Aficamten is a small-molecule cardiac myosin inhibitor designed to treat hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an inherited disease of the sarcomere resulting in excessive cardiac contractility. The first-in-class cardiac myosin inhibitor, mavacamten, improves symptoms in obstructive HCM. Here we present aficamten, a selective small-molecule inhibitor of cardiac myosin that diminishes ATPase activity by strongly slowing phosphate release, stabilizing a weak actin-binding state. Binding to an allosteric site on the myosin catalytic domain distinct from mavacamten, aficamten prevents the conformational changes necessary to enter the strongly actin-bound force-generating state. In doing so, aficamten reduces the number of functional myosin heads driving sarcomere shortening. The crystal structure of aficamten bound to cardiac myosin in the pre-powerstroke state provides a basis for understanding its selectivity over smooth and fast skeletal muscle. Furthermore, in cardiac myocytes and in mice bearing the hypertrophic R403Q cardiac myosin mutation, aficamten reduces cardiac contractility. Our findings suggest aficamten holds promise as a therapy for HCM.

In vitro and in vivo preclinical pharmacokinetic characterization of aficamten, a small molecule cardiac myosin inhibitor.

Aficamten, a small molecule selective inhibitor of cardiac myosin, was characterised in preclinical in vitro and in vivo studies.Protein binding in human plasma was 10.4% unbound and ranged from 1.6% to 24.9% unbound across species. Blood-to-plasma ratios ranged from 0.69 to 1.14 across species. Aficamten hepatic clearance in human was predicted to be low from observed high metabolic stability in vitro in human liver microsomes. Aficamten demonstrated high permeability in Caco-2 cell monolayers.Aficamten in vivo clearance was low across species at 8.8, 2.1, 3.3, and 11 mL/min/kg in mouse, rat, dog, and monkey, respectively. The volume of distribution was low-to-high ranging from 0.53 in rat to 11 L/kg in dog. Oral bioavailability ranged from 41% in monkey to 98% in mouse.Aficamten was metabolised in vitro to eight metabolites with hydroxylated metabolites M1a and M1b predominating. CYP phenotyping indicated multiple CYPs (2C8, 2C9, 2D6, and 3A4) contributing to the metabolism of aficamten.Human clearance (1.1 mL/min/kg) and volume of distribution (6.5 L/kg) were predicted using 4-species allometry employing 'rule-of-exponents'. A predicted 69 hour half-life is consistent with observed half-life in human Phase-1.No CYP-based drug-drug interaction liability as a precipitant was predicted for aficamten.

Pharmacokinetics, mass balance, tissue distribution, metabolism, and excretion of [14C]aficamten following single oral dose administration to rats.

The pharmacokinetics, metabolism, excretion, mass balance, and tissue distribution of [14C]aficamten were evaluated following oral administration of an 8 mg/kg dose in Sprague Dawley rats and in a quantitative whole-body autoradiography study in Long Evans rats.[14C]Aficamten accounted for ∼80% and a hydroxylated metabolite (M1) accounted for ∼12% of total radioactivity in plasma over 48-h (AUC0-48). Plasma tmax was 4-h and the t1/2 of total plasma radioactivity was 5.8-h.Tissues showing highest Cmax exposures were myocardium and semitendinosus muscle.Most [14C]aficamten-derived radioactivity was excreted within 48-h post-administration. Mean cumulative recovery in urine and faeces over 168-h was 8.3% and 90.7%, respectively.In urine and bile, unchanged aficamten was detected at <0.1 and <0.2% of dose, respectively; however, based on total radioactivity excreted in urine (8.0%) and bile (51.7%), approximately 60% of dose was absorbed.[14C]Aficamten was metabolised by hydroxylation with subsequent glucuronidation where the most abundant metabolite recovered in bile was M5 (35.2%), the oxygen-linked glucuronide of hydroxylated aficamten (M1a). The major metabolite detected in faeces was a 1,2,4-oxadiazole moiety ring-cleaved metabolite (M18, 35.3%), shown to be formed from the metabolism of M5 in incubations with rat intestinal contents solution.

