Inhibition of Human Caspases by Peptide-based and Macromolecular Inhibitors

TLDR

Studies with peptide‑based and macromolecular caspase inhibitors have underscored the enzymes’ central role in inflammation and apoptosis, yet incomplete knowledge of inhibitor selectivity has obscured clear interpretation. The study aims to characterize the selectivity of selected peptide‑based inhibitors and the cowpox serpin CrmA toward ten human caspases. The authors evaluated the binding and inhibition of a panel of peptide aldehydes—Ac‑WEHD‑CHO, Ac‑DEVD‑CHO, Ac‑YVAD‑CHO, Boc‑IETD‑CHO, and Boc‑AEVD‑CHO—chosen for optimal tetrapeptide recognition motifs, against these caspases. The aldehydes displayed a wide range of selectivities and potencies, with dissociation constants from 75 pm to >10 µm, while the broad‑specificity halomethyl ketone VAD fluoromethyl ketone showed second‑order inactivation rates from 2.9 × 10² m⁻¹ s⁻¹ (caspase‑2) to 2.8 × 10⁵ m⁻¹ s⁻¹ (caspase‑1); these findings matched predictions from substrate specificity studies, and the cowpox serpin CrmA proved a potent (<20 nM) selective inhibitor of Group I and most Group III caspases, suggesting viral evasion of apoptosis and inflammation.

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,<i>t</i>-butoxycarbonyl-IETD-CHO, and <i>t</i>-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 μm. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 × 10²m⁻¹ s⁻¹ for caspase-2 to 2.8 × 10⁵m⁻¹ s⁻¹ for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (<i>K</i> _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.