M24B aminopeptidase inhibitors selectively activate the CARD8 inflammasome

Abstract

Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.

Extended Data Fig. 1 ∣. Activation of the NLRP1 and CARD8 inflammasomes.

The proteasome-mediated degradation of the NT fragments of NLRP1 and CARD8 release the CT fragments from autoinhibition. DPP8/9 inhibitors and several other danger signals (for example, pathogen proteases) accelerate the rate of NT degradation. If the rate of degradation is slow (top), CT fragments are restrained in ternary complexes consisting of the CT fragment, a full-length (FL) PRR, and DPP9. Disruption of the ternary complex (for example, by VbP) can release the CT fragments to form an inflammasome. If the rate of degradation is fast (bottom), sufficient CT fragments are released to overwhelm the DPP9 ternary complex checkpoint and to form an inflammasome. NLRP1 is activated by a similar overall mechanism, but only CARD8 is shown for clarity. Key differences between NLRP1 and CARD8 are discussed in the text.

Extended Data Fig. 2 ∣. Certain Xaa-Pro dipeptides inhibit DPP9 and activate CARD8.

(a) Inhibition of recombinant DPP9 by the twenty XP dipeptides in an AP-AMC cleavage assay. (b) Inhibition of AP-AMC cleavage activity in HEK 293 T cell lysates (pretreated with 10 μM sitagliptin to inhibit any DPP4 activity) by indicated compounds. (c) The indicated cell types were immunoblotted for proteins involved in VbP-induced pyroptosis. MV4;11 and THP-1 cells express CARD8, whereas N/TERT-1 and HEKa cells express NLRP1. Asterisks indicate background bands. (d,e) The indicated MV4;11 cells were treated with compounds (1 mM) and monitored for PI uptake over 12 h (d) or analyzed by CTG and CTF after 24 h (e). (f) MV4;11 cells were treated with XP-OMes (1 mM) for 14 h before CTG and CTF analyses. (g) The indicated AML cell lines were treated with VP-OMe (dose range = 5mM-19.5 μM, 2-fold dilution) for 24 h before CTG analysis. (h) Primary resting CD3+ T-cells were treated with VP-OMe (1 mM), IP-OMe (1 mM) or VbP (10 μM) for 18 h before immunoblot analysis. Data is representative of two independent experiments. (i) J774.1 macrophages were treated with VP-OMe (1 mM) or VbP (2 μM) for 24 h before assaying for LDH release. (j) The indicated RAW264.7 cells were treated with VP-OMe or IP-Ome (5 mM, 24 h) before CTF and CTG analyses. Data in d, e, and g (n = 4) and a,b, f, i, and j (n = 3) are means ± SEM of replicates. All data except where indicated, including immunoblots, are representative of three or more independent experiments.

Extended Data Fig. 3 ∣. CQ31 releases CQ04 in cells and causes pyroptosis.

(a) The indicated MV4;11 cells were treated with CQ04 or CQ31 for 24 h before assessing cell viability by CTG. Data are means ± SEM of 3 biological replicates. (b) The indicated MV4;11 cells were treated with CQ31 (16 μM) or VbP (16 μM), incubated for 4 h, and stained with PI. PI uptake was recorded for 12 h. Data are means ± SEM of 10 replicates. a and b are representative of three or more independent experiments. (c) HEK 293T cells were treated with vehicle control (DMSO) or CQ31(10 μM) for 24 h before intracellular metabolites were extracted. Methyl ester and free acid of CQ31 were measured by LC-MS and confirmed against pure standards.

Extended Data Fig. 4 ∣. CQ31 selectively activates the CARD8 inflammasome.

