Résumé
Aminoglycoside‐phosphotransferases (APHs) are a class of bacterial enzymes that mediate acquired resistance to aminoglycoside antibiotics. Here we report the identification of small molecules counteracting aminoglycoside resistance in Enterococcus casseliflavus . Molecular dynamics simulations were performed to identify an allosteric pocket in three APH enzymes belonging to 3′ and 2′′ subfamilies in which we then screened, in silico , 12,000 small molecules. From a subset of only 14 high‐scored molecules tested in vitro , we identified a compound, named here EK3 , able to non‐competitively inhibit the APH(2′′)‐IVa, an enzyme mediating clinical gentamicin resistance. Structure‐activity relationship (SAR) exploration of this hit compound allowed us to identify a molecule with improved enzymatic inhibition. By measuring bacterial sensitivity, we found that the three best compounds in this series restored bactericidal activity of various aminoglycosides, including gentamicin, without exhibiting toxicity to HeLa cells. This work not only provides a basis to fight aminoglycoside resistance but also highlights a proof‐of‐concept for the search of allosteric modulators by using in silico methods.