Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen
AffiliationThe Department of Cellular & Molecular Medicine, University of Arizona
MetadataShow full item record
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).
JournalJournal of Biological Chemistry
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/).
Collection InformationThis 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 firstname.lastname@example.org.
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.
NoteOpen access journal
VersionFinal published version
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/).
- Human cardiac myosin-binding protein C restricts actin structural dynamics in a cooperative and phosphorylation-sensitive manner.
- Authors: Bunch TA, Kanassatega RS, Lepak VC, Colson BA
- Issue date: 2019 Nov 1
- High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin-myosin structure and function.
- Authors: Guhathakurta P, Prochniewicz E, Grant BD, Peterson KC, Thomas DD
- Issue date: 2018 Aug 3
- The myosin-binding protein C motif binds to F-actin in a phosphorylation-sensitive manner.
- Authors: Shaffer JF, Kensler RW, Harris SP
- Issue date: 2009 May 1
- N-terminal extension in cardiac myosin-binding protein C regulates myofilament binding.
- Authors: Bunch TA, Lepak VC, Kanassatega RS, Colson BA
- Issue date: 2018 Dec
- A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin.
- Authors: Bunch TA, Lepak VC, Bortz KM, Colson BA
- Issue date: 2021 Mar 1