Interactions between Gepotidacin and Escherichia coli Gyrase and Topoisomerase IV: Genetic and Biochemical Evidence for Well-Balanced Dual-Targeting

Antimicrobial resistance is a global threat to human health. Therefore, efforts have been made to develop new antibacterial agents that address this critical medical issue. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial in clinical development. Recently, phase III clinical trials for gepotidacin treatment of uncomplicated urinary tract infections caused by uropathogens, including Escherichia coli, were stopped for demonstrated efficacy. Because of the clinical promise of gepotidacin, it is important to understand how the compound interacts with its cellular targets, gyrase and topoisomerase IV, from E. coli. Consequently, we determined how gyrase and topoisomerase IV mutations in amino acid residues that are involved in gepotidacin interactions affect the susceptibility of E. coli cells to the compound and characterized the effects of gepotidacin on the activities of purified wild-type and mutant gyrase and topoisomerase IV. Gepotidacin displayed well-balanced dual-targeting of gyrase and topoisomerase IV in E. coli cells, which was reflected in a similar inhibition of the catalytic activities of these enzymes by the compound. Gepotidacin induced gyrase/topoisomerase IV-mediated single-stranded, but not double-stranded, DNA breaks. Mutations in GyrA and ParC amino acid residues that interact with gepotidacin altered the activity of the compound against the enzymes and, when present in both gyrase and topoisomerase IV, reduced the antibacterial activity of gepotidacin against this mutant strain. Our studies provide insights regarding the well-balanced dual-targeting of gyrase and topoisomerase IV by gepotidacin in E. coli.

A ntimicrobial resistance is a growing medical concern.
−14 Of these, the antibacterial that has progressed the furthest in clinical trials is gepotidacin, 15−22 a novel first-in-class triazaacenaphthylene bacterial topoisomerase inhibitor (Figure 1).
Recently, enrollment into gepotidacin phase III clinical trials (EAGLE-2 and EAGLE-3) for the treatment of uncomplicated urinary tract infections caused by uropathogens, including Escherichia coli, was stopped early for demonstrated efficacy following a recommendation by the Independent Data Monitoring Committee. 23These findings support the potential clinical use of gepotidacin in the treatment of human infections.In these phase III trials, "gepotidacin demonstrated noninferiority to nitrofurantoin, an existing first-line treatment for uncomplicated urinary tract infections, in patients with a confirmed uncomplicated urinary tract infection and a uropathogen susceptible to nitrofurantoin.Additionally, in the EAGLE-3 phase III trial, gepotidacin demonstrated statistically significant superiority versus nitrofurantoin." 24Gepotidacin is also currently in a phase III clinical trial for the treatment of uncomplicated urogenital gonorrhea. 16epotidacin targets both DNA gyrase and topoisomerase IV.−30 Gyrase controls DNA under-and overwinding and works ahead of replication forks and transcription complexes to remove positive supercoils that accumulate as a result of these DNA processes.−34 Although topoisomerase IV can relax negative and positive DNA supercoils, its primary function is to decatenate, or remove tangles between, daughter chromatids that are created during replication and eliminate knots that are generated during recombination. 10,28,30,35,36To carry out their catalytic activities, these enzymes use a doublestranded DNA passage mechanism. 6,9,28,30,36,37During this reaction, gyrase and topoisomerase IV generate a transient double-stranded DNA break in one segment of DNA and pass an intact double helix through the DNA gate.To maintain genomic integrity during this process, the enzymes form covalent bonds between active site tyrosine residues and terminal phosphates generated by enzyme-mediated DNA cleavage.This covalent enzyme-cleaved DNA complex is known as the cleavage complex. 6,9,28,30,36,37−10 This action induces DNA recombination and repair pathways and the SOS response.,10 Both effects of fluoroquinolones are capable of inducing bacterial cell death.However, at the present time, the relative contributions of DNA cleavage and enzyme inhibition to cell death induced by fluoroquinolones and other topoisomerase poisons are not well-defined.5 These residues were first described as Ser83 in the A subunit of E. coli gyrase and an acidic residue (Asp87) that is four amino acids away from Ser83.46−48 Fluoroquinolone resistance is characterized by mutations in the anchoring amino acid residues.6,8−10 This resistance is further facilitated by two issues.First, most mutations in the amino acids that anchor the water−metal ion bridge retain high catalytic activity 44,49,50 and do not affect the viability of the bacteria in the absence of drugs.47 Second, although both gyrase and topoisomerase IV are targeted by fluoroquinolones, in the vast majority of species, this targeting is not well-balanced.9,47 Consequently, a single point mutation in gyrase is often sufficient to induce a level of fluoroquinolone resistance that ) modifies the charge of the residue and directly affects the ability of the residue to hydrogen-bond with gepotidacin (pink carbon bonds).(C) Zoomed-in image showing that GyrA P35 is distal from gepotidacin but near the break in the DNA substrate.The GyrA P35 mutation possibly modifies the geometry of the loop spanning GyrA R32 and GyrA K42 �two residues likely responsible for the proper orientation of the double-helix during DNA cleavage.Images were generated using a structure from Protein Data Bank (PDB ID 6RKW) 72 and PYMOL software.
impairs clinical efficacy and allows further resistance mutations to accumulate. 6,9,10,39,47−72 A previous study that characterized the effects of gepotidacin on S. aureus gyrase found that the compound shares many mechanistic features with earlier preclinical candidate novel bacterial topoisomerase inhibitors. 42,54,72However, virtually nothing is known regarding the actions of gepotidacin on topoisomerase IV. 42,51−69 Because of the clinical promise of gepotidacin against urinary tract infections, it is important to characterize how this compound affects the activities of both gyrase and topoisomerase IV in E. coli.Moreover, the roles that the two enzymes play in the antibacterial activity of the compound and the mechanism of target-mediated resistance need to be further clarified.Consequently, we examined the antibacterial activity of gepotidacin against E. coli strains and the effects of gepotidacin on wild-type (WT) and mutant gyrase and topoisomerase IV.Gepotidacin displayed well-balanced dualtargeting in cells, which is consistent with a potential low propensity for target-mediated resistance in this species. 75The compound inhibited the DNA supercoiling and decatenation activities of purified WT gyrase and topoisomerase IV, respectively, with similar sub-μM IC 50 values, further supporting the well-balanced targeting of these enzymes.Under a variety of conditions, gepotidacin enhanced only single-stranded DNA cleavage mediated by gyrase and topoisomerase IV.Finally, mutations in E. coli gyrase and topoisomerase IV amino acid residues that are involved in gepotidacin−target interactions diminished the activity of the compound against both enzymes.These results provide valuable insights into the mechanism of action of this clinically important first-in-class triazaacenaphthylene antibacterial.

