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. 2025 Jul 2;5(4):565–581. doi: 10.1021/acsbiomedchemau.4c00146

A Potent Inhibitor of Caspase‑8 Based on the IL-18 Tetrapeptide Sequence Reveals Shared Specificities between Inflammatory and Apoptotic Initiator Caspases

Christopher M Bourne †, Nicole R Raniszewski †, Ashutosh B Mahale †, Madhura Kulkarni †, Patrick M Exconde †, Sherry Liu †, Winslow Yost ‡, Tristan J Wrong †, Robert C Patio †, Matilda Kardhashi †, Teni Shosanya †, Mirai Kambayashi †, Bohdana M Discher †, Igor E Brodsky ‡, George M Burslem †,§,*, Cornelius Y Taabazuing †,*
PMCID: PMC12371506  PMID: 40860029

Abstract

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes, such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine the factors that dictate specificity. We recently discovered that human inflammatory caspases (caspases-1, -4, and -5) cleave the cytokines IL-1β and IL-18 in a sequence-dependent manner. Here, we report the development of a new peptide-based probe and inhibitor derived from the tetrapeptide sequence of IL-18 (LESD). The LESD-based inhibitor showed a strong preference for caspase-8 with an IC50 of 50 nM, and was more potent in vitro than the commonly used zIETD-FMK inhibitor, which is considered the most selective and potent caspase-8 inhibitor. We further demonstrated that the LESD-based inhibitor prevents caspase-8 activation during Yersinia pseudotuberculosis infection in primary bone marrow-derived macrophages. In addition, we systematically characterized the selectivity and potency of known substrates and inhibitors of the apoptotic and inflammatory caspases using standardized activity units of each caspase. Our findings reveal that VX-765, a known inhibitor of caspases-1 and -4, also inhibits caspase-8 (IC50 = 1 μM). Even when specificities are shared, the caspases exhibit different efficiencies and potencies for shared substrates and inhibitors. Altogether, we report the development of new tools that will facilitate the study of caspases and their roles in biology.

Keywords: caspase substrates, caspase inhibitors, inflammasomes, pyroptosis, apoptosis, in vitro kinetics, Yersinia pseudotuberculosis

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Introduction

Caspases are a family of related cysteine proteases that play key roles in regulating programmed cell death pathways, such as apoptosis and pyroptosis. − Caspases are activated by diverse stimuli but are structurally related, and their activation triggers the proteolysis of what is estimated to be thousands of substrates to execute cell death. − Caspases that induce apoptosis are generally referred to as apoptotic caspases, and those that induce pyroptosis are known as inflammatory caspases.

The inflammatory caspases, caspases-1, -4, and -5 in humans and caspases-1 and -11 in mice, are activated in response to diverse pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). A subset of germline-encoded pattern-recognition receptors (PRRs) detects PAMPs and DAMPs and assembles into multiprotein complexes known as inflammasomes. , Assembly of the canonical inflammasome leads to caspase-1 dimerization, triggering its autoproteolytic activation. − Activated caspase-1 then cleaves and activates the interleukin family of cytokines, IL-1β and IL-18, and the pore-forming protein gasdermin D (GSDMD) to induce pyroptotic cell death. − In contrast to the canonical inflammasome pathway that utilizes pattern-recognition receptors, caspases-4/5 in humans and caspase-11 in mice bind directly to lipopolysaccharide (LPS) from Gram-negative bacteria, triggering their oligomerization and autoproteolytic activation. We recently discovered that caspase-11 activation and autoprocessing precede and are required to form the caspase-11 noncanonical inflammasome, but whether this extends to other inflammasomes remains unknown. Active caspases-4 and -5 cleave and activate IL-18 but cleave and inactivate IL-1β. − It has also been reported that active caspase-4 can cleave and activate IL-1β, but this likely occurs with slower kinetics compared to the processing event that gives rise to the inactive species of IL-1β. , Mouse caspase-11 cannot cleave IL-18 but similarly cleaves and inactivates IL-1β. In this manner, the inflammatory caspases act as initiators and executioners that induce their own activation, permitting the cleavage of other downstream substrates.

Unlike the inflammatory caspases that act as both initiators and executioners, the apoptotic caspases have specialized roles and are classified into initiator (caspases-2, -8, -9, and -10) and executioner (caspases-3, -6, and -7) caspases. ,, Like inflammatory caspases, apoptotic initiator caspases are activated by multiprotein signaling complexes, which induce their autoproteolytic activation. − Once activated, initiator caspases are thought to have a limited substrate repertoire, which includes the executioner caspases. ,,, Initiator caspases activate executioner caspases, which have a broader substrate profile and subsequently cleave thousands of substrates to induce apoptosis. − ,,,

All caspases preferentially cleave peptide bonds after an aspartic acid residue in the P1 position. − Caspases are structurally related with conserved active sites and overlapping substrate specificities. ,,,− As a result, it has been challenging to develop selective reagents to probe the biology of specific caspases. Extensive efforts have been made to develop peptide-based reporter substrates and inhibitors, which have had limited success in producing truly selective reagents, at least using natural amino acids. − Some success has been achieved using unnatural amino acids to develop selective inhibitors of caspases-2, -8, -9, and -10, offering a promising avenue for developing selective caspase reagents. − Another strategy undertaken to develop selective caspase reagents has been to develop small molecule inhibitors, which generally relies on identifying and optimizing hits from screening a fragment-based library of covalent cysteine-reactive molecules. , This approach has led to the development of a selective caspase-6 inhibitor, but specific small molecule inhibitors of other caspases remain a challenge.

Although the caspases are all structurally similar, and peptide-based inhibitors and substrates display limited specificity in vitro, they have distinct protein substrates in cells, likely conferred by protein-binding sites that are distinct from the active site, known as exosites. ,,− However, the molecular basis of substrate recognition by different caspases is not completely understood. We recently discovered that inflammatory caspases (caspases-1, -4, -5, and -11) recognize and cleave IL-1β and IL-18 in a sequence-dependent manner. Notably, we demonstrated that substituting the amino acid recognition motif found in IL-1β (YVHD) with that found in IL-18 (LESD) facilitates IL-1β binding and activation by caspases-4 and -5, suggesting that they preferentially recognize the LESD motif. We also demonstrated that IL-18 has a stronger interaction with caspases-4 and -5 compared to caspase-1, suggesting that peptide-based inhibitors using the LESD scaffold may be specific for caspases-4 and -5 and would be an invaluable tool for studying noncanonical inflammasome biology. Furthermore, apoptotic caspases have been reported to cleave and inactivate IL-1β and IL-18, but whether they also recognize the tetrapeptide sequence adjacent to the cleavage site remains unclear.

Here, we designed and synthesized a new fluorogenic substrate, N-acetyl-Leu-Glu-Ser-Asp-aminomethylcoumarin (Ac-LESD-AMC), and an inhibitor, N-acetyl-Leu-Glu-Ser-Asp-chloromethylketone (Ac-LESD-CMK). This novel fluorogenic substrate reported the activity of inflammatory caspases and the initiator apoptotic caspases-8 and -10, while the inhibitor potently blocked their activity. Surprisingly, the Ac-LESD-CMK inhibitor was most potent against caspase-8 and its homologue, caspase-10. Using in vitro kinetics, we characterized apoptotic and inflammatory caspases at the same activity units with different peptide substrates and inhibitors, revealing key insights into the selectivity profiles of caspases. We demonstrate that our new LESD inhibitor inhibits the proteolysis of inflammatory and apoptotic caspase substrates in cells. Notably, the LESD inhibitor blocked the biologically important substrate processing mediated by caspase-8 and caspase-1, activated by Yersinia pseudotuberculosis infection. Altogether, we have developed new chemical probes that will facilitate the study of both apoptotic and inflammatory caspase biology.

