Mavacamten stabilizes the auto-inhibited state of two-headed cardiac myosin

Abstract

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an auto-inhibited state of two-headed cardiac myosin, not found in the isolated S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single ATP turnover kinetics. A two-headed myosin fragment exhibits distinct auto-inhibited ATP turnover kinetics compared to a single-headed fragment. Mavacamten enhanced this auto-inhibition. It also enhanced auto-inhibition of ADP release. Furthermore, actin changes the structure of the auto-inhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.\n\nSignificance StatementSmall-molecule allosteric effectors designed to target and modulate striated and smooth myosin isoforms for the treatment of disease show promise in preclinical and clinical trials. Beta-cardiac myosin is an especially important target, as heart disease remains a primary cause of death in the U.S. One prevalent type of heart disease is hypertrophic cardiomyopathy (HCM), which is hypothesized to result from dysregulated force generation by cardiac myosin. Mavacamten is a potent cardiac myosin ATPase activity inhibitor that improves cardiac output in HCM animal models. Our results show that mavacamten selectively stabilizes a two-head dependent, auto-inhibited state of cardiac myosin in solution. The kinetics and energetics of this state are consistent with the auto-inhibited super-relaxed state, previously only observed in intact sarcomeres.