| Methicillin-resistant Staphylococcus aureus (MRSA) infections constitute a serious health threat worldwide, and novel antibiotics are urgently needed. The enoyl-ACP reductase (saFabI) is essential for the S. aureus fatty acid biosynthesis and, hence, serves as an attractive drug target. We have obtained a series of snapshots of this enzyme, which provide a mechanistic picture of ligand and inhibitor binding including a dimer-tetramer transition combined with extensive conformational changes. Significantly, our results reveal key differences in ligand binding and recognition compared to orthologous proteins. The remarkable observed protein flexibility rationalizes our finding that saFabI is capable of efficiently reducing branched-chain fatty acid precursors. Importantly, branched-chain fatty acids represent a major fraction of the S. aureus cell membrane and are crucial for its in vivo fitness. Our discovery thus addresses a long-standing controversy regarding the essentiality of the fatty acid biosynthesis pathway in S. aureus and validates saFabI as a drug target.;Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Using our new, efficient and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. Remarkably, we identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Due to its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 hours. By a similar approach, we also rigorously characterized the structure-activity relationship for pyridone-based inhibitors of saFabI, which have superior pharmacokinetics and a distinct pharmacodynamic mechanism of action.;Finally, we explored the translation of residence time to the post-antibiotic effect (PAE). The PAE is the persistent suppression of microbial growth following drug exposure and removal. Just as higher affinity binding is expected to translate to a lower dose needed to inhibit cell growth, we hypothesized that residence time is an important and sometimes predominant factor in the PAE. We uniquely examined PAE effects for a structurally similar series of compounds with different residence times---our diphenyl ether-based inhibitors of saFabI. By integrating target turnover and vulnerability, we achieved a rigorous understanding of the factors that both limit and facilitate the translation of drug-target kinetics to prolonged cellular effects. |