Results

Recombinant Caspases Cleave IL-18 and IL-1β in a Tetrapeptide Sequence-Dependent Manner

We previously reported that inflammatory caspases cleave IL-1β and IL-18 in a sequence-dependent manner, demonstrating that the LESD tetrapeptide sequence in IL-18 is recognized and processed by caspases-1, -4, and -5. Because apoptotic caspases have been reported to cleave and inactivate IL-1β and IL-18, ,, we first wanted to determine if the tetrapeptide motif similarly regulates the processing of IL-18 and IL-1β by apoptotic caspases. To test this, we expressed wild-type IL-18 (IL-18 WT) or IL-18 where the LESD tetrapeptide at position 33-36 was mutated to AAAD (IL-18 33AAAD36), wild-type IL-1β (IL-1β WT), or IL-1β in which the tetrapeptide sequence at position 113-116 was mutated to LESD (IL-1β 113LESD116) in HEK 293T cells. Lysates from these cells were incubated with 0.25 activity units/μL of recombinant caspases purchased from a commercial vendor (Enzo Life Sciences) for either 1 or 24 h. As previously reported, caspases-1, -4, -5, and -11 processed human IL-18 WT into the active p18 fragment, while caspases-3 and -6 generated the inactivating p15 fragment (Figure A). , IL-18 is released by macrophages in response to infection with Yersinia pseudotuberculosis, which induces caspase-8 activation and caspase-8-dependent caspase-1 activation. ,, Given their ability to cleave shared substrates, which of these caspases is responsible for the cleavage and release of IL-18 during Yersinia infection has remained unclear but was presumed to be mediated by caspase-1. Consistent with this, we did not observe caspase-8 processing of IL-18 WT in vitro (Figure A). As anticipated, IL-18 cleavage by inflammatory caspases was tetrapeptide sequence-dependent, as processing of IL-18 33AAAD36 into the active p18 fragment was significantly inhibited (Figure A). Caspases-1, -3, -4, -5, and -6 processed IL-1β WT into the inactive p27 fragment, and only caspase-1 was able to generate the active p17 species (Figure B). Even though IL-18 harboring the LESD sequence was only processed by inflammatory caspases, caspases-1, -4, -5, -6, -8, and -10 all processed IL-1β with the tetrapeptide sequence at position 113-116 mutated to LESD (IL-1β 113LESD116) into the active p17 species after 24 h (Figure B), suggesting that some apoptotic caspases also recognize the tetrapeptide sequence but fail to cleave the native YVHD sequence of IL-1β. To confirm that processing was direct, we purified the cytokines from HEK 293T lysates and incubated them with the recombinant caspases (Figure S1). Consistent with the results from lysates, we observed the same processing events using purified cytokines, indicating direct processing by the caspases (Figure S1). These findings indicate that the tetrapeptide sequence of IL-1β regulates its processing by both inflammatory and apoptotic caspases. Curiously, although apoptotic caspases could process the LESD sequence in IL-1β, they failed to cleave this sequence in IL-18, but the reason for this currently remains unclear. This may be due to the presence of an exosite on IL-1β that is recognized by apoptotic caspases, but future studies are needed to delineate the mechanistic basis of these specificities.

1.

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Recombinant caspases cleave IL-18 and IL-1β in a tetrapeptide sequence-dependent manner. (A) HA-tagged IL-18 WT and IL-18 with the tetrapeptide sequence at position 33-36 mutated to AAAD (IL-18 33AAAD36) were expressed in HEK 293T cells, and lysates were mixed with 0.25 activity units/μL of indicated caspases for 1 or 24 h before immunoblot analysis. (B) IL-1β WT and IL-1β with the tetrapeptide sequence at position 113-116 mutated to LESD (IL-1β 113LESD116) were expressed in HEK 293T cells and treated as in (A). Data are representative of three independent experiments.

An LESD-Based Fluorogenic Substrate Is Selective for Initiator Caspases Except Caspase-2

The observation that replacement of the endogenous YVHD cleavage sequence in IL-1β with LESD expanded the range of caspases able to process this substrate to caspases-4, -5, -6, -8, and -10, in addition to caspase-1, suggests that a fluorogenic substrate or inhibitor based on the LESD sequence may report on the activity of these caspases. We synthesized a new fluorogenic substrate (Ac-LESD-AMC) for testing (Figure A). We incubated equivalent activity units (0.25 activity units/μL) of recombinant enzymes with the Ac-LESD-AMC substrate (Figure C–K) and used the linear rate of hydrolysis at varying substrate concentrations to generate Michaelis–Menten kinetic parameters. An example of the linear rates for caspase-1 is illustrated in Figure B. The extrinsic initiator caspases-8 and -10 (Figure C,D) and inflammatory caspases-1, -4, -5, and -11 (Figure E–H) efficiently cleaved the Ac-LESD-AMC substrate. We did not detect processing of Ac-LESD-AMC by executioner caspases-3, -6, and -7 (Figure I–K), suggesting that Ac-LESD-AMC is specific to initiator and inflammatory caspases. Using the kinetic parameters, we determined the relative catalytic efficiency (k cat/K M) for all caspases with Ac-LESD-AMC (Figure L). Despite no IL-18 processing in vitro by caspase-8, Ac-LESD-AMC was most efficiently hydrolyzed by caspase-8, which was ∼10-fold faster than the human inflammatory caspases-1, -4, and -5. Caspase-10, a homologue of caspase-8, also efficiently cleaved the Ac-LESD-AMC probe. Of the caspases that cleaved the Ac-LESD-AMC probe, mouse caspase-11 was the most inefficient at cleaving Ac-LESD-AMC (∼100-fold slower than caspase-8), in agreement with the notion that caspase-11 does not cleave mouse IL-18, which harbors an LESD motif at the cleavage site. Although caspase-11 does not process mouse IL-18, it is able to process human IL-18 but less efficiently than caspase-4, and recent structural studies imply that this may be regulated by active site sterics. − We also tested the ability of caspases to process other well-established substrates, ,,,,, including Ac-DEVD-AMC, Ac-YVAD-AMC, Ac-LEHD-AMC, and Ac-WEHD-AMC, for the extrinsic initiator caspases-8 and -10 (Figures S2 and S3), the inflammatory caspases-1, -4, -5, and mouse-11 (Figures S4–S7), and the executioner caspases-3, -6, and -7 (Figures S8–S10). The kinetic values are summarized in Table .

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An LESD-based probe is selective for initiator caspases. (A) Chemical structure of the Ac-LESD-AMC fluorogenic tetrapeptide probe. (B) Representative linear rates of hydrolysis by 0.25 activity units/μL of caspase-1 with varying concentrations of the Ac-LESD-AMC substrate. Relative fluorescence units (RFU). (C–K) Michaelis–Menten kinetic profiles of Ac-LESD-AMC substrate cleavage by 0.25 activity units/μL of recombinant apoptotic initiator caspases-8 and -10 (C,D), inflammatory caspases-1, -4, -5, and -11 (E–H), and apoptotic executioner caspases-3, -6, and -7 (I–K). Data are means ± SEM of three independent experiments with two technical replicates per experiment. (L) Relative catalytic efficiency, represented as k cat/K M, of caspases for Ac-LESD-AMC calculated from in vitro Michaelis–Menten curves. The graph depicts values calculated from three independent experiments with the Ac-LESD-AMC probe. Data were fitted using the Michaelis–Menten equation in GraphPad Prism.

1. Summary Data for Probe Kinetics of Each Caspase with Ac-LESD-AMC, Ac-YVAD-AMC, Ac-DEVD-AMC, Ac-LEHD-AMC, and Ac-WEHD-AMC Probes .