(a) Viability of cell lines after treatment with CQ31 for 24 h as assessed by CTF. (b) Human resting T-cells were treated with CQ31 (10 μM) or VbP (10 μM) for 24 h before assessing cell viability by CTG and pyroptotic and apoptotic markers by immunoblotting. Data is representative of two independent experiments. (c) N/TERT-1 immortalized keratinocyte cells were treated with CQ31 (10 μM) or VbP (10 μM), incubated for 1 h, and stained with PI. PI uptake was assessed over 14 h. (d) HEKa immortalized keratinocyte cells were treated with CQ31 (10 μM) or VbP (10 μM), before assaying for LDH release. (e) HEK 293 T cells stably expressing CASP1 and GSDMD were transfected with plasmids expressing NLRP1 and ASC and treated with CQ31 (10 μM) or VbP (10 μM) for 24 h. GSDMD cleavage was assessed by immunoblotting. (f) RAW264.7 cells were treated with CQ31 (100 μM) or VbP (2 μM) for 24 h, before assaying for LDH release. (n = 3) (g) J774.1 cells were treated with various doses of CQ31 before cell viability was measured by CTG and CTF. Data in a (n = 4), b,d,f (n = 3), and c,g (n = 6) are means ± SEM of the indicated replicates. All data except where indicated, including immunoblots, are representative of three or more independent experiments.

Extended Data Fig. 5 ∣. CQ31-induced pyroptosis is DPP8/9 dependent.

(a,b) Indicated THP-1 cells were treated with VP-OMe (1 mM, 14 h) (a) or varying doses of CQ31 or VbP (b) before assaying cell viability by CTG and CTF assays. (c) HEK 293T cells were treated with vehicle control (DMSO), CQ31 (10 μM), or VbP (10 μM) for 6 h before intracellular metabolites were extracted, and dipeptide concentrations were measured by LC-MS. Data in a (n = 4) and b,c (n = 3) are means ± SEM of the indicated replicates. Data in a and b are representative of three or more independent experiments.

Extended Data Fig. 6 ∣. PEPD knockout cells are sensitive to CQ31.

(a) The indicated MV4;11 cells were treated with compounds (16 μM) for 4 h before monitoring for PI uptake. Data is mean ± SEM of 10 biological replicates. (b) The indicated MV4;11 cells were treated with varying doses of CQ31 before assessing cell viability by CTG and CTF assays. (c) The indicated MV4;11 cells were all treated with VX-765 (50 μM) to prevent pyroptosis. Cells were then co-treated with DMSO (control) or CQ31 (10 μM). Intracellular metabolites were extracted and dipeptide concentrations were measured by LC-MS. Data are means ± SEM of the 3 biological replicates, unless indicated otherwise. Data in a and b are representative of three or more independent experiments.

Extended Data Fig. 7 ∣. CQ31 also targets XPNPEP1.

(a) Representative TMT-labeled peptides used to quantify the enrichment of the indicated proteins by CQ73. (b) CETSA analysis of CQ04 (0.5, 5 μM) and CQ31 (0.5, 5 μM) in THP-1Cas9 lysates is representative of three or more independent experiments.

Extended Data Fig. 8 ∣. Dual PEPD and XPNPEP1 inhibition induces pyroptosis.

(a) XPNPEP1/PEPD knockout THP-1 cells were treated with the indicated concentrations of CQ31 for 48 h prior to CTG analysis. (b) The indicated THP-1 cells were treated with VbP (10 μM) or CQ31 (20 μM) for 48 h prior to evaluating supernatants for IL-1β levels by ELISA and evaluating lysates and supernatants for IL-1β cleavage by immunoblotting. (c) Evaluation of XPNPEP1 and PEPD levels in MV4;11 cells stably expressing Cas9 and treated with sgRNAs targeting XPNPEP1. (d) The indicated cells were treated with CQ31 for 24 h before LDH release was evaluated. (e) The indicated MV4;11 cells were treated with VbP (10 μM) or CQ31 (20 μM) for 48 h before LDH release and immunoblot analyses. PEPD/XPNPEP1 knockout MV4;11 cells were generated with sgPEPD_2 and sgXPNPEP1. Data are means ± SEM of biological replicates. *** p < 0.001, ** p < 0.01 by two-sided Students t-test. (f) The indicated MV4;11 cells were treated with CQ31 for 48 h before assessing cell viability by CTF. (g) Inhibition of AP-AMC cleavage in HEK 293 T lysates by methyl esters of IPI or VPI tripeptides. (h) The indicated MV4;11 cells were treated with methyl esters of IPI or VPI for 24 h before CTG analysis. Data in b-h are means ± SEM of 3 replicates. All data, including immunoblots, are representative of three or more independent experiments.

Extended Data Fig. 9 ∣. Schematic comparing CQ31-induced and VbP-induced activation inflammasome activation.

