Discovery of a Novel and Potent Class of F. tularensis Enoyl-Reductase (FabI) Inhibitors by Molecular Shape and Electrostatic Matching

Abstract

Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with sub-micromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented.

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

“…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.…”

“…With the 1 st generation benzimidazole compounds, we initially focused on halogen substituents to the N1 phenyl group, principally due to a known halogen bond interaction between FabI and triclosan, the stereotypical FabI inhibitor, 14 which suggested that the halogen-substituted phenyl group could make a similar interaction. However our structure of 1 bound to FtFabI demonstrates this not to be the case.…”

Discovery of Mycobacterium tuberculosis α-1,4-Glucan Branching Enzyme (GlgB) Inhibitors by Structure- and Ligand-based Virtual Screening

“…Structure and ligand-based virtual screening approaches have been used previously to design inhibitors for important targets, such as FabI and M. tuberculosis malate synthase (46,47). It has been shown that virtual screening involving the fusion of structure-and ligand-based methods is an effective tool to achieve a higher hit rate and provide wider structural coverage of chemical space (30), and a combination of these methodologies was used in this study.…”

Identification of Bacillus anthracis PurE inhibitors with antimicrobial activity

“…30 MBC values were estimated using the standard method of testing all negative MIC wells (no visible growth) for evidence of viable bacteria by transferring an aliquot of MIC dilution culture media to antibiotic-free agar plates. Medium (100 lL) from each negative (clear) well in MIC assays was streaked onto LB agar plates, and the plates were incubated for 72 h for detection of bacterial growth.…”