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

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.