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.
Highlights
We previously showed that actin structural dynamics are restricted by C0-C2 binding [6, 7]
The recently described TR-F assay, which monitors cMyBPC’s amino terminal C0-C2 domains binding to actin [15], successfully identified the first three compounds capable of inhibiting C0-C2 interactions with actin. Characterization of this inhibition using a new TR-FRET assay, Isothermal titration calorimetry (ITC), and Transient phosphorescence anisotropy (TPA) indicates that these three compounds bind directly to C0-C2, inhibiting its ability to bind to actin
Much remains to be learned about the effects of these compounds on cardiac muscle contractility, but the approach used in this work clearly indicates that the TR-F assay is suitable for screening thousands of compounds to identify cMyBP-C-binding Hits capable of altering its function
Summary
At 1 μM FMAL-actin plus 2 μM TMR-C0-C2 (the same conditions as the original screen and for the dose–response curves in Fig. 3A), 20 μM suramin and ATA and 50 μM NF023 almost completely eliminated TR-FRET measured binding of both nonphosphorylated and phosphorylated C0-C2 (Fig. 5D). We tested this further by monitoring compound effects on Factin’s structural dynamics by using TPA of actin labeled at Cys-374 with erythrosine iodoacetamide (ErIA) (Fig. 6A).
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