RESUMEN
Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention.
Asunto(s)
Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas , Oxadiazoles , Oxadiazoles/química , Oxadiazoles/farmacología , Histona Desacetilasa 6/antagonistas & inhibidores , Histona Desacetilasa 6/metabolismo , Histona Desacetilasa 6/química , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Cristalografía por Rayos X , Cinética , Unión Proteica , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
BACKGROUND: In the search of genetic determinants of Duchenne muscular dystrophy (DMD) severity, LTBP4, a member of the latent TGF-ß binding protein family, emerged as an important predictor of functional outcome trajectories in mice and humans. Nonsynonymous single-nucleotide polymorphisms in LTBP4 gene associate with prolonged ambulation in DMD patients, whereas an in-frame insertion polymorphism in the mouse LTBP4 locus modulates disease severity in mice by altering proteolytic stability of the Ltbp4 protein and release of transforming growth factor-ß (TGF-ß). Givinostat, a pan-histone deacetylase inhibitor currently in phase III clinical trials for DMD treatment, significantly reduces fibrosis in muscle tissue and promotes the increase of the cross-sectional area (CSA) of muscles in mdx mice. In this study, we investigated the activity of Givinostat in mdx and in D2.B10 mice, two mouse models expressing different Ltbp4 variants and developing mild or more severe disease as a function of Ltbp4 polymorphism. METHODS: Givinostat and steroids were administrated for 15 weeks in both DMD murine models and their efficacy was evaluated by grip strength and run to exhaustion functional tests. Histological examinations of skeletal muscles were also performed to assess the percentage of fibrotic area and CSA increase. RESULTS: Givinostat treatment increased maximal normalized strength to levels that were comparable to those of healthy mice in both DMD models. The effect of Givinostat in both grip strength and exhaustion tests was dose-dependent in both strains, and in D2.B10 mice, Givinostat outperformed steroids at its highest dose. The in vivo treatment with Givinostat was effective in improving muscle morphology in both mdx and D2.B10 mice by reducing fibrosis. CONCLUSION: Our study provides evidence that Givinostat has a significant effect in ameliorating both muscle function and histological parameters in mdx and D2.B10 murine models suggesting a potential benefit also for patients with a poor prognosis LTBP4 genotype.
Asunto(s)
Distrofia Muscular de Duchenne , Animales , Carbamatos , Modelos Animales de Enfermedad , Haplotipos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Ratones , Ratones Endogámicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genéticaRESUMEN
Histone deacetylase 6 (HDAC6) is a peculiar HDAC isoform whose expression and functional alterations have been correlated with a variety of pathologies such as autoimmune disorders, neurodegenerative diseases, and cancer. It is primarily a cytoplasmic protein, and its deacetylase activity is focused mainly on nonhistone substrates such as tubulin, heat shock protein (HSP)90, Foxp3, and cortactin, to name a few. Selective inhibition of HDAC6 does not show cytotoxic effects in healthy cells, normally associated with the inhibition of Class I HDAC isoforms. Here, we describe the design and synthesis of a new class of potent and selective HDAC6 inhibitors that bear a pentaheterocyclic central core. These compounds show a remarkably low toxicity both in vitro and in vivo and are able to increase the function of regulatory T cells (Tregs) at well-tolerated concentrations, suggesting a potential clinical use for the treatment of degenerative, autoimmune diseases and for organ transplantation.