RESUMO
Cardiac troponin is a regulatory protein complex located on the sarcomere that regulates the engagement of myosin on actin filaments. Low-molecular weight modulators of troponin that bind allosterically with the calcium ion have the potential to improve cardiac contractility in patients with reduced cardiac function. Here we propose an approach to the rational design of troponin modulators through the combined use of solution nuclear magnetic resonance and isothermal titration calorimetry methods. In contrast to traditional approaches limited to calcium and activator-bound troponin structures, here we analyzed the structural and thermodynamic impact of an activator in the context of the troponin functional cycle. This led us to propose a rationale for developing an efficacious troponin activator.
Assuntos
Cálcio , Miocárdio , Actinas/metabolismo , Cálcio/metabolismo , Humanos , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Termodinâmica , Tropomiosina/metabolismo , Troponina/químicaRESUMO
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.
Assuntos
Bibliotecas de Moléculas Pequenas/farmacologia , Miosinas de Músculo Liso/antagonistas & inibidores , Miosinas de Músculo Liso/química , Actinas/metabolismo , Sítio Alostérico , Animais , Cristalografia por Raios X , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Moleculares , Relaxamento Muscular , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ligação Proteica/efeitos dos fármacos , RatosRESUMO
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 â¼2.7-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.
Assuntos
Cálcio/fisiologia , Imidazóis/farmacologia , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Pirazinas/farmacologia , Adenosina Trifosfatases/fisiologia , Animais , Bovinos , Feminino , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiologia , Camundongos Endogâmicos C57BL , Fibras Musculares de Contração Rápida/fisiologia , Miofibrilas/fisiologia , Coelhos , Ratos Sprague-Dawley , Troponina C/fisiologiaRESUMO
Nanomedicines (NMs) offer new solutions for cancer diagnosis and therapy. However, extension of progression-free interval and overall survival time achieved by Food and Drug Administration-approved NMs remain modest. To develop next generation NMs to achieve superior anticancer activities, it is crucial to investigate and understand the correlation between the physicochemical properties of NMs (particle size in particular) and their interactions with biological systems to establish criteria for NM optimization. Here, we systematically evaluated the size-dependent biological profiles of three monodisperse drug-silica nanoconjugates (NCs; 20, 50, and 200 nm) through both experiments and mathematical modeling and aimed to identify the optimal size for the most effective anticancer drug delivery. Among the three NCs investigated, the 50-nm NC shows the highest tumor tissue retention integrated over time, which is the collective outcome of deep tumor tissue penetration and efficient cancer cell internalization as well as slow tumor clearance, and thus, the highest efficacy against both primary and metastatic tumors in vivo.
Assuntos
Antineoplásicos/química , Nanomedicina , Tamanho da Partícula , Animais , Antineoplásicos/uso terapêutico , Humanos , Células MCF-7 , Camundongos Nus , Nanoconjugados , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Dióxido de Silício/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Insuficiência Cardíaca Sistólica/fisiopatologia , Músculo Esquelético/efeitos dos fármacos , Condicionamento Físico Animal , Pirimidinas/farmacologia , Troponina/metabolismo , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Bovinos , Feminino , Insuficiência Cardíaca Sistólica/complicações , Insuficiência Cardíaca Sistólica/metabolismo , Contração Muscular/efeitos dos fármacos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologia , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Coelhos , Ratos Sprague-Dawley , Teste de Desempenho do Rota-RodRESUMO
Members of the kinesin-8 motor class have the remarkable ability to both walk towards microtubule plus-ends and depolymerise these ends on arrival, thereby regulating microtubule length. To analyse how kinesin-8 multitasks, we studied the structure and function of the kinesin-8 motor domain. We determined the first crystal structure of a kinesin-8 and used cryo-electron microscopy to calculate the structure of the microtubule-bound motor. Microtubule-bound kinesin-8 reveals a new conformation compared with the crystal structure, including a bent conformation of the α4 relay helix and ordering of functionally important loops. The kinesin-8 motor domain does not depolymerise stabilised microtubules with ATP but does form tubulin rings in the presence of a non-hydrolysable ATP analogue. This shows that, by collaborating, kinesin-8 motor domain molecules can release tubulin from microtubules, and that they have a similar mechanical effect on microtubule ends as kinesin-13, which enables depolymerisation. Our data reveal aspects of the molecular mechanism of kinesin-8 motors that contribute to their unique dual motile and depolymerising functions, which are adapted to control microtubule length.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Cinesinas/genética , Microtúbulos/metabolismo , Modelos Moleculares , Ligação ProteicaRESUMO
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.
