Inhibition of the bacterial enoyl reductase FabI by triclosan: a structure-reactivity analysis of FabI inhibition by triclosan analogues.
To explore the molecular basis for the picomolar affinity of triclosan for FabI, the enoyl reductase enzyme from the type II fatty acid biosynthesis pathway in Escherichia coli, an SAR study has been conducted using a series of triclosan analogues. Triclosan (1) is a slow, tight-binding inhibitor of FabI, interacting specifically with the E.NAD(+) form of the enzyme with a K(1) value of 7 pM. In contrast, 2-phenoxyphenol (2) binds with equal affinity to the E.NAD(+) (K(1) = 0.5 microM) and E.NADH (K(2) = 0.4 microM) forms of the enzyme and lacks the slow-binding step observed for triclosan. Thus, removal of the three triclosan chlorine atoms reduces the affinity of the inhibitor for FabI by 70,000-fold and removes the preference for the E.NAD(+) FabI complex. 5-Chloro-2-phenoxyphenol (3) is a slow, tight-binding inhibitor of FabI and binds to the E.NAD(+) form of the enzyme (K(1) = 1.1 pM) 7-fold more tightly than triclosan. Thus, while the two ring B chlorine atoms are not required for FabI inhibition, replacement of the ring A chlorine increases binding affinity by 450,000-fold. Given this remarkable observation, the SAR study was extended to the 5-fluoro-2-phenoxyphenol (4) and 5-methyl-2-phenoxyphenol (5) analogues to further explore the role of the ring A substituent. While both 4 and 5 are slow, tight-binding inhibitors, they bind substantially less tightly to FabI than triclosan. Compound 4 binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3.2 and 240 nM, respectively, whereas compound 5 binds exclusively to the E.NADH enzyme complex with a K(2) value of 7.2 nM. Thus, the ring A substituent is absolutely required for slow, tight-binding inhibition. In addition, pK(a) measurements coupled with simple electrostatic calculations suggest that the interaction of the ring A substituent with F203 is a major factor in governing the affinity of analogues 3-5 for the FabI complex containing the oxidized form of the cofactor.
Sivaraman
Sharada
S
Department of Chemistry, SUNY at Stony Brook, Stony Brook, New York 11794-3400, and Department of Pharmacological Sciences, SUNY at Stony Brook, Stony Brook, New York 11794-8651.
Sullivan
Todd J
TJ
Johnson
Francis
F
Novichenok
Polina
P
Cui
Guanglei
G
Simmerling
Carlos
C
Tonge
Peter J
PJ
D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects* / Q000201:enzymology; D018360:Crystallography, X-Ray; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D009154:Mutation; D009243:NAD / Q000737:chemistry; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014260:Triclosan / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D009243:NAD; D014260:Triclosan; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)