Structural and Enzymatic Analyses Reveal the Binding Mode of a Novel Series of Francisella tularensis Enoyl Reductase (FabI) Inhibitors

Abstract

Due to structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.

Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

“…Data collection and refinement statistics for these structures are given in Table 1. As seen with our previous FtFabI co-crystal structure with 1 15 , these inhibitors bind in the presence of NADH, and both the NADH and the inhibitor are present in each monomer. As the structures of the different chains in each of the enzyme/compound complexes were geometrically restrained to be identical during refinement, we focus our further discussion on the features of chain A in each complex.…”

“…Recently, our laboratory reported the identification and structural characterization of a novel series of benzimidazole FabI inhibitors as a new chemical scaffold with promising enzyme and antibacterial activity. 14, 15 We now report the structural and biological evaluation of several second generation benzimidazole compounds with low nanomolar enzyme inhibition and promising antibacterial activity, not only against F. tularensis , but also against the more prevalent pathogen, S. aureus and MRSA. Our co-crystal structures demonstrate the binding modes of these second generation inhibitors in FtFabI and lay a solid foundation for analyzing strategies to improve pharmacokinetic properties while maintaining FabI inhibition.…”

“…In our prior studies reporting hit identification, structural and enzymatic analyses of the first-generation benzimidazole FabI inhibitors, 14, 15 the initial SAR was constructed primarily by testing commercially available benzimidazole analogs, resulting in a limited understanding of the structure-activity relationship. We now report activities from synthetic analogs of our prior best hit, compound 1 (Figure 1), and find that the second generation compounds display enhanced enzymatic inhibitory activity, along with significantly improved antibacterial activity.…”

“…Larger groups at this position were not tested, as the crystal structure of 1 bound to FtFabI (PDB ID 3UIC) 15 demonstrates that large groups cannot be accommodated at this position. The addition of a methyl group at this position results in a chiral center, but only the racemic mixture of 2 has been tested to date.…”

“…However our structure of 1 bound to FtFabI demonstrates this not to be the case. 15 We now investigated the replacement of halogen substituents with other small, lipophilic groups, including methyl and methoxy groups. Compound 7 , substituted with a meta-methyl and para-methoxy group, demonstrated that the activity is not dependent on halogen substitution at these positions, as the inhibitory activity was retained relative to the other compounds.…”