RESUMEN
Triclosan is used widely as an antibacterial agent in dermatological products, mouthwashes, and toothpastes. Recent studies imply that antibacterial activity results from binding to enoyl (acyl carrier protein) reductase (EACPR, EC 1.3.1.9). We first recognized the ability of triclosan to inhibit EACPR from Escherichia coli in a high throughput screen where the enzyme and test compound were preincubated with NAD(+), which is a product of the reaction. The concentration of triclosan required for 50% inhibition approximates to 50% of the enzyme concentration, indicating that the free compound is depleted by binding to EACPR. With no preincubation or added NAD(+), the degree of inhibition by 150 nM triclosan increases gradually over several minutes. The onset of inhibition is more rapid when NAD(+) is added. Gel filtration and mass spectrometry show that inhibition by triclosan is reversible. Steady-state assays were designed to avoid depletion of free inhibitor and changes in the degree of inhibition. The results suggest that triclosan binds to E-NAD(+) complex, with a dissociation constant around 20-40 pM. Triclosan follows competitive kinetics with respect to NADH, giving an inhibition constant of 38 pM at zero NADH and saturating NAD(+). Uncompetitive kinetics are observed when NAD(+) is varied, giving an inhibition constant of 22 pM at saturating NAD(+). By following regain of catalytic activity after dilution of EACPR that had been preincubated with triclosan and NAD(+), the rate constant for dissociation of the inhibitor (k(off)) is measured as 1.9 x 10(-4) s(-1). The association rate constant (k(on)) is estimated as 2.6 x 10(7) s(-1) M(-1) by monitoring the onset of inhibition during assays started by addition of EACPR. As expected, the ratio k(off)/k(on) = 7.1 pM is similar to the inhibition constants from the steady-state studies. The crystal structure of E. coli EACPR in a complex with coenzyme and triclosan has been determined at 1.9 A resolution, showing that this compound binds in a similar site to the diazaborine inhibitors. The high affinity of triclosan appears to be due to structural similarity to a tightly bound intermediate in catalysis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Triclosán/farmacología , Antiinfecciosos Locales/química , Antiinfecciosos Locales/farmacología , Unión Competitiva , Catálisis , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli , Acido Graso Sintasa Tipo II , Cinética , Espectrometría de Masas , Modelos Químicos , NAD/metabolismo , NAD/farmacología , Oxidorreductasas/metabolismo , Relación Estructura-Actividad , Triclosán/químicaRESUMEN
Members of a series of 2,2'-bipyridines have been synthesized and tested as inhibitors of prolyl hydroxylase (EC 1.14.11.2). The structure-activity relationships seen with [2,2'-bipyridine]-5-carboxylic acid (4) closely resemble those of pyridine-2-carboxylic acid (2). Accordingly, [2,2'-bipyridine]-5,5'-dicarboxylic acid (11, IC50 = 0.19 microM) is the most potent inhibitor of its type yet reported. However, 2,2'-bipyridines lacking a 5-carboxylate are poor inhibitors. These contrasting structure-activity relationships are discussed in terms of net anionic charge, iron chelation, and the availability of alternative putative binding modes at a single binding site in each catalytic subunit.
Asunto(s)
2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Ácidos Nicotínicos/síntesis química , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , 2,2'-Dipiridil/síntesis química , 2,2'-Dipiridil/farmacología , Sitios de Unión , Estructura Molecular , Ácidos Nicotínicos/farmacología , Relación Estructura-ActividadRESUMEN
N-Oxaloglycine (3) is an alpha-ketoglutarate (1) analogue that is a competitive inhibitor of prolyl 4-hydroxylase (EC 1.14.11.2). A study of the structure-activity relationships of some other oxalo derivatives shows that substitution on the glycine moiety modulates activity stereoselectively and that if the omega-carboxylate is homologated or replaced by either acylsulfonamides or anilide, then activity is sharply reduced. This sensitivity to these changes is contrasted with the relative insensitivity of another putative alpha-ketoglutarate analogue, pyridine-2,5-dicarboxylic acid (2), and the implication is discussed that compounds of both series are unlikely to bind to prolyl hydroxylase in the same way even though both inhibit the enzyme competitively.