RESUMEN
Ryanodine receptors (RyRs) are important ligand-gated Ca2+ channels; their excessive activation leads to Ca2+ leakage in the sarcoplasmic reticulum that may cause neurological diseases. In this study, three series of novel potent RyR1 inhibitors based on dantrolene and bearing semicarbazone and imidazolyl moieties were designed and synthesized, and their biological activity was evaluated. Using a single-cell calcium imaging method, the calcium overload inhibitory activities of 26 target compounds were tested in the R614C cell line, using dantrolene as a positive control. The preliminary investigation showed that compound 12a suppressed Ca2+ release as evidenced by store overload-induced Ca2+release (SOICR) (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%) at 0.1 µM, 3 µM and 10 µM, respectively. Docking simulation results showed that compound 12a could bind at the active site of the RyR1 protein. The Morris water-maze test showed that compound 12a significantly improved the cognitive behavior of AD-model mice. Further studies on the structural optimization of this series of derivatives are currently underway in our laboratory.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Bloqueadores de los Canales de Calcio/síntesis química , Fármacos Neuroprotectores/síntesis química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Semicarbazonas/síntesis química , Animales , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio , Dantroleno/química , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Imidazoles/química , Masculino , Ratones , Simulación del Acoplamiento Molecular , Prueba del Laberinto Acuático de Morris , Fármacos Neuroprotectores/farmacología , Unión Proteica , Conformación Proteica , Semicarbazonas/farmacología , Análisis de la Célula Individual , Relación Estructura-ActividadRESUMEN
Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial results showing that dantrolene inhibited Ca2+ release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single RyR channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene's action was lost during the purification procedure of RyR. Recently, Mg2+ was demonstrated to be the essential factor for dantrolene to inhibit Ca2+ release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca2+ release and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca2+ release experiments demonstrated that the effect of dantrolene and Mg2+ was cooperative and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 µM dantrolene reduced RyR channel open probability by â¼50% in the presence of 3 mM free Mg2+ and 1 mM ATP, whereas channel activity further decreased to â¼20% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that supports the direct, Mg2+-dependent mechanism of dantrolene's action and suggests that dantrolene also requires ATP to inhibit RyR.