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

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.

Human cardiac myosin-binding protein C phosphorylation- and mutation-dependent structural dynamics monitored by time-resolved FRET

“…The ability to monitor structural changes in C0-C2 using a recently developed FLTPR instrument makes possible the screening of large compound libraries for compounds that bind to human C0-C2 and mimic the effects of phosphorylationmediated structural dynamics. We have recently used similar technology to screen for compounds that bind C0-C2 and interfere with its actin binding activity [48].…”

“…S2). Our recent work using single-cysteine C0-C2 Cys225 labeled with an acceptor probe to follow binding to donor-labeled F-actin found saturable actin binding that was reduced upon phosphorylation [48]. Therefore, C0-C2 Cys225.Cys330 is a functional biosensor for HTS and uncovering mechanistic details of cMyBP-C molecular structure and the effects of phosphorylation in C1-M-domain dynamics.…”

“…We have reported similar levels of percent change and Z'-factors in another lifetime-based assay [25] that was further optimized to screen for, and identify, the first small-molecule compounds that modulate the cMyBP-C-actin interaction [48]. Before screening a library of smallmolecule compounds, similar optimization will be performed to maximize the effectiveness of C0-C2 Cys225.Cys330 as a HTS biosensor.…”

N-terminal cardiac myosin-binding protein C interactions with myosin and actin filaments using time-resolved FRET

“…Binding of C0-C2 to actin brings the acceptor close enough to the donor to result in resonance energy transfer from donor to acceptor probes. This was detected as a change in lifetime (the time it takes for the donor intensity to decay from its maximum following excitation, to ~37% (1/e)) in the FLTPR (25). Here, we further develop the cMyBP-C actin interaction TR-FRET assays and, for the first time establish an assay to accurately measure interactions between cMyBP-C and myosin.…”

“…We recently developed robust high-throughput fluorescence lifetime-based assays to study the dynamic structure of cMyBP-C (23) and the binding interactions between cMyBP-C and F-actin in normal and diseased states (24). Using one of these assays we have screened 1,280 pharmacologically active compounds and have identified the first three drugs that bind to cMyBP-C and abolish its interactions with F-actin (25). We further characterized these drugs effects on C0-C2-actin binding using a TR-FRET assay (25) that we developed further in this research.…”