1. Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin-actin interaction.
- Author
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Roopnarine O and Thomas DD
- Subjects
- Actins drug effects, Adenosine Triphosphatases drug effects, Adenosine Triphosphatases metabolism, Animals, Cardiac Myosins drug effects, Cardiac Myosins physiology, Cattle, Fluorescence, High-Throughput Screening Assays methods, Kinetics, Muscle Contraction physiology, Myosin Subfragments drug effects, Myosin Subfragments metabolism, Myosins drug effects, Myosins metabolism, Physics, Protein Binding, Pyrenes chemistry, Rabbits, Small Molecule Libraries pharmacology, Actins chemistry, Actins metabolism, Cardiac Myosins metabolism
- Abstract
Actin-myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K
1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin-myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure., Competing Interests: Conflict of interest D. D. T. holds equity in, and serves as President of, Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study. The authors declare no conflicts of interest in regard to this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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