High-throughput screen using fluorescence lifetime detects compounds that modulate myosin-binding protein C interactions with actin

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.

Emerging Concepts of Mechanisms Controlling Cardiac Tension: Focus on Familial Dilated Cardiomyopathy (DCM) and Sarcomere-Directed Therapies

“…Although many recent papers continue to refer to "modulation of sarcomere contractility" with small molecules acting at sarcomere proteins as a new or recent therapeutic opportunity [73][74][75][76], research in this area began more than 40 years ago. Solaro and Ruegg introduced a research direction in development of agents with the idea that small molecules can enter the intracellular space and bind directly to myofilament regulatory proteins inducing an increase in sarcomere response to Ca 2+ [77].…”

“…In view of the versatility of cMyBP-C actions in controlling cardiac contraction and dynamics, it is not surprising that efforts are underway to modulate its function with small molecules. Initial studies defining high throughput approaches with use fluorescence lifetime measurements of the actin-cMyBP-C interaction have been reported [76]. Early evidence provides an approach with identification of lead compounds with specificity for cardiac N-terminal C0-C2 regions of cMyBP-C with or without phosphorylation.…”