Using iron to catch a ride - synthetic siderophores as molecular 'Trojan Horses' to visualize and treat MDR bacterial pathogens

Abstract

The rise in multidrug-resistant, bacterial infections, together with a shallow industrial discovery pipeline, urgently calls for novel diagnostic and therapeutic strategies. Bacterial cells, particularly Gram-negative pathogens with their double-layered cell wall, closely resemble a fortress that restricts the accumulation of small molecules. However, microbial transporters ensure a sufficient nutrient supply during infection of a host organism and act as gateways into the pathogen, e.g. for ferric iron, which plays a crucial role in microbial metabolism and growth. Siderophores, small bacterial molecule chelators, sequester Fe3+ from host proteins and are transported by bacterial, TonB-dependent transporters (TBDTs). Like a molecular “Trojan Horse”, synthetic siderophore mimics can hijack the siderophore transport system and actively translocate diagnostic or therapeutic payloads over the impervious bacterial membrane and accumulate at their site of action. This thesis expanded and evaluated the potential of synthetic and natural siderophores for the visualization and antibiotic therapy of MDR bacteria in cellular and in vivo. The structure of the DOTAM triscatecholate siderophore was adapted for an application as a bacteria-specific, gallium-68 labeled PET tracer for the detection of bacterial infections in vivo. Two tracers showed good in vitro, radiochemical and pharmacokinetic properties in vivo and selectively accumulated at the site of infection vs. a site of sterile inflammation. Similarly, chemiluminescent dioxetanes were attached to siderophores to yield a panel of siderophore dioxetane probes that detected Gram-positive and Gram-negative bacterial pathogens. The best compound exhibited superior stability in bacterial supernatant, detected low bacterial counts and even intracellular bacteria in infected lung epithelial cells. In an attempt to enhance the accumulation in Gram-negative bacteria and thus restore the activity of antibiotics used only against Gram-positive bacteria (e.g. lipopeptides, ansamycins, macrolides), chelators were conjugated via covalent and cleavable linker systems, to yield potent drug conjugates. Studies on siderophore receptor mutants of E. coli and P. aeruginosa, including transcriptomic and proteomic investigations, contributed information on the involved siderophore transporters as well as on the mechanistic response upon siderophore and conjugate addition. Peptide siderophore conjugates that target the TonB-dependent transport of ferric chelates in Pseudomonas successfully inhibited bacterial growth. This proof-of-concept established TonB as a novel target in antimicrobial therapy. The design, synthesis and biological evaluation of novel diagnostic and therapeutic siderophore conjugates represents an important milestone towards a clinical usage of this approach against MDR ESKAPE bacteria.