Small Molecules Functioning on Myo?laments as Treating Heart and Skeletal Muscle Illnesses
Khulud Alsulami *
and Steven Marston
Imperial Center for Translational and Experimental Medicine, Cardiovascular Division, National Lung and heart Institute, Imperial College London, London W12 0NN, United kingdom [email protected] kingdom
National Center for Pharmaceutical Technology, King Abdulaziz City for Science, Riyadh 11461, Saudi Arabia
* Correspondence: [email protected]
?
Current address: National Center for Pharmaceutical Technology,
King Abdulaziz City for Science, Riyadh 11461, Saudi Arabia.
Received: 23 November 2020 Recognized: 11 December 2020 Printed: 16 December 2020
Abstract: Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) would be the at their peak types of the chronic and progressive pathological condition referred to as cardiomyopathy. These illnesses have di?erent aetiologies however, they share the feature of haemodynamic abnormalities, generally because of disorder within the contractile proteins that comprise the contractile unit referred to as sarcomere. Up to now, medicinal treatments aren’t disease-speci?c and rather concentrate on handling the signs and symptoms, without addressing the condition mechanism. Earliest attempts at improving cardiac contractility by modulating the sarcomere not directly (inotropes) led to undesirable e?ects. In comparison, individuals sarcomere directly, helped by high-throughput screening systems, could identify small molecules having a superior therapeutic value in cardiac muscle disorders. Herein, a comprehensive literature overview of 21 small molecules forwarded to ?ve di?erent targets was conducted. An easy scoring system was produced to evaluate the appropriateness of small molecules for therapy by evaluating them in eight di?erent criteria. The majority of the compounds unsuccessful because of insufficient target speci?city or poor physicochemical qualities. Six compounds was out, showing a possible therapeutic value in HCM, DCM or heart failure (HF). Omecamtiv Mecarbil and Danicamtiv (myosin activators), Mavacamten, CK-274 and MYK-581 (myosin inhibitors) and AMG 594 (Ca -sensitiser) are small molecules that allosterically modulate troponin or myosin. Omecamtiv Mecarbil demonstrated limited e?cacy in phase III GALACTIC-HF trial, while, is a result of phase III EXPLORER-HCM trial were lately printed, indicating that Mavacamten reduced left ventricular out?ow tract (LVOT) obstruction and diastolic disorder and improved the status of patients with HCM. A singular group of small molecules referred to as “recouplers”was reported to focus on a phenomenon termed uncoupling generally present in familial cardiomyopathies but hasn’t progressed beyond preclinical work. To conclude, the contractile apparatus is really a promising target for brand new drug development.
Keywords: contractility sarcomere cardiomyopathy crossbridge cycle therapeutics drug trials
1. Introduction
Cardiovascular illnesses really are a major reason for morbidity and mortality worldwide. Particularly, cardiomyopathies and ischemic heart illnesses are the most typical reasons for a chronic and progressive pathological condition termed heart failure [1,2]. Cardiomyopathy is really a term that describes abnormalities of heart muscle contractility, covering a heterogeneous selection of aetiologies [3].
Int. J. Mol. Sci. 2020, 21, 9599 doi:10.3390/ijms21249599 world wide web.mdpi.com/journal/ijms
Dilated cardiomyopathy (DCM) is characterised by cardiac dilatation and impaired contractility (reduced ejection fraction and cardiac output), and 20-50% of cases can be because of inherited mutations, associated with over 40 cardiac genes [4]. Heart failure with preserved ejection fraction (HFpEF) is really a heterogeneous clinical syndrome, which in lots of patients is characterised by impairment from the left ventricle’s capability to relax and ?ll during diastole, leading to insu?cient bloodstream ?ow to satisfy our body’s needs. HFpEF is believed to some?ect roughly three million people in america and it is connected with signi?cant morbidity and mortality. Both in HFrEF and HFpEF, the main abnormality is generally away from the contractile apparatus. Hypertrophic cardiomyopathy (HCM) is characterised by left ventricular hypertrophy and hypercontractility [5,6]. It will always be brought on by mutations of genes encoding sarcomeric proteins [5].
Current medicinal treatment tricks of HCM, DCM and heart failure (HF) mostly are centred on handling the signs and symptoms in addition to minimising disease progression however, these strategies aren’t disease-speci?c given that they target neurohormonal system and excitation-contraction coupling as the fundamental disease mechanism remains untreated.
Cardiomyopathy is essentially because of abnormal contractility therefore, individuals contractile apparatus of cardiac muscle is crucial. Now that we know enough concerning the mechanisms of contractility and it is Ca -regulation and modulation by phosphorylation and mutations so that you can de?ne appropriate targets for prescription drugs to relieve the abnormalities of cardiomyopathies. The present focus of cardiac muscle studies is in direction of developing new therapeutic approaches that act on the contractile apparatus or its regulators and therefore, theoretically, avoid most of the side e?ects of current treatments.
We’ve identified five classes of small molecule activity which have possibility of various cardiomyopathies (see Figure 1). Hypertrophic cardiomyopathy (HCM) is manifested as hypercontractility of cardiac muscle and for that reason ought to be targeted by myosin inhibitors or Ca -desensitisers [7,8]. A broader selection of contractile abnormalities can be found in myopathies characterised by insufficient cardiac muscle contractility for example HFrEF, HFpEF or DCM. In skeletal muscles, hypocontractile illnesses occur because of mutations resulting in hereditary skeletal muscle myopathies [9]. In cardiac muscle, however, hypocontractility connected with heart failure is much more complex in the nature which is unlikely that people could target its forms using a single compound. Presently, hypocontractility research is incorporated in the direction
of myosin activation and Ca
sensitisation of thin filament which may be suitable for a
small selection of cardiomyopathies. Within this study, we’ve centered on the results of small molecules on dilated cardiomyopathy (DCM), both familial and idiopathic. Lastly, there has been reports of the phenomenon termed Uncoupling that’s connected with a few installments of DCM and HCM and could be reversed by recoupling agents [10,11].
