RESUMO
The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in âºDG of TA muscle biopsies in the evaluation of potential therapeutics.
Assuntos
Western Blotting , Distroglicanas , Músculo Esquelético , Distrofia Muscular do Cíngulo dos Membros , Humanos , Distroglicanas/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Western Blotting/métodos , Glicosilação , Masculino , FemininoRESUMO
Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of ß-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from ß-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Benzilaminas/química , Miosinas Cardíacas/química , Subfragmentos de Miosina/química , Sarcômeros/química , Uracila/análogos & derivados , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Miosinas Cardíacas/metabolismo , Cardiomegalia/metabolismo , Bovinos , Subfragmentos de Miosina/metabolismo , Sarcômeros/metabolismo , Uracila/químicaRESUMO
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes in response to mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a potentially novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.
Assuntos
Distrofia Muscular Animal , Distrofia Muscular de Duchenne , Camundongos , Animais , Cães , Distrofia Muscular de Duchenne/metabolismo , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofina/genética , Contração Muscular/fisiologia , Modelos Animais de Doenças , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismoRESUMO
In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.
Assuntos
Aminoácido Oxirredutases/metabolismo , Aminopropionitrilo/farmacologia , Anticorpos/farmacologia , Regulação Alostérica , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/imunologia , Aminoácidos/metabolismo , Ligação Competitiva , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Cinética , Metais/farmacologia , Fragmentos de Peptídeos/isolamento & purificação , Proteína-Lisina 6-Oxidase/metabolismo , Pirrolidinas/metabolismo , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Espermina/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Benzilaminas/administração & dosagem , Miosinas Cardíacas/antagonistas & inibidores , Cardiomiopatia Hipertrófica Familiar/tratamento farmacológico , Contração Miocárdica/efeitos dos fármacos , Cadeias Pesadas de Miosina/antagonistas & inibidores , Sarcômeros/efeitos dos fármacos , Uracila/análogos & derivados , Animais , Benzilaminas/química , Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica Familiar/patologia , Cardiomiopatia Hipertrófica Familiar/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Mutação , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Cadeias Pesadas de Miosina/genética , Ratos , Uracila/administração & dosagem , Uracila/químicaRESUMO
We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-beta (TGF-beta) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor beta-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.
Assuntos
Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/efeitos dos fármacos , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Aminopropionitrilo/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Lactonas/farmacologia , Camundongos , Camundongos Nus , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Polienos/farmacologia , RNA Interferente Pequeno/genética , Transcrição Gênica , Transfecção , Transplante HeterólogoRESUMO
We report the design, synthesis, and optimization of the first, selective activators of cardiac myosin. Starting with a poorly soluble, nitro-aromatic hit compound (1), potent, selective, and soluble myosin activators were designed culminating in the discovery of omecamtiv mecarbil (24). Compound 24 is currently in clinical trials for the treatment of systolic heart failure.
RESUMO
Escherichia coli CspA is a small all-beta-sheet protein that folds fast (tau = 4 ms) via an apparent two-state mechanism. Our previous studies have shown that a large aromatic cluster on the surface of the protein participates in the rate-limiting step of folding and thus may be part of the folding nucleus of this protein. To obtain a more detailed picture of molecular events at the peptide backbone during unfolding and folding of CspA, we used native state hydrogen exchange and nuclear magnetic resonance spectroscopy (NMR). The experiments with native CspA were performed over a range of pH values from low pH, where exchange is governed by a rapid equilibrium before chemical exchange (EX2 exchange), to high pH, where exchange is dictated by the rate of unfolding (EX1 exchange). Rates of folding and unfolding were determined for 11 residues. The distribution of rates of folding within the structure of CspA suggests that hairpin turns, including one near the aromatic cluster, may nucleate the folding of CspA.