Caspase LESD-AMC YVAD-AMC DEVD-AMC LEHD-AMC WEHD-AMC
Relative k cat/K M
1 5.01 ± 0.41 23.9 ± 2.79 1.41 ± 0.43 45.36 ± 8.42 334 ± 48.0
3 ND ND 482 ± 76.5 4.61 ± 0.18 1.33 ± 0.04
4 1.44 ± 0.18 0.58 ± 0.08 1.27 ± 0.18 10.8 ± 1.02 10.6 ± 0.33
5 4.65 ± 0.43 7.88 ± 0.73 ND 17.6 ± 2.00 21.6 ± 2.38
6 ND ND 13.3 ± 2.24 8.93 ± 0.70 2.28 ± 0.31
7 ND ND 561 ± 81.2 1.07 ± 0.17 0.31 ± 0.03
8 59.2 ± 3.26 1.84 ± 0.84 310 ± 60.4 150 ± 25.9 14.3 ± 2.92
9 0.15 ± 0.02 N/A ND 0.86 ± 0.10 0.35 ± 0.08
10 10.4 ± 1.30 ND 7.20 ± 1.02 12.3 ± 3.06 0.91 ± 0.23
11 0.42 ± 0.02 ND ND 5.19 ± 0.77 6.97 ± 0.68
V max (RFU/min)
1 465.55 ± 10.25 258.15 ± 6.21 44.68 ± 15.51 2702.91 ± 85.48 3496.13 ± 121.4
3 ND ND 940.88 ± 31.57 288.98 ± 33.2 93.42 ± 8.73
4 2546.06 ± 156.63 1668.58 ± 190.22 862.73 ± 31.55 2821.37 ± 159.35 6565.47 ± 587.59
5 703.19 ± 24.07 555.56 ± 19.58 ND 1207.5 ± 40.91 1372.07 ± 49.43
6 ND ND 455.67 ± 64.18 791.57 ± 16.74 208.71 ± 17.47
7 ND ND 1266.91 ± 38.79 139.05 ± 12.48 32.33 ± 4.07
8 590.08 ± 30.37 19.15 ± 4.44 218.27 ± 2.74 1101.52 ± 37.65 279.84 ± 9.36
9 208.50 ± 45.00 N/A ND 139.43 ± 8.11 38.80 ± 1.35
10 898.63 ± 57.69 ND 476.26 ± 38.71 1043.38 ± 77.53 130.96 ± 19.73
11 211.01 ± 15.01 ND ND 1192.55 ± 30.79 1428.54 ± 73.6
K M (μM)
1 93.9 ± 6.26 11.2 ± 1.64 47.5 ± 30.8 63.7 ± 11.2 10.9 ± 1.39
3 ND ND 2.07 ± 0.36 62.4 ± 5.40 69.8 ± 4.44
4 1867 ± 388 3095 ± 715 702 ± 76.3 264 ± 21.6 626 ± 70.6
5 154 ± 13.5 72.4 ± 9.44 ND 70.1 ± 6.28 64.8 ± 5.47
6 ND ND 35.3 ± 5.12 90.1 ± 8.93 94.7 ± 12.7
7 ND ND 2.36 ± 0.36 138 ± 30.0 102 ± 4.21
8 9.98 ± 0.37 ND 0.75 ± 0.12 7.80 ± 1.32 22.0 ± 6.04
9 1513.83 ± 441.20 N/A ND 167.00 ± 23.86 127.32 ± 36.01
10 87.9 ± 5.38 ND 70.3 ± 14.6 96.4 ± 23.3 154 ± 20.2
11 503 ± 58.4 ND ND 242 ± 41.4 211 ± 33.8
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a

Graphs depicting the raw data are shown in Figures , , and S2–S11.

b

The caspase-9 preparation was different from the other caspases tested. Not assessed (N/A), not detected (ND).

To test the activity of caspase-9, we purchased full-length caspase-9, which autoproteolyzed into the p35 and p10 fragments, and incubated 1 activity unit/μL enzyme with Ac-LESD-AMC, Ac-DEVD-AMC, Ac-LEHD-AMC, and Ac-WEHD-AMC (Figure S11A–D). We observed that Ac-LEHD-AMC was most efficiently cleaved (relative k cat/K M= 0.86) followed by Ac-WEHD-AMC (relative k cat/K M= 0.35), and then Ac-LESD-AMC (relative k cat/K M = 0.15) (Figure S11E, Table ). Recombinant caspase-9 did not cleave the Ac-DEVD-AMC substrate. Caspase-2 is also an initiator caspase, but it has specificity for 5 amino-acid peptide substrates and is unlikely to cleave any of the tetrapeptide substrates tested.

Direct comparison of the catalytic efficiencies for a given probe between caspases is challenging, as each caspase was tested at an unknown concentration normalized for activity. However, a comparison of different substrates for the same caspase can be made. The Ac-LESD-AMC probe was among the most efficiently cleaved substrates for caspases-8 and -10 within 3-fold of Ac-LEHD-AMC, their preferred substrate (Figure A,B, Table ). Caspase-5 also efficiently cleaved Ac-LESD-AMC, within 5-fold of its preferred substrate Ac-WEHD-AMC (Figure E, Table ), while the other inflammatory caspases-1, -4, and mouse caspase-11 preferred Ac-WEHD-AMC, ∼10-fold or more than Ac-LESD-AMC (Figure C–F). All of the executioner caspases preferred the Ac-DEVD-AMC substrate and failed to cleave Ac-LESD-AMC (Figure G–I). Altogether, our data suggest that the initiator caspases-8 and -10, and the inflammatory caspase-5, have the highest catalytic efficiency for Ac-LESD-AMC. Importantly, caspases have distinct substrate specificities, and when substrate specificities overlap, the rates of hydrolysis often differ between caspases. Our data also suggest that other factors, such as the recently reported exosites, ,,− enzyme-substrate binding sites that are distinct from the active site, may play a major role in dictating protein substrate specificities, as the peptide-based substrate profiles do not always recapitulate the protein substrate specificities.

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Evaluation of caspase specificities for peptide-based substrates. (A–I) Catalytic efficiency, represented as k cat/K M of caspases with different tetrapeptide substrates probes. In vitro kinetics were fitted by using the Michaelis–Menten function in GraphPad Prism to generate the kinetic parameters used to calculate k cat/K M values for recombinant apoptotic initiator caspases-8 and -10 (A,-B), inflammatory caspases-1, -4, -5, and -11 (C–F), and apoptotic executioner caspases-3, -6, and -7 (G–I). Data are means ± SEM of three independent experiments. ND (not detected).

Selectivity Profile of Peptide-Based Caspase Inhibitors

Given that the Ac-LESD-AMC substrate was most efficiently processed by caspase-8 and -10, followed by inflammatory caspase-5, we reasoned that we could use this peptide to develop a potent inhibitor for caspase-8. We therefore synthesized a new substrate peptide with a C-terminal chloromethylketone warhead (Ac-LESD-CMK), a classical method for the inhibition of cysteine proteases, and tested the ability of this new inhibitor against commercially available caspase-8 inhibitors, zLEHD-FMK, and zIETD-FMK, as well as the caspase-1/4 inhibitor VX-765, in vitro (Figure ). , The chemical structures of the inhibitors tested are depicted in Figure A. Each inhibitor was tested under saturating conditions of the most efficiently processed substrate for each specific caspase. For example, caspase-1 inhibition was assessed using Ac-WEHD-AMC as a substrate, and caspase-3 inhibition was assessed using Ac-DEVD-AMC as a substrate. The IC50 values for each caspase and inhibitor are summarized in Table . Caspase-8 was most effectively inhibited by Ac-LESD-CMK (IC50 = 50 nM), followed by zLEHD-FMK (IC50 = 70 nM), and then zIETD-FMK (IC50 = 350 nM) (Figure B, Table ). VX-765 also inhibited caspase-8 (IC50 = 1 μM), despite being marketed as a caspase-1 inhibitor. Caspase-10 was also most effectively inhibited by Ac-LESD-CMK (IC50 = 520 nM), followed by zLEHD-FMK (IC50 = 3.59 μM) and zIETD-FMK (IC50 = 5.76 μM), but weakly inhibited by VX-765 (IC50 = 42 μM) (Figure C, Table ). VX-765 was the most efficient inhibitor for caspase-1 (IC50 = 530 nM) (Figure D, Table ). Ac-LESD-CMK was the most potent inhibitor of caspase-5 tested (IC50 = 2 μM) (Figure E, Table ). Broadly, Ac-LESD-CMK and VX-765 weakly inhibited the executioner caspases, while zLEHD-FMK and zIETD-FMK were the two most efficient inhibitors tested for the executioner caspases (Figure F–H, Table ). Taken together, these data demonstrate that Ac-LESD-CMK is a potent inhibitor of caspases-8, -10, and -5. Notably, the commercially available caspase-8 inhibitor zIETD-FMK is less potent for caspase-8 compared to Ac-LESD-CMK and does not seem to exhibit any preference, as it also blocked the activity of the executioner caspases (caspases-3, -6, and -7) in the low micromolar concentration range. VX-765 inhibited caspase-1, as expected, but also blocked caspase-8 activity. zLEHD-FMK was among the most potent inhibitors for all caspases, with seemingly no preference for any specific caspase.

4.

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Selectivity profile of peptide-based caspase inhibitors. Each caspase was incubated with a saturating concentration of its preferred peptide substrate in the presence of either the Ac-LESD-CMK, zLEHD-FMK, zIETD-FMK, or VX-765 inhibitors at indicated concentrations. Substrate cleavage rates were determined at each inhibitor concentration and normalized to the no inhibitor condition for each run. (A) Chemical structures of Ac-LESD-CMK, zLEHD-FMK, zIETD-FMK, and VX-765 inhibitors. (B) Caspase-8 inhibition was assessed using 200 μM Ac-LEHD-AMC substrate. (C) Caspase-10 inhibition was assessed using 200 μM Ac-LEHD-AMC substrate. (D) Caspase-1 inhibition was assessed using 200 μM Ac-WEHD-AMC substrate for activity. (E) Caspase-5 inhibition was assessed using 200 μM Ac-WEHD-AMC substrate. (F) Caspase-3 inhibition was assessed using 100 μM Ac-DEVD-AMC substrate. (G) Caspase-6 inhibition was assessed using 100 μM Ac-DEVD-AMC substrate. (H) Caspase-7 inhibition was assessed using 100 μM Ac-DEVD-AMC substrate. Data were fitted using the [inhibitor] vs normalized response function in GraphPad Prism. Data are means ± SEM of three independent experiments.