Already, some promising small molecules happen to be developed, like the myosin activator Omecamtiv Mecarbil, to deal with HF and DCM, and myosin inhibitor Mavacamten, to deal with HCM. Both drugs have proven the viability of the approach for treating many forms of cardiomyopathy.
Within this review, we investigate potential therapeutic targets within the cardiac and skeletal muscle contractile apparatus and also the actions of small molecules that act on contractile apparatus. Then we o?er an exam of the pros and cons of those as treatments.
Figure 1. Five potential therapeutic targets within the contractile apparatus for small molecules. Original figure is made by utilizing BioRender.com. The supply of myosin heads for interaction could be ameliorated via
myosin activators or alleviated by myosin inhibitors. Similarly, affinity of troponin C towards Ca
ions
could be elevated (Ca -sensitisers) or decreased (Ca -desensitisers). Protein kinase A (PKA)-dependent
phosphorylation of TnI was discovered to be lost in certain types of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), but it may be restored via Recouplers. Figure was produced by utilizing Biorender.com.
2. Contractile Activators as Treating Heart Failure and Muscular Myopathies
HFrEF is connected with structural or functional abnormalities of cardiomyocytes which, as a result, trigger neurohormonal axis activation and cardiac remodelling as compensatory mechanisms that ultimately lead to chronic heart failure and dying [12,13]. Current therapies, including β-blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), mineralocorticoid receptor antagonists and angiotensin receptor-neprilysin inhibitors, act not directly to perturb these compensatory mechanisms having a minimal capacity to boost cardiac function [14-16]. It’s been hypothesised that direct activation from the contractile proteins will be a more e?ective treatment approach.
2.1. Cardiac Muscle Ca -Sensitisers (or Positive Inotropes)
Early attempts at developing cardiac agents were centered on ?nding compounds that may improve cardiac output identi?erectile dysfunction as positive inotropes. Positive inotropes happen to be studied for many years and they may be categorised into either “calcium mobilisers”, which act by elevating the magnitude of calcium ions entering the myocytes or “calcium sensitisers”, which boost the sensitivity of myo?laments towards Ca ions. It’s been suggested that the effective inotropic drug could be one which increases contractility by directly growing Ca sensitivity separate from excitation-contraction coupling (EC-coupling) and adrenergic system. Ca -sensitisers that do something about troponin are thought like a subclass of inotropic agents. Several drugs with Ca sensitising activity happen to be tested within the clinic during the last 3 decades.
Levosimendan is really a positive inotrope that may improve cardiac contractility without growing oxygen need for the myocardium unlike most inotropes [17,18]. Levosimendan binds towards the hydrophobic patch from the N-domain of cardiac troponin C with EC50 of roughly 1 μM [19]. Regrettably, levosimendan also is able to activate ATP sensitive K funnel additionally to inhibiting III isoform of PDE enzyme [20]. Several large numerous studies of levosimendan in heart failure patients were conducted, summarised in Table 1. Although levosimendan seems to become more e?ective than dobutamine in acute situations it is not discovered to be of worth in lengthy-term treatments.
-sensitiser but additionally a PDE inhibitor produced by Boehringer
Ingelheim Pharma KG. The pimobendan in congestive heart failure (PICO) trial in 1996 says pimobendan contributed in elevated chance of mortality when compared with placebo (Table 1) [21,22]. Nowadays, pimobendan qualifies only to treat heart failure in dogs [23,24].
Bepridil is definitely an dental Ca -sensitiser that is another calmodulin (CaM) antagonist, Ca funnel blocker (negative inotrope) and potassium funnel blocker. It’s still marketed in US, Japan, Belgium, France and Ireland indicated for Angina pectoris [25]. Papadaki et al. demonstrated that bepridil uncoupled troponin I phosphorylation from alterations in Ca sensitivity in addition to enhancing Ca sensitivity [10].
MCI-154 (Senazodan) is really a PDE III inhibitor and Ca -sensitiser produced by Mitsubishi Pharma Corporation in Japan. It exerts its positive inotropic and chronotropic e?ects by binding straight to troponin C. Senazodan was tested in ?ve trials within the 1990s and early 2000s, and throughout individuals trials, the little molecule demonstrated favourable haemodynamic pro?le, when compared with dobutamine nonetheless, no data were printed after that about its development [26].
EMD57033 is really a thiadiazinone derivative along with a positive inotrope that boosts the pressure from the contraction without altering intracellular the Ca transient and it has minimal PDE inhibition activity [27]. EMD57033 functions like a Ca -sensitiser by binding towards the hydrophobic pocket from the C-domain of troponin C resulting in an inadequate cTnC-TnI interaction [18]. Baudenbacher et al. demonstrated the left transfer of log Ca versus. relative pressure in rodents was connected having a greater inclination towards ventricular arrhythmias [28]. Like a “pure”Ca -sensitiser, EMD57033 needs to be an excellent inotrope but because of bioavailability problems, it is not tested within the clinic.
AMG 594 is definitely an allosteric cardiac troponin activator that’s claimed to become direct and speci?c, produced by Cytokinetics to treat HF [29]. Conference proceedings reported that AMG 594 functions exclusively around the sarcomere by sensitising cardiac troponin to Ca ions, resulting in more myosin heads engaging with actin ?laments, and much more contractile pressure being generated [30]. A phase I medical trial of AMG 594 was completed on August 2020, but there aren’t any available data to date.
