Biology subjects

Bunch, T. A.

Publications and source records attributed to Bunch, T. A..

3 recordsLinked to original sources

bioRxiv2023

Drug discovery for heart failure targeting myosin-binding protein C

Cardiac MyBP-C (cMyBP-C) interacts with actin-myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces binding of cMyBP-C to actin or myosin, is often decreased in heart failure (HF) patients, and is cardioprotective in model systems 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 labeled actin with fluorescein-5-maleimide (FMAL), and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethyl rhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding and/or structural changes in the protein complex. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fskC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish feasibility for discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

biophysics↗

bioRxiv2022

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

Myosin binding protein-C (cMyBP-C) is a sarcomeric protein responsible for normal contraction and relaxation of the heart. We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to resolve the interactions of cardiac myosin and F-actin with cMyBP-C, focusing on the N-terminal region. The results imply roles of these bound protein complexes in myocardial contraction, with particular relevance to {beta}-adrenergic signaling, heart failure and hypertrophic cardiomyopathy (HCM). N-terminal cMyBP-C domains C0 through C2 (C0-C2) contain binding regions for interactions with both thick (myosin) and thin (actin) filaments. Phosphorylation by protein kinase A (PKA) in the cMyBP-C motif (M-domain) regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C and either myosin or actin. We engineered intermolecular pairs of labeling sites between donor-labeled myosin regulatory light chain (V105C) or F-actin (C374) and cMyBP-C (S85C in C0, C249 in C1, or P330C in M-domain) to detect interactions. Phosphorylation reduced the interaction of cMyBP-C to both myosin and actin. Further insight was gained from evaluating cMyBP-C HCM mutations T59A, R282W, E334K, and L349R, which revealed increases in myosin-FRET, increases or decreases in actin-FRET, and perturbations of phosphorylation effects. These findings elucidate binding of cMyBP-C to myosin or actin under physiological and pathological conditions, providing new molecular insight into the modulatory role of these protein-protein interactions in cardiac muscle contractility. Further, these findings suggest that the TR-FRET assays are suitable for rapid and accurate determination of quantitative binding for screening physiological conditions and compounds that affect cMyBP-C interactions with myosin or F-actin for therapeutic discovery. Significance StatementHypertrophic cardiomyopathy (HCM) is a heritable heart disease involving mutations in genes encoding cardiac muscle proteins. Investigating the underlying molecular mechanisms of HCM mutations provides critical insight into the clinical outcomes and can translate into life-saving therapies. A leading cause of inherited HCM are mutations found in cardiac myosin binding protein-C (cMyBP-C), which binds to both myosin and actin to finely-tune contractility. Efforts in elucidating the details of cMyBP-C interactions with myosin and actin have been limited due to standard techniques that are low-throughput and labor-intensive. We have developed a set of Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assays that report the phosphorylation-sensitive binding of N-terminal cMyBP-C to myosin or actin in a high-throughput plate reader format. We detect altered binding due to phosphorylation and unique changes in HCM mutant cMyBP-C binding to myosin versus actin. Our results are informative for developing precision medicine screening assays and new therapies for HCM.

biophysics↗

bioRxiv2021

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

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-Cs 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.

biophysics↗