Gepotidacin Displays Balanced Dual-Targeting of
Gyrase and Topoisomerase IV in E. coli Cells.Drugs that act against both gyrase and topoisomerase IV, such as fluoroquinolones, can differentially target these enzymes with regard to antibacterial activity. 6,7,9,10−78 Although gyrase is usually the primary lethal target of these drugs, dual-targeting, in which the action of the drug against either enzyme is sufficient for lethality, has been reported. 77,78rugs that equally target gyrase and topoisomerase IV have an obvious advantage in that target-mediated resistance should be observed only when mutations in both enzymes occur simultaneously.In E. coli, gyrase is the primary lethal target of ciprofloxacin. 47,79revious studies suggest that in contrast to fluoroquinolones, some topoisomerase-targeted compounds display well-balanced dual-targeting of gyrase and topoisomerase IV in E. coli. 75,80hereas E. coli cells carrying either the GyrA D82G or ParC D79G mutation displayed WT susceptibility toward NBTI 5463, a compound that differs from gepotidacin in the moieties utilized for interactions with DNA (left-hand side), protein-binding pocket (right-hand side), and the key aspartic acid (basic amine), cells that carried both mutations were >100-fold less susceptible to the compound. 80This compound has a similar binding site to gepotidacin but was not progressed to the clinic.A comparable finding was reported for gepotidacin in cells that carried the GyrA D82N and ParC D79N mutations. 75Although the MIC (minimum inhibitory concentration) value of the compound against cells carrying the individual mutations was within twofold of that of the WT strain, the MIC value in cells carrying both mutations rose to more than 256 times that of WT.
To further explore the potential well-balanced dual-targeting of gepotidacin in E. coli, we examined strains that encoded the GyrA P35L and ParC D79N mutations.Gepotidacin is bactericidal against E. coli cells 81 and was lethal to the E. coli strains used in the present study.The compound displayed an MIC of 0.125 μg/mL against the parental TOP10 WT strain (Table 1).
Unlike fluoroquinolones, no spontaneous individual mutations in E. coli gyrase or topoisomerase IV that resulted in reduced susceptibility to gepotidacin were generated in cellular in vitro frequency of resistance studies. 82Therefore, to further explore the antibacterial activity of gepotidacin, we generated an E. coli TOP10 strain that carried the ParC D79N mutation.On the basis of structural studies, ParC D79 is predicted to interact with the basic amine group of gepotidacin. 54,72,83The presence of ParC D79N increased the frequency of spontaneous resistance to an earlier related compound, GSK203815, under selective pressure.This allowed for the construction of a strain carrying mutations in both gyrase and topoisomerase IV (GyrA P35L and ParC D79N , respectively).Structural and modeling studies predict that GyrA P35 may be involved in DNA binding/bending. 83onsequently, we engineered three isogenic E. coli TOP10 strains, TOP10-1 (GyrA P35L ), TOP10-2 (ParC D79N ), and TOP10-3 (GyrA P35L , ParC D79N ) (Table 1), to evaluate their susceptibility to gepotidacin.
The MIC values for gepotidacin against the strains carrying individual mutations in gyrase (TOP10-1) or topoisomerase IV (TOP10-2) were the same as that seen with the WT TOP10 parent strain (MIC = 0.125 μg/mL).However, the MIC value for gepotidacin against the TOP10-3 strain that carried mutations in both enzymes was 128-fold higher (MIC = 16 μg/mL).These data suggest that the GyrA P35L and ParC D79N mutations confer decreased target sensitivity to gepotidacin but that simultaneous mutations in both enzymes are required for reduced cellular susceptibility to the compound.Thus, it appears that gyrase and topoisomerase IV are dual-targets for gepotidacin in E. coli and that this compound targets both enzymes in a well-balanced fashion (i.e., gepotidacin is equally capable of inhibiting the growth of E. coli cells through its actions against either gyrase or topoisomerase IV).E. coli TOP10-1 cells carrying the GyrA P35L mutation displayed an MIC value for ciprofloxacin that was eightfold higher than that observed with WT cells (Table 1; MIC = 0.012 μg/mL compared to the WT MIC of 0.0015 μg/mL).Little or no change was observed in the TOP10-2 strain that carried the topoisomerase IV ParC D79N mutation alone (MIC = 0.002 μg/ mL), and fluoroquinolone susceptibility in the TOP10-3 double mutant strain was similar to that of the singly mutated TOP10-1 strain.These results are in line with gyrase being the primary cytotoxic target of fluoroquinolones in E. coli.Consistent with an earlier study, 75,80 these findings indicate that fluoroquinolones may still maintain efficacy against some strains carrying mutations that affect cellular susceptibility to gepotidacin.
Gepotidacin Is a Potent Catalytic Inhibitor of WT E. coli Gyrase and Topoisomerase IV.The cellular studies provide strong evidence that gyrase and topoisomerase IV are both targeted by gepotidacin in a well-balanced fashion.However, the effects of gepotidacin on E. coli type II enzymes have never been reported.Therefore, we examined the effects of gepotidacin on the catalytic activities of WT E. coli gyrase and topoisomerase IV.
Gepotidacin inhibited DNA supercoiling (i.e., the conversion of relaxed to negatively supercoiled plasmid) catalyzed by E. coli gyrase at submicromolar concentrations (IC 50 = 0.32 ± 0.17 μM) (Figure 2, left panel, blue) (a summary of data for gepotidacin is shown in Table 2).Gepotidacin was also a potent inhibitor of topoisomerase IV-catalyzed decatenation (IC 50 = 0.34 ± 0.09 μM) (Figure 2, right panel, blue; Table 2).The similar IC 50 values for the inhibition of both enzyme activities support the well-balanced dual-targeting of gepotidacin in E. coli cells.