Ca -sensitisers that do something about troponin are thought as subclass of inotropic agents. In principle, creating a “pure”Ca -sensitiser that may enhance cardiac contractility with no?ecting EC-coupling or compromising cardiac energetics would avoid the majority of the defects of current compounds. In clinical practice, bepridil, pimobendan, MCI-154 and levosimendan have unsuccessful as treating chronic HF mainly due to the o?-target e?ects of these drugs, particularly PDE inhibition that has been enhanced arrhythmia [28,31].
The only real well-researched apparently pure Ca -sensitiser is EMD57033 with preclinical data, suggesting that the idea of troponin Ca sensitisation might be viable however, its bioavailability problems have avoided any studies. The brand new troponin activator produced by Cytokinetics (AMG 594) claims to become a “pure”and selective cardiac troponin activator, although little data happen to be printed, and phase I medical trial only has lately completed. Based on the preclinical data, AMG 594 appears to become enhancing cardiac contractility individually of EC coupling inside a similar
method to Omecamtiv [30]. It ought to be noted that myo?lament Ca
The introduction of direct myosin activators was fuelled by hypothesising that direct activation of cardiac sarcomere can improve cardiac performance with an additional benefit to be in addition to the usual neurohormonal response and cardiac remodelling [35,36]. Selective activation from the actin and myosin interaction aims to prevent disadvantages of classic inotropic agents that enhance cardiac contractility but, simultaneously, increase oxygen demand, heartbeat and intracellular calcium transient that are associated with hypotension, arrhythmias and mortality [31,33,37,38].
2.2.1. Omecamtiv Mecarbil
A higher-throughput screening (HTS) close to 40,000 small molecules, utilizing a myo?brillar ATPase screen, brought towards the discovery of Omecamtiv Mecarbil (OM) [36,39]. OM (formerly referred to as CK-1827452 and AMG-423) was created by Cytokinetics together with Amgen like a novel, allosteric cardiac myosin activator. The molecular mechanism initially suggested through the Cytokinetics group was that OM binds the catalytic S1 domain from the myosin, causing four-fold acceleration from the phosphate (Pi) releasing step thus, enhancing duty ratio [35,40]. However, when the compound grew to become open to 3rd party researchers several inconsistencies within the model put together. Nagy et al. noticed that OM elevated the Ca sensitivity at concentrations of .1 μM and greater in permeabilized rodent cardiomyocytes. In addition, activation was biphasic and concentrations above 1 μM, OM inhibited pressure production [41]. Furthermore, Liu et al. shown that OM reduced the rate of crossbridge cycles within an in vitro motility assay, using porcine ?bres [42].
The molecular mechanism was described by Woodsy et al. they demonstrated that OM caused a ten-fold decrease in how big the significant stroke (from 5.4 nm close to at 10 μM) in addition to ?ve-fold prolongation within the actomyosin attachment duration [43]. These observations take into account the inhibitory and Ca -sensitising e?ect of OM when you are a cooperative activator from the thin ?lament [43,44]. Overall, it’s obvious since OM exerts its action by recruiting more crossbridge cycles rather of altering their dynamics which can be defined as “more hands pulling around the rope”.
A preclinical type of pigs with left ventricular disorder demonstrated that treatment with OM
plays a role in elevated myocardial O2
consumption [45]. It’s been contended the elevation of
myocardial O2 demand, an indication of cardiac ischemia, was because of the administration of high concentrations of OM [46]. Similarly, Teerlink et al. reported within their ?rst-in-man trial that indications of myocardial ischemia emerged at plasma concentrations above 1200 ng/mL [47]. This can be a negative property of OM because it plays a role in narrow its therapeutic window.
There has been several numerous studies of OM which are detailed in Table 2. OM continues to be studied in healthy men, patients with chronic systolic HF and patients with acute HF. Phase I trials were conducted in healthy participants to evaluate pharmacokinetics and pharmacodynamics of OM in intravenous and dental formulations. To date, OM continues to be well-tolerated and elevation in ejection fraction and cardiac output was observed. However, in one of these simple trials (ATOMIC-HF) the main (relief of dyspnoea) and secondary endpoints weren’t met that has brought some to question the need for further trials [48,49]. Lately, results of a big randomised, placebo-controlled phase III Global Method of Lowering Adverse Cardiac Outcomes through Improving Contractility in Heart Failure (GALACTIC-HF) trial were printed (see Table 2) [50]. The main effects were an amalgamated of the heart-failure event or cardiovascular dying (whichever comes ?rst). Secondary outcomes incorporated cardiovascular dying, alternation in within the total symptom score around the Might Cardiomyopathy Questionnaire (KCCQ) from baseline to week 24, ?rst hospitalisation for heart failure or dying. A signi?cant yet modest decrease in the incidence from the composite primary effects were proven in 37% from the OM group as well as in 39.1% from the placebo group (95% disadvantage?dence interval (CI), .86 to .99 p = .03). Furthermore, the trial didn’t show any signi?cant improvement within the secondary outcomes and also the incidences of myocardial ischemia, ventricular arrhythmias and dying were similar both in OM and placebo groups. It’s important to note that the greater treatment bene?t was recommended in patients with LVEF of 28% or fewer (New You are able to Heart Association (NYHA) type of III or IV).
Danicamtiv (formerly referred to as MYK-491) may be the lead candidate myosin activator inside a programme produced by MyoKardia (now offered to Bristol Meyers Squibb) together with Sano? to treat systolic heart failure, and speci?cally DCM. The little molecule is doing OM and presently just ?nished a phase II medical trial. MyoKardia claims that “MYK-491 directly activates cardiac actomyosin, enhancing step one from the pressure-producing chemotherapy-mechanical cycle by Boosts the rate of Pi release and accessibility to myosin-heads” studies around the molecular mechanism haven’t been printed but it’s recommended the mechanism is comparable to OM [51,52].
