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Updated: Nov 4, 2025
Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays
Published on: February 4, 2021
Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput
Thomas A Bunch1, Piyali Guhathakurta2, Victoria C Lepak1
1Department of Cellular & Molecular Medicine, University of Arizona, Tucson Arizona, USA.
Insights
Researchers developed a new fluorescence assay to find drugs targeting cardiac myosin-binding protein C (cMyBP-C) interactions with actin. This screen identified three compounds that inhibit this binding, offering potential heart failure therapeutics.
Area of Science:
- Biochemistry
- Cardiovascular Biology
- Pharmacology
Background:
- Cardiac myosin-binding protein C (cMyBP-C) regulates cardiac contractility by interacting with actin and myosin.
- Phosphorylation of cMyBP-C disfavors these interactions and is cardioprotective, with decreased phosphorylation observed in heart failure.
- cMyBP-C represents a potential therapeutic target for heart failure.
Purpose of the Study:
- To develop and validate a high-throughput screening assay for identifying small-molecule inhibitors of actin-cMyBP-C binding.
- To identify novel compounds that modulate cMyBP-C's interaction with actin, potentially mimicking phosphorylation effects.
Main Methods:
- A novel fluorescence lifetime-based assay utilizing Alexa Fluor 568-labeled actin was developed.
- A high-throughput screen of 1280 compounds was performed to identify inhibitors of the interaction between actin and the cMyBP-C N-terminal fragment (C0-C2).
- Hit validation was conducted using time-resolved FRET (TR-FRET), isothermal titration calorimetry (ITC), and transient phosphorescence anisotropy (TPA).
Main Results:
- The fluorescence lifetime assay successfully identified three reproducible hit compounds: suramin, NF023, and aurintricarboxylic acid.
- These compounds inhibited actin-C0-C2 binding in the micromolar range and also blocked binding of phosphorylated C0-C2.
- Further biophysical assays confirmed that the compounds bind to cMyBP-C, not actin, and inhibit the actin-C0-C2 interaction.
Conclusions:
- The developed fluorescence lifetime assay is a validated high-throughput method for detecting compounds that affect cMyBP-C-actin binding.
- This assay provides a novel tool for discovering pharmacologically active agents targeting cMyBP-C, a key regulator in cardiac function and heart failure.
- Identified compounds represent potential leads for developing new heart failure therapeutics.
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.
