NLRP3 inflammasomes
NLR family, pyrin domain-containing 3 (NLRP3) inflammasome: MCC950 sodium is a potent and selective small-molecule inhibitor of NLRP3; it blocks canonical and noncanonical NLRP3 activation at nanomolar concentrations (no specific IC₅₀/Ki values provided) and does not inhibit AIM2, NLRC4, or NLRP1 inflammasomes[1]

At nanomolar doses, MCC950 inhibits both conventional and non-canonical NLRP3 activation. MCC950 selectively prevents NLRP3 activation, but not that of AIM2, NLRC4, or NLRP1. Using human monocyte-derived macrophages (HMDM) and mouse bone marrow-derived macrophages (BMDM), the impact of MCC950 on NLRP3 inflammasome activation was investigated. MCC950 exhibits an inhibitory capacity of 8.1 nM in HMDM, compared to its about 7.5 nM IC50 in BMDM. Moreover, MCC950 dose-dependently reduced IL-1β secretion but not TNF-α secretion. MCC950 selectively inhibits NLRP3 activation and IL-1β release upon stimulation of non-canonical pathways, which is caused by caspase-11. Even at a 10 μM dose, MCC950 did not prevent Salmonella typhimurium-stimulated IL-1β and TNF-α production from NLRC4. When Salmonella enterica serovar Typhimurium is present, MCC950 does not prevent caspase-1 activation or IL-1β processing. Treatment with MCC950 basically has no effect on the production of pro-caspase-1 and pro-IL-1β in cell lysates [1].

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Inhibition of NLRP3 inflammasome activation in bone marrow-derived macrophages (BMDM): MCC950 sodium (1-1000 nM) dose-dependently reduced IL-1β production in BMDM stimulated with LPS + ATP or LPS + nigericin (canonical NLRP3 stimuli), with no significant effect on TNF-α production. Western blot confirmed that MCC950 sodium inhibited caspase-1 cleavage and IL-1β maturation in BMDM treated with LPS + ATP, while glyburide (a non-specific NLRP3 inhibitor) showed weaker inhibition. For noncanonical NLRP3 activation (Pam3CSK4 + transfected LPS), MCC950 sodium also reduced IL-1β production in C57BL/6 BMDM but had no effect on Nlrp3⁻/⁻ or casp11⁻/⁻ BMDM, confirming NLRP3 specificity. MCC950 sodium did not affect LDH release (cell death marker) in BMDM, indicating no cytotoxicity[1]
- Selectivity for NLRP3 inflammasome: MCC950 sodium had no inhibitory effect on IL-1β production in BMDM stimulated with LPS + S. typhimurium (NLRC4 inflammasome activator) or LPS + transfected Poly(dA:dT) (AIM2 inflammasome activator), while parthenolide (non-specific inflammasome inhibitor) and Bayer compound (AIM2 inhibitor) effectively blocked IL-1β production in these models. MCC950 sodium also did not inhibit TLR signaling (LPS or Poly(A:U) induced TNF-α production) or NLRP3 priming (LPS-induced NLRP3 protein expression in BMDM)[1]
- Mechanism of NLRP3 inhibition: MCC950 sodium blocked NLRP3-dependent ASC oligomerization in BMDM stimulated with LPS + nigericin (detected by cross-linking assay and Western blot), and reduced ASC speck formation in ASC-cerulean reporter cells treated with nigericin, LeuLeu-Ome, or lethal toxin (measured by live cell imaging and FACS). It did not block K⁺ efflux, Ca²⁺ flux (ATP-induced Ca²⁺ flux in BMDM measured by FLIPR TETRA), or direct NLRP3-ASC interactions (co-immunoprecipitation in HEK-293T cells transfected with FLAG-NLRP3/HA-NLRP3 and ASC)[1]
- Inhibition of NLRP3 activation in human PBMC: MCC950 sodium reduced IL-1β production in PBMC isolated from individuals with Muckle-Wells syndrome (MWS, E313K NLRP3 mutation) stimulated with LPS, confirmed by Western blot showing reduced caspase-1 cleavage and IL-1β maturation[1]

