Co-targeting Metabolic Neighbours Constraints Bacterial Adaptive Evolution

Antibiotic resistance often arises through genetic changes that reduce drug susceptibility, with mutation representing one of the primary mechanisms. Here, we report a dual targeting strategy that restricts these escape routes by simultaneously inhibiting two metabolic neighbours in folate biosynthesis pathway. To establish a general framework for limiting adaptive escape in bacteria, we screened the folate pathway to identify enzymes with shared conserved structural features. This analysis identified dihydroneopterin aldolase (folB) and dihydroneopterin triphosphate 2'-epimerase (folX) as optimal dual targets due to their shared pocket architecture and their roles early in the folate pathway. We then screened a large chemical space of FDA-approved small molecules to identify candidates predicted to bind both folB and folX, followed by experimental validation. We identified Arbutin showing the strongest growth inhibition in E. coli and emerging as the potent dual-target inhibitor, validated by metabolic rescue assays that confirmed direct inhibition of both folB and folX. Experimental evolution further revealed that populations exposed to Arbutin exhibit constrained adaptive trajectories and minimal increases in tolerated drug concentration, consistent with the evolutionary bottleneck imposed by dual-enzyme inhibition. Together, these results establish a framework for developing evolution-resistant antibacterials by targeting structurally convergent enzymes within metabolic pathways.