Abstract
Addressing the growing problem of antibiotic resistance requires the development of new drugs with novel antibacterial targets. FtsZ has been identified as an appealing new target for antibacterial agents. Here, we describe the structure-guided design of a new fluorescent probe (BOFP) in which a BODIPY fluorophore has been conjugated to an oxazole-benzamide FtsZ inhibitor. Crystallographic studies have enabled us to identify the optimal position for tethering the fluorophore that facilitates the high-affinity FtsZ binding of BOFP. Fluorescence anisotropy studies demonstrate that BOFP binds the FtsZ proteins from the Gram-positive pathogens Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae with Kd values of 0.6–4.6 µM. Significantly, BOFP binds the FtsZ proteins from the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii with an even higher affinity (Kd = 0.2–0.8 µM). Fluorescence microscopy studies reveal that BOFP can effectively label FtsZ in all the above Gram-positive and Gram-negative pathogens. In addition, BOFP is effective at monitoring the impact of non-fluorescent inhibitors on FtsZ localization in these target pathogens. Viewed as a whole, our results highlight the utility of BOFP as a powerful tool for identifying new broad-spectrum FtsZ inhibitors and understanding their mechanisms of action.
Highlights
Addressing the global antibiotic resistance problem requires the development of new drug chemotypes and the identification of new antibacterial drug targets
We have previously developed prodrugs of benzamide filamentous temperature-sensitive Z (FtsZ) inhibitors (PC190723 and TXA707) that are highly efficacious against infections caused by methicillin-resistant Staphylococcus aureus (MRSA)[9,10,11]
This work describes the structure-guided design of a fluorescent FtsZ-targeting probe (BOFP) consisting of an oxazole-benzamide FtsZ inhibitor conjugated to a BODIPY fluorophore
Summary
Addressing the global antibiotic resistance problem requires the development of new drug chemotypes and the identification of new antibacterial drug targets. FtsZ is a “druggable” target whose function can be disrupted by small molecule targeting of a single site on the protein While this latter property is appealing for a target protein, it introduces the potential for the development of resistance via single mutations[7,8,11,12,13,14,21,22]. Advancing the development of new FtsZ inhibitors that can target a more expansive array of both Gram-positive and Gram-negative bacterial pathogens requires tools that allow us to screen for FtsZ inhibition in a broad range of bacterial species. Fluorescence anisotropy studies demonstrate that BOFP can target the FtsZ proteins from a broad range of Gram-positive pathogens (including S. aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae) with high affinity (Kd values in the range of 1.0 to 3.5 μM at 25 °C). Our results indicate that BOFP can serve as a powerful tool for identifying new broad-spectrum FtsZ inhibitors and understanding their mechanisms of action
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