Danicamtiv binds selectively to human cardiac myosin isoform without binding to skeletal or smooth muscle isoforms inducing the elevation of ATPase turnover rate ( 85% in ventricular myo?brils) and elevated Ca sensitivity ( .35 pCa unit) [53]. In really treated male beagle dogs with caused HF (n = 7), administration of Danicamtiv prolonged SET and improved LVEF additionally to cardiac output [53].
Danicamtiv continues to be studied in one-climbing dose phase IIa trial in 40 patients with chronic and stable HFrEF (see Table 2). The little molecule caused a serving-dependent elevation in left ventricular stroke volume (LVSV) and a rise in Occur medium and concentrations. No alterations in diastolic bloodstream pressure or heartbeat were reported aside from a small reduction systolic bloodstream pressure in high concentration cohort. Furthermore, no indications of cardiac ischemia were reported in the doses used [53]. Many of these answers are rather much like OM however, the e?ects of chronic treatment haven’t been studied.
EMD57033 has additionally been suggested like a myosin activator, additionally to as being a positive inotrope and Ca -sensitiser functioning on cardiac troponin [27]. However, research conducted recently implies that EMD57033 is definitely an e?ective preserver of myosin activity instead of an activator [27,61].
2.2.4. Assessment of Myosin Activators
Investigating the literature brought to ?nding reports on three compounds that could have cardiac myosin activation qualities, and just a couple of individuals compounds (Omecamtiv Mecarbil and Danicamtiv) went to the advanced stages of drug development, using the ?rst in phase III and also the latter making up ground. Both Omecamtiv Mecarbil and Danicamtiv happen to be formulated in dental and intravenous dosage that is essential for further drug development.
Regardless of the encouraging outcomes of Omecamtiv Mecarbil like a myosin activator in vitro so that as a contractile activator in vivo in animal studies, the little molecule shows a restricted effectiveness in numerous studies [48,56]. It’s been asked whether ongoing trials with Omecamtiv Mecarbil are useful [49]. Nonetheless, Omecamtiv Mecarbil continues to be granted steps for success designation like a potential new strategy to heart failure in the US Fda (Food and drug administration) [62]. Using the limited data to date, it seems that Danicamtiv functions in the same manner as OM.
Current therapeutic options to treat HFrEF happen to be effective in lessening mortality rates which sets the bar high for just about any new therapy. All available treating HFrEF are centered on lowering the strain on the center, thus preserving the center function only without improving its mechanical output. Myosin activators have the benefit of acting on the contractile apparatus to boost cardiac contractility by prolonging systolic ejection time (SET), a house that can’t be based in the conventional positive inotropes. The issue remains, will direct activation from the contractile apparatus constitute bene?t for any lengthy-term management of chronic HF? Phase III GALACTIC-HF medical trial was conducted for approximately 22 several weeks and demonstrated a small decrease in the incidence of the composite of HF-event or dying because of cardiovascular causes. With your results it appears that myosin activators (OM speci?cally) are unlikely replacing current standard therapies in the near future.
Although myosin activators increase contractility inside a selective manner which may be useful for treating systolic heart failure, their Ca sensitising e?ect, that might promote diastolic disorder and arrhythmias, limits the expectations of the future therapeutic worth. It is really an inevitable results of the cooperative allosteric mechanism of regulating contractility the compounds do something about. This cooperative allosteric mechanism will probably be accountable for the diastolic disorder observed at high doses that may limit the therapeutically safe selection of doses. Indeed, a narrow therapeutic window is implied by the requirement for dose titration in many studies.
In many aspects, the e?ect of OM mimics the e?ect of mutations that create hypertrophic cardiomyopathy. Particularly, both increase myo?lament Ca -sensitivity by similar amounts resulting in hypercontractility at the fee for possible diastolic disorder that has been enhanced arrhythmia [7,35,43].
Within their book Therapeutic techniques for managing heart failure (2000), Silber and Katz described the failing heart as “a sick, tired horse pulling a wagon up a high hill”, which is a superb example to see heart failure and it is therapeutic choices for enhancing the horse and wagon “up the hill”[63]. The presently used therapies are mainly according to “unloading the wagon”as their mode of action although myosin activators can be “whipping the horse”, which might not be therapeutically beneficial for lengthy-term treatment.
Omecamtiv Mecarbil continues to be studied inside a spectrum of aetiologies of HF, unlike Danicamtiv, that has been promoted as potential therapy for DCM. This enhances the question, would a far more precise target for example familial DCM, particularly if brought on by mutations in contractile proteins, result in more and better effective outcomes? Simultaneously, will it be useful to focus on such a part of HFrEF patients?
-sensitisers
Skeletal muscle hereditary myopathies are characterised by muscle weakness. They may be because of abnormalities from the contractile apparatus or a decrease in the density of muscle innervation, the speed of neuromuscular junction activation or even the e?ciency of synaptic transmission. It had been suggested that the small-molecule fast-skeletal-troponin activator, would increase muscle strength in myopathy because of mutations in contractile proteins but may be helpful by amplifying the response of muscle when neural input is otherwise reduced secondary to neuromuscular disease [64]. You could do because, unlike cardiac muscle, skeletal muscle has the benefit of being regenerative thus muscle activation can promote muscle growth [65]. A screen for Ca sensitising agents that concentrate on fast skeletal muscle troponin by Cytokinetics produced the next compounds: Tirasemtiv (or CK-2017357), Reldesemtiv (or CK-2127107) and CK-2066260.
