Tania Szal

• This dissertation focuses on the identification and characterization of efflux pump inhibitors targeting the main tripartite efflux pump in Escherichia coli, AcrAB-TolC. Tripartite efflux pumps are integral membrane complexes that confer antimicrobial resistance to Gram-negative bacteria by extruding antibiotics. Inhibiting efflux systems with small molecules represents a promising strategy for extending the spectrum of antibiotics, and restoring antibiotic susceptibility in multidrug-resistant bacteria. However, no efflux pump inhibitors have been approved for clinical use so far. Two substances, LP-115 and carmofur, that represent a basis for the development of novel efflux pump inhibitors were discovered, while postulated AcrA inhibitors were shown to be non-specific binders. LP-115 was identified employing an in silico repurposing screen targeting the outer membrane factor TolC followed by microbiological validation and deconstruction of a hit compound into fragments. Binding to TolC and AcrB was confirmed using MST, and a ligand-induced destabilization of the efflux pump complex assembly was observed using dynamic light scattering. Cryo-EM provided detailed molecular insights into the binding site at the AcrA-TolC interface. Our results suggest that LP-115 is an efflux pump inhibitor with a novel mechanism of action that consists of disrupting the AcrAB-TolC efflux pump assembly. Carmofur was identified employing a microbiological repurposing screen focusing on antimicrobial potentiating effects, followed by microbiological and biophysical characterization of the interaction with the isolated efflux pump subunits using microscale thermophoresis, nano differential scanning fluorimetry, and dynamic light scattering. The synergistic activity of carmofur in combination with an AcrAB-TolC substrate was TolC-dependent and specific binding to TolC was observed. Thus, carmofur could be used as starting point for the development of novel efflux pump inhibitors.