Identification of lead compounds targeting the dTDP-L-rhamnose biosynthesis pathway using Streptococcus pyogenes

Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS) and Streptococcus mutans. Both pathogens require dTDP-L-rhamnose for the production of a structurally similar rhamnose-containing polysaccharide in their cell wall. Via heterologous expression in S. mutans, we confirm that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes, respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified amino acids in the catalytic site of these enzymes. Bio-layer interferometry was used to identify inhibitory lead compounds that bind directly to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA in a concentration-dependent manner. One of the identified compounds, Ri03, inhibited growth of GAS as well as several other streptococcal pathogens with an MIC50 of 120-410 μM. Ri03 displayed no cytotoxity in U937 monocytic cells up to a concentration of 15 mM. We therefore conclude that Ri03 can serve as a lead compound for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.