Background:The FabI inhibitor CG400549 is a promising new anti-staphylococcal drug candidate with recently validated human efficacy. Results: We revealed the molecular determinants conferring S. aureus FabI selectivity to rationally design a compound with an improved antibacterial activity spectrum. Conclusion:The 4-pyridone PT166 represents a critical step toward Gram-negative and mycobacterial coverage. Significance: We provide an approach to expand the spectrum of antimicrobial activity.

Summary The newly discovered FabV enoyl-ACP reductase, which catalyzes the last step of the bacterial fatty acid biosynthesis (FAS-II) pathway, is a promising but unexploited drug target against the re-emerging pathogen Yersinia pestis. The structure of the Y. pestis FabV in complex with its cofactor reveals that the enzyme features the common architecture of the short chain dehydrogenase reductase superfamily, but contains additional structural elements which are mostly folded around the usually flexible substrate binding loop, thereby stabilizing it in a very tight conformation that seals the active site. The structures of FabV in complex with NADH and two newly developed 2-pyridone inhibitors provide first insights for the development of new lead compounds, and suggest a mechanism by which the substrate binding-loop opens to admit the inhibitor, a motion that could also be coupled to the interaction of FabV with the acyl-carrier-protein substrate.

Slow-onset enzyme inhibitors are the subject of considerable interest as an approach to increase potency of pharmaceutical compounds by extending the residence time of the inhibitor on the target (the lifetime of the drug-receptor complex). However, rational modulation of residence time presents significant challenges since it requires additional mechanistic insight, such as the nature of the transition state for post-binding isomerization. Our previous work, based on X-ray crystallography, enzyme kinetics and molecular dynamics simulation, suggested that the slow step in inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA involves a change in conformation of the substrate binding loop from an open state in the initial enzyme-inhibitor complex to a closed state in the final enzyme-inhibitor complex. Here, we use multidimensional free energy landscapes for loop isomerization to obtain a computational model for the transition state. The results suggest that slow-onset inhibitors crowd key side chains on helices that slide past each other during isomerization, resulting in a steric clash. The landscapes become significantly flatter when residues involved in the steric clash are replaced by alanine. Importantly, this lower barrier can be increased by rational inhibitor redesign to restore the steric clash. Crystallographic studies and enzyme kinetics confirm the predicted effects on loop structure and flexibility, as well as inhibitor residence time. These loss and regain of function studies validate our mechanistic hypothesis for interactions controlling substrate binding loop isomerization, providing a platform for future design of inhibitors with longer residence time and better in vivo potency. Similar opportunities for slow-onset inhibition via the same mechanism are identified in other pathogens.

The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent. These studies were performed with the T276S FabV variant. In the present work, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20->100 fold). In addition, while T276S ypFabV generally displays higher affinity for 2-pyridone inhibitors compared to the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in gain of slow-onset inhibition for the 4-pyridone PT156.