Fig 2: SK56 inhibits pyroptosis by directly targeting GSDMD pore function.a, Representative TEM images of cytomembranes from THP-1 cells (top) and representative biolayer interferometry traces from biotin–SK56 (bottom) incubated with PBS, 1 μg ml−1 LPS or 1 μg ml−1 LPS + 10 μM nigericin (top) or PBS, GSDMD-NT protein or cytomembranes from THP-1 cells incubated with PBS (CPBS), 1 μg ml−1 LPS (CLPS) or 312 nM–5 μM LPS + nigericin (CL + N) for 2 h (n = 2 repeats); scale bar, 200 nm. b, GSDMD immunoblotting of immunoprecipitated SUMO–SK56 in THP-1 cells treated with LPS + nigericin as in a; n = 2 repeats; IB, immunoblot; IP, immunoprecipitate. c, Representative live-cell images of mouse wild-type BMDMs transfected with BFP–GSDMD-NT or GSDMD-CT–BFP plasmids and incubated with 15 µM FITC–SK56 at 0, 40 and 80 min after LPS + nigericin treatment; scale bar, 2 µm. Arrowheads indicate colocalization; n = 2 cells. d, MST showing the binding affinity of SK56 to mature GSDMA–GSDME (n = 1 repeat). e,f, Representative images (e) and quantification of fluorescence (f) in PDA nanoparticle hydrogel incubated with PBS, 15 µM SK56, 5 μg ACE2 or 1 μM GSDMD-NT for 45 min or 1 μM GSDMD-NT for 30 min and then incubated with 15 μM SK56 for an additional 15 min; scale bar, 70 µm; n = 3 repeats. g, Cell viability in THP-1 cells incubated with PBS, 1 μg ml−1 LPS or LPS + nigericin in addition to PBS, 1.5 µM SK56 or 1.5 µM SK56 mutant peptides for 120 min; n = 4 repeats. h, Docking assay showing SK56–GSDMD-NT interaction. Critical residues (Arg 22–Glu 174 electrostatic interaction, Met 29–Pro 103 hydrophobic interaction and Tyr 26–Thr 63 hydrogen bond) are indicated. i, Proteomic analysis showing differentially expressed proteins (DEPs) in THP-1 cells treated with PBS, LPS + nigericin or LPS + nigericin + 15 µM SK56 for 90 min; n = 3 samples. Data in f and g were analyzed by two-tailed Student’s t-test and are shown as mean ± s.d.Source data

Fig 3: SK56 inhibits pyroptosis by ESCRT and reduces DC phagocytosis of the pyroptotic cytomembrane and IL-1β release.a, Representative images of CHMP4–GFP puncta (arrowheads; left) and percentage of CHMP4 speckle+ (top right) or Annexin V+ (bottom right) cells in BMDMs incubated with PBS, 15 µM SK56, 2 mM EDTA or EDTA + SK56 at 2 h after the addition of 1 μg ml−1 LPS + 10 μM nigericin; scale bar, 50 µm; n = 5 repeats. b, Immunoblots (top) and quantification (bottom) showing GSDMD-NT in the supernatant from THP-1 cells treated with PBS, 30 µM DSF, 15 µM SK56 or 15 µM SK56 at 120 min after LPS + nigericin treatment; n = 3 repeats. c, Representative images (left) and percentage of GSDMD-NT–BFP/cytomembrane-CellMask Orange+ (right) cells in calcein-AM-labeled (green) mouse wild-type BMDCs incubated with 2 μg ml−1 pyroptotic cytomembrane fragments from mouse wild-type BMDMs transfected with a GSDMD-casp–BFP construct and incubated with LPS + nigericin (PCFBFP), 20 µM SK56 (PCFBFP + SK56) or 20 µM SK56scrambled (synthetic SK56 scrambled peptide; PCFBFP + SK56scrambled), pyroptotic cytomembrane fragments from mouse Gsdmd−/− BMDMs incubated with LPS + nigericin and PBS (PCFGsdmd−/−), cytomembranes from wild-type BMDMs incubated with PBS (NCF) or pyroptotic cytomembrane fragments from mouse BMDMs incubated with LPS + nigericin and 10 µg ml−1 BFP (PCF + BFP), 10 µg ml−1 GSDMD-NT–BFP or 10 µg ml−1 BFP for 2 h; green, calcein-AM+ BMDCs; red, CellMask Orange+ NCF, PCF or PCFGsdmd−/−; blue, GSDMD-NT–BFP, BFP or PCFBFP. The white arrow indicates cytomembrane outside BMDCs, and the red arrow indicates phagocytosed cytomembrane; scale bar, 25 μm; n = 3 repeats. d, ELISA of secreted (left) and cell-associated (right) IL-1β from wild-type BMDCs treated with PBS, 10 μg ml−1 BFP, 1 μg ml−1 GSDMD-NT, 20 µM SK56, SK56 + GSDMD-NT, 120 µM oxPAPC, SK56 + oxPAPC or 2 μg ml−1 pyroptotic cytomembrane fragments from mouse wild-type or Gsdmd−/− BMDMs as in c (NCF, PCF, PCF + SK56, PCFGsdmd−/−, PCFGsdmd−/− + SK56), treated or not treated with Pam3 for 12 h; n = 4 repeats. All data are shown as mean ± s.d., and P values were determined by two-tailed Student’s t-test; NS, not significant (P > 0.05).Source data