(a) Weak DPP8/9 inhibition selectively activates CARD8, whereas strong DPP8/9 inhibition activates both NLRP1 and CARD8. (b) Strong DPP8/9 inhibitors (for example, VbP) are required to compete with the neo-N-terminus of the NLRP1^CT fragment for the DPP8/9 active site and destabilize the ternary complex. (c) CARD8 does not directly interact with the DPP8/9 active site, and thus strong inhibitors are not needed to disrupt that interaction. It has not yet been established precisely how DPP8/9 inhibitors impact the CARD8-DPP9 ternary complex in cells. Red dots in b and c represent the DPP9 active site serine.

Fig. 1 ∣. Esterified Xaa-Pro dipeptides selectively activate the CARD8 inflammasome.

a, Domain organizations of human NLRP1 and CARD8. Both proteins undergo autoproteolysis between the ZU5 and UPA subdomains of the FIIND. b, Inhibition of recombinant DPP9 activity by the indicated compounds. IC₅₀ values are shown in parentheses. c, The indicated MV4;11 cells were treated with compounds (1 mM) and monitored for PI uptake. F_t, Fluorescence measurement at time t; F₀, fluorescence measurement at time 0; F_max, maximum fluorescence measurement obtained in the experiment. d,e, MV4;11 (d) or THP-1 (e) cells were treated with XP-OMes (1 mM) or VbP (2 μM) for 14 h (d) or 24 h (e) before LDH release and immunoblot analyses. f, MV4;11 cells were treated with VP-OMe (2 mM), IP-OMe (1 mM), bortezomib (bort; 1 μM) and/or VX-765 (50 μM) as indicated for 6 h before LDH release and immunoblot analyses. ***P < 0.001, **P < 0.01 by two-sided Students t-test. NS, not significant. The indicated significant P values are as follows: VP-OMe versus DMSO = 0.0054, IP-OMe versus DMSO = 0.0020. g, N/TERT-1 immortalized keratinocytes were treated with XP-OMes (1 mM) or VbP (10 μM) for 16 h before LDH release and immunoblot analyses. h, HEK 293T cells were transfected with plasmids expressing FLAG-tagged NLRP1 and GFP-tagged ASC and treated with XP-OMes (1 mM) or VbP (10 μM) for 24 h. ASC speck formation was assessed by fluorescence microscopy. Representative images and average cells with specks (%) ± s.e.m. are shown. Scale bar, 100 μm. h, RAW264;7 cells were treated with XP-OMes (1 mM) or VbP (2 μM) for 24 h before LDH release and immunoblot analyses. Data in b (n = 4), c (n = 4) and d-i (n = 3) are presented as means ± s.e.m. of replicates. All data, including immunoblots, are representative of three or more independent experiments.

Fig. 2 ∣. CQ04 and CQ31 inhibit PEPD.

a, Schematic of DPP8/9 and PEPD activity. b, Structures of CQ04 and CQ31. c, Inhibition of recombinant PEPD activity. IC₅₀ values are shown in parentheses. d, Evaluation of PEPD activity (AP cleavage) in control and PEPD^−/− THP-1 cell lysates treated with the indicated compounds. The release of free alanine in c and d was determined using an alanine assay kit (Sigma). Data in c and d are presented as means ± s.e.m. of three replicates. e, CETSA analyses of the indicated compounds (10 μM) in HEK 293T lysates. All data, including immunoblots and CETSA, are representative of three or more independent experiments.

Fig. 3 ∣. CQ31 selectively activates the CARD8 inflammasome.