MCC950 lessens the production of interleukin-1p (IL-1β) and lessens the severity of multiple sclerosis disease model experimental autoimmune encephalomyelitis (EAE). MCC950 pretreatment decreased serum levels of IL-1β and IL-6, but did not significantly lower TNF-α levels. Mice treated with MCC950 had a delayed onset of EAE and a decreased severity. Mice brain monocytes sacrificed on day 22 were subjected to intracellular cytokine staining and FACS analysis, which revealed that the MCC950-treated mice had more CD3+ T cells that produced IL-17 and IFN-γ than the PBS-treated mice. There is a slight decrease in frequency. The CD4+ and γδ+ subpopulations of CD3+ T cells also have a decrease in the number of cells generating IFN-γ, particularly IL-17 [1].

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Attenuation of experimental autoimmune encephalomyelitis (EAE) in mice: C57BL/6 mice with EAE (multiple sclerosis model) treated with MCC950 sodium showed significantly reduced clinical scores compared with PBS-treated controls. Flow cytometry of brain mononuclear cells isolated on day 22 post-EAE induction revealed that MCC950 sodium decreased the percentage of live IL-17⁺/IFN-γ⁺ CD3⁺ T cells, CD3⁺CD4⁺ T cells, and CD3⁺γδ TCR⁺ cells, indicating suppression of neuroinflammation[1]
- Reduction of systemic inflammation in mice: C57BL/6 mice pre-treated with MCC950 sodium had lower serum IL-1β levels (but not TNF-α or IL-6) 2 hours after intraperitoneal (i.p.) LPS injection (Mann-Whitney test, P ≤ 0.05, n=3)[1]
- Rescue of neonatal lethality in CAPS mouse model: Nlrp3A³⁵⁰VneoR × LysMCre mice (NLRP3 gain-of-function mutation, CAPS model) treated with MCC950 sodium from day 9 had higher body weight and survival rate (up to day 45, drug withdrawn at day 28) compared with PBS-treated mutant mice (unpaired two-tailed t-test, P ≤ 0.005 for body weight at day 9; survival: MCC950 n=5 vs PBS n=9). Plasma IL-18 levels (a NLRP3 inflammasome product) were reduced in MCC950 sodium-treated mutant mice at day 9, and IL-18 levels rebounded 14 days after drug withdrawal. MCC950 sodium had no effect on NLRP1 mutant (Nlrp1aQ593P) mice, confirming NLRP3 specificity[1]
- Amelioration of diabetic encephalopathy (DEP) in db/db mice: db/db mice (type 2 diabetes model) treated with MCC950 sodium for 8 weeks showed improved insulin sensitivity (reduced fasting blood glucose, improved oral glucose tolerance test [OGTT] and insulin tolerance test [ITT] curves, lower AUC for glucose in OGTT/ITT). Behavioral tests demonstrated that MCC950 sodium reversed anxiety-like behaviors (increased time in lighted box and transitions in light/dark box), depression-like behaviors (reduced immobility time in forced swim test [FST] and tail suspension test [TST]), and cognitive dysfunction (reduced escape latency in Morris water maze [MWM] hidden-platform test, increased target crossings and time in target quadrant in probe trial) compared with vehicle-treated db/db mice. Western blot and caspase-1 activity assay showed that MCC950 sodium reduced NLRP3, ASC, and IL-1β protein levels, and decreased caspase-1 activity in the hippocampus of db/db mice. Plasma IL-1β levels were reduced, while plasma insulin levels were increased in MCC950 sodium-treated db/db mice; plasma and hippocampal TNF-α levels showed no significant changes[2]

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.

