Actin-binding compounds that affect the kinetics of the interaction of cardiac myosin with actin

We measured 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 microsecond structural dynamics of actin and the steady-state activity of actin-activated myosin ATPase. In the present study, we have further characterized their mechanisms of action by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin, and is restored upon release of S1 by MgATP. We measured the effects of compounds under equilibrium and steady-state conditions, as affected by S1 and ATP, and also under transient conditions, in stopped-flow experiments following rapid addition of ATP to S1-bound PFA. We observe that these compounds affect the early steps of the myosin ATPase cycle to different extents (mild, moderate, and severe). The compounds decrease the equilibrium constant for the formation of the collision complex and the rate constant for subsequent isomerization to the ternary complex, indicating increased ATP affinity and trapping of ATP in the myosin active site. These compound effects on actin structure inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for possible treatment of hypercontractile forms of hypertrophic cardiomyopathy (HCM). This work helps to elucidate the mechanisms of action of these compounds, several of which are currently used therapeutically, and it sets the stage for future HTS campaigns on a larger scale, to discover new drugs for treatment of heart failure.