{"doi":"10.1101/2023.10.25.563785","title":"Chemoproteomics identifies proteoform-selective caspase-2 inhibitors","abstract":"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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":401320,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9528,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":424811,"name":"Samuel Ofori","orcid":"0000-0001-6921-9543","position":1,"is_corresponding":false},{"id":1178786,"name":"Ernest Armenta","orcid":null,"position":2,"is_corresponding":false},{"id":995433,"name":"Nikolas R. Burton","orcid":"0000-0003-2887-8846","position":3,"is_corresponding":false},{"id":686857,"name":"Lisa M. Boatner","orcid":"0000-0003-0757-4982","position":4,"is_corresponding":false},{"id":1178292,"name":"Evan E. Takayoshi","orcid":"0000-0002-6163-8708","position":5,"is_corresponding":false},{"id":1178787,"name":"Marina Faragalla","orcid":null,"position":6,"is_corresponding":false},{"id":735854,"name":"A. Ning Zhou","orcid":"0000-0002-1746-4210","position":7,"is_corresponding":false},{"id":1178293,"name":"Ky Tran","orcid":"0000-0002-7962-4435","position":8,"is_corresponding":false},{"id":557702,"name":"Jeremy Shek","orcid":"0000-0002-5419-060X","position":9,"is_corresponding":false},{"id":868699,"name":"Tianyang Yan","orcid":"0000-0002-4941-9828","position":10,"is_corresponding":false},{"id":710327,"name":"Heta S. Desai","orcid":"0000-0003-4362-1707","position":11,"is_corresponding":false},{"id":377194,"name":"Keriann M. Backus","orcid":"0000-0001-8541-1404","position":12,"is_corresponding":false},{"id":377192,"name":"José O. Castellón","orcid":"0000-0003-4907-5655","position":0,"is_corresponding":true}],"reference_count":100,"raw_metadata":null,"created_at":"2026-07-19T01:20:12.316631Z","pmid":"37961563","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}