Assuntos
Contração Miocárdica , Troponina , Animais , Masculino , Ratos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Troponina/metabolismoRESUMO
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.
Assuntos
Miosinas Cardíacas , Cardiomiopatia Hipertrófica , Contração Miocárdica , Animais , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/metabolismo , Humanos , Contração Miocárdica/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/genética , Modelos Animais de Doenças , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Camundongos , Cristalografia por Raios X , Mutação , Sarcômeros/metabolismo , Sarcômeros/efeitos dos fármacos , Actinas/metabolismo , Modelos Moleculares , Camundongos Transgênicos , Conformação ProteicaRESUMO
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.
Assuntos
Contração Miocárdica , Humanos , Animais , Contração Miocárdica/efeitos dos fármacos , Doenças Cardiovasculares/tratamento farmacológico , Ratos , Relação Estrutura-Atividade , Masculino , Descoberta de Drogas , Troponina/metabolismo , Camundongos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Sulfonamidas/farmacocinética , Sulfonamidas/química , Sulfonamidas/uso terapêutico , Sulfonamidas/síntese químicaRESUMO
Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients (N = 54) to those of non-ICU patients undergoing thoracic surgery (N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.
Assuntos
Diafragma , Contração Muscular , Miosinas , Respiração Artificial , Músculos Respiratórios , Humanos , Animais , Miosinas/metabolismo , Diafragma/metabolismo , Diafragma/fisiopatologia , Músculos Respiratórios/metabolismo , Ratos , Masculino , Unidades de Terapia Intensiva , Pessoa de Meia-Idade , Feminino , Idoso , Hibernação/fisiologia , Actinas/metabolismoRESUMO
Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.
Assuntos
Doenças Musculares , Sarcômeros , Animais , Humanos , Cálcio/metabolismo , Contração Muscular , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Sarcômeros/metabolismo , Troponina I/genética , Troponina I/metabolismo , Peixe-Zebra/metabolismoRESUMO
Titin-truncating variants (TTNtv) are the single largest genetic cause of dilated cardiomyopathy (DCM). In this study we modeled disease phenotypes of A-band TTNtv-induced DCM in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using genome editing and tissue engineering technologies. Transcriptomic, cellular, and micro-tissue studies revealed that A-band TTNtv hiPSC-CMs exhibit pathogenic proteinopathy, sarcomere defects, aberrant Na+ channel activities, and contractile dysfunction. These phenotypes establish a dual mechanism of poison peptide effect and haploinsufficiency that collectively contribute to DCM pathogenesis. However, TTNtv cellular defects did not interfere with the function of the core contractile machinery, the actin-myosin-troponin-Ca2+ complex, and preserved the therapeutic mechanism of sarcomere modulators. Treatment of TTNtv cardiac micro-tissues with investigational sarcomere modulators augmented contractility and resulted in sustained transcriptomic changes that promote reversal of DCM disease signatures. Together, our findings elucidate the underlying pathogenic mechanisms of A-band TTNtv-induced DCM and demonstrate the validity of sarcomere modulators as potential therapeutics.
Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Humanos , Miócitos Cardíacos/patologia , Sarcômeros , Células-Tronco Pluripotentes Induzidas/patologia , Conectina/genética , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Contração MiocárdicaRESUMO
The present study examined the effect of dietary genistein, a soy isoflavone, on breast cancer patients who take tamoxifen, an antiestrogen treatment, using a preclinical model. The interaction of various doses of genistein with tamoxifen on the growth of estrogen receptor-positive breast cancer MCF-7 cells was investigated by subcutaneously injecting MCF-7 cells into the flank of ovariectomized athymic mice. Animals were randomized into eight experimental groups with 10-13 mice per group: control (C), estrogen (E) (0.08 mg E implant), tamoxifen (T) (3 mg T implant), estrogen + tamoxifen (E + T), tamoxifen + 500 p.p.m. genistein (T + G500), estrogen + tamoxifen + 250 p.p.m. genistein (E + T + G250), estrogen + tamoxifen + 500 p.p.m. genistein (E + T + G500) and estrogen + tamoxifen + 1000 p.p.m. genistein (E + T + G1000). Treatment of tamoxifen significantly reduced the estrogen-induced MCF-7 tumor prevalence and tumor size. This inhibitory effect of tamoxifen was significantly negated by the low doses of dietary genistein (250 and 500 p.p.m.), whereas the 1000 p.p.m. genistein did not have the same effect. Cells harvested from tamoxifen-treated tumors retained estrogen responsiveness of their progenitor MCF-7 cells, indicating that the abrogating effect of genistein on tamoxifen-treated tumor growth was not caused by a diminished tamoxifen response but directly by genistein. The low doses of dietary genistein abrogated the inhibitory effect of tamoxifen potentially by acting on the tumor cell proliferation/apoptosis ratio and the messenger RNA (mRNA) expression of cyclin D1 in addition to regulating the mRNA expression of progesterone receptor. Therefore, data from the current study suggest that caution is warranted regarding the consumption of dietary genistein by breast cancer patients while on tamoxifen therapy.
