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MCC950 closes the active conformation of NLRP3 to an inactive state - PubMed

MCC950 closes the active conformation of NLRP3 to an inactive state

Ana Tapia-Abellán et al. Nat Chem Biol. 2019 Jun.

Erratum in

Abstract

NLRP3 (NOD-like receptor pyrin domain-containing protein 3) is an innate immune sensor that contributes to the development of different diseases, including monogenic autoinflammatory syndromes, gout, atherosclerosis, and Alzheimer's disease. The molecule sulfonylurea MCC950 is a NLRP3 inflammasome inhibitor with potential clinical utility. However, the mechanism of action of MCC950 remains unknown. Here, we characterize the mechanism of action of MCC950 in both wild-type and autoinflammatory-related NLRP3 mutants, and demonstrate that MCC950 closes the 'open' conformation of active NLRP3.

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Conflict of interest statement

Competing financial interests. The authors declare no competing interests.

Figures

Figure 1
Figure 1. MCC950 closes the conformation of autoinflammatory-associated gain-of-function NLRP3.

a,b, BRET signal in HEK293T cells expressing wild-type or p.D305N YFP-NLRP3-Luc as indicated, incubated for 24 h with different concentrations of MCC950 (a) or for different times with MCC950 (10 μM) (b). Vehicle control data is concentration zero (a) and time zero (b). Centre values represent mean and error bars the SEM; for exact n number see Methods, section Statistics and Reproducibility. c, BRET signal (bottom) and immunoblots for NLRP3, IL-1β, GSDMD and β-actin (top) from immortalized Nlrp3 -/- macrophages expressing YFP-NLRP3-Luc p.T350M or p.D305N, treated for 16 h with doxycycline (1 μg/ml) and LPS (100 ng/ml) alone or in combination with MCC950 (10 μM). Blots are representative of n=2 independent experiments with similar result (full blots are shown in Supplementary Figure 10a) and for BRET assays centre values represent mean and error bars the SEM; for exact n number see Methods, section Statistics and Reproducibility; Mann-Whitney test, two-tailed, ***p<0.0001 (U=0). d, Quantification of the number of mutant NLRP3-YFP oligomers per HEK293T cell incubated or not for 24 h with MCC950 (10 μM). Data are representative of n=3 independent experiments and centre values represent mean and error bars the SEM from 1000-1500 cells. e, Percentage of ASC-specking monocytes identified by time of flight assay (left) and ELISA for IL-1β release (right) from PBMCs isolated from healthy donors (white) and individuals with CAPS carrying the NLRP3 p.D305N (grey) after 4h incubation with LPS (1 μg/ml). Centre values represent mean and error bars the SEM; for exact n number see Methods, section Statistics and Reproducibility. f,g, Inhibition of the percentage of ASC-specking monocytes (f) and IL-1β release (g) from PBMCs after 4h incubation with LPS (1 μg/ml) alone (for CAPS samples) or followed by nigericin (5 μM, 30 min, for healthy individuals) in the presence of different concentrations of MCC950. Centre values represent mean and error bars the SEM; for exact n number see Methods, section Statistics and Reproducibility.

Figure 2
Figure 2. MD simulations of MCC950 targeting the NLRP3 NACHT domain and MCC950 action on the NLRP3 triple walker B mutant.

a, Root mean square deviation during the 100 ns MD for NLRP3 (dark grey), complexed MCC950 (light grey) or NLRP3+MCC950 system (black) models. b, BRET signal in HEK293T cells expressing YFP-NLRP3-Luc p.D305N (white, n= 8 independent experiments) or p.D302A/D305N/E306A (grey, n= 6 independent experiments) incubated for 24 h with MCC950 (10 μM). Centre values represent mean and error bars the SEM; Mann-Whitney test, two-tailed, ***p<0.0001 (U=0) and ns p= 0.2403 (U=10).

Figure 3
Figure 3. MCC950 affects NLRP3 conformation upon activation.

a-c, BRET signal for YFP-NLRP3-Luc in HEK293T cells treated or not with nigericin (10 μM) or in HEK293T cells stable expressing P2X7 receptor treated with ATP (3 mM) (a), in the presence or absence of MCC950 (10 μM) incubated 35 min before nigericin or ATP stimulation (red trace in c). In b 15 min after nigericin stimulation, cells were washed, then MCC950 (10 μM) or vehicle was added and BRET was recorded (a representative trace for the initial response to nigericin is included to compare). Mean (black or red line) ±SEM (grey shadow) are shown; for exact n number see Methods, section Statistics and Reproducibility; Mann-Whitney test, two-tailed for each time comparing vehicle with MCC950, ***p<0.0008 (for each time point, exact p and U value are annotated in supplementary datasheet for figure 3). d, Quantification of the number of wild type NLRP3-YFP oligomers per HEK293T cells incubated or not for 30 min with MCC950 (10 μM) and then stimulated or not for 30 min with nigericin (10 μM). Data are representative of n=3 independent experiments and centre values represent mean and error bars the SEM from 1000-1500 cells. e, HEK293T cells transfected with scramble or NEK7 siRNA and then YFP-NLRP3-Luc BRET kinetic was measured before and after nigericin (10 μM) treatment in the absence (blue and dashed trace) or presence of MCC950 (10 μM, red trace). n=11 independent experiments, and mean (blue, red or dashed trace) ±SEM (grey shadow) are shown. Mann-Whitney test, two-tailed for each time comparing vehicle vs MCC950 (*p<0.05; **p≤0.005; ***p≤0.0002) or comparing scramble vs NEK7 siRNA (#p<0.05); for each time point, exact p and U value are annotated in supplementary datasheet for figure 3. f, Representative maximum intensity fluorescence images of HEK293T cells expressing NLRP3-YFP and NEK7 incubated or not for 30 min with MCC950 (10 μM) and then stimulated for another 30 min with nigericin (10 μM). NLRP3, green; NEK7, red; nuclei, blue DAPI; bar 10 μm; arrowheads denote NLRP3 and NEK7 co-localization. Images are representative of n=3 independent experiments with similar result.

Comment in

  • Walking over the inflammasome.

    Gorka O, Neuwirt E, Groß O. Gorka O, et al. Nat Chem Biol. 2019 Jun;15(6):552-553. doi: 10.1038/s41589-019-0292-8. Nat Chem Biol. 2019. PMID: 31086328 No abstract available.

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