A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases

Abstract

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.

Figure 1. MCC950 inhibits NLRP3 inflammasome activation in response to canonical and non-canonical NLRP3 stimuli

(a) MCC950 structure. (b) Production of IL-1β and TNF-α from BMDM stimulated with LPS and ATP and treated with MCC950 (1-1,000 nM) measured by ELISA. Cytokine level is normalized to DMSO control treated cells. Data are expressed as mean ± S.E.M. of six independent experiments carried out in triplicate. Non-linear regression analysis was performed and Log [M] MCC950 vs. normalized response (variable slope) curve is presented. (c) Western blots of cell lysates and supernatants from BMDM stimulated with LPS and ATP and treated with MCC950 or glyburide. These results are representative of five independent experiments. (d–g) Production of IL-1β, TNF-α, IL-1α and LDH from BMDM stimulated with LPS and nigericin and treated with MCC950 as measured by ELISA (d–f) and LDH assay (g). Data are expressed as mean ± S.E.M. of three independent experiments carried out in triplicate. (h–j) Production of IL-1β, IL-1α and LDH from BMDM from the indicated genotypes stimulated with Pam3CSK4 and transfected LPS and treated with MCC950 as measured by ELISA (h,i) and LDH assay (j). Data are expressed as mean ± S.E.M. of three (Nlrp3^−/−), four (casp11^−/−) or six (C57BL/6) independent experiments.

Figure 2. MCC950 does not inhibit NLRC4, AIM2, TLR signalling or priming of NLRP3

(a) Production of IL-1β and TNF-α from BMDM stimulated with LPS and S. typhimurium and treated with MCC950 and parthenolide measured by ELISA. Cytokine level is normalized to DMSO control treated cells. Data are expressed as mean ± S.E.M. of four independent experiments carried out in triplicate. (b) Western blots of cell lysates and supernatants from BMDM stimulated with LPS and S. typhimurium and treated with MCC950 and parthenolide. These results are representative of four independent experiments. (c) Production of IL-1β and TNF-α from BMDM stimulated with LPS and transfected Poly(dA:dT) and treated with MCC950 and Bayer compound measured by ELISA. Cytokine level is normalized to DMSO control treated cells. Data are expressed as mean ± S.E.M. of five independent experiments carried out in triplicate. (d) Western blots of cell lysates and supernatants from BMDM stimulated with LPS and transfected Poly(dA:dT) and treated with MCC950 and Bayer compound. These results are representative of two independent experiments. (e) Western blots of cell lysates from BMDM treated with MCC950 and stimulated with LPS (4 h). These results are representative of three independent experiments. (f) Production of TNF-α from BMDM treated with MCC950 and parthenolide and stimulated with LPS or Poly(A:U) measured by ELISA. Data shown represent mean cytokine level ± S.D. from triplicate determinations and are representative of three independent experiments.

Figure 3. MCC950 blocks NLRP3 dependent ASC oligomerization

(a) Western blots of cell lysates, supernatants and cross-linked cytosolic pellets from BMDM stimulated with LPS and nigericin and treated with MCC950 and parthenolide. These results are representative of three independent experiments. (b) Live cell imaging of ASC-cerulean cells treated with MCC950 and stimulated with ATP. At least five different images were taken of each condition at an original magnification of ×40. Results are representative of three independent experiments. (c) The percentage of ASC-cerulean cells containing an ASC speck after treatment with MCC950 (0.05-10 µM) and stimulation with nigericin, LeuLeu-Ome or lethal toxin. Data shown represent the mean ± S.D. from triplicate measurements.

Figure 4. MCC950 does not block K⁺ efflux, Ca²⁺ flux or direct NLRP3 and ASC interactions

(a) The relative level of intracellular K⁺ determined by ICP-OES in Nlrp3^−/− BMDM stimulated with LPS and nigericin, ATP and SiO₂ and treated with MCC950. (b) IL-1β production from C57BL/6 BMDM stimulated with LPS and nigericin, ATP and SiO₂ measured by ELISA. (a-b) Data are presented as mean ± S.D. from triplicate measurements and are representative of three independent experiments. (c) A trace showing ATP induced Ca²⁺ flux was measured using the FLIPR^TETRA system in C57BL/6 BMDM stimulated with LPS and treated with MCC950. The results are representative of three independent experiments. (d,e) The relative response to ATP induced Ca²⁺ flux in C57BL/6 (d) and Ice^−/− (e) BMDM stimulated with LPS and treated with MCC950 as measured using the FLIPR^TETRA system. Data are presented as mean ± S.E.M. and are representative of three independent experiments. (f,g) Western blots of cell lysates and FLAG-immunoprecipitation samples from HEK-293T cells transfected with FLAG-NLRP3, HA-NLRP3, ASC or empty vector plasmids as indicated and treated with MCC950. These results presented are representative of three independent experiments. (h) The percentage of ASC speck containing live HEK-293T cells transfected with GFP-ASC and NLRP3-mCherry plasmids as indicated, treated with MCC950. and analysed by FACS. Data is presented as mean ± S.E.M. from three independent experiments each carried out in triplicate.

Figure 5. MCC950 is active in vivo and treatment of mice with MCC950 attenuates EAE

(a–c) Serum levels of IL-1β (a), TNF-α (b) and IL-6 (c) measured by ELISA from C57BL/6 mice pre-treated with MCC950 or vehicle control, 2 h post i.p. LPS injection. Data shown are mean values ± S.D. n=3, *P ≤ 0.05 by Mann-Whitney test. (d) Clinical scores after EAE induction from C57BL/6 treated with PBS or MCC950. Mean ± S.D. n=6.(e–g) Representative dot plots from five mice and mean percentages ± S.D. of live IL-17 and IFN-γ secreting CD3⁺ T cells (e), CD3⁺ CD4⁺ T cells (f) or CD3⁺ γδ TCR⁺ cells (g) from brain mononuclear cells isolated on day 22 post EAE induction in C57BL/6 mice treated with PBS or MCC950 are shown. n=5, *P ≤ 0.05 by Mann-Whitney test.

Figure 6. MCC950 inhibits NLRP3 activation in a mouse model of MWS and in MWS PBMC ex vivo

(a) Nlrp3A^350VneoR crossed with LysMCre mice (NLRP3 mutant) treated with PBS or MCC950 at day 9. (b) Weight at day 9. Mean ± S.D. WT PBS and WT MCC950 both n=4, NLRP3 mutant PBS n=3 and NLRP3 mutant MCC950 n=6. **P ≤ 0.005 by unpaired two-tailed t-test. (c) Survival of NLRP3 mutant mice treated with PBS or MCC950 up to day 45 (MCC950 withdrawn at day 28). MCC950 group n=5, PBS group n=9. (d) WT and NLRP3 mutant mice on day 17. (e) Plasma IL-18 concentrations as measured by ELISA at day 9, and again for NLRP3 mutant mice 14 days after drug withdrawal. WT PBS and WT MCC950 both n=4, NLRP3 mutant PBS n=5 and NLRP3 mutant MCC950 and withdrawal both n=4. NLRP1a^Q593P mice (NLRP1 mutant) PBS and MCC950 both n=3. Data shown are mean ± S.D. * P ≤ 0.05 by Mann-Whitney test. (f) Western blots of cell lysates and supernatants from PBMC isolated from an individual with MWS (mutation E313K) stimulated with LPS and treated with MCC950.