Fig 4: Structural analysis of SK56 interaction with the GSDMD-NT pore.a, Polydiacetylene (PDA) nanoparticles solidified by hydrogel were used to determine if SK56 has a repairing effect on preformed holes. Schematic presentation of installing PDA nanoparticles in the network of Poly (ethylene glycol) diacrylate (PEGDA) hydrogel. PDA nanoparticles can be chemically linked to the network of PEGDA hydrogels by photocrosslinking PEGDA monomers and acrylamide-modified PDA nanoparticles via addition polymerization. NPs, nanoparticles. b, MD simulation depicting residue-wise MM-GBSA binding free energy decomposition for SK56-GSDMD-NT interaction. n = 1 run. c, Structural analysis showing SK56-induced surface charge alterations in GSDMD-NT acidic patches (AP1/AP3).

Fig 5: SK56 protects human alveolar organoids and blood leukocytes from pyroptosis-induced damage.a, Representative images from live-cell imaging (see Supplementary Video 4; left) and relative PI stain intensity (top right) and calcein-AM staining (bottom right) in human alveolar organoids (calcein-AM+, green) cocultured with GSDMD-casp–BFP-transfected THP-1 cells treated with LPS + nigericin and incubated with PBS, 30 µM DSF, 25 µM DMF, 20 µM NSA or 15 μM SK56 at 0.5, 4, 8, 12 and 16 h after treatment with LPS + nigericin; scale bar, 40 μm; n = 3 repeats. b, Representative H&E staining (top) and percentage of the infiltration area (bottom) in fixed alveolar organoids + THP-1 cell cocultures as in a treated with PBS or LPS + nigericin together with PBS (L + N + PBS) or 15 μM SK56 at 8 h after the addition of LPS + nigericin; scale bar, 75 μm; n = 5 repeats. c, ELISA showing IL-1β release in the supernatant of alveolar organoids + THP-1 cell cocultures as in a treated with PBS, 15 μM SK56 or LPS + nigericin together with PBS or 15 μM SK56 or at 12 h after LPS + nigericin treatment; n = 4 repeats. d, Representative images (left) and percentage relative to DAPI+ cells (right) of GSDMD-NT+ (green) cells in human blood leukocytes incubated with PBS or LPS + nigericin together with PBS or 15 μM SK56 at 1 h after nigericin + LPS treatment; n = 20 repeats. e, Representative immunoblots of IL-1β and GSDMD-NT in whole human blood leukocytes treated as in d; n = 3 repeats. f, Heat map illustrating inflammatory cytokine profiles in whole human blood treated with normal saline (NSal) and LPS + nigericin together with normal saline (L + N + NS) or 15 μM SK56; *P < 0.05 L + N + SK56 versus L + N + NS, n = 4 samples. Data are shown as mean ± s.d., and P values were calculated by two-tailed Student’s t-test; *P < 0.05.Source data