2. Summary of Caspase Inhibitor IC50 Values for Data Depicted in Figures , S12, and S13 .

Caspase LESD-CMK zLEHD-FMK zIETD-FMK FLTD-CMK VX-765 zVAD-FMK
IC50 (μM)
1 5.67 ± 1.87 0.82 ± 0.13 5.72 ± 0.47 3.38 ± 0.77 0.53 ± 0.09 0.024 ± 0.006
3 54.0 ± 18.0 1.07 ± 0.18 1.88 ± 0.26 56.0 ± 27.7 >100 0.030 ± 0.004
4 59.0 ± 5.77 N/A N/A >100 30.1 ± 2.67 0.32 ± 0.011
5 2.03 ± 0.45 2.31 ± 0.31 19.91 ± 1.19 15.6 ± 1.09 19.5 ± 1.37 0.13 ± 0.011
6 5.08 ± 1.14 0.11 ± 0.06 0.06 ± 0.04 43.4 ± 15.9 >100 0.08 ± 0.01
7 72.0 ± 12.9 3.90 ± 0.29 2.63 ± 0.54 53.5 ± 31.0 >100 0.006 ± 0.001
8 0.05 ± 0.01 0.07 ± 0.01 0.35 ± 0.10 5.22 ± 2.35 1.02 ± 0.19 0.002 ± 0.0003
9 7.45 ± 3.92 0.80 ± 0.32 1.47 ± 0.62 N/A 0.54 ± 0.26 N/A
10 0.52 ± 0.12 3.59 ± 1.27 5.76 ± 1.10 82.8 ± 40.6 42.3 ± 3.53 0.116 ± 0.021
11 70.4 ± 31.0 N/A N/A 25.6 ± 21.6 81.0 ± 2.77 0.004 ± 0.0004
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a

Note that these values were calculated using equivalent activity units of each enzyme, and while comparisons can be made for different inhibitors for a given caspase, they represent relative values when comparing between different caspases.

b

The caspase-9 preparation was different from the other caspases tested. Not assessed (N/A).

We also tested Ac-FLTD-CMK, which is a peptide inhibitor derived from the tetrapeptide sequence of GSDMD, and zVAD-FMK, a broad-spectrum caspase inhibitor based on the tetrapeptide sequence of IL-1β (Figure S12). In all cases, zVAD-FMK was the most potent inhibitor, inhibiting all caspases at low-to-mid nanomolar concentrations (Figure S12, Table ). Ac-LESD-CMK inhibited the extrinsic apoptotic initiator caspases over 100-fold more potently than Ac-FLTD-CMK (Figure S12B,C, Table ). Ac-FLTD-CMK and Ac-LESD-CMK both inhibited caspase-1 at similar IC50 values (3.36 μM vs 5.67 μM, respectively) (Figure S12D, Table ), while both inhibitors weakly inhibited caspase-4 (30 μM vs 59 μM, respectively), although caspase-4 was difficult to saturate (Figure S12E, Table ). In contrast to the other inflammatory caspases, Ac-LESD-CMK efficiently blocked caspase-5 activity (IC50 = 2 μM) better than Ac-FLTD-CMK (IC50 = 15 μM) (Figure S12F, Table ). Broadly, mouse caspase-11 and executioner caspases-3, -6, and -7 were all weakly inhibited by both Ac-FLTD-CMK and Ac-LESD-CMK (Figure S12G–J, Table ). We also tested commercially available caspase-8 inhibitors against caspase-9 and determined that Ac-LESD-CMK was the least potent inhibitor tested (IC50 = 7.5 μM) compared to zLEHD-FMK (IC50 = 0.8 μM), VX-765 (IC50 = 0.54 μM), and zIETD-FMK (IC50 = 1.47 μM) (Figure S13, Table ). In summary, our new Ac-LESD-CMK inhibitor represents the most potent inhibitor with a preference for inhibiting caspase-8 over other caspases in our assays and thus provides a useful tool compound to explore caspase-8 biology.

Peptide Based Caspase Inhibitors Prevent Processing of Inflammatory and Apoptotic Caspase Substrates in Cells

To better understand the cellular activity of Ac-LESD-CMK, we preincubated HCC1806 triple-negative breast cancer cells with 10 μM of either Ac-LESD-CMK, Ac-FLTD-CMK, or zIETD-FMK for 30 min, then stimulated extrinsic apoptosis, which activates caspase-8, using TNF-α. We assessed caspase-8 inhibition by measuring the activity of the downstream substrates caspases-3/7, which are proteolytically activated by caspase-8 (Figure S14). At early time points (2–6 h), we observed inhibition of caspases-3/7 activation by all inhibitors, but both Ac-LESD-CMK and Ac-FLTD-CMK appeared to be less cell-penetrant as they were not as potent as zIETD-FMK (Figure S14A). Immunoblot analysis demonstrated that zIETD-FMK was able to inhibit caspase-3 and PARP processing in cells at 24 h, whereas Ac-LESD-CMK and Ac-FLTD-CMK treated cells still maintained some caspase-3 and PARP processing (Figure S14B).

We next tested the ability of Ac-LESD-CMK to inhibit caspase-8 activation in THP1 cells by inducing extrinsic apoptosis using TRAIL. , We preincubated THP1 monocytes with 25 μM Ac-LESD-CMK, Ac-FLTD-CMK, zLEHD-FMK, zIETD-FMK, or zVAD-FMK and then induced caspase-8 activation with TRAIL. Similar to the results in HCC1806 cells, Ac-LESD-CMK and Ac-FLTD-CMK failed to prevent caspase-8-mediated activation of caspases-3/7 in cells, as evidenced by the lack of decreased signal using the caspases-3/7 dye and PARP and caspase-3 processing (Figure A). In contrast, zLEHD-FMK, zIETD-FMK, or zVAD-FMK appeared to be cell-penetrant and were able to inhibit caspase-3 and PARP processing in cells. zLEHD-FMK and zVAD-FMK were more potent than zIETD-FMK at inhibiting caspases-3/7 activity in cells based on the caspases-3/7 dye.

5.

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Profiling inhibitors in human cells. (A) THP1 monocytes were preincubated with 25 μM of the indicated inhibitors for 30 min, then treated with 100 ng/mL of TRAIL to induce extrinsic apoptosis. The kinetics of caspases-3/7 activation was monitored using a cell-permeable dye and after 24 h, the cells were used for immunoblotting. The p values are shown for t = 24 h and were calculated by two-way ANOVA with Tukey’s multiple comparison test comparing DMSO treatment with inhibitor treated cells. (B–D) THP1 monocytes were differentiated into macrophages using 50 ng/mL of phorbol 12-myristate 13-acetate (PMA) for 2 days. Cells were preincubated with the Ac-LESD-CMK, Ac-FLTD-CMK, or zVAD-FMK inhibitors at 0, 12.5, 25, 50, or 100 μM, or matched DMSO concentrations for 30 min before the addition of 1 μM staurosporine for 6 h to induce intrinsic apoptosis (B), 5 μM nigericin for 3 h to induce canonical inflammasome activation (C) or transfection with 25 μg/mL LPS for 6 h to induce noncanonical inflammasome activation (D). Cell lysis was assessed by Sytox Green uptake. Protein was precipitated from supernatants and analyzed by immunoblotting. Data are means ± SEM of three independent experiments. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 by two-way ANOVA test.

