Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen

Thomas A. Bunch, Piyali Guhathakurta, Victoria C. Lepak, Andrew R. Thompson, Rhye Samuel Kanassatega, Anna Wilson, David D. Thomas, Brett A. Colson

Research output: Contribution to journalArticlepeer-review

Abstract

Cardiac 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.

Original languageEnglish (US)
Article number100840
JournalJournal of Biological Chemistry
Volume297
Issue number1
DOIs
StatePublished - Jul 1 2021

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health grants R37 AG26160 (to D. D. T.) and R01 HL141564 (to B. A. C.) and a University of Arizona Sarver Heart Center ?Novel Research Project Award in the Area of Cardiovascular Disease and Medicine? (to B. A. C). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding Information:
Funding and additional information—This work was supported by National Institutes of Health grants R37 AG26160 (to D. D. T.) and R01 HL141564 (to B. A. C.) and a University of Arizona Sarver Heart Center “Novel Research Project Award in the Area of Cardiovascular Disease and Medicine” (to B. A. C). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2021 THE AUTHORS.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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