Bradley P. Morgan

Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure

A small molecule improves cardiac function by accelerating the transition of myosin into a force-producing state. 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.

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.

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.

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 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.

Significance Defects in myosin function are linked to a number of widespread and debilitating diseases, including asthma, chronic obstructive pulmonary disease, and hypertrophic cardiomyopathy. We report here the discovery of an allosteric site that modulates myosin motor function with high specificity that opens the path toward new therapeutic solutions. Identification of specific antimyosin drugs that significantly alter a motor’s function is an imperative first step toward the development of targeted and effective treatments for such diseases. Highly specific drugs against different members of the superfamily would also provide exquisite tools to investigate in cells their functional role. Additionally, detailed, high-resolution studies of the interaction of drugs with their myosin targets provide insights into the molecular mechanism of motor function. 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.

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.

The Fast Skeletal Troponin Activator, CK-1909178 Reduces Muscle Fatigue in a Model of Peripheral Artery Disease in Situ

CK-1909178 is a member of a class of fast skeletal troponin activators that sensitize skinned skeletal muscle fibers to calcium. In rat muscle preparations in vitro and in situ, CK-1909178 increased sub-tetanic force without altering maximum force. Given that a major cause of muscle fatigue during repeated muscle contraction is reduced myoplasmic Ca2+ due to impaired sarcoplasmic reticulum Ca2+ release, we tested whether increased calcium sensitivity with CK-1909178 would slow the development of fatigue. Rat flexor digitorum brevis muscle was pretreated in vitro with CK-1909178 and stimulated every 3 seconds at a frequency sufficient to achieve 50% of maximum force for 6 min at 30°C. CK-1909178 diminished the extent of fatigue as compared to control (terminal force 29.5±8% vs. 12.7±4%, p<0.001). We next tested whether CK-1909178 treatment would slow the development of muscle fatigue using rat extensor digitorum longus muscle in situ, where the muscle was stimulated via the peroneal nerve. To accelerate the development of muscle fatigue, vascular insufficiency was produced by femoral artery ligation (FAL). Muscle fatigue with FAL and sham ligation in the presence and absence of CK-1909178 was assessed. CK-1909178 was administered as a 5mg/kg intravenous bolus before assessment of fatigue at a frequency adjusted to achieve the same force at 30Hz prior to dosing. FAL resulted in significantly reduced terminal tension as compared to sham (33±4% vs. 77±5%, p<0.01). CK-1909178 administration significantly attenuated FAL-induced fatigue at 10 minutes (61±7% vs. 33±4%, p<0.01). In summary, CK-1909178 increased sub-maximal muscle force development and reduced the extent of fatigue in the presence of limited blood flow in situ. We believe that this mechanism may improve muscle fatigue in diseases where blood flow to muscles is compromised such as intermittent claudication.

The Small Molecule Skeletal Sarcomere Activator, CK-2017357, is a Calcium Sensitizer that Binds Selectively to the Fast Skeletal Troponin Complex

Striated muscle contraction is governed by the release of Ca2+ from the sarcoplasmic reticulum via the sarcomeric calcium sensor, the troponin complex. A trimer consisting of troponins T, I, and C, the complex undergoes calcium-dependent conformational changes that regulate the accessibility of myosin binding sites along actin filaments. We used a high throughput screen to identify compounds that activate the ATPase activity of skinned fast skeletal myofibrils; optimization of the initial hit compounds has resulted in compounds with improved potency and medicinal chemical properties. The most advanced exemplar of this chemical series, CK-2017357, shifts the calcium sensitivity of detergent-skinned fast skeletal myofibrils by >10-fold in a concentration dependent manner. This compound specifically activates fast skeletal myofibrils, with no effect on either slow skeletal or cardiac myofibrils. A reconstituted sarcomere assay using combinations of fast skeletal, slow skeletal, and cardiac components demonstrates that the activity of CK-2017357 requires the presence of fast skeletal troponin. Isothermal titration calorimetry indicates the compound binds directly to fast skeletal troponin with a sub-micromolar dissociation constant, while experiments with the fluorescent calcium chelator Quin-2 demonstrate that CK-2017357 slows calcium dissociation from troponin. Consistent with this ability to stabilize the calcium-troponin complex, CK-2017357 increases sub-maximal force development in vitro and in vivo, suggesting this mechanism may increase power or strength in diseases where muscle function is compromised due to injury, disease or age.