Co-targeting Metabolic Neighbours Constraints Bacterial Adaptive Evolution.

Area of Science:

• Microbiology
• Biochemistry
• Drug Discovery

Background:

• Antibiotic resistance is a growing threat driven by genetic mutations reducing drug efficacy.
• Targeting single enzymes in metabolic pathways allows bacteria to develop resistance through adaptive mutations.
• The folate biosynthesis pathway is crucial for bacterial survival and presents potential targets for novel antimicrobials.

Purpose of the Study:

• To develop a novel dual-targeting strategy to overcome antibiotic resistance.
• To identify conserved structural features in the folate pathway for simultaneous enzyme inhibition.
• To find FDA-approved small molecules that can inhibit two key enzymes, dihydroneopterin aldolase (folB) and dihydroneopterin triphosphate 2'-epimerase (folX).

Main Methods:

• Screened the folate pathway for enzymes with shared conserved structural features.
• Identified folB and folX as optimal dual targets due to their similar pocket architecture.
• Screened FDA-approved small molecules for dual binding affinity to folB and folX, followed by experimental validation.
• Utilized metabolic rescue assays and experimental evolution to confirm inhibition and assess resistance development.

Main Results:

• Arbutin was identified as a potent dual-target inhibitor of folB and folX in *E. coli*.
• Arbutin demonstrated significant bacterial growth inhibition.
• Experimental evolution showed that Arbutin constrains adaptive trajectories, minimizing resistance evolution due to the imposed evolutionary bottleneck.

Conclusions:

• Simultaneously inhibiting structurally convergent enzymes like folB and folX is an effective strategy against antibiotic resistance.
• Arbutin serves as a promising lead compound for developing evolution-resistant antibacterial therapies.
• This framework provides a general approach for designing antibacterials that limit adaptive escape in bacteria.