2.3.1. Tirasemtiv
Tirasemtiv (or CK-2017357) was created by Cytokinetics being an orally administered, highly specific small molecule fast skeletal troponin activator (FSTA) with affinity of 40 nM. Russell et al. suggested that amplifying the sarcomeric reaction to inadequate neuronal input by growing Ca sensitivity to troponin-tropomyosin complex can improve muscular pressure generation and physical performance in patients with neuromuscular disorders for example Myasthenia gravis and amyotrophic lateral sclerosis (ALS). Inside a passive transfer experimental autoimmune myasthenia gravis (PT-EAMG) rat model, Tirasemtiv elevated the pressure of muscle contraction at submaximal nerve stimulation frequencies, elevated grip strength, and decreased muscle fatigability [64].
Hansen et al. reported that Tirasemtiv augmented the skeletal muscle reaction to nerve input in healthy human males, inside a randomised, double-blind, four-period crossover study [66]. Tirasemtiv went through phase II numerous studies in patients with ALS (ClinicaltrialNCT01709149), peripheral vascular disease (NCT011310313) and myasthenia gravis (NCT01268280). Ale Tirasemtiv to pass through the bloodstream-brain barrier (BBB) contributed in adverse occasions for example dizziness and fatigue.
Nonetheless, this year, Tirasemtiv was granted steps for success designation in the American Food and drug administration additionally towards the orphan drug status for ALS in Europe and also the US [67]. Despite everything, in 2017, Cytokinetics made the decision to suspend the introduction of Tirasemtiv because of the negative is a result of the force-ALS trial [68].
2.3.2. Reldesemtiv
Reldesemtiv (formerly referred to as CK-2127107 or CK-107) is really a next-gen, orally available FSTA developed also by Cytokinetics together with Astellas Pharma having a potential bene?t in improving skeletal muscle function and physical performance in neuromuscular disorders for example spine muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS), additionally to muscle fatigue in chronic obstructive lung disease (Chronic obstructive pulmonary disease) [69]. In phase I numerous studies in healthy human, Reldesemtiv demonstrated signi?cant elevation in tibialis anterior muscular response inside a dose-dependent fashion that seems to become better than Tirasemtiv [70]. Is a result of Strength-ALS trial established that there wasn’t any record signi?cance in the primary endpoint that is vary from baseline in slow vital capacity (SVC) after 12 days of dosing with different pre-speci?erectile dysfunction dose-response relationship (p = .11) [71].
CK-2066260 is another Tirasemtiv-structural analogue FSTA produced by Cytokinetics, together with Astellas, as part of skeletal muscle activator research programme. Unlike Tirasemtiv and Reldesemtiv, CK-2066260 continues to be tested on nemaline myopathy patients with nebulin mutations [72,73].
These recently developed skeletal muscle troponin-speci?c Ca -sensitisers offer an interesting
comparison with cardiac Ca
2
-sensitisers. Skeletal muscle is resistant against arrhythmia and it is capable
of regenerating therefore, Ca -sensitisers are safer as well as can stimulate nerve and muscle growth thus potentially alleviating an array of neuromuscular disorders. Nonetheless, preclinical potential
Int. J. Mol. Sci. 2020, 21, 9599 12 of 30
hasn’t converted into effective trials yet because the lead compound, Tirasemtiv, has been frozen and it is analogue Reldesemtiv unsuccessful in meeting its endpoints within the Strength-ALS trial (Table 3).
2.3.3. Piperine
Nogara et al. recommended that myosin activation of fast skeletal muscles could be of therapeutic bene?t for weight problems and diabetes type 2. Transferring myosin heads in the super-relaxed (SRX) towards the disordered-relaxed (DRX) condition can boost the metabolism from the whole body by 2-4 MJ each day [74,75]. Furthermore, Nogara et al’s study found a ?uorescent probe on regulatory light chain (RLC) of myosin that shows shorter wavelengths upon the transition in the SRX towards the DRX. It was employed for a higher throughput screening a library well over 600 compounds which led to identifying Piperine a naturally sourced alkaloid obtained from pepper like a appropriate candidate [74,76].
Like a cardiac myosin activator, Piperine fails because of the insufficient speci?city for skeletal muscle and o? target e?ects. It’s conceivable that further development from piperine directed at cardiac muscle SRX destabilisers may uncover a helpful compound.
Hypertrophic cardiomyopathy is really a hypercontractile disease many known mutations that create HCM have been in thick ?lament proteins, myosin and MyBP-C [82,83]. It’s, therefore, valuable to ?nd small molecules that hinder cardiac myosin within the contractile apparatus for targeted treatment HCM [84-86].
HCM could be split into two groups: obstructive hypertrophic cardiomyopathy (HOCM or oHCM), where the left ventricular out?ow tract (LVOT) is obstructed or non-obstructive hypertrophic cardiomyopathy (nHCM), that is characterised by the lack of LVOT resting (<30 mm Hg) [87]. Pharmacological treatment options for patients with HOCM and nHCM include non-vasodilating β-receptor blockers titrated to maximum tolerated dose, anti-arrhythmic drug disopyramide as an add-on treatment (for HOCM) or non-dihydropyridine calcium channel blockers [88]. The current therapeutic options lack speci?city and they have modest e?cacy in controlling LVOT gradients as they do not target the main cause for the disease which is the hypercontracting sarcomere.
3.1. Ca -Desensitisers
Ca -desensitisers that act on troponin are a group of small molecules that, theoretically, aim to treat HCM by desensitising the thin ?lament toward Ca ions, thus reducing contractility.
