NLRP3

At nanomolar doses, MCC950 prevents both conventional and non-canonical NLRP3 activation. AIM2, NLRC4, and NLRP1 activation are not specifically inhibited by MCC950, whereas NLRP3 is. Using mouse bone marrow-derived macrophages (BMDM) and human monocyte-derived macrophages (HMDM), the impact of MCC950 on NLRP3 inflammasome activation was investigated. MCC950 exhibits an inhibitory capacity of around 7.5 nM in BMDM and 8.1 nM in HMDM. Moreover, MCC950 reduces IL-1β secretion in a dose-dependent manner but not TNF-α secretion. MCC950 stimulates the non-canonical pathway first, then selectively inhibits caspase-11-mediated NLRP3 activation and IL-1β release. MCC950 failed to suppress Salmonella Typhimurium-induced NLRC4-stimulated IL-1β and TNF-α production, even at a dosage of 10 µM. MCC950 had no effect on the processing of IL-1β or caspase-1 activation in response to Salmonella typhimurium. Pro-caspase-1 and pro-IL-1β production in cell lysates is not considerably impacted by MCC950 treatment [1].

MCC950 attenuates the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis, and decreases the production of interleukin-1p (IL-1β). Pretreatment with MCC950 lowers serum concentrations of IL-1β and IL-6 but does not significantly lower TNF-α levels. MCC950 treatment lessens the severity and postpones the development of EAE in mice. When comparing MCC950-treated animals to PBS-treated mice, intracellular cytokine labeling and FACS analysis of brain mononuclear cells from mice killed on day 22 reveal slightly lower frequencies of CD3+ T cells that produce IL-17 and IFN-γ. There is a decrease in the quantity of CD4+ and γδ+ sub-populations of CD3+ T cells that produce IFN-γ, and in particular, IL-17 generating cells[1].

Inflammasome activation assays [1]
BMDM were seeded at 5 ×0 105/ml or 1 × 106/ml, HMDM at 5 × 105/ml and PBMC at 2 × 106/ml or 5 ×0 106/ml in 96 well plates. The following day the overnight medium was replaced and cells were stimulated with 10 ng/ml LPS from Escherichia coli serotype EH100 (ra) TLRgrade™ for 3 h. Medium was 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 were 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 2000™ (Invitrogen) (3–4 h), 200 µg/ml MSU (overnight) and 10 µM nigericin (1 h) or S. typhimurium UK-1 strain (M.O.I. 20) obtained from Dr. Sinead Corr, Trinity College Dublin, Ireland (2 h). Cells were also stimulated with 25 µg/ml Polyadenylic-polyuridylic acid (4 h). For non-canonical inflammasome activation cells were primed with 100 ng/ml Pam3CSK4 for 4 h, medium was removed and replaced with SFM containing DMSO or MCC950 and 2 µg/ml LPS was transfected using 0.25% FuGENE® for 16 h. Supernatants were removed and analysed using ELISA kits according to the manufacturer’s instructions. LDH release was measured using the CytoTox96® non-radioactive cytotoxicity assay.[1]

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Time of Flight Imflammasome Evaluation (TOFIE) assay[1]
HEK293T cells (4 × 105 /ml) were transfected in 24 well plates using Lipofectamine 2000™ with the following plasmids: pEF6 human ASC-GFP, pEF6 human C-mCherry or empty vector control. 1 h post transfection cells were treated with DMSO or MCC950 (0.1–50 µM). 15 h post transfection cells were removed and suspended in DPBS containing 1% FCS and 2 mM EDTA. Cells were analysed using a Gallios™ flow cytometer and using FlowJo software. Live cells were gated on GFP and Cherry expression (when co-transfected). The percentage of ASC speck containing cells was determined by analysing the height and width of the GFP pulse area (low width:area and high height:area). This analysis is described in detail in by Sester et al.