Because the Ac-LESD-AMC lacked good cell penetrance, we preincubated differentiated THP1 macrophages with varying concentrations of Ac-LESD-CMK, Ac-FLTD-CMK, or zVAD-FMK inhibitors for 30 min and activated intrinsic apoptosis using staurosporine (Figure B), the caspase-1 inflammasome using nigericin (Figure C), and the caspases-4/5 inflammasomes using LPS (Figure D). , In agreement with the inhibition potency and pan-caspase inhibition profile observed in vitro, zVAD-FMK was the most potent dose-dependent inhibitor of apoptotic and pyroptotic cell death. zVAD-FMK dose-dependently inhibited cell death, caspase-3 processing into the active p19 and p17 species, and PARP cleavage when apoptosis was induced with staurosporine (Figure B). At all concentrations tested, zVAD-FMK inhibited cell death and GSDMD, IL-1β, caspase-1, and caspase-4 activation upon nigericin and LPS treatment, respectively (Figure C,D). Consistent with our in vitro data, Ac-LESD-CMK did not significantly inhibit apoptosis activated by staurosporine, which activates caspase-9 to induce intrinsic apoptosis. Caspase-3 and PARP processing were poorly attenuated with Ac-FLTD-CMK and Ac-LESD-CMK inhibitors, even at high doses (Figure B).

At high doses, Ac-FLTD-CMK and Ac-LESD-CMK inhibited cell death with LPS treatment (Figure D) but had a modest impact on cell death with nigericin treatment (Figure C). However, both Ac-FLTD-CMK and Ac-LESD-CMK prevented GSDMD, IL-1β, and caspase-1 activation. Ac-FLTD-CMK appeared to be more potent at inhibiting GSDMD, IL-1β, and caspase-1 processing in cells compared to Ac-LESD-CMK, as GSDMD and IL-1β processing were completely abrogated at all concentrations of Ac-FLTD-CMK tested, whereas only the highest concentration of Ac-LESD-CMK inhibited GSDMD and caspase-1 processing (Figure C,D). Altogether, our data suggest that Ac-LESD-CMK inhibits activation of caspase-8-dependent cell death and modestly inhibits cell death triggered by inflammasome activation, demonstrating its utility for studying caspase-8 biology in cells.

We next wanted to determine the activity of the inhibitors in a cellular context during pathogenic infection. We thus employed a Yersinia pseudotuberculosis (Yptb) model, wherein infection leads to caspase-8-mediated processing of caspase-1, which then cleaves and activates GSDMD. , We treated Mlkl knockout (KO) mouse bone marrow-derived macrophages (mBMDMs) with 100 μM Ac-LESD-CMK, Ac-FLTD-CMK, or zVAD-FMK for 30 min, then infected them with Yptb and tracked the kinetics of cell death by measuring the uptake of Sytox Green dye over time (Figure A). As anticipated, zVAD-FMK abrogated all cell death, and Ac-LESD-CMK more significantly attenuated cell death compared to Ac-FLTD-CMK (Figure A), suggesting that Ac-LESD-CMK inhibited caspase-8 activation in cells. Consistent with this, immunoblot analysis demonstrated that Ac-FLTD-CMK failed to prevent caspase-8-mediated activation of caspase-1 and subsequent GSDMD cleavage, but both zVAD-FMK and Ac-LESD-CMK abrogated caspase-1 processing into the mature p20 species and GSDMD processing (Figure B). Since Ac-LESD-CMK is a potent inhibitor of caspase-8 activity and can also inhibit caspase-1, this likely explains why it is a better inhibitor than Ac-FLTD-CMK during Yptb infection.

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Ac-LESD-CMK inhibits caspase-8 activation in cells. (A) Mouse Mlkl KO BMDMs were pretreated with 100 μM of the indicated inhibitors for 30 min before Yptb (MOI = 20) infection. The kinetics of cell death were tracked by measuring Sytox Green uptake using an Incucyte. Representative images are depicted below, and the quantification is depicted above. (B) Immunoblot of Yptb-infected Mlkl KO BMDMs 2.5 h postinfection. Data are means ± SEM of three independent experiments. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 by two-way ANOVA with Dunnett’s multiple comparison test comparing Yptb treatment with no inhibitor to Yptb treatment with inhibitors.

Conclusion

The caspases are all structurally similar, making it challenging to develop specific active site inhibitors. Many inhibitors have been reported, but at high doses, most have off-target activity for other caspases. Thus, understanding the selectivity and concentrations needed to target certain caspases over others will aid in uncovering their roles in biology. In the present study, we have systematically characterized the relative catalytic efficiency and determined the half-maximal inhibitory concentrations of peptide-based substrates and inhibitors of inflammatory and apoptotic caspases. We tested each caspase at the same activity units (as opposed to enzyme concentration) and compared their activities for a given substrate or inhibitor. A limitation of this approach is that activity units are determined using the optimal substrate specific to that caspase, sometimes performed at different temperatures. Without the enzyme concentrations, exact kinetic parameters cannot be measured, and while a comparison can be made for different substrates or inhibitors for a given caspase, bias may be introduced when comparing between caspases. Therefore, we interpret our catalytic parameters to be relative.

As anticipated from prior work using positional scanning peptide libraries to determine the specificity of caspases, ,,, Ac-YVAD-AMC, a peptide substrate based on the sequence of IL-1β, was the only substrate that was specifically cleaved by the human inflammatory caspases (caspases-1, -4, -5), but caspases-4 and -5 are unable to cleave this sequence on the native IL-1β substrate, suggesting that additional factors confer specificity for protein substrates. Indeed, it is now known that exosites, alternative binding sites distinct from the active sites, play a critical role in substrate recognition by inflammatory and apoptotic caspases. ,,− Also in agreement with prior work, the apoptotic caspases exhibited significantly higher catalytic efficiency for the Ac-DEVD-AMC substrate compared to sequences based on pyroptotic substrates. However, there were differences in the catalytic efficiency among the apoptotic caspases for Ac-DEVD-AMC, implying that they may have differences in their ability to process protein substrates.

The observation that substrates and inhibitors derived from inflammatory substrate sequences conferred some selectivity for the inflammatory caspases, and our observation that the LESD sequence in IL-18 played a critical role in IL-18 recognition by the noncanonical inflammasomes (caspases-4/5), prompted us to develop a specific substrate and inhibitor pair of the inflammatory caspases. Although our new substrate and inhibitor targeted the inflammatory caspases, they were most potent for caspase-8 and caspase-10 compared with other substrates and inhibitors tested for those caspases. Notably, this inhibitor did not target caspase-9 and will be a useful tool to distinguish the contributions of extrinsic and intrinsic apoptosis pathways. Caspase-8 is essential for development and plays a role in regulating apoptosis and necroptosis, and has recently been implicated in pyroptosis as well. , The existence of a potent and preferential inhibitor of caspase-8 will facilitate studies of the function of caspase-8 activity in various biological pathways in which caspase-8 is the initiating or dominant protease activated. For example, Yersinia infection of macrophages triggers robust activation of caspase-8, which leads to caspase-1 activation due to pathogen blockade of NF-κB signaling. ,, We found that the LESD inhibitor robustly prevented caspase-8 activation and the activation of downstream responses. Interestingly, caspase-8 is thought to negatively regulate a constitutively active inflammatory state in neutrophils, and its inhibition drives the production of cytokines and chemokines that promote neutrophil mobilization and antibacterial defense in vivo, suggesting that caspase-8 inhibition may have therapeutic benefits in certain contexts.

Except for Ac-YVAD-AMC, all other substrates and inhibitors targeted both apoptotic and pyroptotic caspases and, in particular, caspase-8. Even VX-765, which is developed to be a specific caspase-1 inhibitor, inhibited caspase-8, and caspase-8 had high catalytic efficiency against inflammatory peptide substrates such as Ac-WEHD-AMC and Ac-LEHD-AMC. It is possible that, because of the pleiotropic effects it has on biology, caspase-8 serves as a backup pathway for the inflammatory caspases and may have overlapping substrate specificities. Consistent with this idea, caspase-8 was demonstrated to form an inflammasome with ASC and NLRC4 when caspase-1 is defective. We did not detect in vitro cleavage of IL-1β and IL-18 by caspase-8, but the LESD-based tetrapeptide substrate and inhibitor do target caspase-8. Other factors, such as exosites, subcellular localization, and the presence of pathogenic factors, may also influence the substrate repertoire and activity of caspases in cells.

It was somewhat surprising that the LESD-based inhibitor appeared to display a preference for caspase-8. Several factors can account for this, such as conformational changes upon substrate binding, steric effects, and hydrophobic and electrophilic interactions at the active site between the peptide substrate and residues in the caspase-8 active site. For example, mouse caspase-11 lacks key electrophilic interactions with the LESD sequence and harbors an elongated loop, compared to the human caspase-4 ortholog, that sterically occludes IL-18 and prevents its processing. Prior work on caspase-9 suggests that, in addition to the sequence, the local context and three-dimensional positioning and interactions influence substrate processing. Some sequences can be recognized but are not processed unless the correct local environment is present. This might explain why other caspases, such as caspases-4/5 and -6, are unable to process the YVHD sequence in IL-1β but are able to process IL-1β when the YVHD sequence is substituted with LESD. Structural studies are needed to better understand the basis of the selectivity of LESD for caspase-8.

