RESUMEN
LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.
Asunto(s)
Aminopeptidasas/química , Azepinas/farmacología , Proteínas 14-3-3 , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/genética , Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Sitios de Unión , División Celular , Línea Celular Tumoral , Clonación Molecular , Cobalto/química , Cristalografía por Rayos X , Ciclohexanos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Inhibidores Enzimáticos/farmacología , Ácidos Grasos Insaturados/farmacología , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Metionil Aminopeptidasas , Modelos Químicos , Modelos Moleculares , Péptidos/química , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteoma , ARN Interferente Pequeño/metabolismo , Sesquiterpenos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Alanine racemase (EC 5.1.1.1) catalyzes the interconversion of alanine enantiomers, and thus represents the first committed step involved in bacterial cell wall biosynthesis. Cycloserine acts as a suicide inhibitor of alanine racemase and as such, serves as an antimicrobial agent. The chemical means by which cycloserine inhibits alanine racemase is unknown. Through spectroscopic assays, we show here evidence of a pyridoxal derivative (arising from either isomer of cycloserine) saturated at the C4' carbon position. We additionally report the L- and D-cycloserine inactivated crystal structures of Bacillus stearothermophilus alanine racemase, which corroborates the spectroscopy via evidence of a 3-hydroxyisoxazole pyridoxamine derivative. Upon the basis of the kinetic and structural properties of both the L- and D-isomers of the inhibitor, we propose a mechanism of alanine racemase inactivation by cycloserine. This pathway involves an initial transamination step followed by tautomerization to form a stable aromatic adduct, a scheme similar to that seen in cycloserine inactivation of aminotransferases.