RESUMEN
Phosphatidylinositol-4-kinase IIIα (PI4KIIIα) is an essential host cell factor for hepatitis C virus (HCV) replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for small-molecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIIIα inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIIIα inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4IIIα-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4IIIα inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIIIα as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIIIα, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIIIα inhibitors for treatment of chronic HCV infection.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Replicación Viral , 1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Animales , Antivirales/farmacología , Línea Celular , Análisis Mutacional de ADN , Farmacorresistencia Viral , Inhibidores Enzimáticos/farmacología , Femenino , Genes Esenciales , Hepatocitos/enzimología , Hepatocitos/virología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
An effort aimed at exploring structural diversity in the N-pyrazole-N'-naphthylurea class of p38 kinase inhibitors led to the synthesis and characterization of N-phenyl-N'-naphthylureas. Examples of these compounds displayed excellent inhibition of TNF-alpha production in vitro, as well as efficacy in a mouse model of lipopolysaccharide induced endotoxemia. In addition, perspective is provided on the role of a sulfonamide functionality in defining inhibitor potency.
Asunto(s)
2-Naftilamina/análogos & derivados , Inhibidores de Proteínas Quinasas/farmacología , Urea/análogos & derivados , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , 2-Naftilamina/química , Animales , Química Orgánica/métodos , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Concentración 50 Inhibidora , Lipopolisacáridos/metabolismo , Ratones , Modelos Químicos , Estructura Molecular , Factor de Necrosis Tumoral alfa/metabolismo , Urea/químicaRESUMEN
We report on a series of N-pyrazole, N'-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5'-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Inhibidores Enzimáticos/síntesis química , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Naftalenos/síntesis química , Pirazoles/síntesis química , Urea/análogos & derivados , Urea/síntesis química , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Unión Competitiva , Línea Celular , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Fluorescencia , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Naftalenos/química , Naftalenos/farmacología , Unión Proteica , Pirazoles/química , Pirazoles/farmacología , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/biosíntesis , Urea/química , Urea/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Discovery of the pyrazole-naphthyl urea class of p38 MAP kinase inhibitors typified by the clinical candidate BIRB 796 has encouraged further exploration of this particular scaffold. Modification to the part of the inhibitor that occupies the adenine/ATP binding site has resulted in a new way to obtain potent inhibitors that possess favorable in vitro and in vivo properties.