RESUMEN
Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Girasa de ADN/química , Quinolinas/química , Quinolinas/farmacología , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II , Antibacterianos/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Arginina/metabolismo , Ácido Aspártico/metabolismo , Sitios de Unión , Dominio Catalítico , Ciprofloxacina/química , Ciprofloxacina/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , División del ADN , Girasa de ADN/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Diseño de Fármacos , Resistencia a Medicamentos , Escherichia coli/enzimología , Manganeso/metabolismo , Modelos Moleculares , Conformación Proteica , Quinolinas/metabolismo , Quinolonas/química , Quinolonas/metabolismo , Relación Estructura-ActividadRESUMEN
Understanding the quality of a screening collection is the first step to improving it and, as a result, the quality of the screening process. This article outlines how this issue was approached at GlaxoSmithKline and some of the hurdles that needed to be overcome to achieve success. The article focuses specifically on the necessary software and hardware infrastructure needed, and at some of the extra benefits of such a project in terms of data mining and data modelling.
Asunto(s)
Técnicas Químicas Combinatorias/métodos , Industria Farmacéutica/métodos , Almacenamiento y Recuperación de la Información/métodos , Cromatografía Líquida de Alta Presión , Técnicas Químicas Combinatorias/organización & administración , Computadores , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Control de Calidad , Programas InformáticosRESUMEN
Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.
Asunto(s)
Citocromo P-450 CYP2D6/química , Secuencia de Aminoácidos , Ácido Aspártico/química , Sitios de Unión , Monóxido de Carbono/química , Cristalografía por Rayos X , Ácido Glutámico/química , Hemo/química , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Programas Informáticos , Fracciones Subcelulares , Especificidad por SustratoRESUMEN
The design of conformationally restricted eight-membered ring diketones as transition state mimics of the mechanism of action of cyclotheonamides on serine proteases is described. Two target compounds are prepared from mutilin, derived from the natural product pleuromutilin. Compound 3 shows significant inhibition of plasmin and urokinase in enzyme rate assays, but an analogue 4 in which the amide moiety has been omitted does not. An X-ray crystal structure of the diketone 3 confirms the conformational predictions made by molecular modelling.
Asunto(s)
Cetonas/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Hidrocarburos Aromáticos con Puentes/síntesis química , Hidrocarburos Aromáticos con Puentes/farmacología , Diseño de Fármacos , Cetonas/química , Cetonas/farmacología , Cinética , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Compuestos Policíclicos/química , Compuestos Policíclicos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-ActividadRESUMEN
A convenient two-step homologation of both aliphatic and aromatic ketones to the corresponding carboxylic acid has been developed. First ketones were converted to epoxynitriles with the Darzens reaction. Second, a Lewis acid mediated rearrangement of these epoxynitriles with lithium bromide was achieved to give homologated secondary alkanoic acids (as well as aryl-alkanoic) in good yields. The mechanism and the scope of the rearrangement reaction were investigated. This strategy constitutes a two-step homologation of ketones to secondary carboxylic acids.