RESUMEN
Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.
Asunto(s)
Músculo Esquelético/metabolismo , Miosinas del Músculo Esquelético/efectos de los fármacos , Miosinas del Músculo Esquelético/genética , Adulto , Animales , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Línea Celular , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Masculino , Ratones , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Espasticidad Muscular/genética , Espasticidad Muscular/fisiopatología , Músculo Esquelético/fisiología , Miosinas/efectos de los fármacos , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas , Ratas , Ratas Wistar , Miosinas del Músculo Esquelético/metabolismoRESUMEN
Blebbistatin is a widely used inhibitor of myosin 2 that enables the study of a broad range of cytoskeleton-related processes. However, blebbistatin has several limitations hindering its applicability: it is fluorescent, poorly water soluble, cytotoxic, and prone to (photo)degradation. Despite these adverse effects, being the only available myosin 2-specific inhibitor, blebbistatin is rather a choice of necessity. Blebbistatin has been modified to improve its properties and some of the new compounds have proven to be useful replacements of the original molecule. This review summarizes recent results on blebbistatin development. We also discuss the pharmacological perspectives of these efforts, as myosins are becoming promising drug target candidates for a variety of conditions ranging from neurodegeneration to muscle disease, wound healing, and cancer metastasis.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Compuestos Heterocíclicos de 4 o más Anillos , Enfermedades Musculares , Miosinas/antagonistas & inhibidores , Neoplasias , Enfermedades Neurodegenerativas , Cicatrización de Heridas/efectos de los fármacos , Animales , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Enfermedades Musculares/tratamiento farmacológico , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Miosinas/metabolismo , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patologíaRESUMEN
Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.
Asunto(s)
Absorción Fisicoquímica , Descubrimiento de Drogas , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Miosinas/antagonistas & inhibidores , Animales , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/toxicidad , Humanos , Simulación de Dinámica Molecular , Miosinas/química , Conformación Proteica , Ratas , Distribución TisularRESUMEN
We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, ß-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg(2+)-dependent manner (0.3-9.0 mm free Mg(2+)) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg(2+) in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg(2+) in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg(2+) coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg(2+) concentrations, demonstrating that the ADP release rate constant is slowed by Mg(2+) in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg(2+) reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg(2+) inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg(2+)-dependent alterations in actin binding. Overall, our results suggest that Mg(2+) reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.
Asunto(s)
Actinas/metabolismo , Adenosina Difosfato/metabolismo , Magnesio/fisiología , Proteínas Motoras Moleculares/metabolismo , Miosinas/metabolismo , Animales , Cinética , Miocardio/metabolismo , Unión Proteica , Conejos , PorcinosRESUMEN
Our aim was to elucidate the physical background of internal friction of enzyme reactions by investigating the temperature dependence of internal viscosity. By rapid transient kinetic methods, we directly measured the rate constant of trypsin 4 activation, which is an interdomain conformational rearrangement, as a function of temperature and solvent viscosity. We found that the apparent internal viscosity shows an Arrhenius-like temperature dependence, which can be characterized by the activation energy of internal friction. Glycine and alanine mutations were introduced at a single position of the hinge of the interdomain region to evaluate how the flexibility of the hinge affects internal friction. We found that the apparent activation energies of the conformational change and the internal friction are interconvertible parameters depending on the protein flexibility. The more flexible a protein was, the greater proportion of the total activation energy of the reaction was observed as the apparent activation energy of internal friction. Based on the coupling of the internal and external movements of the protein during its conformational change, we constructed a model that quantitatively relates activation energy, internal friction, and protein flexibility.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Sustitución de Aminoácidos , Elasticidad , Activación Enzimática , Fricción , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Transición de Fase , Conformación Proteica , Temperatura , Termodinámica , Tripsina/química , Tripsina/genética , Tripsina/metabolismo , ViscosidadRESUMEN
