Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid.
Structural and genetic studies indicate that the antibacterial compound triclosan, an additive in many personal care products, is an inhibitor of EnvM, the enoyl reductase from Escherichia coli. Here we show that triclosan specifically inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis and a target for the antitubercular drug isoniazid. Binding of triclosan to wild-type InhA is uncompetitive with respect to both NADH and trans-2-dodecenoyl-CoA, with K(i)' values of 0.22+/-0.02 and 0.21+/-0.01 microM, respectively. Replacement of Y158, the catalytic tyrosine residue, with Phe, reduces the affinity of triclosan for the enzyme and results in noncompetitive inhibition, with K(i) and K(i)' values of 36+/-5 and 47+/-5 microM, respectively. Consequently, the Y158 hydroxyl group is important for triclosan binding, suggesting that triclosan binds in similar ways to both InhA and EnvM. In addition, the M161V and A124V InhA mutants, which result in resistance of Mycobacterium smegmatis to triclosan, show significantly reduced affinity for triclosan. Inhibition of M161V is noncompetitive with K(i)' = 4.3+/-0.5 microM and K(i) = 4.4+/-0.9 microM, while inhibition of A124V is uncompetitive with K(i)' = 0. 81 +/- 0.11 microM. These data support the hypothesis that the mycobacterial enoyl reductases are targets for triclosan. The M161V and A124V enzymes are also much less sensitive to isoniazid compared to the wild-type enzyme, indicating that triclosan can stimulate the emergence of isoniazid-resistant enoyl reductases. In contrast, I47T and I21V, two InhA mutations that occur in isoniazid-resistant clinical isolates of M. tuberculosis, show unimpaired inhibition by triclosan, with uncompetitive inhibition constants (K(i)') of 0.18+/-0.01 and 0.12+/- 0.01 microM, respectively. The latter result indicates that InhA inhibitors targeted at the enoyl substrate binding site may be effective against existing isoniazid-resistant strains of M. tuberculosis.
Parikh
S L
SL
Department of Chemistry and Graduate Programs in Biophysics and Molecular and Cellular Biochemistry, State University of New York at Stony Brook, New York 11794-3400, USA.
Xiao
G
G
Tonge
P J
PJ
D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D001665:Binding Sites; D004352:Drug Resistance, Microbial / Q000235:genetics / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007538:Isoniazid / Q000494:pharmacology*; D007700:Kinetics; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000235:genetics; D014260:Triclosan / Q000494:pharmacology*
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D014260:Triclosan; D010088:Oxidoreductases; C085257:InhA protein, Mycobacterium; D007538:Isoniazid