Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin-actin interaction

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.

Automated summary

The study found that ten actin-binding compounds affect the interaction between cardiac myosin subfragment 1 (S1) and pyrene-labeled F-actin (PFA), increasing ATP affinity for actin-bound myosin and slowing the transition of actin-myosin interaction in the early ATPase cycle. This work elucidates the mechanisms of action for these compounds, providing valuable insights for potential treatments of hypercontractile forms of cardiomyopathy and future drug discovery efforts for heart failure.