Currently, zIETD-FMK is thought to be a selective caspase-8 inhibitor. However, we observed that Ac-LEHD-FMK was more potent than zIETD-FMK at inhibiting caspase-8 activity, in line with a prior report indicating that caspase-8 processed an LEHD-based probe 4.5-fold faster than the corresponding IETD probe. However, the assumption that substrate selectivity should match inhibitor selectivity is inaccurate, as our data indicate that caspase-6 is inhibited by an LESD-based inhibitor despite being unable to cleave the Ac-LESD-AMC substrate. Ac-LEHD-FMK was not selective and exhibited potent inhibition of other caspases, such as caspases-1, -3, and -7. Notably, our LESD-based inhibitor was also more potent at inhibiting caspase-8 compared to zIETD-FMK and was not as potent at inhibiting other caspases like Ac-LEHD-FMK, indicating that under our assay conditions, it is both potent and relatively selective for caspase-8. Although Ac-LESD-AMC was exceptionally potent in vitro, it appeared to be less potent in cells, as high doses were needed to achieve cellular inhibition. This is likely due to a lack of cell penetrance, and a more cell-penetrant version is needed to facilitate biological studies. This can likely be achieved by benzylating the N-terminus and having a methyl ester on the C-terminal carboxylate, as inhibitors with these functional groups (zIETD, zVAD, and zLEHD) exhibited better cell penetrance compared to Ac-LESD-CMK and Ac-FLTD-CMK, which lack these functional groups.

A peptide inhibitor based on the sequence of GSDMD, Ac-FLTD-CMK, was reported to be specific for inflammatory caspases. We observed a similar trend in the inhibition profile, but the IC50 values we determined were higher in magnitude. For example, Yang et al. reported an IC50 value of 0.0467 μM for caspase-1, whereas we measured an IC50 value of 3.38 μM. This is likely because they used 50 nM of enzyme and 5 μM of the Ac-WEHD-AMC substrate for activity when measuring the IC50 value, whereas we used 0.25 activity units of caspase-1 and 200 μM Ac-WEHD-AMC substrate in our study. We also discovered that Ac-FLTD-CMK inhibits caspase-8, but this inhibition is 100-fold weaker than that of our Ac-LESD-CMK inhibitor. Overall, our findings support the known selectivity of peptide substrates and inhibitors and reaffirm seemingly forgotten insights, such as the finding that VX-765 inhibits caspase-8 potently. VX-765 was originally reported to inhibit caspase-1 (K i = 0.8 nM), caspase-4 (K i < 0.6 nM), and caspase-8 (K i = 100 nM), but over the years, it has been primarily thought of as a caspase-1 inhibitor. Our findings are consistent with this trend, except for caspase-4, which had a high IC50 value in our assays, probably reflecting differences in experimental conditions. We anticipate that this database of substrate and inhibitor preferences for caspases assayed at the same activity units, along with our new probes for caspase-8, will prove useful for selecting pharmacological tools to facilitate future studies into the function of caspases in biology.

Method Details

Recombinant Caspase Assays

All in vitro assays using recombinant caspases were performed in 20 μL reactions containing caspase assay buffer (20 mM PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, pH 7). We used 0.25 activity units/μL of each indicated caspase. For cytokine cleavage assays, 10 μg of each cytokine was transfected into 10 cm plates of HEK 293T cells using FuGENE HD Transfection Reagent (Promega, E2312) according to the manufacturer’s protocol. After 24 h, cells were harvested and lysed by sonication in 10 s pulses for 30 s at 30% amplitude in PBS and centrifuged at 12,000 g to remove cell debris. Lysates were diluted 10× in caspase assay buffer and aliquoted for incubation with caspases or further purified as previously described before performing cleavage assays. At the indicated time points, samples were mixed 1:1 with 2× Licor Protein Sample Loading Buffer (Neta Scientific, 928-40004), boiled at 95 °C for 10 min, and then analyzed by SDS-PAGE and immunoblotting. Antibodies and reagents used are listed in Table .

3. Antibodies and Reagents Table.

Reagent or Resource Source Identifier
Antibodies
GSDMD Rabbit polyclonal Ab Novus Biologicals Cat#NBP2-33422; RRID:AB_2687913
GAPDH Rabbit monoclonal Ab Cell Signaling Tech Cat#2118S; RRID:AB_561053
CASP1 p12/10 Rabbit monoclonal Ab Abcam Cat#ab179515; RRID:AB_2884954
CASP4 Rabbit polyclonal Ab Cell Signaling Tech Cat#4450S; RRID:AB_1950386
hIL-1β Goat Polyclonal Ab RPC1&D systems Cat#AF-201-NA; RRID:AB_354387
hIL-18 Goat Polyclonal Ab RPC2&D systems Cat#AF2548; RRID:AB_562603
PARP Rabbit polyclonal Ab Cell Signaling Tech Cat#9542S; RRID:AB_2160739
HA Rabbit monoclonal Ab Cell Signaling Tech Cat#3724S; RRID:AB_1549585
Cleaved Caspase-3 (Asp175) (5A1E) Rabbit monoclonal Ab Cell Signaling Tech Cat#9664; RRID:AB_2070042
Anti-Caspase-1 (p20) (Casper-1) (mCasp1) AdipoGen AG-20B-0042-C100 RRID:AB_2490248
Anti-GSDMD (mGSDMD) Abcam ab209845 RRID:AB_2783550
Cleaved Caspase-9 (Asp330) Cell Signaling 52873S RRID:AB_2799423
IRDye 800CW antirabbit LI-COR Cat#926-32213; RRID:AB_621848
IRDye 800CW antimouse LI-COR Cat#926-32212; RRID:AB_621847
IRDye 800CW antigoat LI-COR Cat#926-32214; RRID:AB_621846
IRDye 680CW antirabbit LI-COR Cat#926-68073; RRID:AB_10954442
IRDye 680CW antimouse LI-COR Cat#926-68072; RRID:AB_10953628
IRDye 680CW antigoat LI-COR Cat#926-68074; RRID:AB_10956736
Chemicals, peptides, and recombinant proteins
LPS-EB Ultrapure InvivoGen Cat#tlrl-3pelps
VX-765 Apexbio Technology LLC Cat#50-101-3604
zVAD-FMK Enzo Life Sciences Cat#NC9471015
FuGENE HD Promega Cat#2311
Phorbol 12-myristate 13-acetate (PMA) Promega Cat#V1171
Staurosporine MedChemExpress Cat#HY-15141
TNF-α InvivoGen Cat#Rcyc-htnfa
TRAIL RPC3&D systems Cat#75-TL-010/CF
Nigericin (sodium salt) Cayman Chemical Cat#11437
CellEvent Caspase-3/7 Dye Invitrogen Cat#C10423
Ac-LESD-AMC This study N/A
Ac-DEVD-AMC Fischer Scientific Cat#ALX-260-031-M005
Ac-YVAD-AMC Fischer Scientific Cat#ALX-260-024-M005
Ac-LEHD-AMC Fischer Scientific Cat#ALX-260-080-M005
Ac-WEHD-AMC Fischer Scientific Cat#ALX-260-057-M005
Ac-LESD-CMK This study N/A
Ac-FLTD-CMK Neta Scientific Cat#CAYM-34469-1
zLEHD-FMK Fisher Scientific S7313
zIETD-FMK InvivoGen inh-ietd
Caspase-1 (human), (recombinant) Enzo Life Sciences Cat#BML-SE168-5000
Caspase-3 (human), (recombinant) Enzo Life Sciences Cat#BML-SE169-5000
Caspase-4 (human), (recombinant) Enzo Life Sciences Cat#BML-SE176-5000
Caspase-5 (human), (recombinant) Enzo Life Sciences Cat#BML-SE171-5000
Caspase-6 (human), (recombinant) Enzo Life Sciences Cat#BML-SE170-5000
Caspase-7 (human), (recombinant) Enzo Life Sciences Cat#BML-SE177-5000
Caspase-8 (human), (recombinant) Enzo Life Sciences Cat#BML-SE172-5000
Caspase-9 (human), (recombinant) Enzo Life Sciences Cat#BML-SE173-5000
Caspase-10 (human), (recombinant) Enzo Life Sciences Cat#BML-SE174-5000
Caspase-11 (mouse), (recombinant) Enzo Life Sciences Cat#BML-SE155-5000
Caspase-9 (full-length) (human), (recombinant) Enzo Life Sciences Cat#BML-SE240
Critical commercial assays
DC Protein Assay kit Bio-Rad Cat#5000111
Experimental models: Cell lines
THP1 Clare Bryant N/A
HEK 293T ATCC RRID:CVCL_0063
HCC1806 ATCC CRL-2335
Recombinant DNA
pLEX_307 IL-1β_HA Exconde et al., 2023 N/A
pLEX_307 IL-1β 113LESD116_HA Exconde et al., 2023 N/A
pLEX_307 IL-18_HA Exconde et al., 2023 N/A
pLEX_307 IL-18 33AAAD36_HA Exconde et al., 2023 N/A
Software and algorithms
GraphPad Prism Version 10.3.1 GraphPad Software GraphPad Prism (RRID:SCR_002798)
Empiria Studio LI-COR Inc. Empiria Studio (RRID:SCR_022512)
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In Vitro Substrate Kinetics