Assuntos
Antineoplásicos Hormonais/antagonistas & inibidores , Dieta , Genisteína/farmacologia , Tamoxifeno/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Primers do DNA , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estrogênios/sangue , Genisteína/administração & dosagem , Camundongos , Camundongos NusRESUMO
Modulation of sarcomere contractility represents a new therapeutic opportunity for the treatment of heart failure by directly targeting the thick and thin filament proteins of the sarcomere to increase cardiac muscle contraction. This study compared the effect of 2 small molecules (M and T) that selectively alter myosin thick filament (M) or troponin thin filament (T) activity on overall cardiac muscle mechanics. This study revealed key differences related to the mechanism utilized by M and T to increase contractile force generation and suggests that targeting different proteins within the sarcomere may result in differentiating therapeutic profiles.
RESUMO
BACKGROUND: Current heart failure therapies unload the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropes (ie, calcitropes) stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. A new class of agents-myotropes-activates the sarcomere directly, independent of calcium. We hypothesize that a novel myotrope TA1 increases contractility without the deleterious myocardial energetic impact of a calcitrope dobutamine. METHODS: We determined the effect of TA1 in bovine cardiac myofibrils and human cardiac microtissues, ex vivo in mouse cardiac fibers and in vivo in anesthetized normal rats. Effects of increasing concentrations of TA1 or dobutamine on contractile function, phosphocreatine and ATP concentrations, and ATP production were assessed by 31P nuclear magnetic resonance spectroscopy on isolated perfused rat hearts. RESULTS: TA1 increased the rate of myosin ATPase activity in isolated bovine myofibrils and calcium sensitivity in intact mouse papillary fibers. Contractility increased dose dependently in human cardiac microtissues and in vivo in rats as assessed by echocardiography. In isolated rat hearts, TA1 and dobutamine similarly increased the rate-pressure product. Dobutamine increased both developed pressure and heart rate accompanied by decreased phosphocreatine-to-ATP ratio and decreased free energy of ATP hydrolysis (ΔG~ATP) and elevated left ventricular end diastolic pressure. In contrast, the TA1 increased developed pressure without any effect on heart rate, left ventricular end diastolic pressure, phosphocreatine/ATP ratio, or ΔG~ATP. CONCLUSIONS: Novel myotrope TA1 increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long-term survival, myotropes may represent a superior alternative to traditional inotropes in heart failure management.
Assuntos
Dobutamina , Insuficiência Cardíaca , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Dobutamina/farmacologia , Metabolismo Energético , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Contração Miocárdica , Miocárdio/metabolismo , Fosfocreatina/metabolismo , Ratos , Troponina/metabolismoRESUMO
Troponin regulates the calcium-mediated activation of skeletal muscle. Muscle weakness in diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy occurs from diminished neuromuscular output. The first direct fast skeletal troponin activator, tirasemtiv, amplifies the response of muscle to neuromuscular input. Tirasemtiv binds selectively and strongly to fast skeletal troponin, slowing the rate of calcium release and sensitizing muscle to calcium. We report the solution NMR structure of tirasemtiv bound to a fast skeletal troponin C-troponin I chimera. The structure reveals that tirasemtiv binds in a hydrophobic pocket between the regulatory domain of troponin C and the switch region of troponin I, which overlaps with that of Anapoe in the X-ray structure of skeletal troponin. Multiple interactions stabilize the troponin C-troponin I interface, increase the affinity of troponin C for the switch region of fast skeletal troponin I, and drive the equilibrium toward the active state.