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Caspase-1 activity assay in cell/ tissue lysates: Cell lysates (BMDM, hippocampal tissue) or supernatants were incubated with a caspase-1-specific substrate (containing p-nitroaniline) at 37°C for a specific time (e.g., 1-2 hours). The release of p-nitroaniline was measured by spectrophotometry at 405 nm, and caspase-1 activity was calculated as the absorbance value normalized to protein concentration or control group. MCC950 sodium treatment reduced caspase-1 activity in NLRP3-activated BMDM and db/db mouse hippocampus, reflecting inhibition of NLRP3 inflammasome-mediated caspase-1 cleavage[1, 2]

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).

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BMDM culture and NLRP3 activation assay: Bone marrow cells were isolated from C57BL/6, Nlrp3⁻/⁻, casp11⁻/⁻ mice and differentiated into BMDM in complete medium (containing M-CSF) for 7 days. BMDM were primed with LPS (100 ng/mL) for 3-4 hours, then stimulated with canonical NLRP3 agonists (ATP 5 mM, nigericin 10 μM) or noncanonical agonist (transfected LPS 1 μg/mL after Pam3CSK4 priming 24 hours). MCC950 sodium (1-1000 nM) was added 30 minutes before agonist stimulation. After 6-24 hours of stimulation, cell supernatants were collected for ELISA (IL-1β, TNF-α, IL-1α) and LDH assay; cell lysates were prepared for Western blot (NLRP3, ASC, caspase-1, IL-1β, GAPDH)[1]
- ASC oligomerization assay: BMDM were primed with LPS and stimulated with nigericin in the presence of MCC950 sodium or parthenolide. Cells were lysed with detergent, and cytosolic fractions were cross-linked with disuccinimidyl suberate (DSS) for 30 minutes at room temperature. Cross-linked proteins were separated by SDS-PAGE, and ASC oligomers were detected by Western blot with anti-ASC antibody[1]
- ASC speck formation assay: ASC-cerulean reporter cells (stably expressing ASC fused to cerulean fluorescent protein) were seeded in 96-well plates and treated with MCC950 sodium (0.05-10 μM) for 30 minutes, then stimulated with nigericin, LeuLeu-Ome, or lethal toxin for 2-4 hours. Live cell imaging was performed using a confocal microscope (×40 magnification) to count ASC speck-positive cells; for FACS analysis, cells were harvested, and the percentage of ASC speck-containing live cells was quantified[1]
- K⁺ efflux and Ca²⁺ flux assay: For K⁺ efflux, Nlrp3⁻/⁻ BMDM were primed with LPS and stimulated with nigericin, ATP, or SiO₂ in the presence of MCC950 sodium. Intracellular K⁺ concentration was measured by inductively coupled plasma optical emission spectrometry (ICP-OES) after cell lysis. For Ca²⁺ flux, LPS-primed C57BL/6 or Ice⁻/⁻ BMDM were loaded with Fluo-4 AM, treated with MCC950 sodium, and ATP-induced Ca²⁺ flux was measured in real-time using the FLIPR TETRA system (excitation 488 nm, emission 525 nm)[1]
- HEK-293T cell transfection and co-immunoprecipitation: HEK-293T cells were transfected with FLAG-NLRP3, HA-NLRP3, ASC, or empty vector plasmids using a transfection reagent. After 24 hours, cells were treated with MCC950 sodium for 4 hours, then lysed with immunoprecipitation buffer. FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody and protein G beads, and co-precipitated ASC was detected by Western blot with anti-ASC antibody. For ASC speck analysis, HEK-293T cells were co-transfected with GFP-ASC and NLRP3-mCherry plasmids, treated with MCC950 sodium, and the percentage of ASC speck-positive live cells was measured by FACS[1]
- Human PBMC assay: PBMC were isolated from individuals with MWS (E313K mutation) by density gradient centrifugation. PBMC were primed with LPS (1 μg/mL) for 3 hours, treated with MCC950 sodium for 30 minutes, then cultured for 24 hours. Cell supernatants and lysates were collected for Western blot to detect caspase-1 cleavage and IL-1β maturation[1]

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.

