Breaking glycolysis: allosteric hotspots for multi-target drug repurposing

Glycolysis is essential for cellular energy production, making its enzymes attractive antimicrobial targets. Traditional strategies targeting conserved catalytic sites risk host toxicity due to limited species specificity. Allosteric sites-spatially distinct, evolutionarily divergent regulatory regions-offer selective inhibition but are challenging to detect experimentally. Here, we systematically mapped allosteric sites across all ten glycolytic enzymes of Staphylococcus aureus and Plasmodium vivax using a multi-scale computational framework combining elastic network models, residue interaction networks, and complementary machine-learning algorithms. High-confidence allosteric sites were identified in fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, and phosphoglycerate mutase, revealing diverse regulatory architectures, from interfacial network hubs to hinge-mediated dynamic control. Experimentally validated sites in pyruvate kinase and phosphofructokinase further reinforced the predictive framework. Comparative analysis with human homologs confirmed pronounced species-specific divergence, supporting selective targeting. Virtual screening 1,615 FDA-approved compounds across all ten enzymes identified multi-target ligands exhibiting amphiphilic, interface-stabilizing architectures capable of coordinated glycolytic modulation. Binding analyses revealed a balanced contribution of polar and hydrophobic interactions, consistent with robust allosteric modulation. This pathway-wide, network-informed approach demonstrates the feasibility of selective disruption of bacterial glycolysis and provides a blueprint for rational polypharmacology and next-generation antimicrobial design.