3.1.1. Green Tea Catechins (EGCg and ECg)
Consumption of green tea has been linked to a lower risk of cardiovascular diseases in several studies mainly due to the presence of biologically active compounds in green tea are the polyphenols known as Catechins [89-91]. Epigallocatechin-3-gallate (EGCg) is the most widely studied catechin which has been reported as a Ca -desensitiser [92]. Tadano et al. also reported that epicatechin gallate (ECg) shares the direct Ca desensitisation property with EGCg through binding to troponin C. In skinned cardiac muscle ?bres, EGCg showed a greater desensitisation e?ect than ECg and cardiac-selective molecular action. In isolated working hearts of an HCM mouse model with increased Ca sensitivity, EGCg restored cardiac output by improving diastolic dysfunction suggesting a potential therapeutic bene?t in HCM. When applied to isolated cardiomyocytes of guinea pig HCM model, EGCg showed a poor potency as it requires 30-100 μM to desensitise the myo?lament, while at lower concentrations (<1 μM), signi?cant o?-target e?ects were observed [93].
3.1.2. Nebivolol
Nebivolol is a β-adrenergic receptor antagonist that also has a Ca desensitisation activity at EC50 of 10 μM [94,95]. Nebivolol selectively desensitised permeabilised cardiac muscle from Mybpc3-targeted knock-in (KI) cardiomyopathy mouse model without resulting in any signi?cant change in contractility [95]. The drug also had a negative impact on shortening in the cardiomyocyte, while causing a slower contraction and relaxation [96].
Troponin based Ca -desensitisers have not been researched much and have not gone beyond in vitro studies for various reasons. EGCg and related compounds are known to act promiscuously in vivo with multiple actions that preclude their use outside in vitro situations and no therapeutic bene?t should be anticipated [97-99]. Nebivolol, on the other hand, seems to be a more promising desensitiser as it, so far, ticked all the boxes needed in a good desensitiser. The small molecule is cardio-speci?c that e?ectively worked in animal models and already approved as a β-blocker, accordingly, it has an established safety pro?le. Since β-blockers are currently the ?rst line of treatment in HCM therefore, having a compound that acts as β-blocker and Ca -desensitiser may act synergistically to treat HCM.
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Protein kinase A (PKA)-mediated phosphorylation of MyBP-C and troponin I modulate the Ca switch within the contractile apparatus. The absence of modulation of Ca sensitivity by troponin I phosphorylation results in blunted response to adrenergic stimulation in a phenomenon known as uncoupling. In familial cardiomyopathies, mutations in thin ?lament proteins often associated with the loss of this modulation, thus uncoupling is postulated to contribute to the HCM or DCM phenotype [10,11,100,101]. In support of this, in vivo transgenic mouse studies showed that uncoupling leads to heart failure under stress [102].
Recouplers are a novel category of small molecules that demonstrated the ability to reverse the uncoupling of troponin I phosphorylation and Ca sensitivity. Compounds that are shown to be e?ective as recouplers, to date, include EGCg, Silybin B, Dehydrosilybin B, resveratrol, novobiocin [97]. As compounds under this category are in early stages of research, our assessment system seemed inapplicable.
3.3. Myosin Inhibitors
The concept of utilising myosin inhibitors in biochemical and physiological studies has been around for a long time. Early myosin II inhibitors include of 2,3-butanedione monoimine (BDM), N-benzyl-p-toluene sulphonamide (BTS) and blebbistatin of these, only blebbistatin has been considered as a drug prototype [103-106]. Recently a new range of unrelated cardiac muscle speci?c myosin inhibitors has been developed as potential treatment for HCM.
3.3.1. Blebbistatin and Its Analogues
Blebbistatin is a myosin II inhibitor that has been widely used as a research tool in various areas such as muscle physiology, cancer, cell migration and di?erentiation [107]. Structural and functional studies showed that the inhibitory e?ect of blebbistatin is due its ability to stabilise myosin heads of the thin ?lament in SRX thus decreasing the number of active force producing myosin heads [108].
The majority of myosin II isoforms are inhibited by blebbistatin with the highest a?nity to skeletal muscle myosin II (EC50 0.1-5 mM) and intermediate a?nity for cardiac and non-muscle myosin II
isoforms (EC
50
1-10 mM) ruling out blebbistatin as a cardiac compound [107]. Multiple derivatives
of blebbistatin have been developed in order to improve its physicochemical and pharmacological properties [109], but improved speci?city has not been achieved yet.
3.3.2. Mavacamten
The journey of developing Mavacamten as a small molecule for treatment of HCM started with a hypothesis that excess sarcomere power can be the primary cause of HCM and thus, the pathological phenotype of HCM could be alleviated by normalising the hyperdynamic sarcomeric power [110-112]. A chemical screening for compounds with the ability to reduce actin-activated ATPase rate of myosin by MyoKardia yielded MYK-461 or “Mavacamten”.
Transient kinetic analyses showed that Mavacamten decreases the rate of inorganic phosphate (Pi) release, the rate-limiting step of the chemomechanical cycle without altering the rate of ADP release in actin-activated state. Mavacamten binds to myosin where it stabilises the super-relaxed (SRX) conformation [113,114]. In mouse cardiac and bovine myo?brils, treatment with Mavacamten showed that Mavacamten reduced ATPase activity (EC50 0.3 μM in mouse) [112]. Similarly, a dose-dependent reduction in fractional shortening (FS) without a?ecting calcium transient was observed in isolated rat cardiomyocytes (EC50 0.18 μM) [112].
