Inhibition of the Bacterial Enoyl Reductase FabI by Triclosan: A Structure-Reactivity Analysis of FabI Inhibition by Triclosan Analogues † Sharada Sivaraman, § Todd J. Sullivan, § Francis Johnson, §,# Polina Novichenok, § Guanglei Cui, § Carlos Simmerling, § and Peter J. Tonge* ,§ 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 Received April 17, 2003 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 µM) and E‚NADH (K 2 ) 0.4 µM) 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. Introduction The pathway for fatty acid biosynthesis (FAS) in Escherichia coli is a paradigm for understanding type II FAS systems in other bacteria and has been exten- sively studied. The differences in protein sequences and active site organization between the human and bacte- rial FAS systems make it possible for specific inhibitors to be designed against the bacterial FAS enzymes, and thus this system has been validated as an excellent drug target for the design of antimicrobial agents. 1-3 FabI, the NADH-dependent trans-2-enoyl-ACP re- ductase (ENR) in E. coli, is the key regulator of fatty acid biosynthesis. ENRs have been shown to be inhib- ited by diazaborines, isoniazid, triclosan, and more recently by other new classes of novel inhibitors. 4-7 Triclosan (Scheme 1) (5-chloro-2-(2,4-dichloro-phenoxy)- phenol) is a broad-spectrum biocide that has been in use for over 30 years, mainly as a component of antimicro- bial wash products in health-care settings. More re- cently, triclosan has found extensive use in consumer products such as toothpaste, mouthwashes, deodorants, hand soaps, and lotions. It is also incorporated in children’s toys, cutting boards, and the plastic film used to wrap meat products. 8 Until recently, it was thought that triclosan, being a small hydrophobic molecule, was absorbed via diffusion into the bacterial cell wall and that nonspecific disruption of the cell wall was the mechanism by which triclosan exhibited its antibacterial † This work was supported by NIH Grant AI44639 to P.J.T. P.J.T. is an Alfred P. Sloan Research Fellow. * To whom correspondence should be addressed. Telephone: (631) 632 7907. Fax: (631) 632 7960. E-mail: peter.tonge@sunysb.edu. § Department of Chemistry. # Department of Pharmacological Sciences. Scheme 1 509 J. Med. Chem. 2004, 47, 509-518 10.1021/jm030182i CCC: $27.50 © 2004 American Chemical Society Published on Web 01/06/2004