Western blotting[1]
Cell lysates were prepared by direct lysis in 50 µl 5 ィ Laemmli sample buffer. The protein content of supernatants was concentrated using StrataClean™ resin according to the manufacturer’s instructions. The protein samples were resolved on 15% SDS-PAGE gels and transferred onto polyvinylidene diflouride (PVDF) membrane using a wet transfer system. Membranes were blocked in 5% (w/v) dried milk in TBS-T (50 mM Tris/HCL, pH 7.6, 150 mM NaCl and 0.1% (v/v) Tween-20) for 1 h at room temperature (RT). Membranes were incubated with primary antibody diluted in 5% (w/v) dried milk in TBS-T, followed by incubation with the appropriate horseradish peroxidise (HRP) conjugated secondary antibody diluted in 5% (w/v) dried milk in TBS-T for 1 h. Membranes were developed using 20 ィ LumiGLO® chemilluminescent reagent. Membranes were stripped using Restore™ PLUS western blot stripping buffer before being reprobed.[1]
PBMC from individuals with CAPS were seeded at 2 ×0 106/ml in 12 well plates and then primed with 1 µg/ml LPS for 3 h. Medium was replaced with SFM containing MCC950 (5–1,000 nM). After 45 min, cell culture supernatants and cell lysates were collected. Samples were resolved using Novex® Tris-Glycine Gel Systems.[1]

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Fluorescent Imaging Plate Reader (FLIPR) Ca2+ analysis[1]
BMDM (3 × 104/well) were loaded for 30 min at 37 °C with a no wash calcium dye (Molecular Devices) in physiological salt solution (PSS; composition NaCl 140 mM, glucose 11.5 mM, KCl 5.9 mM, MgCl2 1.4 mM, NaH2PO4 1.2 mM, NaHCO3 5 mM, CaCl2 1.8 mM, HEPES 10 mM) containing 0.1% BSA. Cells were then transferred to the FLIPRTETRA fluorescent plate reader and Ca2+ responses measured using a cooled CCD camera with excitation at 470–495 nM and emission at 515–575 nM. Camera gain and intensity were adjusted for each plate to yield a minimum of 1,000 arbitrary fluorescence units (AFU) baseline fluorescence. Prior to addition of MCC950, 10 baseline fluorescence readings were taken, followed by fluorescent readings every second for 300 seconds following sample addition and a further 300 seconds following addition of either PSS or ATP (500 µM).

In vivo LPS challenge[1]
C57BL/6 mice were injected intraperitoneally (i.p.) with 50 mg/kg MCC950 or vehicle control (DMSO/PBS) 1 h h before i.p. injection of 10 mg/kg LPS Escherichia coli 055:B5 or PBS. After for 2 h mice were sacrificed and serum levels of IL-1β, TNF-α and IL-6 were measured by ELISA.[1]

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Induction and Assessment of EAE[1]
C57BL/6 mice were immunized subcutaneously with 150 µg of MOG peptide 35–55 (GenScript) emulsified in CFA containing 4 mg/ml (0.4.mg/mouse) of heat-killed MTB (Chondrex). Mice were injected i.p. with 500 ng pertussis toxin (PT: kaketsuken) on days 0 and 2. MCC950 was 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 were administered vehicle (PBS) at the same time points. Mice were observed for clinical signs of disease daily (unblinded). Disease severity was 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., Experiments were performed under license (BI00/2412) from The Irish Medicine Board and with approval from the Trinity College Dublin BioResources Ethics Committee.[1]

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FACS analysis of EAE[1]
On day 22 post immunization mononuclear cells were isolated from whole brains of perfused mice with EAE, following homogenisation and centrifugation on a Percoll gradient. Mononuclear cells (MNC) (2 × 106/ml) were stimulated for 4 h with PMA (10 ng/ml) and ionomycin (1 µg/ml) in the presence of brefeldin A (5 µg/ml). Cells were washed in PBS and re-suspended in 50 µL PBS with 1:1,000 LIVE/DEAD® Fixable Aqua Dead Cell Stain kit for 20 min. Surface stains for CD3 (145-2c11) (0.5 µl/106 cells), CD4 (RM4-5) (0.5 µl/106 cells) and γδ TCR (GL3) (1 µl/106 cells) (eBioscience) were added and cells were incubated for a further 20 mins. Cells were then fixed with 2% paraformaldehyde and washed in PBS twice, before being intracellularly stained for IL-17 or IFN-γ in permeabilization buffer (0.2% saponin in PBS + 1% FBS). Flow cytometric analysis of MNC was performed using a BD LSRFortessa™ and analysed with FlowJo software. MNC were first gated on live CD3+ T cells followed by CD4 expression, γδ TCR expression or cytokine production.[1]