Simple and consistent chiral HPLC methods for the efficient separation of enantiomeric blebbistatin derivatives, namely parent compound blebbistatin and derivatives 4-nitroblebbistatin, 4-aminoblebbistatin, 4-dimethylaminoblebbistatin, and 4-t-butylblebbistatin were developed using cellulose tris(3,5-dimethylphenylcarbamate) as a stationary phase (Lux cellulose-1 column). Blebbistatin, 4-aminoblebbistatin, and 4-dimethylaminoblebbistatin racemates were well-separated in normal-phase HPLC conditions while 4-nitroblebbistatin and 4-t-butylblebbistatin were effectively separated in both normal- and reversed-phase HPLC conditions. Furthermore, the order of elution of enantiopure compounds was found to be independent of mobile phase compositions and conditions used, and solely depends on the interaction between the enantiomer and the chiral stationary phase. We found that despite the chiral center being present far from the D-ring in the blebbistatin structure, the D-ring substitutions prominently affect the chiral separation. Ex vivo racemization studies of the most popular blebbistatin derivative (S)-(-)-4-aminoblebbistatin in rat blood and brain tissues revealed that the compound does not convert into the inactive enantiomer. This confirms that (S)-(-)-4-aminoblebbistatin is a useful tool compound in cellular and molecular biology studies without the risks of racemization and degradation effects.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos , Vertebrados , Animales , Cromatografía Líquida de Alta Presión , Ratas , EstereoisomerismoRESUMEN
BACKGROUND: Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values) and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs) were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. RESULTS: PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. CONCLUSIONS: This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that, except for few specific cases, the shapes of the binding pockets have relatively low weights in the determination of the affinity profiles of proteins. Since the MAF profile is closely related to the target specificity of ligand binding sites we can conclude that the shape of the binding site is not a pivotal factor in selecting drug targets. Nonetheless, based on strong specific associations between certain MAF profiles and specific geometric descriptors we identified, the shapes of the binding sites do have a crucial role in virtual drug design for certain drug categories, including morphine derivatives, benzodiazepines, barbiturates and antihistamines.
Asunto(s)
Sitios de Unión/genética , Preparaciones Farmacéuticas/metabolismo , Unión Proteica/fisiología , Conformación Proteica , Proteínas/genética , Proteínas/metabolismo , Análisis Factorial , Humanos , Análisis de Componente Principal , Unión Proteica/genética , Relación Estructura-Actividad Cuantitativa , Sensibilidad y Especificidad , Bibliotecas de Moléculas PequeñasRESUMEN
It has been shown that not only calcium but also strong binding myosin heads contribute to thin filament activation in isometrically contracting animal fast-twitch and cardiac muscle preparations. This behavior has not been studied in human muscle fibers or animal slow-twitch fibers. Human slow-twitch fibers are interesting since they contain the same myosin heavy chain isoform as the human heart. To explore myosin-induced activation of the thin filament in isometrically contracting human slow-twitch fibers, the endogenous troponin complex was exchanged for a well-characterized fast-twitch skeletal troponin complex labeled with the fluorescent dye N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (fsTn-IANBD). The exchange was ≈70% complete (n = 8). The relative contributions of calcium and strong binding cross-bridges to thin filament activation were dissected by increasing the concentration of calcium from relaxing (pCa 7.5) to saturating levels (pCa 4.5) before and after incubating the exchanged fibers in the myosin inhibitor para-aminoblebbistatin (AmBleb). At pCa 4.5, the relative contributions of calcium and strong binding cross-bridges to thin filament activation were ≈69 and ≈31%, respectively. Additionally, switching from isometric to isotonic contraction at pCa 4.5 revealed that strong binding cross-bridges contributed ≈29% to thin filament activation (i.e., virtually the same magnitude obtained with AmBleb). Thus, we showed through two different approaches that lowering the number of strong binding cross-bridges, at saturating calcium, significantly reduced the activation of the thin filament in human slow-twitch fibers. The contribution of myosin to activation resembled that which was previously reported in rat cardiac and rabbit fast-twitch muscle preparations. This method could be applied to slow-twitch human fibers obtained from the soleus muscle of cardiomyopathy patients. Such studies could lead to a better understanding of the effect of point mutations of the cardiac myosin head on the regulation of muscle contraction and could lead to better management by pharmacological approaches.
RESUMEN
Technological resources for sustained local control of molecular effects within organs, cells, or subcellular regions are currently unavailable, even though such technologies would be pivotal for unveiling the molecular actions underlying collective mechanisms of neuronal networks, signaling systems, complex machineries, and organism development. We present a novel optopharmacological technology named molecular tattooing, which combines photoaffinity labeling with two-photon microscopy. Molecular tattooing covalently attaches a photoreactive bioactive compound to its target by two-photon irradiation without any systemic effects outside the targeted area, thereby achieving subfemtoliter, long-term confinement of target-specific effects in vivo. As we demonstrated in melanoma cells and zebrafish embryos, molecular tattooing is suitable for dissecting collective activities by the separation of autonomous and non-autonomous molecular processes in vivo ranging from subcellular to organism level. Since a series of drugs are available for molecular tattoo, the technology can be implemented by a wide range of fields in the life sciences.