Mavacamten is shown to have selectivity of >4-fold for cardiac myosin, with IC50 values of 490 nM in the bovine system, 711 nM in the human system, and 2140 nM in the rabbit system[1].

Mavacamten treatment lowers FS from 52±3% to 38±7%. Mavacamten treatment lowers FS from 81±7% to 60±13%, which is a 25% relative reduction. There is a linear relationship between FS and Mavacamten plasma concentrations across all assays; for every 100 ng/mL rise in Mavacamten concentration, FS is lowered by 4.9%[2]. By lowering the cardiac myosin heavy chain's adenosine triphosphatase activity, mavacamten decreases contractility. In mice with heterozygous human mutations in the myosin heavy chain, chronic Mavacamten treatment inhibits the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression[3].

Steady-state characterization [1]
ATPase measurements were conducted using a coupled enzyme system utilizing pyruvate kinase and lactate dehydrogenase. Unless otherwise stated, the buffer system used in all experiments was 12 mm Pipes, 2 mm MgCl2, 1 mm DTT at pH 6.8 (PM12 buffer). All steady-state experiments were carried out at 20 °C using a SpectraMax 384Plus plate reader, and rates were recorded using the SoftMax Pro software package. For all steady-state experiments, data were collected in triplicate and averaged, with n = 3. The value for n refers to the number of individual experiments performed. All data analysis of the steady-state systems were conducted using GraphPad Prism.

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Transient kinetic characterization[1]
Transient kinetic experiments were performed using a stopped-flow apparatus (Hi-Tech Scientific, SF-61 DX2) to determine the effects of mavacamten on myosin association and dissociation from actin filaments, phosphate (Pi) release, and 2′-(or-3′)-O-(N-methylanthraniloyl)-ADP (mant-ADP/ATP) release by myosin. For each data point, transient traces were collected in triplicate and averaged for each experiment, with n = 3. All transient experiments were performed with either varying amounts of mavacamten or single concentrations of mavacamten at varying substrate concentrations to determine a concentration-dependent change in each kinetic parameter, and control experiments were carried out with 2% DMSO final. For mant-ATP or mant-ADP experiments, fluorescence emission was measured through a 400-nm cutoff filter with excitation at 365 nm. The increase in fluorescence upon myosin binding mant-ATP or decrease in fluorescence after release of mant-ADP was monitored as previously described.[1]
The rates of Pi release were measured using the bacterial phosphate-binding protein (PBP) modified with 7-diethyl-amino-3-[[[2-(maleimidyl)ethyl]amino]carbonyl] coumarin (MDCC) dye prepared according to Brune et al. The stopped flow instrument was set up in double mix mode. In this configuration nucleotide free myosin-S1 was mixed with ATP at a 1:1 molar ratio and aged for 2 s to allow for complete hydrolysis. The myosin-nucleotide complex was then rapidly mixed with actin plus MDCC-PBP, and the fluorescence increase due to phosphate binding was measured through a 455-nm cutoff filter with excitation at 425 nm. This system was used to measure the effect of mavacamten by varying the concentration of compound in all syringes and comparing the data to a DMSO control. Before data collection, contaminating phosphate was removed from the system by soaking with a “Pi-mop,” which consisted of purine nucleoside phosphorylase and 7-methylguanosine at concentrations of 1 units/ml and 0.5 mm, respectively. This Pi-mop was also present in all solutions at concentrations of 0.1 units/ml purine nucleoside phosphorylase and 0.25 mm 7-methylguanosine to remove any residual phosphate.[1]
Myosin-S1 association to pyrene-actin filaments was monitored by the quenching of pyrene fluorescence that occurs upon S1 binding to pyrene-actin. The kinetics of ATP-induced acto-S1 dissociation was measured by monitoring the increase in pyrene fluorescence upon mixing pyrene-acto-S1 with increasing concentrations of ATP. Pyrene fluorescence was measured using a 400-nm cutoff filter with excitation at 360 nm. This interaction was also used to monitor the transition from the weakly to strongly bound state of myosin to actin. Briefly, bovine cardiac myosin-S1 and ATP were mixed under single turnover conditions and allowed to age for 2 s to hydrolyze the ATP to ADP-Pi. This mixture was then mixed with pyrene actin in a 1:1 ratio with myosin and 1 mm ADP to shift the equilibrium to the strongly bound state. The quenching of pyrene actin was monitored with varying concentrations of mavacamten, and the reaction amplitudes were analyzed.[1]

Cardiac myofibrils were prepared as previously described. Bovine cardiac tissue was harvested, placed immediately on wet ice, and shipped overnight, and the left ventricle and septum were dissected, frozen in liquid nitrogen, and stored at −80 °C. Human tissue was procured from BioReclamations IVT, and myofibrils were prepared on the day of receipt. Cardiac and skeletal myosin S1 was prepared using a chymotryptic digestion of full-length myosin prepared from bovine cardiac left ventricle and rabbit psoas muscle, respectively. Bovine cardiac HMM was prepared according to Margossian and Lowey. Human cardiac myosin subfragment-1 was expressed in differentiated murine C2C12 myotubes using an adenovirus infection method. The recombinant product utilized a 6×-histidine tag on the essential light chain for initial purification on Ni2+-resin with further purification by anion exchange and size exclusion chromatography. All myofibril and myosin-S1 preparations were brought to 10% sucrose, snap-frozen in liquid nitrogen, and stored at −80 °C. Actin was prepared from a bovine cardiac acetone powder (Pel Freez Biologicals) according to the method of Spudich and Watt. Pyrene actin was prepared according to the method of Criddle et al.[1]

[1]. Kawas RF, et al. A small-molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle. J Biol Chem. 2017 Oct 6;292(40):16571-16577.
[2]. Stern JA, et al. A Small Molecule Inhibitor of Sarcomere Contractility Acutely Relieves Left Ventricular Outflow Tract Obstruction in Feline Hypertrophic Cardiomyopathy. PLoS One. 2016 Dec 14;11(12):e0168407.
[3]. Green EM, et al. A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice. Science. 2016 Feb 5;351(6273):617-21

C15H19N3O2

273.336

273.1477

C, 65.91; H, 7.01; N, 15.37; O, 11.71

1642288-47-8

Mavacamten-d6;2453251-18-6;Mavacamten-d1;2453251-02-8;Mavacamten-d5;2453251-00-6

White to off-white solid powder

2.1

61.4Ų

O=C1N(C(C)C)C(C=C(N[C@H](C2=CC=CC=C2)C)N1)=O

RLCLASQCAPXVLM-NSHDSACASA-N

InChI=1S/C15H19N3O2/c1-10(2)18-14(19)9-13(17-15(18)20)16-11(3)12-7-5-4-6-8-12/h4-11,16H,1-3H3,(H,17,20)/t11-/m0/s1

(S)-3-isopropyl-6-((1-phenylethyl)amino)pyrimidine-2,4(1H,3H)-dione

MYK 461; SAR-439152; MYK-461; SAR 439152; SAR439152; MYK461; Mavacamten

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~304.87 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.61 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)