In Figure 2, it is not obvious why gepotidacin and ciprofloxacin do not suppress 100% of enzyme catalysis.However, it is likely because the DNA supercoiling and decatenation assays are steady state rather than equilibrium (as in the DNA cleavage assays), and any gyrase or topoisomerase IV molecule that completes its catalytic cycle before a drug molecule binds or following drug dissociation will result in an apparent low level of remaining activity that cannot be overcome.
Despite the fact that ciprofloxacin displayed MIC values lower than those of gepotidacin against E. coli cells (Table 1), this enhanced antibacterial activity was not reflected in in vitro enzyme assays.The IC 50 value for ciprofloxacin in gyrasecatalyzed supercoiling assays (0.33 ± 0.12 μM) (Figure 2, left panel, orange) was similar to that observed for gepotidacin.In addition, ciprofloxacin was less potent against topoisomerase IVcatalyzed decatenation (i.e., the conversion of a catenated network of DNA circles to monomers) (IC 50 = 2.47 ± 0.48 μM) (Figure 2, right panel, orange; Table 2) consistent with gyrase being the primary target of ciprofloxacin in E. coli.It is not known why the MIC values for ciprofloxacin against E. coli cells are lower than those of gepotidacin.It could be due to enhanced uptake or decreased efflux or differences in the metabolism of the fluoroquinolone and the triazaacenaphthylene.Alternatively, fluoroquinolone-induced DNA damage may be more lethal to bacterial cells.

Gepotidacin Induces Single-Stranded DNA Breaks Generated by WT E. coli Gyrase and Topoisomerase IV.
Gepotidacin is a potent enhancer of DNA cleavage [i.e., the conversion of negatively supercoiled plasmid to nicked (singlestranded scission) or linear (double-stranded scission) DNA products] mediated by E. coli gyrase (Figure 3).Similar to the case of other NBTIs, gepotidacin induced only single-stranded DNA breaks.CC 50 (concentration at which 50% maximal DNA cleavage was observed) was 2.18 ± 0.77 μM, with levels of single-stranded DNA breaks maxing out at ∼20.1% of the initial    2).Gepotidacin was even more active against E. coli topoisomerase IV, inducing nearly 40% single-stranded DNA cleavage with a CC 50 value of 0.02 ± 0.004 μM (Figure 3, top right panel; Table 2).Note that this high level of DNA cleavage is inflated by the high baseline levels of scission mediated by E. coli topoisomerase IV in the absence of drugs (∼8% single-stranded and ∼15% doublestranded DNA breaks).Again, no enhancement of the doublestranded DNA cleavage was observed.
For comparison, the effects of ciprofloxacin on gyrase/ topoisomerase IV-mediated DNA cleavage are shown in Figure 3 (bottom left and right panels, respectively).As reported previously, 42,52,55,84 ciprofloxacin induced both single-and double-stranded DNA breaks, with the latter being more prevalent.In contrast, gepotidacin strongly enhanced only single-stranded DNA breaks.
Although no enhancement of topoisomerase IV-mediated double-stranded DNA cleavage was observed in the presence of gepotidacin, the high baseline DNA cleavage activity of this enzyme allowed us to further examine the effects of gepotidacin on DNA scission.As seen in Figure 3 (top right panel), gepotidacin suppressed the ability of E. coli topoisomerase IV to generate double-stranded DNA breaks.This suppression has been reported previously for gepotidacin with S. aureus gyrase 54 and for some other NBTIs. 53,55The reason for this suppression is unknown.However, it is believed that gepotidacin and related molecules induce sufficient distortion in the active site of gyrase/ topoisomerase IV following cleavage of one DNA strand that it prevents the enzyme from cleaving the second strand. 42,53nfortunately, the low baseline levels of DNA cleavage generated by E. coli gyrase (Figure 3, top left panel), even in the presence of divalent metal ions such as Ca 2+ that often enhance enzyme-mediated DNA cleavage, 49,53,85−87 did not allow us to examine this suppression of gyrase-generated doublestranded DNA breaks.
Two additional experiments were carried out to further assess the effects of gepotidacin on single-stranded gyrase/topoisomerase IV-mediated DNA cleavage.In the first, the ability of gepotidacin to induce single-versus double-stranded DNA scission at high concentrations and at long time courses was examined (Figure 4).The conditions used for gyrase (top panel) were 50 and 200 μM gepotidacin (5 and 20 times the concentration necessary for maximal cleavage) at incubation times of 20 and 120 min (one-and sixfold longer than a normal cleavage assay).The conditions used for topoisomerase IV (bottom panel) were 10 and 200 μM gepotidacin (10 and 200 times the concentration necessary for maximal cleavage) at incubation times of 10 and 60 min (one-and sixfold longer than a normal cleavage assay).Under all circumstances, only singlestranded DNA breaks were induced by gepotidacin.
In the second experiment, the effects of 1.5 mM ATP on DNA cleavage were examined.Although the high energy cofactor is not required for DNA cleavage, it is necessary to support the overall catalytic activity of gyrase and topoisomerase IV. 9,28,88,89 ATP binding drives the closing of the N-terminal protein gate and the DNA strand passage event, whereas ATP hydrolysis drives enzyme turnover. 9,28,88,89In some cases, ATP has enhanced the effects of topoisomerase poisons on the  stimulation of DNA scission, and in others, it has impaired the actions of these compounds. 41,55As seen in Figure 5, ATP had little effect on the ability of gepotidacin to induce DNA cleavage by E. coli gyrase (left panel) or topoisomerase IV (right panel).Levels of single-stranded cleavage were maintained; no doublestranded break enhancement was observed; and suppression of double-stranded DNA cleavage by E. coli topoisomerase IV was seen.Therefore, in the bacterial cell, which contains millimolar concentrations of ATP, 90 gepotidacin should maintain its activity against gyrase and topoisomerase IV-mediated DNA cleavage and induce only enzyme-mediated single-stranded DNA breaks.
