Chemoproteomics identifies proteoform-selective caspase-2 inhibitors

Castellon, J. O.; Ofori, S.; Armenta, E.; Burton, N.; Boatner, L. M.; Takayoshi, E. E.; Faragalla, M.; Zhou, A.; Tran, K.; Shek, J.; Yan, T.; Desai, H. S.; Backus, K. M.

doi:10.1101/2023.10.25.563785

bioRxiv · 10.1101/2023.10.25.563785

Chemoproteomics identifies proteoform-selective caspase-2 inhibitors

Castellon, J. O.·Ofori, S.·Armenta, E.·Burton, N.·Boatner, L. M.·Takayoshi, E. E.·Faragalla, M.·Zhou, A.·Tran, K.·Shek, J.·Yan, T.·Desai, H. S.·Backus, K. M.

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Abstract

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all twelve human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive non-catalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify caspase contributions to initiation of intrinsic apoptosis, supports compensatory caspase-9 activity in the context of caspase-2 inactivation. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target non-conserved and non-catalytic cysteine residues.

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Castellon, J. O., Ofori, S., Armenta, E., Burton, N., Boatner, L. M., Takayoshi, E. E., Faragalla, M., Zhou, A., Tran, K., Shek, J., Yan, T., Desai, H. S., Backus, K. M.. 2023-10-26. Chemoproteomics identifies proteoform-selective caspase-2 inhibitors. https://doi.org/10.1101/2023.10.25.563785

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