In vivo e?ects of long term Mavacamten treatment were investigated in mouse models of HCM expressing α-cardiac myosin heavy chain mutations and showed diminution of ?brosis and myocyte disarray [112]. A Feline model of HCM showed that IV treatment with Mavacamten resulted in reduction of cardiac contractility, indicated by reduced fractional shortening (p = 0.01), and left ventricular
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out?ow tract (LVOT) pressure gradient (p = 0.0007) [115]. Similarly, Del Rio et al. assessed acute and chronic cardiac responses of dogs to Mavacamten. In acute studies, Mavacamten reduced inotropic indices (p-value < 0.05) whilst maintaining systemic pressure (MBP: 107 ± 6 to 109 ± 5 mmHg) [116].
Pharmacokinetic properties were extensively studied by Grillo et al. showing that Mavacamten can be administered via both oral and intravenous routes. Notably, Mavacamten has a high volume of distribution (Vd = 9.5 L/kg), plasma clearance of 0.51 mL/min/kg and half-life (t1/2 ) of 9 days [117].
Clinical trials on Mavacamten are detailed in Table 4. Earlier clinical trials (PIONEER-HCM and PIONEER-OLE) targeted patients with HOCM, followed by MAVERICK-HCM, a trial designed for patients with nHCM. In the PIONEER-HCM trial, Mavacamten was well-tolerated in the two cohorts of patients with HOCM [118]. Moreover, PIONEER-OLE is an open-label extension trial in patients from PIONEER-HCM and it showed reduced LVOT obstruction and improve exercise capacity without cardiac-related adverse events [119]. MAVERICK-HCM, on the other hand, was conducted on patients with nHCM and was designed to evaluate the dosing and safety of Mavacamten. Adverse events reported in the trial in 90% of Mavacamten group while in 68% in the placebo group [120]. The most commonly reported adverse events were palpitations, dizziness and fatigue [120]. Moreover, the trial showed reduction in cardiac markers N-terminal pro b-type natriuretic peptide (NT-proBNP) and Cardiac troponin I (cTnI) [120].
The results of phase III clinical trial (EXPLORER-HCM) were presented in European Society of Cardiology virtual congress on August 2020 [121]. EXPLORER-HCM was the largest placebo-controlled randomised clinical trial in HCM with 251 patients from 13 countries. The primary endpoint was an elevation in peak oxygen consumption (pVO2 ) by 1.5 mL/kg per min or greater and at least one NYHA class reduction OR a 3.0 mL/kg per min or greater pVO2 increase without NYHA class worsening [122]. The composite primary endpoint was met in 37% of Mavacamten group versus 17% of the placebo group (p = 0.0005). Moreover, complete abolition of all LVOT gradients (resting and post-exercise) was achieved in 57% patients in Mavacamten group. In general, Mavacamten was associated with improvement in exercise capacity, LVOT obstruction and NYHA functional classi?cation with reduction in plasma NT-proBNP and cTnI.
Further developments by MyoKardia have yielded compounds that may have a better pharmacological pro?le than Mavacamten. Preclinical pharmacodynamics data of MYK-581 were presented at the American Heart Association Scienti?c Sessions 2019 [123]. MYK-224 is another new Mavacamten analogue. There are no available preclinical data on MYK-224 besides what is stated on MyoKardia’s website [124]. A Phase I clinical trial of MYK-224 has been initiated to assess safety, tolerability and pharmacokinetics of MYK-224 in healthy participants. The trial was suspended on May 2020 in response to the COVID-19 pandemic and resumed recruitment again in August 2020 [125].
CK-274 is described as a next generation, oral cardiac myosin inhibitor developed by Cytokinetics to treat HCM. Its mechanism is not detailed but is likely to be the same as Mavacamten. CK-274 was studied in bovine cardiac myo?brils, using the ATPase assay as described in Malik et al., for OM which resulted in identifying a cardiac-speci?c inhibitory e?ect (EC50 1.26 μM) with little e?ect on Ca -sensitivity [35,137,138]. Using healthy male Sprague Dawley (SD) rats, cardiac contractility was assessed in vivo via single oral doses ranging from 0.5 to 4 mg/kg at multiple time points after administering the single dose [138]. Echocardiography results indicated that CK-274 caused a FS reduction in a dose-dependent manner that peaked at 1 hour. CK-274 decreased FS in a similar manner in both WT and R403Q (HCM mutation) transgenic mice [139]. Currently, CK-274 is under investigation in REDWOOD-HCM trial (Table 4) for patients with HOCM which has been temporarily suspended due to COVID-19 pandemic [140]. All available data come from unpublished conference proceedings.
3.3.4. Assessment of Cardiac Myosin Inhibitors
Blebbistatin is the prototype direct myosin inhibitor and further investigation led to identifying 5 compounds with therapeutic potential in cardiac muscle. There are other small molecules with myosin inhibition properties excluded, such as BTS, BDM and others as they only target myosin isoforms that are found in fast skeletal muscle ?bres or due to poor physicochemical and pharmacological properties.
Compared to Omecamtiv Mecarbil, Mavacamten seems to be developing in the process of drug development faster with more favourable results. In particular, the e?ect of Mavacamten especially in HOCM seems to be largely independent of HCM genotype which is valuable in a heterogeneous disease such as HCM [121,141]. The reason for the success of Mavacamten vs. OM is most likely related to the disease targeted. The abnormality in HCM is well understood and con?ned to the sarcomere and stabilising the SRX presents a speci?c mechanism, whereas the abnormalities targeted by OM are much more di?use.
The positive results of PIONEER-HCM, MAVERICK-HCM and EXLORER-HCM show Mavacamten to be an e?ective treatment for nHCM and HOCM in patients with a mean age of around 50. Additionally, Mavacamten could be especially bene?cial in younger patients to minimise cardiac remodelling and avoid invasive surgical interventions. Moreover, it has been hypothesised that Mavacamten could be valuable as a long-term sole treatment without the need for β-blockers and calcium channel blockers. Two clinical trials are currently progressing (see Table 4). VALOR-HCM is an ongoing trial that will investigate the impact of Mavacamten in younger HOCM patients who are eligible for septal reduction therapy, while MAVA- LTE trial aims to study the long-term e?ect of Mavacamten for up to ?ve years.
