We aim to develop a high-throughput screening (HTS) platform to identify modulators of cardiac myosin-binding protein C (cMyBP-C). The clinical heterogeneity and the complexity of cardiomyopathies make it unlikely that a single therapy will address all aspects of the disease, leaving a critical need for mechanism-specific, cardiac-targeted modulators of contractility. cMyBP-C is an attractive therapeutic target for correcting muscle dysfunction in heart disease; however, no modulators that directly bind to cMyBP-C and alter cardiac muscle function have been reported. Phosphorylation of cMyBP-C is cardioprotective and modulates its interactions with actin and myosin. Drugs that mimic the functional effect of phosphorylation, or otherwise alter cMyBP-C binding to actin or myosin, represent a novel therapeutic strategy. Cardiac myosin inhibitors such as mavacamten and aficamten are approved for obstructive hypertrophic cardiomyopathy (HCM) but are not suitable for non-obstructive HCM, highlighting the urgent need for alternative treatments. To address this gap, we established a dual-wavelength, time-resolved fluorescence resonance energy transfer (FRET)-based HTS to identify small molecules that bind to cMyBP-C, and modulate its interactions. Actin was labeled at C374 with fluorescein-5-maleimide (donor) and the cMyBP-C C0-C2 fragment at C249 with tetramethyl rhodamine (acceptor). We monitored FRET between the donor and acceptor in the presence and absence of compounds. The HTS microplates were then re-screened for effects of the compounds on the environmentally sensitive fluorescence lifetime of the acceptor, indicating direct binding to C0-C2. Screening of a 50,000-compound library yielded several candidates that altered cMyBP-C interactions with actin or myosin in secondary assays. This robust structural screening approach offers an innovative alternative to myosin ATPase activity-based HTS and provides a path toward mechanism-specific therapies for cardiomyopathies.
Guhathakurta et al. (Sun,) studied this question.