Discovery of Nelutroctiv (CK-136), a Selective Cardiac Troponin Activator for the Treatment of Cardiovascular Diseases Associated with Reduced Cardiac Contractility.

Cardiac myosin activation has been shown to be a viable approach for the treatment of heart failure with reduced ejection fraction. Here, we report the discovery of nelutroctiv (CK-136), a selective cardiac troponin activator intended for patients with cardiovascular conditions where cardiac contractility is reduced. Discovery of nelutroctiv began with a high-throughput screen that identified compound 1R, a muscle selective cardiac sarcomere activator devoid of phosphodiesterase-3 activity. Optimization of druglike properties for 1R led to the replacement of the sulfonamide and aniline substituents which resulted in improved pharmacokinetic (PK) profiles and a reduced potential for human drug-drug interactions. In vivo echocardiography assessment of the optimized leads showed concentration dependent increases in fractional shortening and an improved pharmacodynamic window compared to myosin activator CK-138. Overall, nelutroctiv was found to possess the desired selectivity, a favorable pharmacodynamic window relative to myosin activators, and a preclinical PK profile to support clinical development.

Cardiac myosin inhibitor, CK-586, minimally reduces systolic function and ameliorates obstruction in feline hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) remains the most common cardiomyopathy in humans and cats with few preclinical pharmacologic interventional studies. Small-molecule sarcomere inhibitors are promising novel therapeutics for the management of obstructive HCM (oHCM) patients and have shown efficacy in left ventricular outflow tract obstruction (LVOTO) relief. The objective of this study was to explore the 6-, 24-, and 48-hour (h) pharmacodynamic effects of the cardiac myosin inhibitor, CK-586, in six purpose-bred cats with naturally occurring oHCM. A blinded, randomized, five-treatment group, crossover preclinical trial was conducted to assess the pharmacodynamic effects of CK-586 in this oHCM model. Dose assessments and select echocardiographic variables were assessed five times over a 48-h period. Treatment with oral CK-586 safely ameliorated LVOTO in oHCM cats. CK-586 treatment dose-dependently eliminated obstruction (reduced LVOTOmaxPG), increased measures of systolic chamber size (LVIDs Sx), and decreased select measures of heart function (LV FS% and LV EF%) in the absence of impact on heart rate. At all tested doses, a single oral CK-586 dose resulted in improved or resolved LVOTO with well-tolerated, dose-dependent, reductions in LV systolic function. The results from this study pave the way for the potential use of CK-586 in both the veterinary and human clinical setting.

Cardiac Troponin Activator CK-963 Increases Cardiac Contractility in Rats.

Novel cardiac troponin activators were identified using a high throughput cardiac myofibril ATPase assay and confirmed using a series of biochemical and biophysical assays. HTS hit 2 increased rat cardiomyocyte fractional shortening without increasing intracellular calcium concentrations, and the biological target of 1 and 2 was determined to be the cardiac thin filament. Subsequent optimization to increase solubility and remove PDE-3 inhibition led to the discovery of CK-963 and enabled pharmacological evaluation of cardiac troponin activation without the competing effects of PDE-3 inhibition. Rat echocardiography studies using CK-963 demonstrated concentration-dependent increases in cardiac fractional shortening up to 95%. Isothermal calorimetry studies confirmed a direct interaction between CK-963 and a cardiac troponin chimera with a dissociation constant of 11.5 ± 3.2 µM. These results provide evidence that direct activation of cardiac troponin without the confounding effects of PDE-3 inhibition may provide benefit for patients with cardiovascular conditions where contractility is reduced.

Effects of Aficamten on cardiac contractility in a feline translational model of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease in humans and cats and lacks efficacious pharmacologic interventions in the preclinical phase of disease. LV outflow tract obstruction (LVOTO) is commonly observed in HCM-affected patients and is a primary driver of heart failure symptoms and reduced quality of life. Novel small-molecule cardiac myosin inhibitors target actin-myosin interactions to alleviate overactive protein interactions. A prospective, randomized, controlled cross-over study was performed to evaluate pharmacodynamic effects of two doses (0.3 and 1 mg/kg) of a next-in-class cardiac myosin inhibitor, aficamten (CK-3773274, CK-274), on cardiac function in cats with the A31P MYBPC3 mutation and oHCM. Dose-dependent reductions in LV systolic function, LVOT pressure gradient, and isovolumetric relaxation times compared to baseline were observed. Promising beneficial effects of reduced systolic function warrant further studies of this next-in-class therapeutic to evaluate the benefit of long-term administration in this patient population.