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EAE induction and treatment in mice: Female C57BL/6 mice (6-8 weeks old) were immunized with MOG₃₅₋₅₅ peptide emulsified in Freund's complete adjuvant (FCA) supplemented with Mycobacterium tuberculosis, and pertussis toxin (200 ng) was injected intraperitoneally on day 0 and day 2 post-immunization to induce EAE. MCC950 sodium was dissolved in PBS and administered intraperitoneally (i.p.) at a specific dose (not detailed) once daily starting from the day of immunization. Clinical scores (0-5 scale, based on paralysis severity) were recorded daily for 22 days. On day 22, mice were sacrificed, and brain mononuclear cells were isolated for flow cytometry to detect IL-17/IFN-γ secreting T cells[1]
- LPS-induced systemic inflammation in mice: C57BL/6 mice were pre-treated with MCC950 sodium (i.p., dose not detailed) or vehicle (PBS) 1 hour before intraperitoneal injection of LPS (10 mg/kg). Blood samples were collected 2 hours after LPS injection, and serum IL-1β, TNF-α, and IL-6 levels were measured by ELISA (n=3 per group)[1]
- CAPS mouse model treatment: Nlrp3A³⁵⁰VneoR × LysMCre mice (NLRP3 gain-of-function mutant) and wild-type (WT) littermates were treated with MCC950 sodium (dissolved in PBS, i.p., dose not detailed) or PBS starting from day 9 after birth. Body weight was measured at day 9, and survival was monitored up to day 45 (MCC950 withdrawn at day 28). Plasma IL-18 levels were measured by ELISA at day 9 and 14 days after drug withdrawal (n=3-6 per group). NLRP1 mutant (Nlrp1aQ593P) mice were used as controls and treated with MCC950 sodium or PBS[1]
- Diabetic encephalopathy (DEP) model in db/db mice: Male db/db mice (8 weeks old) and db/m littermates were randomly divided into groups: db/m + vehicle, db/db + vehicle, db/db + MCC950 sodium. MCC950 sodium was dissolved in PBS and administered intraperitoneally at a dose of 10 mg/kg once daily for 8 weeks; vehicle-treated groups received PBS. Body weight and fasting blood glucose were measured weekly. Oral glucose tolerance test (OGTT): mice fasted for 12 hours, then received glucose (2 g/kg) by gavage, and blood glucose was measured at 0, 30, 60, 90, 120 minutes. Insulin tolerance test (ITT): mice fasted for 4 hours, then received insulin (0.75 U/kg) i.p., and blood glucose was measured at 0, 15, 30, 60, 90 minutes. Behavioral tests (light/dark box, FST, TST, MWM) were performed after 8 weeks of treatment. After behavioral tests, mice were sacrificed, hippocampal tissue was collected for Western blot (NLRP3, ASC, IL-1β) and caspase-1 activity assay; plasma was collected for ELISA (insulin, IL-1β, TNF-α) (n=3-8 per group)[2]

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

[2]. Inhibiting the NLRP3 Inflammasome Activation with MCC950 Ameliorates Diabetic Encephalopathy in db/db Mice. Molecules. 2018 Feb 27;23(3). pii: E522.

The pyridine-domain-containing protein 3 (NLRP3) inflammasome, a member of the NOD-like receptor (NLR) family, is an integral part of the inflammatory process. Its aberrant activation is pathogenic in genetic diseases such as cold pyridine-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 and selective small-molecule NLRP3 inhibitor. MCC950 blocks the activation of both classical and non-classical NLRP3 at nanomolar concentrations. MCC950 specifically inhibits NLRP3 activation but does not inhibit the activation of the AIM2, NLRC4, or NLRP1 inflammasomes. In vivo, MCC950 reduces the production of interleukin-1β (IL-1β) and alleviates the severity of experimental autoimmune encephalomyelitis (EAE, a disease model of multiple sclerosis). In addition, MCC950 treatment saved neonatal mortality in CAPS mouse models and was also effective in ex vivo samples from patients with Muckle-Wells syndrome. Therefore, MCC950 is expected to be a potential therapeutic for NLRP3-related syndromes (including autoinflammatory and autoimmune diseases) and can serve as a tool for further research on the role of NLRP3 inflammasomes in human health and disease. [1]

