Development of a spectrophotometric assay for high-throughput screening and mechanistic characterization of glucose-1-phosphate thymidylyltransferase inhibitors

Abstract

The sugar β-L-rhamnose is often a conserved component of bacterial polysaccharide capsules, an extracellular structure featured in many drug-resistant, pathogenic bacteria. The β-L-rhamnose biosynthetic enzymes (RmlA-D) have therefore become key targets in antibiotic development. However, inhibitor discovery for these enzymes, particularly α-D-glucose-1-phosphate thymidylyltransferase, has been hindered by a lack of robust activity assays. To address this challenge, we report a continuous, coupled-enzyme assay for screening α-D-glucose-1-phosphate thymidylyltransferase inhibitors, using Streptococcus pneumoniae Cps2L as a model enzyme. This platform was used to characterize ligand binding to Cps2L, identifying uridine diphosphate-α-D-glucose and pyrophosphate as substrates of the reverse pyrophosphorylase reaction, and to explore inhibition by thymidine diphosphate-β-L-rhamnose (TDP-Rha). A rapidly reversible binding mechanism at the allosteric site was identified for TDP-Rha (IC₅₀ = 45.1 ± 2.3 µM) using progress curve shape analysis and an engineered E253D Cps2L variant. Offering high-throughput capabilities and detailed kinetic resolution, the developed assay is well-suited for inhibitor discovery and mechanistic characterization.