Thomas A. Keating

Two independent series of inhibitors of Helicobacter pylori glutamate racemase (MurI) were characterized for their kinetic mechanism, and one was used to generate resistant mutants in vitro. Mutant MurI enzymes from these strains were characterized by structural, genetic, kinetic and biophysical methods. Both inhibitor series, pyrazolopyrimidinediones and benzodiazepines, are uncompetitive with respect to the glutamate substrate, and the resistance mutations were found to act by reducing the affinity of MurI for substrate, thereby reducing the pool of enzyme-substrate complex available for binding inhibitor, while still allowing sufficient glutamate racemase activity for peptidoglycan construction. Uncompetitive inhibitors of a single-substrate, single-product enzyme are rare, and this work gives insight into an remarkable resistance mechanism. This article will discuss the projected clinical impact of H. pylori MurI resistance on these types of inhibitors.

There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity. TK-666 has potent, broad-spectrum Gram-positive microbiological activity (including activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate in vivo efficacy against S. aureus in a murine infected-thigh model. This work presents the first validation of TMK as a compelling antibacterial target and provides a rationale for pursuing novel clinical candidates for treating Gram-positive infections through TMK.