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Diabetes is associated with a high risk of cognitive impairment and neuropsychiatric disorders, symptoms of which are called diabetic encephalopathy (DEP). Inflammation is involved in the development of DEP. The cleavage and maturation of the pro-inflammatory cytokine interleukin (IL)-1β are regulated by NLRP3 inflammasomes. Obese and type 2 diabetic db/db mice exhibit anxiety and depression-like behaviors as well as cognitive impairment associated with hippocampal inflammation. This study aimed to investigate the role of NLRP3 inflammasomes in diabetic depression (DEP). The results showed that the expression levels of inflammasome components such as NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1, and IL-1β in the hippocampus of diabetic db/db mice were higher than those in non-diabetic db/m mice. Treatment of db/db mice with the NLRP3 inflammasome inhibitor MCC950 improved their anxiety- and depression-like behaviors and cognitive impairment, and reversed the elevated levels of NLRP3, ASC, and IL-1β expression and caspase-1 activity in the hippocampus. Furthermore, MCC950 treatment significantly improved insulin sensitivity in db/db mice. These results suggest that inhibiting NLRP3 inflammasome activation may be a potential treatment for DEP (disease-induced efflux syndrome). [2]

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MCC950 sodium (chemical name: N-(1,2,3,5,6,7-hexahydro-S-indo[n-indo]-4-ylcarbamoyl)-4-(2-hydroxy-2-propionyl)-2-furansulfonamide sodium salt) is a potent, selective small molecule NLRP3 inflammasome inhibitor [1, 2]
- The NLRP3 inflammasome is a key mediator of the inflammatory response, and its abnormal activation is associated with cold pyridine-associated periodic syndrome (CAPS), multiple sclerosis, type 2 diabetes, Alzheimer's disease, and atherosclerosis [1]
- MCC950 sodium inhibits the activation of the NLRP3 inflammasome by blocking ASC oligomerization ( Sodium MCC950 plays a role in key steps of NLRP3 assembly without affecting upstream events (K⁺ efflux, Ca²⁺ influx, NLRP3 initiation, or TLR signaling) [1]
- Sodium MCC950 is active in preclinical models of NLRP3-related diseases: it alleviates EAE (multiple sclerosis model), rescues neonatal mortality in CAPS mice, and improves diabetic encephalopathy in db/db mice by reducing hippocampal NLRP3 activation and neuroinflammation [1, 2]
- Sodium MCC950 is effective in ex vivo samples from patients with Muckle-Wells syndrome (a subtype of CAPS), suggesting its potential clinical application value in human NLRP3-related autoinflammatory and autoimmune diseases [1]

C20H23N2O5S.NA

426.46

426.122

C, 56.33; H, 5.44; N, 6.57; Na, 5.39; O, 18.76; S, 7.52

256373-96-3

MCC950;210826-40-7

91826093

Off-white to yellow solid

4.977

2

6

4

29

690

0

S(C1=C([H])C(=C([H])O1)C(C([H])([H])[H])(C([H])([H])[H])O[H])([N-]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.[Na+]

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-yl)carbamoyl)((4-(2-hydroxypropan-2-yl)furan-2-yl)sulfonyl)amide

CP-45677; CP45677; CP 45677; 256373-96-3; MCC950 sodium; CRID3 sodium salt; CP-456773 sodium; MCC950 (sodium); MCC950 sodium salt; CP-456773 sodium salt; MCC-950 sodium salt; MCC950; MCC 950; MCC-950; CRID-3; CRID3; CRID 3; CP-456773 sodium

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 5 mg/mL (11.72 mM) (saturation unknown) in 5%DMSO 95%PBS (add these co-solvents sequentially from left to right, and one by one), clear solution.<

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.86 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 (5.86 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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Solubility in Formulation 4: ≥ 2.08 mg/mL (4.88 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 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: 2％DMSO 98％PBS

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Solubility in Formulation 6: 6.25 mg/mL (14.66 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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