Identification of specific tethered inhibitors for caspase-5

The development of highly selective small molecule inhibitors for individual caspases, a class of cysteine-dependent aspartate-specific proteases, has been challenging due to conservation of the active site. Previously, we discovered an allosteric site at the dimer interface of caspases-3, -7, and -1 using disulfide trapping. Here, we show this approach can generate selective tethered ligands and inhibitors for caspase-5, which is remarkable considering its high sequence similarity to caspase-1. Among the 62 hits of a screen of ∼15 000 thiol-containing fragments, a naphthyl-thiazole-containing molecule was identified that selectively inhibited and labeled the allosteric cysteine in the p10 subunit of caspase-5, but caused very little inhibition or labeling of caspase-1. Interestingly, some of allosteric tethered compounds to caspase-5 did not inhibit its enzymatic activity, suggesting that thiol-labeling itself is not sufficient to drive inhibition. These studies validate an allosteric site on caspase-5 and provide a useful starting point to develop selective compounds to probe the role of caspase-5 separate from caspase-1 in the innate immune response.