{"doi":"10.1101/2021.03.24.436879","title":"High-throughput screen using fluorescence lifetime detects compounds that modulate myosin-binding protein C interactions with actin","abstract":"ABSTRACT Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac contractility. These interactions are regulated by cMyBP-C phosphorylation. Heart failure patients often have decreased cMyBP-C phosphorylation and phosphorylation in model systems appears to be cardioprotective for heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation and/or perturb its interactions with actin/myosin. We have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites. When combined with cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified 3 reproducible Hit compounds that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by a novel actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that the Hit compounds bind to cMyBP-C but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C’s actin binding function. TPA, TR-FRET, and ITC can then be used to understand the mechanism by which the compounds alter cMyBP-C interactions with actin.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":223585,"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":0,"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":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":431508,"name":"Piyali Guhathakurta","orcid":null,"position":1,"is_corresponding":false},{"id":734384,"name":"Victoria C. Lepak","orcid":null,"position":2,"is_corresponding":false},{"id":825068,"name":"Andrew R. Thompson","orcid":"0000-0003-0917-1255","position":3,"is_corresponding":false},{"id":733819,"name":"Rhye‐Samuel Kanassatega","orcid":"0000-0001-9007-6524","position":4,"is_corresponding":false},{"id":524429,"name":"Anna Wilson","orcid":null,"position":5,"is_corresponding":false},{"id":417547,"name":"David D. Thomas","orcid":"0000-0002-8822-2040","position":6,"is_corresponding":false},{"id":733820,"name":"Brett A. Colson","orcid":"0000-0002-5721-4320","position":7,"is_corresponding":false},{"id":733818,"name":"Thomas A. Bunch","orcid":"0000-0002-6237-1427","position":0,"is_corresponding":true}],"reference_count":19,"raw_metadata":null,"created_at":"2026-07-18T23:54:11.061295Z","pmid":null,"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":[]}