Gepotidacin Induces Stable DNA Cleavage Complexes with WT E. coli Gyrase and Topoisomerase IV.In general, drugs that induce more stable cleavage complexes are more lethal to cells. 91Therefore, we used a persistence assay 44,91,92 to assess the stability of gyrase− and topoisomerase IV−DNA cleavage complexes formed in the presence of gepotidacin.In this assay, ternary enzyme−DNA−drug complexes were formed at high concentrations of enzyme and DNA (and when present, saturating concentrations of gepotidacin or ciprofloxacin; see Figure 6 legend for details) and then diluted 20-fold into buffer that did not contain the divalent cation necessary to sustain DNA cleavage.Following dilution, ternary complexes that dissociate are highly unlikely to reform. 44,91,92The stability of these complexes is monitored by the loss of single-stranded DNA cleavage generated in the presence of gepotidacin and compared with double-stranded DNA cleavage in the absence of compounds or in the presence of ciprofloxacin.As seen in Figure 6, gepotidacin induced the formation of very stable cleavage complexes with E. coli gyrase (left panel) and topoisomerase IV (right panel).With both enzymes, the half-life (T 1/2 ) was well in excess of 120 min.In contrast, in the absence of the drug, the T 1/2 of cleavage complexes was less than 5 s.Postdilution times for the data points for cleavage complexes formed in the absence of drug in Figure 6 ranged between 5 and 15 s.Levels of cleavage complex remaining at 5 s in the absence of drug were 18.9 and 13.6% for gyrase and topoisomerase IV, respectively.A previous rapid quench kinetic study estimated that the half-life of cleavage complexes formed with E. coli gyrase and topoisomerase IV in the absence of drugs was 0.16 and 0.21 s, respectively. 93profloxacin stabilized cleavage complexes compared to the no-drug experiments (T 1/2 = 3.6 and 5.7 min with gyrase and topoisomerase IV, respectively).However, the lifetimes of these complexes were substantially shorter than those seen with gepotidacin.
Gepotidacin Competes with Ciprofloxacin for Activity Against WT E. coli Gyrase and Topoisomerase IV.Crystallography and cryo-EM structural studies place gepotidacin and fluoroquinolones in the active site of gyrase and topoisomerase IV. 9,42,43,45,72,94 Two fluoroquinolone molecules interact in the cleavage complex and insert at sites of cleavage on both strands of the double-helix.In contrast, a single gepotidacin molecule binds midway between the two scissile DNA bonds in a pocket between the two A subunits of the bacterial type II enzymes.Although fluoroquinolones and gepotidacin do not interact with the same amino acid residues, modeling and competition studies suggest that these two antibacterial classes cannot occupy the gyrase/topoisomerase IV active site at the same time. 54Similar results have been reported for fluoroquinolones and other NBTIs. 53,54herefore, to determine whether gepotidacin and fluoroquinolones can simultaneously act on E. coli gyrase and topoisomerase IV, competition studies were carried out between gepotidacin and ciprofloxacin (Figure 7).In this assay, cleavage complexes with gyrase or topoisomerase IV were formed in the presence of a mixture of 50 or 10 μM ciprofloxacin, respectively, and increasing concentrations of gepotidacin (0−100 or 0−25 μM, respectively).The levels of fluoroquinolone used in these assays reflect the concentrations at which ciprofloxacin induced maximal levels of double-stranded breaks.Because gepotidacin induces only single-stranded breaks, competition was monitored by the loss of double-stranded DNA breaks, which could have  Gepotidacin was a potent competitor of ciprofloxacin with both gyrase (Figure 7, left panel) and topoisomerase IV (right panel) and decreased levels of double-stranded DNA breaks by 50% at concentrations of 5.66 ± 2.7 and 0.66 ± 0.97 μM, respectively.These values are ∼3and ∼33-fold higher than the CC 50 values for gepotidacin against the two enzymes (2.18 ± 0.77 μM for gyrase and 0.02 ± 0.004 μM for topoisomerase IV; Table 2), respectively, suggesting that the decrease in doublestranded breaks results from the competition between gepotidacin and ciprofloxacin rather than a suppression of double-stranded breaks by gepotidacin.
The decrease in ciprofloxacin-induced double-stranded breaks was accompanied by a concomitant rise in singlestranded breaks induced by gepotidacin (CC 50 values for gepotidacin-induced DNA cleavage in the presence of ciprofloxacin were 4.63 ± 3.7 and 0.13 ± 0.09 μM for gyrase and topoisomerase IV, respectively).These values are ∼2and ∼6-fold higher than the respective CC 50 values for gepotidacin calculated in the absence of ciprofloxacin (Table 2).Taken together, these results indicate that gepotidacin and ciprofloxacin cannot simultaneously act on the E. coli type II topoisomerases and confirm that the affinity of gepotidacin for these enzymes is considerably higher than that of the fluoroquinolone.
Effects of the E. coli GyrA P35L Enzyme Mutation on Gepotidacin Activity.Virtually nothing is known about the biochemical mechanisms by which cells lose their susceptibility to gepotidacin.A previous report indicated that NBTI 5463 was much less potent against the ATPase activities of gyrase or topoisomerase IV that carried the GyrA D82G or ParC D79G mutation, respectively.However, the basis for this decreased potency was never examined.To address the important issue of resistance, we characterized the interactions of gepotidacin with two enzymes that carried mutations in amino acids that are involved in gepotidacin−target interactions (Figure 1).Gyrase GyrA P35L was designed to include the mutation that was encoded in the TOP10-1 E. coli strain discussed earlier.Formation of inclusion bodies under conditions of overexpression prevented purification of the mutant topoisomerase IV gyrase (ParC D79N ) that was encoded by the TOP10-2 E. coli strain.Therefore, the topoisomerase IV ParC D79G mutant enzyme was engineered for comparative studies.
The effects of gepotidacin on DNA supercoiling mediated by E. coli GyrA P35L are shown in Figure 8 (left panel).As discussed above, GyrA P35 is predicted to be involved in DNA binding/ bending. 83Even though GyrA P35L is associated with decreased sensitivity in E. coli cells in the presence of a simultaneous ParC D79N mutation, the mutation had no effect on the ability of gepotidacin to inhibit gyrase-catalyzed DNA supercoiling (IC 50 = 0.25 ± 0.43 μM, as compared to 0.32 ± 0.17 μM for WT; Figure 8 and Table 2).
