Biology subjects

Guhathakurta, P.

Publications and source records attributed to Guhathakurta, P..

5 recordsLinked to original sources

bioRxiv2024

A FRET assay to quantitate levels of the human β-cardiac myosin interacting heads motif based on its near-atomic resolution structure

In cardiac muscle, many myosin molecules are in a resting or "OFF" state with their catalytic heads in a folded structure known as the interacting heads motif (IHM). Many mutations in the human {beta}-cardiac myosin gene that cause hypertrophic cardiomyopathy (HCM) are thought to destabilize (decrease the population of) the IHM state. The effects of pathogenic mutations on the IHM structural state are often studied using indirect assays, including a single-ATP turnover assay that detects the super-relaxed (SRX) biochemical state of myosin functionally. Here we develop and use a fluorescence resonance energy transfer (FRET) based sensor for direct quantification of the IHM state in solution. The FRET sensor was able to quantify destabilization of the IHM state in solution, induced by (a) increasing salt concentration, (b) altering proximal S2 tail length, or (c) introducing the HCM mutation P710R, as well as stabilization of the IHM state by introducing a dilated cardiomyopathy-causing mutation (E525K). Our FRET sensor conclusively showed that these perturbations indeed alter the structural IHM state. These results establish that the structural IHM state is one of the structural correlates of the biochemical SRX state in solution.

biophysics↗

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

Identification of β-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia

{beta}-III-spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells, and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the cytoskeletal protein {beta}-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small molecule actin-binding modulators of the L253P mutant {beta}-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P {beta}-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay, we screened a 2,684-compound library of FDA-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous co-sedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a SCA5-specific therapeutic. Furthermore, our validated FRET-based in vitro HTS platform is poised for screening large compound libraries for {beta}-III-spectrin ABD modulators.

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↗

bioRxiv2020

Actin-binding compounds, discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

We have used spectroscopic and functional assays to evaluate the effects of a group of actin-binding compounds on striated muscle protein structure and function. Actin is present in every human cell, and its interaction with multiple myosin isoforms and multiple actin-binding proteins is essential for cellular viability. A previous high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay from our group identified a class of compounds that bind to actin and affect actomyosin structure and function. In the current study, we tested their effects on the two isoforms of striated muscle -actins, skeletal and cardiac. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that these effects were different for the two actin isoforms, suggesting a different mode of action. To determine the effects of these compounds on sarcomeric function, we further tested their activity on skeletal and cardiac myofibrils. We found that several compounds affected ATPase activity of skeletal and cardiac myofibrils differently, suggesting different mechanisms of action of these compounds for the two muscle types. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

biophysics↗