Assuntos
Imidazóis/farmacologia , Músculo Esquelético/efeitos dos fármacos , Pirazinas/farmacologia , Troponina C/metabolismo , Troponina I/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Imidazóis/química , Simulação de Acoplamento Molecular , Músculo Esquelético/fisiologia , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Pirazinas/química , Troponina C/química , Troponina I/químicaRESUMO
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.
Assuntos
Miosinas Cardíacas/efeitos dos fármacos , Cardiomiopatia Hipertrófica/tratamento farmacológico , Descoberta de Drogas , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Descoberta de Drogas , Músculo Esquelético/efeitos dos fármacos , Troponina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Relação Estrutura-AtividadeRESUMO
Structural changes in the mitotic arrest deficient protein 2 (Mad2) have been proposed to be essential for spindle checkpoint function. Current models for checkpoint activation propose that a C-Mad2-Mad1 core complex at unattached kinetochores is required for the structural activation through a process involving the interaction of two Mad2 conformers: a closed conformer bound to Mad1 or Cdc20 and an open conformer unbound to these ligands. To gain a molecular understanding of the mechanisms that accelerate the structural transition between the open and closed Mad2 conformations, we constructed a unique in vitro homogeneous Mad2 activity assay that specifically reports C-Mad2-Cdc20 formation. Using this assay we were are able to directly establish that (a) O-Mad2 transforms into a C-Mad2-Cdc20 complex >300-fold slower than unliganded C-Mad2, (b) a stable C-Mad2-Mad1 core complex catalyzes the transformation of O-Mad2 into a Cdc20-bound C-Mad2 complex, (c) a C-Mad2-Cdc20 complex can promote its own transformation of O-Mad2 into a Cdc20-bound C-Mad2 complex, and (d) the binding interaction between unliganded C-Mad2 and Cdc20 cannot be catalyzed by a C-Mad2-Mad1 core complex. Our data are consistent with the "Mad2 template" catalytic model in which a C-Mad2 template facilitates the binding of O-Mad2 to Cdc20 and supports a mechanism of C-Mad2-Cdc20 formation away from Mad1 containing kinetochores. Furthermore, our unique homogeneous Mad2 assay could be translated into a screening platform to identify small molecule drug-like compounds that directly modulate C-Mad2-Cdc20 formation.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Fuso Acromático/química , Fuso Acromático/metabolismo , Sequência de Aminoácidos , Catálise , Proteínas Cdc20 , Polarização de Fluorescência , Humanos , Cinética , Ligantes , Proteínas Mad2 , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Genistein (GEN), a soy isoflavone, stimulates growth of estrogen-dependent human tumor cells (MCF-7) in a preclinical mouse model for postmenopausal breast cancer. Antiestrogens and aromatase inhibitors are frontline therapies for estrogen-dependent breast cancer. We have demonstrated that dietary GEN can negate the inhibitory effect of tamoxifen. In this study, we evaluated the interaction of dietary GEN (at 250-1000 p.p.m. in the American Institute of Nutrition 93 growth diet) and an aromatase inhibitor, letrozole (LET), on the growth of tumors in an aromatase-expressing breast cancer xenograft model (MCF-7Ca) in the presence and absence of the substrate androstenedione (AD). Dietary GEN (250 and 500 p.p.m.) or implanted AD stimulated MCF-7Ca tumor growth. Implanted LET inhibited AD-stimulated MCF-7Ca tumor growth. In the presence of AD and LET, dietary GEN (250, 500 and 1000 p.p.m.) reversed the inhibitory effect of LET in a dose-dependent manner. Uterine wet weight, plasma estradiol (E(2)) levels (enzyme-linked immunosorbent assay) and total plasma GEN and LET levels (liquid chromatography-electrospray/tandem mass spectrometry) were measured. Ki-67 (cellular proliferation), aromatase and pS2 protein expression in tumors were evaluated using immunohistochemical (IHC) analysis. In conclusion, dietary GEN increased the growth of MCF-7Ca tumors implanted in ovariectomized mice and could also negate the inhibitory effect of LET on MCF-7Ca tumor growth. These findings are significant because tumors, which express aromatase and synthesize estrogen, are good candidates for aromatase therapy dietary and GEN can reverse the inhibitory effect of LET on tumor growth and adversely impact breast cancer therapy. Caution is warranted for consumption of dietary GEN by postmenopausal women with estrogen-dependent breast cancer taking LET treatment.