In contrast, the GyrA P35L mutation greatly reduced the levels of gyrase-mediated single-stranded DNA cleavage induced by gepotidacin (Figure 9, left panel; Table 2).The compound induced a maximum of ∼3.4% cleaved DNA (which is only ∼2.6% above the baseline levels of cleavage induced by the enzyme in the absence of drug) as compared to 21.7% with the WT enzyme.These findings strongly suggest that in relation to DNA gyrase, decreased cellular susceptibility to gepotidacin correlates with a diminished effect of the compound on enzymemediated DNA scission as opposed to its effect on the overall catalytic activity.This result further implies that the component of gepotidacin-induced cell killing that is mediated by gyrase in E. coli is due, at least in part, to increased levels of DNA cleavage.
Despite the fact that gepotidacin induced very little DNA cleavage with GyrA P35L , the CC 50 value with the mutant enzyme (0.13 ± 0.42 μM) was even lower than that observed with WT E. coli gyrase (2.18 ± 0.77 μM) (Table 2).Thus, the reduction in the bactericidal activity of gepotidacin associated with the GyrA P35L mutation results from a lower efficacy (i.e., maximal level of DNA cleavage) of the compound rather than a loss of  potency (i.e., decrease in affinity for gepotidacin).This finding implies that the GyrA P35L mutation does not diminish the level of binding of gepotidacin to the gyrase−DNA complex.Coupled with the fact that gepotidacin maintains its ability to inhibit DNA supercoiling by the GyrA P35L enzyme, this mutation likely alters the positioning of gepotidacin in the twofold axis of the enzyme.
Finally, ciprofloxacin maintained a high potency for inhibition of supercoiling with the GyrA P35L mutant enzyme (IC 50 = 0.08 ± 0.32 μM, Figure 8, right panel) but displayed a reduced ability to induce DNA cleavage (Figure 9, right panel).This likely accounts for the eightfold increase in MIC values for ciprofloxacin in the E. coli strain (TOP10-1) harboring the GyrA P35L mutation compared to the wild-type strain (Table 1).
Effects of the E. coli ParC D79G Enzyme Mutation on Gepotidacin Activity.The effects of gepotidacin on DNA decatenation mediated by E. coli ParC D79G are shown in Figure 10 (left panel) and Table 2. Topoisomerase IV ParC D79 is predicted to interact directly with gepotidacin. 54,72,83The presence of the ParC D79G mutation reduced the sensitivity to gepotidacin in this assay by ∼15-fold (IC 50 = 6.30± 4.33 μM, as compared to 0.34 ± 0.09 μM for the WT).In addition, the mutation decreased the ability of gepotidacin to enhance topoisomerase IV-mediated single-stranded cleavage.The maximum level of single-stranded cleavage fell from ∼38% with the WT enzyme (∼28.8%above the baseline) to ∼16% (∼6.2% above the baseline) with the ParC D79G mutant enzyme (Figure 11, left panel).Because both activities of gepotidacin were affected by the ParC D79G mutation, it is not possible to speculate on whether the reduction in the cell-killing activity that is due to gepotidacin effects on topoisomerase IV is caused primarily by the diminished effects on DNA cleavage or the overall catalytic activity of this enzyme.
Finally, ciprofloxacin partially maintained its ability to inhibit decatenation with the ParC D79G mutant enzyme (IC 50 = 5.0 ± 0.92 μM) (Figure 10, right panel) but induced considerably less DNA cleavage than that induced by the WT enzyme (maximal ParC D79G cleavage of ∼2% above the baseline of ∼5%).This implies that ciprofloxacin might have a reduced activity against strains that carry this mutation.However, any decreased sensitivity would likely be obscured by the fact that topoisomerase IV is a secondary cytotoxic target for fluoroquinolones in E. coli.

■ DISCUSSION
Gepotidacin is a first-in-class triazaacenaphthylene antibacterial that is the most clinically advanced novel bacterial topoisomerase inhibitor. 23,113The compound is a potent inhibitor of E. coli gyrase and topoisomerase IV catalytic activity.The finding that gepotidacin displays a similar sub-μM IC 50 value against both enzymes is consistent with its well-balanced dual-targeting in cells.
In contrast to the fluoroquinolones, gepotidacin induces only single-stranded enzyme-mediated DNA cleavage (Figures 3−5). 54,72,83The relative lethality of topoisomerase-induced single-stranded versus double-stranded DNA breaks has yet to be explored.−105 Although interactions between other novel bacterial topoisomerase inhibitors and WT gyrase and topoisomerase IV have been reported, 51−69 the mechanisms underlying resistance to these compounds and to gepotidacin have yet to be explored.The studies presented here show that simultaneous mutations in both GyrA P35 and ParC D79 are required for a decreased susceptibility to gepotidacin in E. coli cells.However, individually, these mutations diminish the activity of the compound against purified gyrase and topoisomerase IV.
−41 Our in vitro enzymology studies provide insights into which of these mechanisms are used by gepotidacin to kill E. coli cells.Indeed, the IC 50 value of gepotidacin for supercoiling with the GyrA P35L mutant was similar to that with WT gyrase, while the levels of gepotidacin-induced DNA scission were considerably lower.The finding that reduced susceptibility tracks with the diminished enhancement of DNA cleavage rather than decreased inhibition of catalytic activity implies that the mechanism by which gepotidacin induces gyrase-mediated cell death is primarily through the generation of single-stranded DNA strand breaks.In contrast to gyrase, the ParC D79G mutation substantially impaired the effects of gepotidacin on both the catalytic and DNA cleavage activities of topoisomerase IV.This result makes it challenging to delineate between these two mechanisms for topoisomerase IV-mediated cell death by gepotidacin at the present time.
Finally, one of the most important attributes of gepotidacin against E. coli cells is its well-balanced dual-targeting.Because fluoroquinolones primarily target gyrase in E. coli, 47,79 a single mutation in GyrA can result in significant resistance to fluoroquinolones.In contrast, gepotidacin requires simultaneous mutations in both gyrase and topoisomerase IV to significantly reduce the susceptibility to the compound.This well-balanced dual-targeting of gepotidacin should significantly reduce the occurrence of resistant mutant E. coli strains and substantially lengthen the clinical usefulness of this compound against urinary tract and other infections caused by this bacterium.