Pharmacokinetics of a single dose of Aficamten (CK-274) on cardiac contractility in a A31P MYBPC3 hypertrophic cardiomyopathy cat model.

Hypertrophic cardiomyopathy (HCM) is the most prevalent cardiac disease in cats and lacks efficacious preclinical pharmacologic intervention, prompting investigation of novel therapies. Genetic mutations encoding sarcomeric proteins are implicated in the development of HCM and small molecule myosin inhibitors are an emerging class of therapeutics designed to target the interaction of actin and myosin to alleviate the detrimental effects of inappropriate contractile protein interactions. The purpose of this study was to characterize the pharmacodynamic effects of a single oral dose of the novel cardiac myosin inhibitor aficamten (CK-274) on cardiac function in purpose bred cats with naturally occurring A31P MYBPC3 mutation and a clinical diagnosis of HCM with left ventricular outflow tract obstruction (LVOTO). Five purpose bred cats were treated with aficamten (2 mg/kg) or vehicle and echocardiographic evaluations were performed at 0, 6, 24, and 48 h post-dosing. High dose aficamten (2 mg/kg) reduced left ventricular fractional shortening (LVFS%) by increasing the LV systolic internal dimension (LVIDs) and reduced isovolumic relaxation time (IVRT) compared with baseline without significant adverse effects. The marked reduction in systolic function and reduced IVRT coupled with an increased heart rate in treated cats, suggest a lower dose may be optimal. Further studies to determine optimal dosing of aficamten are indicated.

Discovery of Reldesemtiv, a Fast Skeletal Muscle Troponin Activator for the Treatment of Impaired Muscle Function.

The discovery of reldesemtiv, a second-generation fast skeletal muscle troponin activator (FSTA) that increases force production at submaximal stimulation frequencies, is reported. Property-based optimization of high throughput screening hit 1 led to compounds with improved free exposure and in vivo muscle activation potency compared to the first-generation FSTA, tirasemtiv. Reldesemtiv demonstrated increased muscle force generation in a phase 1 clinical trial and is currently being evaluated in clinical trials for the treatment of amyotrophic lateral sclerosis.

Discovery of Aficamten (CK-274), a Next-Generation Cardiac Myosin Inhibitor for the Treatment of Hypertrophic Cardiomyopathy.

Hypercontractility of the cardiac sarcomere may be essential for the underlying pathological hypertrophy and fibrosis in genetic hypertrophic cardiomyopathies. Aficamten (CK-274) is a novel cardiac myosin inhibitor that was discovered from the optimization of indoline compound 1. The important advancement of the optimization was discovery of an Indane analogue (12) with a less restrictive structure-activity relationship that allowed for the rapid improvement of drug-like properties. Aficamten was designed to provide a predicted human half-life (t1/2) appropriate for once a day (qd) dosing, to reach steady state within two weeks, to have no substantial cytochrome P450 induction or inhibition, and to have a wide therapeutic window in vivo with a clear pharmacokinetic/pharmacodynamic relationship. In a phase I clinical trial, aficamten demonstrated a human t1/2 similar to predictions and was able to reach steady state concentration within the desired two-week window.

Discovery of Tirasemtiv, the First Direct Fast Skeletal Muscle Troponin Activator.

The identification and optimization of the first activators of fast skeletal muscle are reported. Compound 1 was identified from high-throughput screening (HTS) and subsequently found to improve muscle function via interaction with the troponin complex. Optimization of 1 for potency, metabolic stability, and physical properties led to the discovery of tirasemtiv (25), which has been extensively characterized in clinical trials for the treatment of amyotrophic lateral sclerosis.

