Antibacterial Inhibitors of Gram-Positive Thymidylate Kinase: Structure–Activity Relationships and Chiral Preference of a New Hydrophobic Binding Region

Thymidylate kinase (TMK), an essential enzyme in bacterial DNA biosynthesis, is an attractive therapeutic target for the development of novel antibacterial agents, and we continue to explore TMK inhibitors with improved potency, protein binding, and pharmacokinetic potential. A structure-guided design approach was employed to exploit a previously unexplored region in Staphylococcus aureus TMK via novel interactions. These efforts produced compound <b>39</b>, with 3 nM IC₅₀ against S. aureus TMK and 2 μg/mL MIC against methicillin-resistant S. aureus (MRSA). This compound exhibits a striking inverted chiral preference for binding relative to earlier compounds and also has improved physical properties and pharmacokinetics over previously published compounds. An example of this new series was efficacious in a murine S. aureus infection model, suggesting that compounds like <b>39</b> are options for further work toward a new Gram-positive antibiotic by maintaining a balance of microbiological potency, low clearance, and low protein binding that can result in lower efficacious doses

<i>In Vivo</i> Validation of Thymidylate Kinase (TMK) with a Rationally Designed, Selective Antibacterial Compound

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 <i>Staphylococcus aureus</i> and vancomycin-resistant <i>Enterococcus</i>), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate <i>in vivo</i> efficacy against <i>S. aureus</i> 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