Construction of the Isogenic E. coli Strains with
Mutations in Amino Acids that Are Involved in Gepotidacin Interactions with Gyrase and Topoisomerase IV.Construction of a Kanamycin-Resistant E. coli TOP10-2 (ParC D79N ) Mutant Strain.A kanamycin-resistance marker closely linked to the parC gene was used to introduce the ParC D79N mutation into a temperature-sensitive (ts) E. coli strain at a nonpermissive temperature following selection.Initially, we used E. coli TOP10/pKD46 competent cells grown under 0.04% L(+)-arabinose to induce the expression of λ-Red recombinase from pKD46.The cells also carried a parC D214G temperaturesensitive mutation (unpublished data).PCR products of approximately 5.5 kilobases (kb) were used to transform the E. coli TOP10/pKD46 competent cells by electroporation. 106hese PCR products contained the gene that encoded E. coli ParC D79N (a GAT → AAT mutation had been introduced by site-directed mutagenesis), its flanking sequences, as well as a kanamycin-resistant (KmR) cassette inserted immediately downstream of the parC open reading frame (ORF).The resultant transformants were selected with 50 μg/mL kanamycin on LB plates under a nonpermissive temperature of 42 °C.When the WT parC D214 from the PCR product was exchanged with the chromosomal parC D214G ts mutation by crossover, both the parC D79N mutation and the flanking KmR cassette were also exchanged due to close linkage to the parC D214.Four clones resistant to kanamycin and able to grow at 42 °C were isolated.Genetic characterization by PCR amplification and sequencing of the parC region from the chromosome confirmed that three clones carried the desired parC D79N and the KmRcassette.
Isolation of a Kanamycin-Resistant E. coli TOP10-3 (GyrA P35L , ParC D79N ) Double-Mutant Strain.An overnight culture of the WT E. coli TOP10 strain or the E. coli TOP10-2 (ParC D79N ) mutant strain was plated onto LB plates containing 4X MIC of GSK203815 (an early NBTI, unpublished) 42 and incubated at 37 °C.After incubation for 24−48 h, GSK203815resistant colonies were isolated from the E. coli TOP10-2 mutant strain, but none were found in the WT E. coli TOP10 strain.The resultant GSK203815-resistant colonies were replated on the 4X MIC plates for further purification.Selected mutants were further characterized by MIC analysis against GSK203815 and by PCR amplification and sequencing of the GyrA and GyrB quinolone resistance-determining regions.One of the selected mutants contained the GyrA P35L and ParC D79N mutations.
Isolation of a Kanamycin-Resistant E. coli TOP10-1 (GyrA P35L ) Mutant Strain.An overnight culture of WT E. coli TOP10 was plated onto LB plates containing 20 or 30 μg/mL of a DNA gyrase inhibitor, isoquinoline sulfonamide (IQS, cat.#:B1427, Sigma-Aldrich), and incubated at 37 °C.The E. coli TOP10-3 double-mutant strain described above conferred resistance to the IQS, but the E. coli TOP10-2 (ParC D79N ) mutant strain did not (unpublished data).After incubation for 24 h, resistant colonies were isolated, purified, and characterized by MIC analysis against the IQS and by PCR amplification and sequencing of the GyrA quinolone resistance-determining regions.One of the IQS-resistant mutants contained the GyrA P35L mutation.
DNA, Compounds, and Enzymes.Negatively supercoiled pBR322 DNA was prepared from E. coli using a Plasmid Mega Kit (Qiagen) as described by the manufacturer.Calf thymus topoisomerase I (Invitrogen) treatment of negatively supercoiled pBR322 was used to prepare relaxed plasmid DNA as described previously. 44Kinetoplast DNA (kDNA) was isolated from Crithidia fasciculata as described by Englund. 107epotidacin mesylate (GSK2140944, GlaxoSmithKline Lot no.152392771) was stored at 4 °C as 20 mM aliquots in 100% dimethyl sulfoxide (DMSO).Ciprofloxacin was obtained from Sigma-Aldrich (cat.no.17850, Lot no.1396107) and stored at 4 °C as 40 mM aliquots in 0.1 N NaOH.Initial dilution (1:5) of ciprofloxacin was in 10 mM Tris-HCl, pH 7.9.ATP was obtained from Sigma-Aldrich and stored at −20 °C as 20 mM aliquots in H 2 O.All other chemicals were analytical reagent grade.
All proteins were His-tagged.WT E. coli gyrase subunits (GyrA and GyrB) were expressed and purified as described by Chan et al. 74 or purchased from Profoldin (catalog ID: GDSA100 KE, Lot 112A150130).Mutant E. coli GyrA S83L was made following site-directed mutagenesis of the WT GyrA clone and expressed and purified as described by Dong et al. 108 WT E. coli topoisomerase IV subunits (ParC and ParE) and the ParC S80L mutant were expressed and purified as described by Peng and Marians or by a minor modification by Corbett et al. 109,110 Mutant E. coli GyrA P35L and ParC D79G were customproduced by GenScript and expressed and purified as described by Chan et al. 74 Determination of MIC Values.Antibacterial MIC assays were determined according to Clinical and Laboratory Standards Institute guidelines. 111yrase-Catalyzed DNA Supercoiling.DNA supercoiling assays were based on the procedure of Aldred et al. 112 Assays contained 5 nM E. coli WT gyrase (1:1 GyrA:GyrB ratio), 5 nM relaxed pBR322, and 1.5 mM ATP in a total volume of 20 μL of 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 175 mM KGlu, and 50 μg/mL bovine serum albumin (BSA).Assays with mutant enzymes contained 10 nM GyrA S83L or 15 nM GyrA P35L .The stated enzyme concentrations reflect those of the holoenzyme (A 2 B 2 ).Reactions were carried out in the absence of compound or in the presence of 0−10 μM gepotidacin or ciprofloxacin, and mixtures were incubated at 37 °C for 45 min (WT), 60 min (GyrA S83L ), or 45 min (GyrA P35L ), which represents the minimum time required to supercoil 80−90% of the DNA in the absence of drug.Reactions were stopped by the addition of 3 μL of a mixture of 0.77% sodium dodecyl sulfate (SDS) and 77.5 mM Na 2 EDTA.Samples were mixed with 2 μL of loading buffer [60% sucrose, 10 mM Tris-HCl (pH 7.9), 0.5% bromophenol blue, and 0.5% xylene cyanol FF] and incubated at 45 °C for 2 min before being loaded onto 1% agarose gels in 100 mM Trisborate (pH 8.3) and 2 mM EDTA.Gels were stained with 1 μg/ mL ethidium bromide for 30 min.DNA bands were visualized with medium-range ultraviolet light and quantified by scanning densitometry using a Protein Simple AlphaImager HP (HP-Al) digital imaging system.DNA supercoiling was monitored by the conversion of the relaxed to supercoiled plasmid.IC 50 values represent the concentration of gepotidacin or ciprofloxacin that decreased the supercoiling activity by 50% and were calculated using the analysis "[inhibitor] versus response − variable slope (four parameters) nonlinear least-squares fit" provided by GraphPad Prism 9 using the equation: where Y = the band intensity for the particular reaction, and X = the concentration of the compound.
