James T. Loch

In Vivo 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 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.

Discovery of Selective and Potent Inhibitors of Gram-Positive Bacterial Thymidylate Kinase (TMK).

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.

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 39, 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 39 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.

Acylhydrazones as M1/M3 selective muscarinic agonists

Abstract To improve receptor binding affinity and to investigate functional selectivity of 2,8-dimethyl-1-oxa-8-azaspiro[4.5]decan-3-one acetylhydrazone 2 at muscarinic receptor subtypes, a series of acylhydrazones A was synthesized. The SAR indicates that the binding affinity in the pirenzepine assay (M1) correlates well with lipophilicity. Intrinsic activity (30% of carbachol response) of agonists at M1 remains unchanged. Compounds with n = 0 and 6, where X = NHCO(CH 2 ) n Me, did not inhibit cAMP formation in rat heart membrane (M2). Most of the compounds are more efficacious at the M3 receptor than at M1. The results suggest that the M1 and M3 receptors can better tolerate bulky and long chained substituents than the M2 receptor.

Synthesis and biological activity of enantiomers of a conformationally restricted muscarone analog

Abstract A short and efficient synthesis of both enantiomers of 2,8-dimethyl-1-oxa-8-azaspiro[4.5]-decan-3-one is described. The biological activity of the racemate resides predominantly in the S-enantiomer. While the S-isomer is a full M2-agonist, the R-isomer is devoid of M2 efficacy.