Inhibition of Human Caspases by Peptide-based and Macromolecular Inhibitors

Abstract

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 M. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 ؋ 10 2 M ؊1 s ؊1 for caspase-2 to 2.8 ؋ 10 M ؊1s ؊1 for caspase-1. The results obtained with peptidebased inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (K i < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.Members of the caspase family of cysteine proteases, which at present includes 11 homologues of human origin, are important mediators of both inflammation, where they are involved in the production of several inflammatory cytokines, and apoptosis, where they participate in signaling and effector pathways (for review, see Ref. 1). The evidence for the central role of these enzymes in both of these biological processes was initially obtained using potent peptide-based and macromolecular inhibitors. For example, the finding that Ac-YVAD-CHO, a potent inhibitor of caspase-1, prevented the release of interleukin-1␤ (IL-1␤) 1 from monocytes, suggested that this enzyme was, in fact, the pro-IL-1␤-processing enzyme (2). This was later confirmed with the description of caspase-1-deficient mice, which are defective in the production of this cytokine (3,4). Similarly, the observation that apoptosis could be attenuated by the cowpox serpin CrmA, also known to be a potent caspase-1 inhibitor, provided the first compelling evidence that caspases play an important role in mammalian cell death (5). This has recently been confirmed by several studies, including the description of caspase-3-deficient mice, which have a striking defect in the programmed cell deaths that occur during neuronal development (6).Studies using these and other caspase inhibitors continue to be an important component of the repertoire of scientists investigating these complex biological processes in whole cells and in vivo. Regarding the latter, there are numerous recent re...

Retinoid-related molecules require caspase 9 for the effective release of Smac and the rapid induction of apoptosis

“…The latter is possibly due to the activation of caspase 2, which occurs independently of caspase 9 activity in MX781-treated cells and may provoke the translocation of proapoptotic proteins from the mitochondria. 15,16,64 This assumption is further supported by our observations that Z-VAD-fmk, a poor inhibitor of caspase 2, 65 weakly prevented MX781-mediated release of Smac.…”

Caspase-dependent cleavage and inactivation of the Vav1 proto-oncogene product during apoptosis prevents IL-2 transcription

“…Speci®c processing of the proteins was not observed in cells that were pretreated with the irreversible caspase inhibitors zDEVD-cmk or zVADfmk. The broad-range inhibitor zVAD-fmk inhibits all caspase activities and the peptide zDEVD-cmk displays some speci®city for caspase-3-like caspases (Villa et al, 1997;Garcia-Calvo et al, 1998). In order to ensure that caspase-mediated Vav1 fragmentation is not restricted to a particular cell line, a similar experimental approach was performed for the human B-cell line SKW6.4 (Figure 1b).…”

Catalytic activity of caspase-3 is required for its degradation: stabilization of the active complex by synthetic inhibitors

“…Comparative inhibitor analysis clearly indicates that peptide-derived inhibitors harboring the optimal substrate tetrapeptide-motif for a specific caspase are highly potent and in some cases selective. 16 To date, all synthetic caspase inhibitors, as well as the endogenous, macromolecular inhibitors that have been identified are competitive inhibitors which act by occupying the active site of the enzyme.…”

“…Caspase inhibitors typically contain an electrophilic group or 'warhead,' which binds covalently to the active-site cysteine, 16 and depending on the chemistry of this 'warhead', caspase inhibitors can be characterized as being either reversible inhibitors that include aldehydes, nitriles and ketones or irreversible inhibitors such as diazomethylketones, acyloxymethylketones and halomethylketones. Although in general peptide-based inhibitors show substantial disadvantages, like poor cell penetration and poor metabolic stability in cell-based assays, some classes have proven effective at inhibiting recombinant caspases in vitro, as well as in cellbased systems.…”