As a result of the distribution of FabI and FabK in clinical pathogens, a FabI-directed antibacterial should possess antibacterial activity against those pathogens in which FabI is the sole enoyl-ACP reductase (e.g., S. aureus, H. influenzae, Moraxella catarrhalis, and E. coli) but not against S. pneumoniae, enterococci, or P. aeruginosa, which utilize either FabK or both FabI and FabK. However, inhibitors with dual FabI and FabK inhibitory activities are expected to have antibacterial activities against FabK-utilizing organisms, and the results of the present study support this expectation.
Compound 2 had improved potency against S. aureus FabI compared to the potency of the original screening hit compound, which may account for the increase in antibacterial activity against S. aureus. Antibacterial activity against this pathogen improved further with the increase in inhibitory potency against FabI achieved with compound 3. Neither compound 2 nor 3 demonstrated any detectable activity against FabK, and thus, their lack of activity against E. faecalis and S. pneumoniae is to be expected.
Compound 4 has significantly improved inhibitory potency against FabI. This resulted in impressive potency against a panel of multidrug-resistant strains of S. aureus and S. epidermidis and an increase in potency against other FabI-utilizing organisms such as H. influenzae, E. coli, and M. catarrhalis. However, this compound also possesses some inhibitory potency against FabK, which may explain the antibacterial activity observed against S. pneumoniae and E. faecalis. Although the modes of antibacterial action of compounds 2 to 4 could all be clearly demonstrated to be via FabI in S. aureus and H. influenzae (where appropriate), we were unable to obtain data to illustrate that the mode of antibacterial activity of compound 4 in S. pneumoniae was via FabK, and this evaluation is ongoing.
Compound 4 also possessed activity against a broader range of gram-negative organisms. The studies with the H. influenzae
efflux pump deletion mutants clearly demonstrate that this compound is a substrate for efflux pumps, and this is further illustrated in several other gram-negative organisms when the antibacterial activity of compound 4 was determined in the presence of Phe-Arg beta-naphthylamide dihydrochloride, an efflux pump inhibitor of gram-negative organisms. However, even in the presence of the efflux inhibitor, the MIC of compound 4 was >64 μg/ml for S. marcescens
, S. maltophilia
, and P. aeruginosa
. Although the impermeability of these organisms was likely to be a contributing factor, the lack of activity against P. aeruginosa
may also be a result of the potential presence of a plethora of enoyl-ACP reductases in this organism (7
Compounds 3 and 4 demonstrate the potential clinical utilities of representatives of this novel series, with the efficacy of compound 4 equaling that of amoxicillin-clavulanate in the S. aureus groin abscess infection model. Interestingly, the high levels of protein binding observed with these compounds did not affect their in vivo efficacies, suggesting that sufficient unbound compound was available during the infection process.
This work describes the chemical optimization of the activity of a hit compound obtained from high-throughput screening against S. aureus FabI. The original screening hit compound possessed weak inhibitory activity against FabI and no significant antibacterial activity. Iterative medicinal chemistry and structure-based design improved the inhibitory potency of the original lead compound by >350-fold, and exquisite activity against multidrug-resistant S. aureus has been built into the series. The potential utility of this series is exemplified by compound 4, the MIC90s of which were >500-fold lower that those of each of the commercially available antibiotics tested. Furthermore, since these compounds exhibit activities against both FabK and FabI, the potential also exists to optimize and develop these compounds as broader-spectrum antibacterial agents.
In conclusion, we have described the discovery of a novel series of highly potent FabI-directed antibiotics. These novel FabI- and FabK-directed antibacterials have clear potentials as new therapeutic options for tackling infections caused by multidrug-resistant pathogens and also serve to highlight the potential of the bacterial fatty acid biosynthetic enzymes as a source of novel antibacterial targets for the 21st century.