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FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation

Abstract

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1β) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1β and GSDMD processing, but abrogates pore formation, thereby preventing IL-1β release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

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Fig. 1: High-throughput screen identifies disulfiram as an inhibitor of GSDMD pore formation.
Fig. 2: Disulfiram inhibits pyroptosis and IL-1β secretion.
Fig. 3: Disulfiram inhibition of liposome leakage is mediated primarily by direct inhibition of GSDMD pore formation.
Fig. 4: Disulfiram covalently modifies GSDMD Cys191.
Fig. 5: GSDMD pore formation is the main target of disulfiram.
Fig. 6: Disulfiram protects against LPS-induced sepsis.

Data availability

All relevant data are available in the Source Data or Extended Data of the manuscript.

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Acknowledgements

This work was supported by the US National Institutes of Health (grant nos. DP1HD087988 to H.W.; R01Al139914 to H.W. and J.L.; R01AI123265 to J.L.; and R01 AI142642, R01 AI145274, R01 AI141386, R01HL092020 and P01HL095489 to H.R.L.), National Natural Science Foundation of China (grant no. 31972897), Key Research Program of the Chinese Academy of Sciences (grant no. ZDBS-LY-SM008), Shanghai Municipal Science and Technology Major Project (grant no. 2019SHZDZX02), Rising-Star Program of Shanghai Science and Technology Committee (grant no. 19QA1409800 to X.L.), a grant from FAMRI (no. CIA 123008 to H.R.L.), Cancer Research Institute Irvington Postdoctoral Fellowship Program (to J.J.H.), Charles A. King Trust Postdoctoral Fellowship Program (to J.R., X.L., Z.Z.) and a US DOD Breast Cancer Research Program Breakthrough Fellowship Award (Y.Z.). We thank J. Smith, G. Frey, J. Nale, D. Wrobel and the entire staff of the ICCB-L for their outstanding technical support.

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Contributions

H.W. and J.J.H. conceived the study. J.J.H., S.X. and J.R. optimized the liposome leakage assay. J.J.H performed the high-throughput screen and the validation experiments in vitro. S.X. performed negative staining electron microscopy. X. Liu, Z.Z., J.Z., X. Lou, Y.B., J.W., L.R.H. and V.G.M. performed cellular experiments. X. Liu, Y.Z., L.Z. and H.R.L. carried out studies in mice. X. Luo ran mass spectrometry. J.K. advised on chemistry. H.W. and J.L. supervised the project. H.W., J.J.H., J.L. and X. Liu wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Xing Liu or Judy Lieberman or Hao Wu.

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J.L. and H.W. are cofounders of Ventus Therapeutics. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Optimization and hits from the liposome leakage assay screen.

(a–c) Optimization of the Tb3+/DPA assay. a, GSDMD (2.5 μM) and caspase-11 (2.5 μM) were incubated in liposome solutions at various concentrations in 20 mM HEPES buffer (150 mM NaCl) for 1 h. The concentration of liposome lipids for the screen was set at 50 μM. n = 3 independent experiments. The mean ± s.e.m. is shown. b, Different concentrations of GSDMD and caspase-11 (1:1 ratio) were incubated in liposome (50 μM) solutions for 1 h. The concentration of GSDMD used in the screen was set at 0.3 μM. n = 3 independent experiments. The mean ± s.e.m. is shown. c, Different concentrations of caspase-11 and GSDMD (0.3 μM) were incubated in liposome (50 μM) solutions for 1 h. The concentration of caspase-11 used in the screen was set at 0.15 μM. n = 3 independent experiments. The mean ± s.e.m. is shown. The fluorescence intensity at 545 nm was measured after excitation at 276 nm. d, Hit compounds evaluated in binding and/or cell-based assays. (e) Mouse iBMDMs were pretreated or not with disulfiram (C-23) ranging from 5-40 μM for 1 h before transfection with PBS or poly(dA:dT) and analyzed for cell viability by CellTiter-Glo assay 4 hrs later. Graphs show mean ± s.d; data are representative of three independent experiments with replicates (n = 3) and similar results. Data were analyzed using two-tailed Student’s t-test. **P < 0.01. Source data

Extended Data Fig. 2 The activity of disulfiram in cells is greatly increased by Cu(II).

a, DTC–copper complex formation of disulfiram metabolite diethyldithiocarbamate (DTC) with Cu(II). b, Dose response curves of inhibition of liposome leakage by disulfiram (C-23) or DTC in the presence or absence of Cu(II). n = 3 independent experiments. The mean ± s.e.m. is shown. c, LPS-primed THP-1 were pretreated with C-23 or DTC in the presence or absence of Cu(II) for 1 h before adding nigericin or medium for 2 hrs. Cell death was determined by CytoTox96 assay. n = 3 independent experiments. The mean ± s.e.m. is shown. Source data

Extended Data Fig. 3 Effect of disulfiram on caspase-1 and caspase-11.