a, Viability of cell lines after treatment with CQ31 (24 h), assessed by CTG. b, Immunoblots of lysates from the indicated cell lines treated with CQ31 (10 μM) or VbP (10 μM) for 24 h. c,d, The indicated MV4;11 (c) and THP-1 (d) cells were treated with CQ31 (10 μM) or VbP (10 μM) for 24 h before LDH release and immunoblot analyses. e, MV4;11 cells were treated with CQ31 (10 μM) for 2 h before treating with bortezomib (1 μM) or VX-765 (50 μM) for 6 h before LDH release and immunoblot analyses. f, N/TERT-1 keratinocytes were treated with CQ31 (10 μM) or VbP (10 μM) for 16 h before LDH release and immunoblot analyses. *P < 0.05 by two-sided Students t-test. NS, not significant. The indicated significant P value is as follows: CQ31 versus DMSO = 0.0223. g, HEK 293T cells were transfected with plasmids expressing FLAG-tagged NLRP1 and GFP-tagged ASC and treated with CQ31 (10 μM) or VbP (10 μM) for 24 h. ASC speck formation was assessed by fluorescence microscopy. Representative images and average cells with specks (%) ± s.e.m. are shown. Scale bar, 100 μm. h,i, BMDMs from C57BL/6J mice (h) or RAW264.7 cells (i) were treated with CQ31 (10 μM) or VbP (10 μM) for 24 h or 6 h, respectively, before LDH release and/or immunoblot analyses. Data in a (n = 4), c–g (n = 3) and i (n = 3) are presented as means ± s.e.m. of replicates. All data, including immunoblots, are representative of three or more independent experiments.

Fig. 4 ∣. CQ31 indirectly inhibits DPP8/9 activity.

a,b, The indicated THP-1 cells were treated with VP-OMe (1 mM), IP-OMe (1 mM) (a) or CQ31 (10 μM) (b) and VbP (2 μM) for 24 h before LDH release and immunoblot analyses. c, HEK 293T cells were treated with vehicle control (DMSO), CQ31 (10 μM) or VbP (10 μM) for 24 h. Intracellular metabolites were extracted and dipeptide concentrations were measured by LC-MS. d, HEK 293T cells were treated with the indicated compounds (10 μM, 6 h) before treatment with the DPP4 inhibitor sitagliptin (1 μM, to block any DPP4 activity in the media) for 1 h and assaying for AP-AMC (2.5 μM) cleavage. Data in a–d are presented means ± s.e.m. of three replicates. ***P < 0.001, **P < 0.01, *P < 0.05 by two-sided Students t-test. NS, not significant. Indicated significant P values versus DMSO are as follows: VbP = 0.0087, 8j = 0.0121, CQ31 = 0.0449. e,f, FLAG-tagged NLRP1 and CARD8 from HEK 293T cells (input) were immobilized on anti-FLAG beads and treated with VbP (10 μM) or VP dipeptide (0.5, 5 mM) (e) or CQ31 or CQ04 (10 μM) (f). DPP9 displaced from the complex was evaluated by immunoblotting. Cmpd, compound. All LDH, enzymatic activity, immunoprecipitation and immunoblot data are representative of three or more independent experiments.

Fig. 5 ∣. CQ31 engages a target in addition to PEPD.

a, The indicated MV4;11 cells were treated with VbP (10 μM) or the indicated XP-OMe (1 mM) for 6 h before LDH release and immunoblot analyses. b, WT and PEPD knockout MV4;11 cells were treated with XP-OMes (1 mM, 14 h) before CTG and CTF analyses. c–e, The indicated THP-1 and MV4;11 cells were treated with CQ31 (10 μM), VbP (10 μM) or XP-OMes (1 mM) for 14 h (c) or 24 h (d,e) before LDH release and immunoblot analyses. Data are presented as means ± s.e.m. of three replicates. All data, including immunoblots, are representative of three or more independent experiments.

Fig. 6 ∣. Dual inhibition of XPNPEP1 and PEPD activates CARD8.

a, Structure of CQ73. b, CQ73 inhibits recombinant PEPD activity. The IC₅₀ value is shown in parentheses. c, The indicated MV4;11 cells were treated with CQ73 for 24 h before CTG analysis. d, Schematic of the target identification protocol using the CQ73 probe. e, Scatter plots depicted the proteins enriched by CQ73 and competed by CQ31 as determined by TMT-MS. f, Immunoblots of lysates from HEK 293T cells before (input) and after streptavidin enrichment (IP:biotin) following the scheme in d. g, Schematic of the known enzymatic function of PEPD and XPNPEPs. h, Cladogram depicting all M24 aminopeptidases and their messenger RNA (mRNA) abundances in the indicated cell lines. i, The indicated THP-1 cells were treated with control (DMSO), CQ31(10 μM) or VbP (10 μM) for 48 h before LDH release and immunoblot analyses. Data in b, c and i are presented as means ± s.e.m. of three replicates. Data in b, c and i, including immunoblots, are representative of three or more independent experiments.