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.

Fast skeletal muscle troponin activator CK-2066260 increases fatigue resistance by reducing the energetic cost of muscle contraction.

KEY POINTS: Skeletal muscle fatigue limits performance in various physical activities, with exercise intolerance being a key symptom in a broad spectrum of diseases. We investigated whether a small molecule fast skeletal troponin activator (FSTA), CK-2066260, can mitigate muscle fatigue by reducing the cytosolic free [Ca2+ ] required to produce a given submaximal force and hence decreasing the energy requirement. Isolated intact single mouse muscle fibres and rat muscles in-situ treated with CK-2066260 showed improved muscle endurance., which was accompanied by decreased ATP demand and reduced glycogen usage. CK-2066260 treatment improved in-vivo exercise capacity in healthy rats and in a rat model of peripheral artery insufficiency. In conclusion, we show that the FSTA CK-2066260 effectively counteracts muscle fatigue in rodent skeletal muscle in vitro, in situ, and in vivo. This may translate to humans and provide a promising pharmacological treatment to patients suffering from severe muscle weakness and exercise intolerance. ABSTRACT: Skeletal muscle fatigue limits performance during physical exercise and exacerbated muscle fatigue is a prominent symptom among a broad spectrum of diseases. The present study investigated whether skeletal muscle fatigue is affected by the fast skeletal muscle troponin activator (FSTA) CK-2066260, which increases myofibrillar Ca2+ sensitivity and amplifies the submaximal force response. Because more force is produced for a given Ca2+ , we hypothesized that CK-2066260 could mitigate muscle fatigue by reducing the energetic cost of muscle activation. Isolated single mouse muscle fibres were fatigued by 100 repeated 350 ms contractions while measuring force and the cytosolic free [Ca2+ ] or [Mg2+ ] ([Mg2+ ]i ). When starting fatiguing stimulation at matching forces (i.e. lower stimulation frequency with CK-2066260): force was decreased by ∼50% with and by ∼75% without CK-2066260; [Mg2+ ]i was increased by ∼10% with and ∼32% without CK-2066260, reflecting a larger decrease in [ATP] in the latter. The glycogen content in in situ stimulated rat muscles fatigued by repeated contractions at matching forces was about two times higher with than without CK-2066260. Voluntary exercise capacity, assessed by rats performing rotarod exercise and treadmill running, was improved in the presence of CK-2066260. CK-2066260 treatment also increased skeletal muscle fatigue resistance and exercise performance in a rat model of peripheral artery insufficiency. In conclusion, we demonstrate that the FSTA CK-2066260 mitigates skeletal muscle fatigue by reducing the metabolic cost of force generation.

The Ca2+ sensitizer CK-2066260 increases myofibrillar Ca2+ sensitivity and submaximal force selectively in fast skeletal muscle.

KEY POINTS: We report that the small molecule CK-2066260 selectively slows the off-rate of Ca2+ from fast skeletal muscle troponin, leading to increased myofibrillar Ca2+ sensitivity in fast skeletal muscle. Rodents dosed with CK-2066260 show increased hindlimb muscle force and power in response to submaximal rates of nerve stimulation in situ. CK-2066260 has no effect on free cytosolic [Ca2+ ] during contractions of isolated muscle fibres. We conclude that fast skeletal muscle troponin sensitizers constitute a potential therapy to address an unmet need of improving muscle function in conditions of weakness and premature muscle fatigue. ABSTRACT: Skeletal muscle dysfunction occurs in many diseases and can lead to muscle weakness and premature muscle fatigue. Here we show that the fast skeletal troponin activator, CK-2066260, counteracts muscle weakness by increasing troponin Ca2+ affinity, thereby increasing myofibrillar Ca2+ sensitivity. Exposure to CK-2066260 resulted in a concentration-dependent increase in the Ca2+ sensitivity of ATPase activity in isolated myofibrils and reconstituted hybrid sarcomeres containing fast skeletal muscle troponin C. Stopped-flow experiments revealed a ∼2.7-fold decrease in the Ca2+ off-rate of isolated troponin complexes in the presence of CK-2066260 (6 vs. 17 s-1 under control conditions). Isolated mouse flexor digitorum brevis fibres showed a rapidly developing, reversible and concentration-dependent force increase at submaximal stimulation frequencies. This force increase was not accompanied by any changes in the free cytosolic [Ca2+ ] or its kinetics. CK-2066260 induced a slowing of relaxation, which was markedly larger at 26°C than at 31°C and could be linked to the decreased Ca2+ off-rate of troponin C. Rats dosed with CK-2066260 showed increased hindlimb isometric and isokinetic force in response to submaximal rates of nerve stimulation in situ producing significantly higher absolute forces at low isokinetic velocities, whereas there was no difference in force at the highest velocities. Overall muscle power was increased and the findings are consistent with a lack of effect on crossbridge kinetics. In conclusion, CK-2066260 acts as a fast skeletal troponin activator that may be used to increase muscle force and power in conditions of muscle weakness.

The small-molecule fast skeletal troponin activator, CK-2127107, improves exercise tolerance in a rat model of heart failure.

Heart failure-mediated skeletal myopathy, which is characterized by muscle atrophy and muscle metabolism dysfunction, often manifests as dyspnea and limb muscle fatigue. We have previously demonstrated that increasing Ca(2+) sensitivity of the sarcomere by a small-molecule fast skeletal troponin activator improves skeletal muscle force and exercise performance in healthy rats and models of neuromuscular disease. The objective of this study was to investigate the effect of a novel fast skeletal troponin activator, CK-2127107 (2-aminoalkyl-5-N-heteroarylpyrimidine), on skeletal muscle function and exercise performance in rats exhibiting heart failure-mediated skeletal myopathy. Rats underwent a left anterior descending coronary artery ligation, resulting in myocardial infarction and a progressive decline in cardiac function [left anterior descending coronary artery heart failure (LAD-HF)]. Compared with sham-operated control rats, LAD-HF rat hindlimb and diaphragm muscles exhibited significant muscle atrophy. Fatigability was increased during repeated in situ isokinetic plantar flexor muscle contractions. CK-2127107 produced a leftward shift in the force-Ca(2+) relationship of skinned, single diaphragm, and extensor digitorum longus fibers. Exercise performance, which was assessed by rotarod running, was lower in vehicle-treated LAD-HF rats than in sham controls (116 ± 22 versus 193 ± 31 seconds, respectively; mean ± S.E.M.; P = 0.04). In the LAD-HF rats, a single oral dose of CK-2127107 (10 mg/kg p.o.) increased running time compared with vehicle treatment (283 ± 47 versus 116 ± 22 seconds; P = 0.0004). In summary, CK-2127107 substantially increases exercise performance in this heart failure model, suggesting that modulation of skeletal muscle function by a fast skeletal troponin activator may be a useful therapeutic in heart failure-associated exercise intolerance.