Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques.

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC50 of benzimidazole (-)-1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (-)/(+)-1 and (-)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (-)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (-) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (-) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.

Benzimidazole-Based FabI Inhibitors: A Promising Novel Scaffold for Anti-staphylococcal Drug Development.

The enoyl-ACP reductase (FabI) enzyme is a well validated target for anti-staphylococcal drug discovery and development. With the goal of finding alternate therapeutics for drug-resistant strains of Staphylococcus aureus, such as methicillin-resistant S. aureus (MRSA), our previously published series of benzimidazole-based inhibitors of the FabI enzyme from Francisella tularensis (FtFabI) have been evaluated against FabI from S. aureus (SaFabI). We report here the preliminary structure-activity relationship of this series and the prioritization of compounds toward lead optimization. Mutational studies have identified key residues that contribute toward stabilizing the inhibitors in the active site of FabI. Mutations that do not significantly impact enzyme function but destabilize inhibitor binding are more likely to occur in nature as organisms evolve to evade the action of antibiotics leading to resistance. Identifying these residues provides guidance for minimizing susceptibility to resistance. Additionally, we have identified compounds that elicit antibacterial activity through off-target effects and observe that close analogs can display differing modes of action (on-target vs off-target) and need to be individually evaluated early on to prioritize compounds for lead optimization. Overall, our data suggest that the benzimidazole scaffold is a promising scaffold for anti-staphylococcal drug development.