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.

(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.

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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.

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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 [ 14 C]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. Sutherland et al. (23) synthesized the (6S)-6-fluoro derivative of shikimic acid (Fig.

Potent antagonists of the integrin α(5)β(1), which are RGD mimetics built from tyrosine are described. This letter describes the optimization of in vitro potency obtained by variation of two parts of the molecule, the basic group and the linker between the basic group and the phenyl central core.