Drug discovery for heart failure: Targeting the myosin-binding protein C interactions with actin.

Abstract

We are using novel fluorescence detection technology to seek small-molecule drugs targeting the interaction of myosin-binding protein C (MyBP-C) with actin. Myosin-targeting drugs have been FDA-approved to treat heart failure (HF) and obstructive hypertrophic cardiomyopathy (HCM), to correct contractile dysfunction. However, cardiomyopathies are diverse, and a variety of drugs may be needed in order to provide adequate treatment options for patients. Cardiac MyBP-C (cMyBP-C) modulates actin-myosin interactions, and these interactions are regulated by phosphorylation. HF patients often have decreased cMyBP-C phosphorylation, and phosphorylation in model systems appears to be cardioprotective for HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic phosphorylation and/or perturb its interactions with actin or myosin. We previously used a fluorescence lifetime-based assay to screen a 1280-compound small-molecule library (LOPAC) and identified the first three cMyBP-C binding compounds that inhibit interactions with actin. In the present study, we have used a novel dual-wavelength time-resolved fluorescence resonance energy transfer (FRET)-based high-throughput screen (HTS) to discover small molecules that can specifically bind to cMyBP-C and affect its interactions with actin or myosin. We have labeled actin at C374 with fluorescein-5-maleimide (donor) and the C0-C2 fragment of cMyBP-C at C249 with tetramethyl rhodamine (acceptor), and then monitored FRET between the donor and acceptor in the presence and absence of compounds. The plates of samples were then re-screened for effects of the compounds on the environmentally-sensitive fluorescence lifetime of the acceptor, indicative of binding of compounds to C0-C2. We screened a 3000-compound FDA-approved library and identified several compounds that affected the interactions of cMyBP-C with actin or myosin in secondary assays. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF/HCM.