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
We show that the antibacterial agent, (6S)-6-fluoroshikimate, is a substrate for the shikimate transport system of Escherichia coli because in exchange-diffusion experiments it displaced intracellular [14C]shikimate with the same kinetics as did unlabelled shikimate. Other shikimate analogues were also substrates: as judged by similar experiments or, in the case of (6R)-6-fluoroshikimate, by inference.
Asunto(s)
Antibacterianos/metabolismo , Escherichia coli/metabolismo , Ácido Shikímico/análogos & derivados , Ácido Shikímico/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Difusión , CinéticaRESUMEN
Spontaneous resistance to (6S)-6-fluoro-shikimic acid arose in Escherichia coli and other enterobacteria at high frequencies, between 10(-5) and 10(-4). Two resistant variants of E. coli were tested for their susceptibilities to the diastereomeric compound, (6R)-6-fluoro-shikimate, and both of them had become resistant to this compound as well. (6S)-6-Fluoro-shikimate-resistant variants of E. coli generally failed to transport [14C]shikimate. In E. coli K-12, (6S)-6-fluoro-shikimate resistance cotransduced with his at the same frequency as shiA, a gene locus that governs shikimate transport phenotypes. We propose that the loss of susceptibility to (6S)-6-fluoro-shikimic acid in spontaneous resistant variants is due to the loss of activity of the transport system by which it enters the bacterial cytoplasm.
Asunto(s)
Antibacterianos/farmacología , Enterobacter/efectos de los fármacos , Ácido Shikímico/análogos & derivados , Antibacterianos/farmacocinética , Farmacorresistencia Microbiana , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Ácido Shikímico/farmacocinética , Ácido Shikímico/farmacología , EstereoisomerismoRESUMEN
(6S)-6-Fluoroshikimic acid inhibited the growth of Escherichia coli B on minimal medium (MIC, 0.25 micrograms ml-1), and it protected mice challenged intraperitoneally with the same organism (50% protective dose, 0.06 mg kg of body weight-1). We propose that inhibitors of bacterial aromatic biosynthesis have the potential for use in human medicine.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Ácido Shikímico/análogos & derivados , Ácido Shikímico/antagonistas & inhibidores , Ácido Shikímico/metabolismo , Animales , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/prevención & control , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Ácido Shikímico/farmacología , PorcinosRESUMEN
Membrane preparations were made from a number of Gram-positive and Gram-negative bacterial strains. The ability of the membrane-bound penicillin-binding proteins (PBP) in these preparations to bind [14C]cefotetan has been studied. Increasing amounts of unlabelled benzylpenicillin were added and subsequently [14C]cefotetan was introduced to determine its ability to bind to additional proteins or to displace the pre-bound benzylpenicillin. In the Gram-positive series some quantitative binding differences between the two labelled compounds could be seen. However, in the majority of Gram-negative organisms tested cefotetan exhibited greatest affinity for PBP 3 and none for PBP 2. No new PBPs were detected using [14C]cefotetan, suggesting that the compound exerts its antibacterial effect by a mechanism similar to the other cephamycins. The high minimal inhibitory concentrations (MICs) of cefotetan found for the Gram-positive genera tested are reflected in only one organism by the failure to bind of some of its PBPs. In the majority of cases, permeability is assumed to play the dominant role in the relatively low sensitivity of these organisms to cefotetan.