Cardiac Myosin Strutural Kinetics are Modulated by Myk461

Abstract

The biochemical and structural kinetics of bovine cardiac myosin are perturbed by the putative heart failure therapeutic Myk461. Myosin's lever arm undergoes structural changes that are coupled to its biochemical state during ATPase cycling in muscle. Structural states of myosin's lever arm can be detected directly using the high-precision, high-throughput technique of transient time-resolved FRET revealing kinetics, thermodynamics, and allosteric coupling. We have previously used this technique to investigate mechanochemical coupling in skeletal myosin and cardiac myosin perturbed by the heart failure drug omecamtiv mecarbil. Here we investigate the MyoKardia compound Myk461, in clinical trials for treating hypertrophic cardiomyopathy. We find that the small molecule therapeutic primarily inhibits actomyosin association, both in the absence and presence of nucleotide. This inhibition is also detected in the coupled kinetic steps following actin activation of myosin: the force-producing structural isomerization known as the powerstroke, and the principal free energy-driven biochemical step of Pi release. Other actin-dependent experiments reveal a potent inhibition by Myk461, steady-state ATPase cycling and single-turnover of fluorescent ATP. We also find that the drug's affinity for myosin varies by two-orders of magnitude, dependent on the structural and biochemical state of myosin: binding strongest to actomyosin-ATP and weakest to apo myosin. This work was supported by NIH AR032961 & AR057220 (DDT), American Heart Association Scientist Development Grant (JMM), and Graduate Excellence Fellowship-University of MN (JAR).