Structure-Guided Design of a Fluorescent Probe for the Visualization of FtsZ in Clinically Important Gram-Positive and Gram-Negative Bacterial Pathogens

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.

Targeting the FtsZ Allosteric Binding Site with a Novel Fluorescence Polarization Screen, Cytological and Structural Approaches for Antibacterial Discovery

“…Recently, the crystal structure of SaFtsZ in complex with a fluorescent BODIPY derivative of TXA6101 bound into the interdomain cleft has been reported. Nonetheless, this probe (BOFP, Chart S1) binds to unassembled SaFtsZ in solution without nucleotide and magnesium, as well as to Gram-negative FtsZs, in puzzling contrast with its complex filament crystal structure, the specificity of our probes, and the characteristic selectivity of PC190723 analogues.…”

Targeting the Achilles Heel of FtsZ: The Interdomain Cleft

“…Recently, Ferrer-González et al developed a structure-guided fluorescent benzamide derivative by conjugating BODIPY to an oxazole benzamide FtsZ inhibitor (BOFP). BOFP binds to FtsZ from both Gram positive and Gram negative bacteria with K d s of 0.6–4.6 and 0.2–0.8 μM, respectively ( Ferrer-Gonzalez et al, 2019 ). BOFP binds to the IDC, where the BODIPY moiety interacts with residues I228 and V307 of Bs FtsZ ( Table 4 ).…”

FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics

“…Among the most widely employed techniques for drug screening [ 64 ], fluorescence anisotropy can precisely measure high affinity interactions, and several low volume samples can be simultaneously and rapidly analyzed by using a plate reader equipped with polarizers. Anisotropy has been used to determine the binding affinity between a boron-dipyrromethene (BODIPY)-labeled oxazole-benzamide inhibitor and FtsZs from a variety of Gram-negative and Gram-positive bacteria [ 65 ]. In addition, this technique has been used to determine interactions between FtsZ and 4′,6-Diamidino-2-phenylindole (DAPI), a DNA intercalating dye that is also a modulator of FtsZ assembly [ 66 ], and the guanidinomethyl biaryl compound 13, a broad spectrum bactericidal agent [ 67 ].…”