All fluorogenic substrates were resuspended in DMSO at 40 mM stock concentrations and diluted to the indicated concentrations in a caspase assay buffer. All probe experiments were completed at a 20 μL final volume, with 0.25 activity units per μL of each recombinant caspase (1 activity units per μL for caspase-9). Relative fluorescence units were measured every 5 min using a Cytation 5 plate reader (Biotek). Background fluorescence was subtracted from samples incubated with caspases. At each concentration of substrate, simple linear regressions (y = mx + b) were performed using GraphPad Prism to determine the rate. Rates were plotted against substrate concentration for n = 3 separate runs and fit to a Michaelis–Menten curve (Y = V max × X/(K M + X)) on Prism. Caspase-substrate pairs that resulted in V max values under 20 RFUs/min were considered undetected. For V max, K M, and k cat/K M calculations, curves were generated for each individual experimental replicate, and V max, K M, and k cat values were recorded for each replicate. k cat was determined as the V max value with the total enzyme concentration (ET) set to 1 for all caspases. The mean and standard error of the mean were calculated by compiling the values for each individual run.

In Vitro Inhibition Assays

To determine the inhibition of maximal enzyme activity, each caspase was incubated with its optimal probe substrate at a saturating concentration. Inhibition of maximal caspase activity was determined with 200 μM Ac-WEHD-AMC for caspase-1 and -5, 1000 μM Ac-WEHD-AMC for caspase-4 and -11, 200 μM Ac-LEHD-AMC for caspase-8 and -10, 25 μM Ac-DEVD-AMC for caspase-3 and -7 with ZVAD-FMK, 100 μM Ac-DEVD-AMC for caspase-3, -6, and -7 for all other inhibitors tested, and 200 μM Ac-LEHD-AMC for caspase-9. Rates were determined as described in the In Vitro Substrate Kinetics section. Rates for n = 3 independent runs were plotted against inhibitor concentration. Percent caspase activity was determined by normalizing each independent run, with 0% activity set at 0 and maximum activity set as the sample with no inhibitor added. Replicates were combined, and inhibition curves were calculated for the aggregate data using an [inhibitor] vs normalized response curve on GraphPad Prism (Y = 100/(1 + X/IC50)).

Testing Inhibitors in Cells

THP1 cells were resuspended in RPMI medium containing 50 ng/mL phorbol 12-myristate 13-acetate (PMA). Cells were plated in 96-well plates at a density of 8 × 104 cells/well. After 48 h, the media was replaced with 100 μL per well of Opti-MEM (Thermo Fisher Scientific, 31-985-062) containing Sytox Green (Fisher Scientific, S7020) at 200 nM. Where indicated, cells were treated with Ac-LESD-CMK, Ac-FLTD-CMK, or zVAD-FMK 30 min before the cell death trigger was added (described below). Cells were incubated in the trigger for the indicated time points, and Sytox Green values were read using the Cytation 5 plate reader. Supernatants were harvested and pooled across replicates, and proteins were precipitated. Nigericin (Neta Scientific, 11437) was resuspended in ethanol and added at a final concentration of 5 μM, while staurosporine (MedChemExpress, HY-15141) was resuspended in DMSO and added at a final concentration of 1 μM. The LPS transfection solution was prepared by adding LPS (Cell Signaling Technology, 14011S) (25 μg/mL final concentration) and FuGENE (Promega, E2312) (0.5% final concentration) to Opti-MEM. This solution was gently mixed by flicking and incubated for 15 min at room temperature before addition to each well.

Western Blotting

Protein samples were run on NuPAGE 4–12% Bis-Tris Midi Protein Gel (Thermo Scientific, WG1403BOX) at 175 V. Proteins were transferred to 0.45 μm nitrocellulose membranes (Bio-Rad, 1704271) at 25 V for 7 min using the Bio-Rad Trans-Blot Turbo System. Membranes were blocked using LI-COR Intercept Blocking Buffer (Neta Scientific, 927-70010) for 1 h and stained with primary antibodies at a 1:1000 concentration in 50% LI-COR Blocking Buffer and 50% TBS buffer with 0.1% Tween for 1 h at room temperature or overnight at 4 °C. Membranes were washed 3× for 15 min, followed by incubation with Donkey Anti-Mouse/Rabbit/Goat IgG Polyclonal Antibody (IRDye 800CW) for 1 h at room temperature. Membranes were then washed 3× for 10 min and imaged on an Odyssey M Imaging System (LI-COR Biosciences). Images were analyzed using Empiria Studio version 3.2 (LI-COR), and brightness, contrast, and tone parameters were adjusted uniformly for entire membranes in Adobe Photoshop.

Cell Culture

HEK 293T and HCC1806 cells were purchased from ATCC, and THP1 cells were obtained from the Bryant lab. HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with l-glutamine and 10% fetal bovine serum (FBS). HCC1806 and THP1 cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 with l-glutamine and 10% fetal bovine serum (FBS). Isolated bone marrow cells from 6- to 10-week-old male and female mice were grown at 37 °C and 5% CO2 in 30% macrophage medium [30% L929 fibroblast supernatant and complete Dulbecco’s modified Eagle’s medium (DMEM)]. BMDMs were harvested in cold phosphate-buffered saline (PBS) on day 7 and replated in 10% macrophage medium onto tissue culture (TC)-treated plates. All cells were grown at 37 °C in a 5% CO2 atmosphere incubator.

Incucyte Caspase-3/7 Activity and Sytox Green Uptake Assays

The kinetics of caspase-3/7 activity and Sytox Green uptake were measured in cells using the IncuCyte S3 (Sartorius). HCC1806 or THP1 cells (8 × 104 cells/well) were seeded in black, clear-bottom 96-well plates and treated with apoptotic or inflammatory stimuli as described in the figure legends. The medium was supplemented with either the cell-permeable caspase-3/7 green dye (final concentration: 2 μg/mL) or Sytox Green (final concentration: 0.2 μM). A series of images was collected with a 10× objective at 30 min intervals for 24 h. The number of Sytox Green or caspase-3/7 dye-positive cells in each image was determined using the IncuCyte S3 software with 5 different areas/well. Apoptosis was triggered using TRAIL, staurosporine, or TNFα, and pyroptosis was triggered using either LPS or nigericin. If inhibitors were used, the cells were preincubated with the reported inhibitors for 30 min prior to stimulation.

Cloning

All plasmids were cloned using Gateway technology as previously described. DNA encoding the indicated proteins was inserted between the attR recombination sites and shuttled into modified pLEX_307 vectors (Addgene) using Gateway technology (Thermo Fisher Scientific) according to the manufacturer’s instructions. Proteins expressed from these modified vectors contain an N-terminal attB1 linker (GSTSLYKKAGFAT) after any N-terminal tag or a C-terminal attB2 linker (DPAFLYKVVDI) preceding any C-terminal tag, such as V5 or HA.

Yersinia pseudotuberculosis Primary Murine Bone Marrow-Derived Macrophage Infections

Yersinia pseudotuberculosis (Yptb) infections were performed as previously described. Strain IP2666 was induced before infection by diluting stationary-phase overnight cultures 1:40 in 3 mL of inducing medium [2× yeast extract tryptone (YT) broth, 20 mM sodium oxalate, and 20 mM MgCl2]. Cultures were grown at 28 °C for 1 h and then transferred to 37 °C for 2 h with shaking. Bacterial growth was measured by absorbance at an optical density of 600 nm (OD600) using a spectrophotometer. Bacteria were pelleted, washed, and resuspended in Opti-MEM or 10% macrophage media (10% L929 fibroblast supernatant and complete Dulbecco’s modified Eagle’s medium (DMEM)) for infection. In vitro infections were performed at a multiplicity of infection of 20. Indicated inhibitors were added 30 min prior to bacterial addition, and the plate was incubated at 37 °C. Gentamycin (100 μg/mL) was added 1 h after infection for all infections. The plate was imaged in a Sartorius Incucyte S3.