Topoisomerase IV-Catalyzed DNA Decatenation.DNA decatenation assays were based on the procedure of Aldred et al. 44 Assays contained 1 nM E. coli WT topoisomerase IV (1:1 ParC:ParE ratio), 5 nM kDNA, and 1.5 mM ATP in 20 μL of 40 mM HEPES (pH 7.6), 100 mM KGlu, 10 mM Mg(OAc) 2 , and 25 mM NaCl.Assays with the mutant enzymes contained 1 nM ParC S80L or ParC D79G .The stated enzyme concentrations reflect those of the holoenzyme (A 2 B 2 ).Reactions were carried out in the absence of the compound or in the presence of 0−25 μM gepotidacin or ciprofloxacin, and mixtures were incubated at 37 °C for 20 min (WT), 15 min (ParC S80L ), or 55 min (ParC D79G ), which represents the minimum time required to decatenate 80− 90% of the DNA in the absence of drug.Reactions were stopped by the addition of 3 μL of a mixture of 0.77% SDS and 77.5 mM Na 2 EDTA.Samples were mixed with 2 μL of loading buffer and incubated at 45 °C for 2 min before being loaded onto 1% agarose gels in 100 mM Tris-borate (pH 8.3) and 2 mM EDTA.Gels were stained with 1 μg/mL ethidium bromide for 30 min.DNA bands were visualized as described above and monitored by the conversion of catenated kDNA into decatenated monomers.IC 50 values were calculated using the analysis "[inhibitor] versus response − variable slope (four parameters) nonlinear least-squares fit" provided by GraphPad Prism 9 and the equation as described above.
DNA Cleavage.DNA cleavage assays with E. coli gyrase and topoisomerase IV were based on the procedure of Aldred et al. 100 Reactions with gyrase were carried out in the absence of compound or in the presence of 0−25 μM gepotidacin or 0−100 μM ciprofloxacin.Reactions with topoisomerase IV were carried out in the absence of a compound or in the presence of 0−5 μM gepotidacin or 0−25 μM ciprofloxacin.Reaction mixtures contained 100 nM WT or mutant (GyrA S83L or GyrA P35L ) gyrase (1:1 GyrA:GyrB ratio), or 20 nM WT or mutant (ParC S80L or ParC D79G ) topoisomerase IV (1:1 ParC:ParE ratio).Stated enzyme concentrations reflect those of the holoenzyme (A 2 B 2 ). Assay mixtures contained 10 nM negatively supercoiled pBR322 in a total volume of 20 μL of cleavage buffer [40 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl 2 , and 12.5% glycerol].Some reactions contained 1.5 mM ATP.Samples were incubated at 37 °C for 20 min with gyrase and for 10 min with topoisomerase IV.Enzyme−DNA cleavage complexes were trapped by adding 2 μL of 5% SDS, followed by 2 μL of 250 mM Na 2 EDTA.Proteinase K (Sigma-Aldrich) (2 μL of 0.8 mg/ mL) was added, and mixtures were incubated at 45 °C for 30 min to digest the enzyme.Samples were mixed with 2 μL of loading buffer and incubated at 45 °C for 2 min before being loaded onto 1% agarose gels.Reaction products were subjected to electrophoresis in 40 mM Tris-acetate (pH 8.3) and 2 mM EDTA containing 0.5 μg/mL ethidium bromide.DNA bands were visualized and quantified, as described above.DNA singleor double-stranded cleavage was monitored by the conversion of supercoiled plasmid to nicked or linear molecules, respectively, and quantified by comparison to a control reaction in which an equal amount of DNA was digested with restriction endonuclease EcoRI (New England BioLabs).CC 50 values represent the concentration of gepotidacin or ciprofloxacin that induced 50% of maximal DNA cleavage and were calculated using the analysis "[agonist] versus response − variable slope (four parameters) nonlinear least-squares fit" provided by GraphPad Prism 9 using the equation: where Y = the band intensity for the particular reaction and X = the concentration of the compound.
For assays that monitored competition between gepotidacin and ciprofloxacin, the level of double-stranded DNA cleavage induced by 50 μM (gyrase) or 10 μM (topoisomerase IV) ciprofloxacin in the absence of gepotidacin was set to 100%.As increasing concentrations of gepotidacin were added (0−100 or 0−25 μM, respectively), the decrease in double-stranded and increase in single-stranded breaks were quantified.The maximal levels of single-stranded DNA cleavage were set to 100%.In these experiments, competition IC 50 values were calculated using the analysis "[inhibitor] vs response:variable slope (four parameters) nonlinear least-squares fit" provided by GraphPad Prism 9 described above in the section on gyrase-catalyzed DNA supercoiling.
