Kinetic and Structural Characteristics of the Inhibition of Enoyl (Acyl Carrier Protein) Reductase by Triclosan

Abstract

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.

Structural Elucidation of the Specificity of the Antibacterial Agent Triclosan for Malarial Enoyl Acyl Carrier Protein Reductase

“…The Triclosan Analogs 1 and 2-Recently, the antibacterial activities of several 2-hydroxydiphenylethers as well as hexachlorophene and 2-hydroxydiphenylmethanes were determined (55). Studies with a des-hydroxyl analog of triclosan showed a more than 10,000-fold reduced affinity for E. coli ENR and implicated a critical antibacterial role for the triclosan 2-hydroxy group (55).…”

“…Studies with a des-hydroxyl analog of triclosan showed a more than 10,000-fold reduced affinity for E. coli ENR and implicated a critical antibacterial role for the triclosan 2-hydroxy group (55). Moreover, it was proposed that the ether oxygen of triclosan might be critical to the formation of the ternary complex, because corresponding 2-hydroxydiphenylmethanes did not result in tight binding (59), whereas the replacement of the ether oxygen with sulfur abolished the inhibitory activity (21).…”

Structure of Acyl Carrier Protein Bound to FabI, the FASII Enoyl Reductase from Escherichia coli

“…Triclosan, which binds with picomolar affinity to the E. coli FabI (23,60,61), has been proposed to bind to the enzyme as a substrate analog (62). This hypothesis has gained support from the structure of C16-NAC bound in a stable ternary complex to InhA in the presence of NAD ϩ (27). )…”

Mycobacterium tuberculosisenoyl-acyl carrier protein reductase inhibitors as potential antituberculotics: development in the past decade

“…The nonspecific damage to bacterial cell wall was believed to be the mechanism of antibacterial effect. Triclosan was later proven to target fatty acid synthesis via inhibition of enoylreductase in late 90's 17,18 . However, its use is limited by its poor bioavailability.…”