Synthesis of Ac-LESD-AMC Substrate and Ac-LESD-CMK Inhibitor

General Peptide Synthesis

Solvents and reagents were purchased from commercial suppliers and used without further purification. Analytical LC-MS was performed using a system comprising an Agilent 1260 Infinity II HPLC instrument equipped with an Agilent InfinityLab LC/MSD XT MS detector with electrospray ionization. The system ran with an Agilent ZORBAX SB-C18 RRHT (50 × 4.6 × 1.8 μm) column and gradient elution with two binary solvent systems: MeCN/H2O or MeCN/H2O plus 0.1% formic acid. Preparative HPLC was performed using a system comprising an Agilent 1260 Infinity II HPLC system equipped with an Agilent 1290 Prep Bin Pump, an Agilent Prep-C18 column (250 × 21.2 × 10 μm), and an Agilent prep autosampler and fraction collector. The system ran using a UV diode array detector, and purification was performed using gradient elution with a MeCN/H2O binary solvent system containing 0.05% trifluoroacetic acid.

Ac-LESD-AMC

Inline graphicAc-LESD-AMC was synthesized by manual Fmoc solid-phase peptide chemistry using Fmoc-Asp­(Wang-resin)-AMC. Following resin swelling in DMF, iterative cycles of Fmoc deprotection (20% piperidine in DMF containing 0.1 M HOBt, 3× 5 min), amino acid coupling (4 eq. Fmoc AA, 4 eq. HATU in 4% DIPEA in DMF, 1 h), and washing (3× DMF, 3× DCM) were performed until the sequence was complete. N-terminal acetylation was achieved using 5% acetic anhydride in pyridine. Peptides were deprotected and cleaved from the resin by treatment with trifluoroacetic acid/water/triisopropylsilane/phenol (88:5:5:2) for 1–3 h. The solvent was removed under a flow of nitrogen, and peptides were precipitated with ice-cold ether. Ac-LESD-AMC was subsequently purified by reverse-phase chromatography, eluting with 5–95% acetonitrile in water containing 0.05% trifluoroacetic acid over a C18 column. Peaks containing Ac-LESD-AMC were identified by LC-MS, pooled, and concentrated in vacuo.

Ac-LESD-AMC: m/z [M + H]+ calcd for C30H39N5O12 661.3; found 661.9.

Ac-LESD-CMK

Ac-LE­(tBu)­S­(tBu)–OH was synthesized by manual Fmoc solid-phase peptide chemistry on a 2-chlorotrityl resin. Following resin swelling in DMF, Fmoc-l-Ser­(tBu)–OH was loaded onto the resin (1.2 eq. AA, 2.5 eq. DIPEA in DCM for 1 h), and the resin was capped (3 eq. MeOH, 2.5 eq. DIPEA in DCM for 1 h). The resin then underwent iterative cycles of Fmoc deprotection (20% piperidine in DMF containing 0.1 M HOBt, 3× 5 min), amino acid coupling (4 eq. Fmoc AA, 4 eq. HATU in 4% DIPEA in DMF, 1 h), and washing (3× DMF, 3× DCM) until the sequence was complete. N-terminal acetylation was achieved using 5% acetic anhydride in pyridine. Ac-LE­(tBu)­S­(tBu)–OH was cleaved from the resin in 20% hexafluoroisopropanol (HFIP) in DCM at room temperature for 1 h. The solvent was removed under a flow of nitrogen, and the crude material was used with no further purification.

Separately, Boc-l-aspartic acid β-benzyl ester chloromethylketone 1 (65 mg, 0.18 mmol) (purchased from Santa Cruz Biotechnologies, Cat # sc-285170) was dissolved in 3 mL of DCM at room temperature before the addition of 1 mL of trifluoroacetic acid (TFA) and stirred for 1 h (Scheme ). The solvent and volatiles were removed under reduced pressure. Crude deprotected l-aspartic acid β-benzyl ester chloromethyl ketone 2 was dissolved in 3 mL of tetrahydrofuran (THF) with HATU (68 mg, 1.1 equiv) and DIPEA (440 μL, 5.5 equiv). To this solution, Ac-LE­(tBu)­S­(tBu)–OH peptide (80 mg, 1 equiv) in 1 mL of THF was added. The reaction was stirred at r.t. for 2 h, and the reaction progress was monitored by LC/MS. The solvent and volatiles were removed under reduced pressure, and the product was redissolved in 10 mL of ethyl acetate before 2× 10 mL washes with 0.5 M HCl. Organic extracts were dried over magnesium sulfate and concentrated in vacuo to yield 3, which was used with no further purification in the next step.

1. Synthesis of Ac-LESD-CMK.

1

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To a solution of 3 (127 mg, 1 equiv) in THF (10 mL) under an argon atmosphere, 10% Pd/C (13 mg, 10% w/w) was added, and hydrogen gas was bubbled through the reaction for 2 h. The reaction mixture was filtered over Celite, and the filter cake was washed with THF and MeOH. Solvents and volatiles were removed under reduced pressure, and the residue was dissolved directly in 50% TFA in DCM and stirred at room temperature for 1 h to generate Ac-LESD-CMK. Solvents and volatiles were again removed under reduced pressure. Ac-LESD-CMK was subsequently purified by reverse-phase chromatography, eluting with 5–95% acetonitrile in water containing 0.05% trifluoroacetic acid over a C18 column. Peaks containing Ac-LESD-CMK were identified by LC-MS, pooled, and concentrated in vacuo.

Ac-LESD-CMK: m/z [M + H]+ calcd for C21H33ClN4O10 537.2; found 537.2.

Supplementary Material

bg4c00146_si_001.pdf (16.6MB, pdf)

Acknowledgments

We thank all the members of the Taabazuing lab for their scientific discussions. This work was supported by an NIH R00 Career Transition Award (Grant# 4R00AI148598-03, C.Y.T.), an NIGMS Maximizing Investigator’s Research Award (MIRA) (Grant# 1R35GM155239-01, C.Y.T.), a Center for Translational Chemical Biology Pilot Award from the University of Pennsylvania (C.Y.T. and G.M.B.), and the Colton Center for Autoimmunity at Penn (C.Y.T.). G.M.B. is also supported by the National Institutes of Health (Grant# R35-GM142505). N.R.R. is supported by an NIH Chemistry Biology Interface Training Grant (Grant# T32 GM133398). P.M.E. is supported by the Martin and Pamela Winter Infectious Disease Fellowship. C.M.B. is a Penn Provost Postdoctoral Fellow and is supported by the Burroughs Wellcome Fund (Grant# 1054907). I.E.B. is supported by two NIH R01 grants (Grant# R01AI128530, R01139102A1) and the Mark Foundation.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomedchemau.4c00146.

  • Western blot of purified cytokines incubated with recombinant caspases; kinetic characterization of caspase substrates and inhibitors, including caspase-9; images of caspase-3/7 dye and Western blot of inhibitor treated HCC1806 cells treated with TNF (PDF)

Conceptualization: C.Y.T.; methodology: C.Y.T., G.M.B., C.M.B., N.R.R., W.Y., and I.E.B.; investigation: C.M.B. and N.R.R., Ma.Ku., P.M.E., S.L., W.Y., T.J.W., R.C.P., Ma.Ka., T.S., Mi.Ka., B.M.D., and A.B.M.; visualization: C.Y.T., C.M.B.; funding acquisition: C.Y.T., G.M.B., I.E.B., C.M.B., P.M.E., and N.R.R.; project administration: C.Y.T., G.M.B., and C.M.B.; data curation: C.Y.T. and C.M.B.; formal analysis: C.Y.T., C.M.B., T.J.W., and A.B.M.; resources: C.Y.T., G.M.B., and I.E.B.; supervision: C.Y.T., G.M.B., and I.E.B.; writingoriginal draft: C.Y.T. and C.M.B.; writingreview and editing: C.Y.T., I.E.B., G.M.B., C.M.B., Ma.Ku., N.R.R., P.M.E., A.B.M.

The authors declare no competing financial interest.

Published as part of ACS Bio & Med Chem Au special issue “Juneteenth 2025”.

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