This study investigates the potential of energy‐coupling factor (ECF) transporters as promising anti‐infective targets to combat antimicrobial resistance (AMR). ECF transporters, a subclass of ATP‐binding cassette (ABC) transporters, facilitate the uptake of B‐vitamins across bacterial membranes by utilizing ATP as an energy source. Vitamins are essential cofactors for bacterial metabolism and growth, and they can either be synthesized de novo or absorbed from the environment. These transporters are considered promising drug targets, underscoring the need for further research to harness their medicinal potential and develop selective inhibitors that block vitamin uptake in bacteria. Herein, we focused on the ECF transporter for pantothenate (vitamin B5) from Streptococcus pneumoniae and the ECF transporter for folate (vitamin B9) from Lactobacillus delbrueckii as a reference protein. We also included the energizing module for pantothenate along with both full transporter complexes. Initially, we transformed and purified the transporters, followed by an assessment of their thermal stability under various buffer composition, pH, and salt concentrations. Additionally, we monitored the melting temperature over six days to confirm their stability for further assays. We then measured the binding affinities of six ECF inhibitors using surface plasmon resonance (SPR) and evaluated their inhibitory effects through in vitro assays, including bacterial growth assay, whole‐cell uptake, and transport‐activity assays. After determining cytotoxicity in two human cell lines, we established an in vivo infection model using Galleria mellonella larvae to further validate our findings.
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