TK-666

Thymidylate kinase (TMK) is an essential enzyme in bacterial DNA synthesis. The deoxythymidine monophosphate (dTMP) substrate binding pocket was targeted in a rational-design, structure-supported effort, yielding a unique series of antibacterial agents showing a novel, induced-fit binding mode. Lead optimization, aided by X-ray crystallography, led to picomolar inhibitors of both Streptococcus pneumoniae and Staphylococcus aureus TMK. MICs < 1 μg/mL were achieved against methicillin-resistant S. aureus (MRSA), S. pneumoniae, and vancomycin-resistant Enterococcus (VRE). Log D adjustments yielded single diastereomers 14 (TK-666) and 46, showing a broad antibacterial spectrum against Gram-positive bacteria and excellent selectivity against the human thymidylate kinase ortholog.

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.