A small-molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle

Abstract

Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of β-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from β-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.

Keywords: MYK-461; cardiac hypertrophy; cardiomyopathy; myosin; presteady-state kinetics; sarcomere; small molecule.

Figure 1.

Chemical structure of mavacamten.

Figure 2.

Concentration-dependent response curve of mavacamten (MYK-461) in multiple myofibril-based systems. Myofibrils were assayed at pCa50 values of 6.25, 6.00, and 6.00 for bovine cardiac (1 mg/ml), rabbit skeletal (0.25 mg/ml), and human cardiac (1 mg/ml) respectively. Final assay conditions contained 2% DMSO with varying concentrations of mavacamten. Data were normalized to a DMSO control for each myofibril type assayed and analyzed using a four parameter fit (GraphPad Prism) to determine IC₅₀ values of mavacamten. DMSO control rates of 0.097 ± 0.0002 μm/s, 0.158 ± 0.01 μm/s, and 0.104 ± 0.005 μm/s were measured for bovine cardiac, rabbit skeletal, and human cardiac myofibrils, respectively.

Figure 3.

Mavacamten (MYK-461) decreased in vitro motility-sliding velocities. The final in vitro motility buffer contained 2% DMSO with or without mavacamten at varying concentrations. Sliding velocities were normalized to DMSO only velocities. n = 3 for all data points. For each n, >500 filament trajectories were quantified, and the median velocity from each of the 3 data sets was averaged.

Figure 4.

Concentration-dependent response of mavacamten (MYK-461) in purified actomyosin systems. Bovine cardiac (0.25 μm), rabbit skeletal (0.1 μm), chicken gizzard (0.5 μm), and human cardiac (0.25 μm) myosin-S1 were assayed with a constant concentration of actin (14 μm) at varying concentrations of mavacamten. ATPase rates were normalized to DMSO controls for each myosin type assayed, and EC₅₀ values were calculated using a four-parameter-fit model in Prism (GraphPad). DMSO controls rates of 0.145 ± 0.002 μm/s, 0.121 ± 0.001 μm/s, 0.154 ± 0.001 μm/s, and 0.134 ± 0.002 μm/s were measured for bovine cardiac, rabbit skeletal, chicken gizzard, and human cardiac myosin-S1, respectively.

Figure 5.

Concentration-dependent response of mavacamten (MYK-461) in purified human actomyosin systems. A, human cardiac myosin-S1 (0.25 μm) and all mutants were assayed with a constant concentration of actin (14 μm) at varying concentrations of mavacamten. B, an expanded panel, showing the subtle differences in the potency of mavacamten against all mutant myosins assayed. ATPase rates were normalized to DMSO controls for each myosin type assayed, and EC₅₀ values were calculated using a four-parameter-fit model in Prism (GraphPad). DMSO controls rates of 0.269 ± 0.002 μm/s, 0.142 ± 0.001 μm/s, 0.138 ± 0.002 μm/s, 0.184 ± 0.001 μm/s, 0.135 ± 0.001 μm/s, and 0.078 ± 0.001 μm/s were measured for wild-type, R403Q, R453C, R719W, R723G, and G741R human cardiac myosin-S1, respectively.

Figure 6.

Generalized chemomechanical cycle of myosin. M is defined as myosin, A as actin, and P_i as inorganic phosphate.

Figure 7.

Phosphate release of myosin-S1 with varying concentrations of mavacamten (MYK-461). Myosin-S1 (0.5 μm final) was mixed with ATP (0.5 μm final) and aged for 2 s. This solution was then mixed with actin (14 μm final) and PBP (10 μm final), the fluorescence change upon binding of inorganic phosphate was recorded at an excitation wavelength of 430 nm, and emission was monitored through a 455-nm cutoff filter. Data were been normalized to DMSO controls. IC₅₀ values of 1.85 ± 0.14 μm (bovine cardiac, A) and 1.78 ± 0.069 μm (human cardiac, B) were calculated using a four-parameter-fit model in Prism (GraphPad). A DMSO controls rate of 2.72 ± 0.16 s⁻¹ and 0.257 ± 0.025 s⁻¹ was measured for bovine cardiac and human cardiac myosin, respectively.

Figure 8.

Mant-ADP release experiments were performed in both the basal (A)- and actin-associated (B) states of bovine cardiac myosin-S1, and representative traces are shown. In the basal (actin-free) state, bovine cardiac myosin-S1 (1.25 μm final) was incubated with mant-ADP (1 μm final) in the presence of 2% DMSO or 20 μm mavacamten (MYK-461) and rapidly mixed with 1 mm ATP. The fluorescence decrease of the mant-ADP was followed and fit to a single exponential, yielding mant-ADP release rates of 0.2616 ± 0.01455 s⁻¹ and 0.1402 ± 0.010356 s⁻¹ for DMSO and mavacamten, respectively. In the actin-associated state, bovine cardiac myosin-S1 (1.25 μm final), actin (4 μm final), and mant-ADP (1 μm final) were incubated and rapidly mixed with 1 mm ATP. The rate of actin-associated mant-ADP release was measured to be 128.9 ± 3.10 s⁻¹ and 132.2 ± 3.05 s⁻¹, for DMSO and mavacamten, respectively.

Figure 9.

Actin association measured via pyrene actin in the ADP state. Bovine cardiac myosin-S1 (0.25 μm final) was rapidly mixed with pyrene actin (10 μm final) in an ADP-bound state (1.25 μm final). The pyrene actin fluorescence decrease was recorded and fit to a single exponential. This method was done over a range of pyrene actin concentrations in the presence and absence of mavacamten (MYK-461). It was found that the second order rate constant of myosin-S1 binding was reduced ∼4-fold in the presence of mavacamten.

Figure 10.

Weak to strong transition of myosin-ADP-P_i binding to pyrene actin in the presence of 1 mm ADP. Single turnover conditions in double mix mode are shown. Myosin-S1 (0.5 μm final) and ATP (0.5 μm final) were mixed and aged for 2 s and then mixed with 0.5 μm pyrene actin and 1 mm ADP. Representative traces show that increasing concentrations of mavacamten (MYK-461) reduce the amplitude of pyrene quenching caused by fewer myosin heads interacting with the actin filament.