DNA gyrase is an essential type II topoisomerase that catalyzes the introduction of negative supercoils using the free energy of adenosine triphosphate (ATP) hydrolysis. The enzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), that form a functional A2
heterotetramer required for bacterial viability. The N-terminal domain of the GyrB subunit contains the ATP ase active site, and the C-terminal domain is necessary for the interaction with the GyrA subunit and DNA. The GyrA subunit is involved in the cleavage and reformation of DNA strands needed to supercoil DNA.1–3
The GyrA subunit is targeted by quinolone antibiotics, which have broad-spectrum activity against both gram-positive and gram-negative bacteria. The GyrB subunit is targeted primarily by natural product antibiotics such as the aminocoumarin antibiotics (e.g., novobiocin4
), as well as cyclothialidine. 6
Mutations that confer drug resistance to all 3 antibiotics have been reported.7,8
Mutations associated with both coumarin and cyclothialidine resistance map to the periphery of the ATP binding site of GyrB that hydrolyzes ATP.9
The emergence of bacterial strains resistant to existing antibiotics makes it imperative to develop new classes of antibiotics that take into account these known mutations and, to the extent possible, restrict their mode of action to portions of the enzyme that are conserved by functional necessity. Residues required for coupling ATP hydrolysis to DNA supercoiling in GyrB have been identified using site-directed mutagenesis.9
Along with these extensive mutational data, analysis of high-quality crystal structures suggests the value of pursuing next-generation GyrB inhibitors that target the ATP binding domain.10
The ATP binding site within GyrB is highly conserved across bacterial species and is not present in humans, making it suitable for the development of broad-spectrum antibiotics.
Because the bacterial gyrase holoenzyme has been the subject of multiple drug discovery efforts, many assays exist to measure its activity. Assays used for general studies of the holoenzyme as well as many high-throughput screens measure the ability of the enzyme to convert relaxed DNA into supercoiled DNA. Most of these studies use an assay that couples ATP hydrolysis to nicotinamide adenine dinucleotide (NADH), resulting in a measureable colorimetric change.6,9,10
Similar assays directly measure the total level of supercoiled DNA using agarose gel separations or fluorescent dyes.11–13
Cell-based assays that measure the level of DNA damage have also been used to measure gyrase activity.14
Although all of these assays can be used to measure the activity of the gyrase holoenzyme, they often require multiple addition steps, cannot separate GyrA from GyrB inhibitors, and do not focus on the ATP binding domain. One assay has been described that measures the direct binding of [3
H]dihydronovobiocin to a biotin-labeled 43-kDa fragment of GyrB using a scintillation proximity assay (SP A).15
Although the SP A directly examines the ATP binding domain, an assay that uses fluorescence rather than radioactivity would be better suited for high-throughput screening (HTS).
Fluorescence polarization (FP) is a homogeneous assay that can be used to measure the binding interaction between molecules. 16
FP is based on the principle that a fluorophore excited by polarized light will also emit polarized light. Molecular motion, which is dependent on the size of the molecule, causes depolarization of the light by radiating at a different direction than the incident light. A small unbound fluorescent probe rotates rapidly and maintains low levels of polarization after excitation. If the fluorescent probe binds to a larger molecule, such as a protein, forming a stable complex, the bound probe rotates more slowly and increases the amount of polarized light. Binding is directly related to the polarization level of the sample: an unbound fluorescent probe has low FP, and a bound fluorescent probe has high FP. The FP assay is well suited for measuring the interaction between molecules in real time and is commonly used in HTS.17
This article presents the development and optimization of a novel FP assay to detect competitive inhibitors of the ATP binding domain of Francisella tularensis
GyrB. We have designed and synthesized a novel fluorescent probe by covalently attaching a Texas Red fluorophore to novobiocin (Novo-TRX) guided by the GyrB/novobiocin crystal structure (Protein Data Bank entry 1KIJ).18
Experiments were performed to develop the FP assay and optimize the use of Novo-TRX to measure the competition for binding to the ATP binding domain of F. tularensis
GyrB. We have determined the kinetics of the interaction of Novo-TRX with GyrB as well as the effect of common buffer additives on the interaction. The assay was also validated for use in HTS for inhibitors of the ATP binding domain by screening a small library of Food and Drug Administration (FDA)–approved compounds. This screen identified a known GyrB inhibitor as well as 4 members of the anthracycline family of cancer therapeutics (doxorubicin, idarubicin, epirubicin, and daunorubicin).