Evaluating the Contribution of Transition State Destabilization to Changes in the Residence Time of Triazole Based InhA Inhibitors

A critical goal of lead compound selection and optimization is to maximize target engagement while minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug–target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug–target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA. This led to the discovery of a triazole-containing diphenyl ether with an increased residence time on InhA due to transition-state destabilization rather than ground-state stabilization. In the present work, we evaluate the inhibition of InhA by 14 triazole-based diphenyl ethers and use a combination of enzyme kinetics and X-ray crystallography to generate a structure–kinetic relationship for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug–target complex by up to 3 orders of magnitude. In addition, we identify a novel inhibitor with a residence time on InhA of 220 min, which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug–target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme–inhibitor complex, and demonstrates the important role that on-rates can play in drug–target residence time

Rational Design of Broad Spectrum Antibacterial Activity Based on a Clinically Relevant Enoyl Acyl Carrier Protein Acp Reductase Inhibitor

Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus specific 2 pyridone compound that inhibits the enoyl acyl carrier protein reductase FabI , has recently been shown to possess human efficacy for the treatment of methicillin resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65 fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4 pyridone based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram negative and mycobacterial organism