The only downside of Mavacamten is that it has less favourable pharmacokinetic pro?le which include long t1?2 (≈9 days) and low plasma clearance rate (≈0.51 mL/min/kg) [117]. CK-274 has a much shorter half-life (about 12 h) and MyoKardia has recently developed multiple Mavacamten analogues such as MYK-581 and MYK-224 for shorter half-life which can reduce the time necessary to achieve steady-state concentration.
4. Discussion
In this review, the contractile apparatus of cardiac and skeletal muscle and the small molecules that can target it were investigated, with the objective of identifying the proper small molecule to treat muscle diseases (Figure 2). In the past decade, interest in developing small molecules that can act directly on the contractile apparatus have emerged. This phase of research was suggested as the “fourth wave”of muscle research [65].
The targets we have de?ned do not act on the crossbridge cycle instead, they interfere with the cycle magnitude by “modulating its modulators”, as illustrated in Figure 3. As myopathies are due to either hypercontracting or hypocontracting muscle, modulating the sarcomere at the troponin or myosin level might be more e?ective in treating cardiac muscle disorders. In case of HCM, a classic cardiac muscle gain-of-function disorder, myosin inhibitors and Ca -desensitisers address the defect directly. Experimentally, myosin inhibitors seem promising while developing the appropriate Ca -desensitisers appears to be more challenging. In contrast, HFrEF and DCM have more complex aetiologies. Compensatory mechanisms such as neurohormonal axis activation and cardiac remodelling (i.e., ?brosis and in?ammation) are often associated with HFrEF and DCM leading to chronic heart failure [12,13]. Heart failure also involves abnormalities in myocardial metabolism which can trigger systemic metabolic changes [142]. Nevertheless, enhancing contractility directly via myosin activation and Ca sensitisation have been proposed as a possible approach, using small molecules with better pharmacological pro?les than existing inotropes.
Figure 3. The chemomechanical crossbridge cycle and its regulation via troponin-tropomyosin (thin filament state) and super-relaxed/disordered-relaxed (SRX/DRX) equilibrium. The crossbridge is represented in the blue circle. The availability of actin-binding sites is regulated by the state of thin filament (top left). The equilibrium between blocked (no myosin bound) and closed (weak myosin-binding) is controlled by Ca . Myosin heads regulate the closed-open state in a cooperative fashion. Only thin filament in open state can participate in the chemomechanical cycle. Two small molecules that interact with both transitions are illustrated. The availability of myosin heads is regulated by the SRX/DRX equilibrium, and only myosin heads in DRX can be part of the crossbridge cycle. Four small molecules can regulate the transition, as shown. Figure was created by using Biorender.com as a modified version from References [65,83].
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Out of 21 compounds investigated, only six compounds showed any potential to be of therapeutic value. Omecamtiv Mecarbil and Danicamtiv (myosin activators), Mavacamten, CK-274 and MYK-581 (myosin inhibitors) and lastly AMG 594 (Ca -sensitiser) are small molecules that act allosterically to correct cardiac muscle abnormalities. OM acts by allosterically recruiting more crossbridge cycles and just recently completed phase III GALACTIC-HF clinical trial as a treatment for HF. Danicamtiv is another myosin activator with what appears to be a similar mode of action as OM however, it is targeted more narrowly to DCM as a form of HF. Mavacamten is a ?rst-in-class myosin inhibitor that showed clinical bene?t in patients with HCM proved by the recently published results from phase III EXPLORER-HCM trial. CK-274 is another myosin inhibitor developed by Cytokinetics and currently in phase II trials. MYK-581 is a Mavacamten analogue developed with the aim of improving pharmacokinetic properties. AMG 594 is a novel Ca -sensitiser and troponin activator suggesting a therapeutic bene?t in HF.
4.3. Limitations and Di?culties
The scarcity of printed peer-reviewed data on a few of the compounds within their initial phases of development is an issue to make a vital look at the little molecules. What we should could obtain originated from the businesses by means of abstracts, posters, press announcements or presentations at private conferences. As many of these types of literature are unpublished and non-peer-reviewed, these were largely uninformative with the potential of bias. One other issue would be that the structure from the new molecules isn’t necessarily printed which the little molecules are not always open to organizations for analysis. This will be significant for fundamental investigate the perfect example for that’s the molecular mechanism of action of OM that was not labored out before the drug was open to organizations and it was quite di?erent from Cytokinetics’s original suggested mechanism [35,43].
4.4. Potential Customers
Within the situation of myosin and troponin activators in cardiac muscle disorders, numerous studies indicated moderate improvement in cardiac functions however, whether or not they can switch the current therapies remains unlikely. In comparison, numerous studies demonstrated that myosin inhibitors, as exempli?erectile dysfunction by Mavacamten, are promising small molecules for HOCM. Its success raises the chance that myosin inhibitors might be a curative therapy for HOCM which is now also being suggested as “a unique and precise therapy, to the non-obstructive HCM patients and potentially other similar individuals su?ering from HFpEF”[143]. The subgroup identi?erectile dysfunction for future look at Mavacamten is believed to incorporate roughly 10-20% from the broader HFpEF population.
Funding: These studies received no exterior funding.
Acknowledgments: The authors extend their thanks to the nation’s Lung and heart Institute (NHLI), Imperial College London and King Abdulaziz City for Science (KACST).
Disadvantage?icts of great interest: The authors declare no disadvantage?ict of great interest.
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