Structure-Guided Optimization of Inhibitors of Acetyltransferase Eis from .

The enhanced intracellular survival (Eis) protein of () is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of , we designed and optimized structurally unique thieno[2,3-]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3-]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors as well as their ability to restore the activity of kanamycin in a resistant strain of , in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds . This study showcases how structural information can guide Eis inhibitor design.