略有降低。同时，在 CD4+ 和 γδ+ 亚群中也都减少了IFN-gamma和特别是IL-17 的产生[1]。
Reference:
[1]. Coll RC, et al. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med. 2015 Mar;21(3):248-55. [2]. Gan W, et al. The SGK1 inhibitor EMD638683, prevents Angiotensin II-induced cardiac inflammation and fibrosis by blocking NLRP3 inflammasome activation. Biochim Biophys Acta. 2017 Oct 3;1864(1):1-10. [3]. Zhang XY, et al. Propofol Does Not Reduce Pyroptosis of Enterocytes and Intestinal Epithelial Injury After Lipopolysaccharide Challenge. Dig Dis Sci. 2018 Jan;63(1):81-91. [4]. Qi Y, et al. NLRP3-dependent synaptic plasticity deficit in an Alzheimer’s disease amyloidosis model in vivo. Neurobiol Dis. 2018 Feb 23;114:24-30.
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Cell experiment: |
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]. |
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Animal experiment: |
Mice[1] C57BL/6 mice are immunized subcutaneously with 150 μg of MOG peptide 35-55 emulsified in CFA containing 4 mg/mL (0.4.mg/mouse) of heat-killed MTB. Mice are injected i.p. with 500 ng pertussis toxin (PT: kaketsuken) on days 0 and 2. MCC950 is administered i.p. to mice (10 mg/kg) at induction of the disease, day 0, 1 and 2 and every 2 days thereafter. Control mice are administered vehicle (PBS) at the same time points. Mice are observed for clinical signs of disease daily (unblinded). Disease severity is scored as follows: no clinical signs, 0; limp tail, 1; ataxic gait, 2; hind limb weakness, 3; hind limb paralysis, 4; and tetra paralysis, 5. |
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参考文献: [1]. Coll RC, et al. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med. 2015 Mar;21(3):248-55. |
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