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dc.contributor.authorBunch, T.A.
dc.contributor.authorGuhathakurta, P.
dc.contributor.authorLepak, V.C.
dc.contributor.authorThompson, A.R.
dc.contributor.authorKanassatega, R.-S.
dc.contributor.authorWilson, A.
dc.contributor.authorThomas, D.D.
dc.contributor.authorColson, B.A.
dc.date.accessioned2021-07-20T00:15:26Z
dc.date.available2021-07-20T00:15:26Z
dc.date.issued2021
dc.identifier.citationBunch, T. A., Guhathakurta, P., Lepak, V. C., Thompson, A. R., Kanassatega, R.-S., Wilson, A., Thomas, D. D., & Colson, B. A. (2021). Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen. Journal of Biological Chemistry, 297(1).
dc.identifier.issn0021-9258
dc.identifier.pmid34052227
dc.identifier.doi10.1016/j.jbc.2021.100840
dc.identifier.urihttp://hdl.handle.net/10150/660856
dc.description.abstractCardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here 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 the 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 three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) 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 an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these 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 fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure. © 2021 THE AUTHORS.
dc.language.isoen
dc.publisherAmerican Society for Biochemistry and Molecular Biology Inc.
dc.rightsCopyright © 2021 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.titleCardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen
dc.typeArticle
dc.typetext
dc.contributor.departmentThe Department of Cellular & Molecular Medicine, University of Arizona
dc.identifier.journalJournal of Biological Chemistry
dc.description.noteOpen access journal
dc.description.collectioninformationThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.
dc.eprint.versionFinal published version
dc.source.journaltitleJournal of Biological Chemistry
refterms.dateFOA2021-07-20T00:15:26Z


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Copyright © 2021 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Except where otherwise noted, this item's license is described as Copyright © 2021 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).