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Caspases control regulated cell death (apoptosis), a process that is crucial in the development of multicellular organisms as well as in various diseases. In order to spatiotemporally study apoptosis, we here develop photoactivatable caspase inhibitors. These are based on cysteine-reactive acyloxymethyl ketone electrophiles connected to a peptide targeting caspases. Importantly, the aspartate crucial for recognition by caspases is caged with a photoprotecting group. Ester photocages were found to be labile, and it was critical to have a nitroindoline cage, which forms a stable amide bond with the aspartate side chain. The nitroindoline-protected inhibitors lead to an efficient turn-on of inhibitory activity after irradiation with light. They are applicable in live cells, where they prevent anti-FAS-induced apoptosis only upon irradiation. Overall, these reagents will allow a better understanding of the spatial and temporal dimensions of apoptosis in complex, dynamic systems., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world. While the role of NSAIDs as cyclooxygenase (COX) inhibitors is well established, other targets may contribute to anti-inflammation. Here we report caspases as a new pharmacological target for NSAID family drugs such as ibuprofen, naproxen, and ketorolac at physiologic concentrations both in vitro and in vivo. We characterize caspase activity in both in vitro and in cell culture, and combine computational modeling and biophysical analysis to determine the mechanism of action. We observe that inhibition of caspase catalysis reduces cell death and the generation of pro-inflammatory cytokines. Further, NSAID inhibition of caspases is COX independent, representing a new anti-inflammatory mechanism. This finding expands upon existing NSAID anti-inflammatory behaviors, with implications for patient safety and next-generation drug design., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Dysregulation of apoptosis is associated with several human diseases. The main apoptotic mediators are caspases, which propagate death signals to downstream targets. Executioner caspase-3 is responsible for the majority of cleavage events and its therapeutic potential is of high interest with to date several available active site peptide inhibitors. These molecules inhibit caspase-3, but also homologous caspases. Here, we describe caspase-3 specific inhibitors D3.4 and D3.8, which have been selected from a library of designed ankyrin repeat proteins (DARPins). The crystal structures of D3.4 and mutants thereof show how high specificity and inhibition is achieved. They also show similarities in the binding mode with that of the natural caspase inhibitor XIAP (X-linked inhibitor of apoptosis). The kinetic data reveal a competitive inhibition mechanism. D3.4 is specific for caspase-3 and does not bind the highly homologous caspase-7. D3.4 therefore is an excellent tool to define the precise role of caspase-3 in the various apoptotic pathways., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Caspase inhibition is a promising approach for treating multiple diseases. Using a reconstituted assay and high-throughput screening, we identified a group of nonpeptide caspase inhibitors. These inhibitors share common chemical scaffolds, suggesting the same mechanism of action. They can inhibit apoptosis in various cell types induced by multiple stimuli; they can also inhibit caspase-1-mediated interleukin generation in macrophages, indicating potential anti-inflammatory application. While these compounds inhibit all the tested caspases, kinetic analysis indicates they do not compete for the catalytic sites of the enzymes. The cocrystal structure of one of these compounds with caspase-7 reveals that it binds to the dimerization interface of the caspase, another common structural element shared by all active caspases. Consistently, biochemical analysis demonstrates that the compound abates caspase-8 dimerization. Based on these kinetic, biochemical, and structural analyses, we suggest that these compounds are allosteric caspase inhibitors that function through binding to the dimerization interface of caspases., (Copyright © 2012 Elsevier Inc. All rights reserved.)