RESUMEN
Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aß levels, but did not lower rat brain Aß due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1.
Asunto(s)
Aminas/química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Indoles/síntesis química , Inhibidores de Proteasas/química , Piridinas/síntesis química , Aminas/síntesis química , Aminas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/sangre , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Indoles/química , Indoles/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína , Piridinas/química , Piridinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Antisense RNA is a versatile tool for reducing gene expression. It was used to determine if ahpC, a gene that is involved in defence against oxidative stress and isoniazid (INH) resistance, is important for virulence of Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. Antisense RNA constructs of ahpC were made using different strength promoters in front of a reversed coding sequence of ahpC. These constructs were electroporated into a virulent wild-type M. bovis strain and a moderately virulent INH-resistant M. bovis strain that was catalase/peroxidase-negative. Down-regulation of protein synthesis occurred and this was visualized by immunoblotting. All strains containing antisense RNA were markedly less virulent than their parent strains in guinea pigs. M. bovis with an up-regulated ahpC gene was more resistant to cumene hydroperoxide than its parent strain, which had a wild-type ahpC promoter. These results agree with a model of INH resistance in which overexpression of AhpC compensates in some INH-resistant strains for loss of catalase/peroxidase by maintaining the ability to defend against oxidative stress mediated through organic peroxides. In addition, normal expression of AhpC is crucial for maintaining the virulence of wild-type M. bovis, which has normal catalase/peroxidase levels.