Structure-based design of ligands for vitamin transporters in bacteria

Transport of substrates across biological membranes is essential for life. Energy-coupling factor (ECF) transporters form a distinct type of importers in the family of ATP-binding cassete (ABC) transporters and mediate the uptake of essential nutrients, such as vitamins, in bacteria. ECF transporters are predominantly found in Gram-positive bacteria, including many human pathogens. A significant fraction of these human pathogens lack enzymes for the de novo synthesis of many vitamins transported by ECF transporters, which makes the presence of these transporters essential for those pathogens. Given that eukaryotic cells do not make use of ECF transporters, they represent an attractive target for the development of antibiotics. The aim of this thesis is to shed light on the mechanism of substrate binding and transport of ECF transporters by using modified substrates, which may lead to novel antibiotics. This thesis describes the application of different hit-identification strategies to identify binders of ECF transporters. The design, synthesis and biochemical evaluation of thiamine (vitamin B1) derivatives, allowed the identification of amino acids that are crucial for substrate-binding in ThiT, the substrate-binding subunit (S-component) of an ECF transporter for thiamine. Using dynamic combinatorial chemistry, ligands were identified that reach into an unoccupied pocket of ThiT. A structure-based virtual screening campaign afforded an allosteric inhibitor of vitamin transport setting the stage for the development of novel antibiotics with an unprecedented mode of action