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Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in <jats:italic>E. coli<\/jats:italic> through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant <jats:italic>E. coli<\/jats:italic> mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. 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