(a, b) Time course of caspase-1 (a) and caspase-11 (b) activity in the presence of indicated concentrations of disulfiram. Caspases (0.5 U) were incubated with disulfiram (at indicated concentrations for 1 h before adding Ac-YVAD-AMC (40 μM)). (c,d) Dose response curve of disulfiram in the caspase-1 (a) and caspase-11 (b) activity assay. (e,f) Time course of caspase-1 (e) and caspase-11 (f) activity in the presence of indicated concentrations of disulfiram + Cu(II). Caspases (0.5 U) were incubated with disulfiram + Cu(II) (at indicated concentrations for 1 h before adding Ac-YVAD-AMC (40 μM)). (g,h) Dose response curve of disulfiram + Cu(II) in the caspase-1 (e) and caspase-11 (f) activity assay. (a-h) n = 3 independent experiments. The mean ± s.e.m. is shown. Fluorescence intensity at 460 nm was measured after excitation at 350 nm. Source data

Extended Data Fig. 4 Disulfiram covalently modifies human GSDMD on Cys 191.

a, Disulfiram was preincubated for 1 h with N-acetylcysteine (NAC, 500 μM) or medium before evaluating whether it inhibited pyroptosis of LPS + nigericin treated THP-1 cells. Disulfiram 2-fold dilutions ranged from 5-40 μM. Graphs show mean ± s.d; data are representative of three independent experiments with replicates (n = 3) and similar results. Data were analyzed using two-tailed Student’s t-test. Graphs show the mean ± s.d. and data shown are representative of three independent experiments. **P < 0.01. (b, c) nano-LC-MS/MS spectrum for the peptide containing C191 in human GSDMD. Data are representative of three independent experiments. b, MS/MS spectrum for peptide FSLPGATCLQGEGQGHLSQK modified on cysteine (red) by carbamidomethyl. Protein coverage was 73%. c, MS/MS spectrum for peptide FSLPGATCLQGEGQGHLSQK modified on cysteine (red) by disulfiram. Protein coverage was 72%. Source data

Extended Data Fig. 5 Disulfiram covalently modifies GSDMD Cys191.

a, Sequence alignment of GSDMA3, hGSDMA, mGSDMD and hGSDMD showing Cys residues (highlighted in red). b, GSDMD (0.3 μM) was preincubated with the indicated concentrations of disulfiram (0–5.6 μM) for indicated times (2–90 min) before caspase-11 (0.15 μM) and liposomes (50 μM) were added. n = 3 independent experiments. The mean ± s.e.m. is shown. c, FL mouse GSDMD or wildtype, C192S or C39A GSDMD-NT were transiently expressed in HEK293T cells. Cell death was determined by CytoTox96 cytotoxicity assay 20 hrs after transfection. c, shows the mean ± s.d. of 1 representative experiment of three independent experiments performed. Comparison in (c) was calculated by two-tailed Student’s t-test. *P < 0.05. Source data

Extended Data Fig. 6 Mouse monoclonal antibody recognizes full-length human GSDMD and the GSDMD-NT pore form on immunoblots and by immunofluorescence microscopy.

The monoclonal antibody against GSDMD was generated by immunizing mice with recombinant human GSDMD and boosting with recombinant human GSDMD-NT as described in Methods. a, HEK293T cells were transfected with the indicated plasmids and cell lysates were analysed by immunoblot of reducing gels probed with the indicated antibodies. b, Cell lysates of HCT116, 293 T and THP-1 cells, treated or not with nigericin, were immunoblotted with the indicated antibodies. 293 T cells do not express endogenous GSDMD. c, 293T and THP-1 cells were stained with the anti-GSDMD monoclonal antibody and co-stained with DAPI (blue). 293T cells show no background staining. Data are representative of at least three independent experiments. Source data

Extended Data Fig. 7 Disulfiram protects against LPS-induced sepsis.

(a–c) Mice were pretreated with disulfiram (50 mg/kg) or vehicle (Ctrl) by intraperitoneal injection 24 and 4 hrs before intraperitoneal challenge with 15 mg/kg LPS and followed for survival. Serum IL-1β (a), TNF (b) and IL-6 (c) were measured by Luminex Multiplex Assay (n = 5/group) 6 hrs post LPS challenge. Shown are mean ± s.e.m. Statistical differences between the groups were calculated by multiple t-test. Type I error was corrected by the Holm-Sidak method. Source data

Extended Data Fig. 8 Dose response curve of other compounds in GSDMD-mediated liposome leakage assay.

Dose response curve of necrosulfonamide (a), Bay 11-7082 (b), dimethyl fumarate (DMF) (c), afatinib (d), ibrutinib (e), and LDC7559 (f) in liposome leakage induced by 0.3 μM GSDMD plus 0.15 μM caspase-11. (a-f) n = 3 independent experiments. The mean ± s.e.m. is shown. Source data

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Hu, J.J., Liu, X., Xia, S. et al. FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation. Nat Immunol 21, 736–745 (2020). https://doi.org/10.1038/s41590-020-0669-6

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