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NLRP3 and NLRP1 activating mutation mice[1]
Mice were backcrossed to C57BL/6 at least ten times. Nlrp3A350VneoR mice were provided by Hal M. Hoffman, The University of California, San Diego, U.S.A. and crossed with LysMCre mice (B6.129P2-Lyz2tm1(cre)Ifo/J. MCC950 was administered i.p. (20 mg/kg) every second day starting at day 4 after birth. Mice with an activating mutation in NLRP1, Nlrp1aQ593P were generated on a C57BL/6 background as described previously and administered MCC950 i.p. (20 mg/kg) every second day for 9 days. Blood was collected at the timepoints indicated for analysis of plasma cytokines by ELISA. IL-18 ELISA was performed as described by Westwell-Roper et al. Experiments were performed under AEC Project 2013.011 and were approved by the Animal Ethics Committee of The Walter and Eliza Hall Institute of Medical Research.

[1]. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med. 2015 Mar;21(3):248-55.

The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activation is pathogenic in inherited disorders such as cryopyrin-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease and atherosclerosis. We describe the development of MCC950, a potent, selective, small-molecule inhibitor of NLRP3. 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 interleukin-1β (IL-1β) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescued neonatal lethality in a mouse model of CAPS and was active in ex vivo samples from individuals with Muckle-Wells syndrome. MCC950 is thus a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease.[1]

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Diabetes is associated with a high risk of developing cognitive dysfunction and neuropsychiatric disabilities, and these disease symptomsare termed diabetic encephalopathy (DEP). Inflammation is involved in the development of DEP. The cleavage and maturation of the proinflammatory cytokine interleukin (IL)-1β is regulated by the NLRP3 inflammasome. Obese and type 2 diabetic db/db mice show anxiety- and depression-like behaviors and cognitive disorders associated with hippocampal inflammation. The purpose of this study was to explore the role of NLRP3 inflammasome in DEP. Results showed that expression levels of inflammasome components including NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1, as well as IL-1β in the hippocampus of diabetic db/db mice were higher than those of non-diabetic db/m mice. Treatment of db/db mice with NLRP3 inflammasome inhibitor MCC950 ameliorated anxiety- and depression-like behaviors as well as cognitive dysfunction, and reversed increased NLRP3, ASC, and IL-1βexpression levels and caspase-1 activity in hippocampus. Moreover, MCC950 treatment significantly improved insulin sensitivity in db/db mice. These results demonstrate that inhibition of NLRP3 inflammasome activation may prove to be a potential therapeutic approach for DEP treatment.[2]

C20H24N2O5S

404.48

404.14

210826-40-7

MCC950 sodium;256373-96-3

9910393

White to light yellow solid

1.4±0.1 g/cm3

239 ºC

1.637

3.2

3

5

4

28

684

0

S(C1=C([H])C(=C([H])O1)C(C([H])([H])[H])(C([H])([H])[H])O[H])(N([H])C(N([H])C1=C2C([H])([H])C([H])([H])C([H])([H])C2=C([H])C2C([H])([H])C([H])([H])C([H])([H])C=21)=O)(=O)=O

LFQQNXFKPNZRFT-UHFFFAOYSA-M

InChI=1S/C20H24N2O5S.Na/c1-20(2,24)14-10-17(27-11-14)28(25,26)22-19(23)21-18-15-7-3-5-12(15)9-13-6-4-8-16(13)18;/h9-11,24H,3-8H2,1-2H3,(H2,21,22,23);/q;+1/p-1

sodium;1,2,3,5,6,7-hexahydro-s-indacen-4-ylcarbamoyl-[4-(2-hydroxypropan-2-yl)furan-2-yl]sulfonylazanide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.18 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.18 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.18 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)