Discovery of GBT440, an Orally Bioavailable R-State Stabilizer of Sickle Cell Hemoglobin.

We report the discovery of a new potent allosteric effector of sickle cell hemoglobin, GBT440 (36), that increases the affinity of hemoglobin for oxygen and consequently inhibits its polymerization when subjected to hypoxic conditions. Unlike earlier allosteric activators that bind covalently to hemoglobin in a 2:1 stoichiometry, 36 binds with a 1:1 stoichiometry. Compound 36 is orally bioavailable and partitions highly and favorably into the red blood cell with a RBC/plasma ratio of ∼150. This partitioning onto the target protein is anticipated to allow therapeutic concentrations to be achieved in the red blood cell at low plasma concentrations. GBT440 (36) is in Phase 3 clinical trials for the treatment of sickle cell disease (NCT03036813).

Highly selective inhibition of myosin motors provides the basis of potential therapeutic application.

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the "recovery stroke" transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.

Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases.

Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.

Cardiac myosin activation part 1: from concept to clinic.

Decreased cardiac contractility is a central feature of systolic heart failure and yet we have no effective drugs to improve cardiac contractility. Existing drugs that increase cardiac contractility do so indirectly through signaling cascades and their use is limited by their mechanism-related adverse effects. Direct activation of the cardiac sarcomere to increase cardiac contractility may provide a means to avoid these limitations. Using a reconstituted version of the cardiac sarcomere, we screened a small molecule library and identified several chemical classes that directly activate cardiac myosin. One compound class has been optimized extensively using an iterative process; omecamtiv mecarbil, a small-molecule, selective, cardiac myosin activator is the most advanced exemplar of this novel mechanistic class. It accelerates the transition of myosin into the force-generating state without affecting cardiac myocyte calcium homeostasis. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Initial clinical studies have demonstrated the translation of this mechanism into humans, and further clinical studies of its use in acute and chronic heart failure are planned. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."

Cardiac myosin activation: a potential therapeutic approach for systolic heart failure.

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Discovery of the First Potent and Selective Inhibitor of Centromere-Associated Protein E: GSK923295.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Discovery of omecamtiv mecarbil the first, selective, small molecule activator of cardiac Myosin.

We report the design, synthesis, and optimization of the first, selective activators of cardiac myosin. Starting with a poorly soluble, nitro-aromatic hit compound (1), potent, selective, and soluble myosin activators were designed culminating in the discovery of omecamtiv mecarbil (24). Compound 24 is currently in clinical trials for the treatment of systolic heart failure.

Octahydrophenanthrene-2,7-diol analogues as dissociated glucocorticoid receptor agonists: discovery and lead exploration.

As exemplified by the lead compound 2, octahydrophenanthrene-2,7-diol analogues exhibit the profile of a pathway-selective or "dissociated" agonist of the glucocorticoid receptor (GR), retaining the potent activity that glucocorticoids have for transrepression (as measured by inhibition of IL-1 induced MMP-13 expression) but showing an attenuated capacity for transactivation (as measured in an MMTV luciferase reporter assay). With the guidance of a homology model of the GR ligand binding domain, structural modifications to 2 were carried out that were successful in replacing the allyl and propynyl side chains with groups likely to be more chemically stable and less likely to produce toxic metabolites. Key to success was the introduction of an additional hydroxyl group onto the tricyclic carbon framework of the series.

In vitro and in vivo characterization of 3-[2-[6-(2-tert-butoxyethoxy)pyridin-3-yl]-1H-imidazol-4-yl]benzonitrile hydrochloride salt, a potent and selective NPY5 receptor antagonist.

To investigate the anorectic potential of NPY5 receptor antagonists, we have profiled the in vitro and in vivo properties of 3-[2-[6-(2-tert-butoxyethoxy)pyridin-3-yl]-1H-imidazol-4-yl]benzonitrile hydrochloride salt (1). This compound was found to have excellent NPY5 receptor affinity and selectivity, potent functional antagonism, and good peripheral and central nervous system exposure in rats. This compound attenuated bovine pancreatic polypeptide induced food intake in rats but failed to demonstrate anorectic activity in rodent natural feeding models.