RESUMEN
In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.
Asunto(s)
Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Pseudomonas aeruginosa/enzimología , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Catálisis , Cromatografía Liquida , Cristalografía por Rayos X , Escherichia coli , Expresión Génica , Espectrometría de Masas , Conformación Proteica , Zinc/química , Zinc/metabolismoRESUMEN
We describe a novel series of potent inhibitors of human beta-secretase. These compounds possess the hydroxyethyl amine transition state isostere. A 2.5A crystal structure of inhibitor 32 bound to BACE is provided.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores de Proteasas/química , Sulfonamidas/química , Sulfonas/química , Secretasas de la Proteína Precursora del Amiloide/química , Diseño de Fármacos , Humanos , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Conformación Proteica , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonas/síntesis químicaRESUMEN
We describe an optimized series of acyclic hydroxyethylamine transition state isosteres of beta-secretase that incorporates a variety of P(2) side chains that yield potent inhibitors with excellent cellular activity. A 2.2A crystal structure of compound 13 is shown.