RESUMEN
FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of T m (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.
Asunto(s)
Antituberculosos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Ligasas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/farmacología , Cromatografía en Capa Delgada/métodos , Descubrimiento de Drogas/métodos , Ligasas/genética , Ligasas/metabolismo , Modelos Biológicos , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Unión Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Estudios de Validación como AsuntoRESUMEN
We describe a 21 year old man who was found to have an opening on his external nose since birth. No other clinical and radiological abnormalities were demonstrable elsewhere in his body. The opening is on the lower part of the nose one cm. away from the midline on right side, clinically looking like a sinus. Histological examination of the excised tract showed an organoid structure that was consistent with an"Accessory nose."
