Structure-activity studies of the inhibition of FabI, the enoyl ACP reductase from Escherichia coli by triclosan

Triclosan, a common antibacterial additive, targets FabI, the enoyl reductase enzyme from the type II dissociated fatty acid synthase system in Escherichia coli. The emergence of triclosan-resistant E. coli strains has led to questions about the wisdom of the widespread use of this biocide in the consumer market. We have investigated the structure-activity relationship of FabI inhibition of triclosan by examining the binding of triclosan to the wild-type and mutant FabI enzymes. Triclosan is a slow, reversible, tight binding inhibitor of FabI and binds preferentially to the E·NAD + form of the wild-type enzyme with a K_d value of 3.8 pM. The triclosan binding to three FabI mutants G93V, M159T and F203L, identified by selection for triclosan resistance in E. coli by Levy and coworkers, has been characterized. Triclosan binds to the E·NAD + complex of G93V, M159T and F203L enzymes with K_d's of 0.15 μM, 14 nM and 0.7 nM respectively. We have also characterized the Y156F and A197M mutants in order to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis . As observed for InhA, Y156F FabI has a decreased affinity for triclosan; the inhibitor binds to both E·NAD+ and E·NADH with K_d values of 297 nM and 7 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that specific differences in the loop sequence between the E. coli FabI and the M. tuberculosis InhA cannot account for the 50,000 fold difference in triclosan affinity for the two enzymes.; Triclosan analogs which each present a systematic change in the structure have been tested for their binding affinity against the FabI enzyme. The dramatic changes in affinity shown by the analogs and FabI mutants together with different mechanisms of inhibition provide valuable information for future antibacterial drug design of enoyl reductase inhibitors based on the triclosan skeleton.