Persistence of DNA Cleavage Complexes.Persistence of cleavage complexes was monitored in the absence or presence of gepotidacin or ciprofloxacin using the procedure of Aldred et al. 44 Initial gyrase reaction mixtures contained 500 nM WT gyrase, 50 nM negatively supercoiled pBR322, and 10 μM gepotidacin or 50 μM ciprofloxacin in 20 μL of cleavage buffer.CaCl 2 replaced MgCl 2 to raise baseline levels of cleavage in experiments without compound.Initial topoisomerase IV reaction mixtures contained 100 nM WT topoisomerase IV, 50 nM negatively supercoiled pBR322, and 0.1 μM gepotidacin or 10 μM ciprofloxacin in 20 μL of cleavage buffer.Topoisomerase IV has high basal activity; therefore, CaCl 2 was not used in experiments without compounds.Cleavage complexes were allowed to form at 37 °C for 20 min with gyrase and 10 min with topoisomerase IV, then diluted 20-fold in divalent cation-free cleavage buffer.Samples were removed at time points from 0 to 120 min, and reactions were stopped, processed, and quantified as described above.Levels of gepotidacin-induced singlestranded breaks or ciprofloxacin-induced double-stranded breaks were set to 100% at time zero, as were double-stranded breaks in the absence of compound.Persistence was monitored as a loss of single-or double-stranded scission over time.

Figure 1 .
Figure 1.Structures of the triazaacenaphthylene gepotidacin and the fluoroquinolone ciprofloxacin and the positioning of gepotidacin in the active site of E. coli gyrase.The key pharmacophoric elements of gepotidacin are shown (top left): left-hand side that pi−pi stacks with two central base pairs of the stretched DNA (LHS, blue), central linker (black), basic amine that interacts with E. coli GyrA D82 (equivalent to ParC D79 ; red), and right-hand side that binds in a largely hydrophobic GyrA or ParC pocket that opens up on the dimer interface (RHS, green).The triazaacenaphthylene is the LHS moiety.The quinolone core (red), C6 fluorine (green), and C3/4 keto acid (blue) substituents of ciprofloxacin are highlighted (top right).The structure of gepotidacin bound to E. coli gyrase showing predicted interactions with residues GyrA D82 and GyrA P35 is shown at the bottom.(A) Structure of the ternary complex of gepotidacin, E. coli gyrase, and double-stranded (ds) DNA.Left and right boxes B and C show the locations of GyrA D82 and GyrA P35 , respectively, relative to gepotidacin.(B) Zoomed-in image showing that gepotidacin interacts via a single hydrogen bond with GyrA D82 .The mutation GyrA D82N (ParC D79N) modifies the charge of the residue and directly affects the ability of the residue to hydrogen-bond with gepotidacin (pink carbon bonds).(C) Zoomed-in image showing that GyrA P35 is distal from gepotidacin but near the break in the DNA substrate.The GyrA P35 mutation possibly modifies the geometry of the loop spanning GyrA R32 and GyrA K42 �two residues likely responsible for the proper orientation of the double-helix during DNA cleavage.Images were generated using a structure from Protein Data Bank (PDB ID 6RKW)72 and PYMOL software.

Figure 2 .
Figure 2. Gepotidacin is a potent inhibitor of WT E. coli gyrasecatalyzed DNA supercoiling and WT topoisomerase IV-catalyzed DNA decatenation.The effects of gepotidacin (blue) and ciprofloxacin (orange) on DNA supercoiling and decatenation mediated by gyrase (left) and topoisomerase IV (right), respectively, are shown.Error bars represent the standard deviation (SD) of at least three independent experiments.The gels shown at the top are representative of supercoiling (left) and decatenation (right) assays with gepotidacin.DC represents the fully relaxed (left) or fully catenated (right) DNA control.The mobilities of relaxed, negatively supercoiled, [(−)SC], catenated (Cat), and decatenated (Decat) DNA are indicated.
(P35L) Mutation, or Mutant Topoisomerase IV Enzymes that Carry the ParC S80L (S80L) or ParC D79G (D79G) Mutation a a IC 50 values with gyrase and topoisomerase IV were calculated on the basis of DNA supercoiling and decatenation assays, respectively.Standard deviations (SD) are shown.Net Max Cleavage represents the maximal single-stranded DNA cleavage after subtracting the baseline levels of DNA cleavage.D DNA substrate (Figure 3, top left panel; Table

Figure 3 .
Figure 3. Gepotidacin enhances single-stranded DNA breaks mediated by WT E. coli gyrase and topoisomerase IV.The effects of gepotidacin (blue, top) and ciprofloxacin (orange, bottom) on DNA cleavage mediated by gyrase (left) and topoisomerase IV (Topo IV, right) are shown.Levels of single-stranded (SS, closed circles) and doublestranded (DS, open circles) DNA breaks are shown.Error bars represent the SD of at least three independent experiments.The insets show the effects of low concentrations of gepotidacin and ciprofloxacin on single-and double-stranded DNA cleavage mediated by WT topoisomerase IV.The gels shown at the top are representative DNA cleavage assays with gepotidacin.The mobilities of nicked (Nick or SS), linear (Lin or DS), and negatively supercoiled [(−)SC] are indicated.

Figure 4 .
Figure 4. Gepotidacin enhances only single-stranded breaks mediated by WT E. coli gyrase and topoisomerase IV.The enhancement of gyrase-mediated single-stranded (SS, black) or double-stranded (DS, red) cleavage at 20 min (filled bar) or 120 min (open bar) in the presence of 0, 50, or 200 μM gepotidacin is shown.Bottom panel: The enhancement of topoisomerase IV-mediated single-stranded (black) or double-stranded (red) cleavage at 10 min (filled bar) or 60 min (open bar) in the presence of 0, 10, or 200 μM gepotidacin is shown.Error bars represent the SD of at least three independent experiments.

Figure 5 .
Figure 5. Gepotidacin enhances only single-stranded breaks mediated by WT E. coli gyrase and topoisomerase IV in the presence of ATP.The effects of ATP on gepotidacin-induced single-(SS, closed blue circles) and double-stranded (DS, open blue circles) breaks mediated by gyrase (left panel) and topoisomerase IV (right panel) are shown.DNA cleavage in the absence of ATP (Figure 3) is shown as dashed lines for comparison.Error bars represent the SD of at least three independent experiments.

Figure 6 .
Figure 6.Gepotidacin induces stable single-stranded DNA breaks generated by WT E. coli gyrase and topoisomerase IV.The persistence of DNA cleavage complexes mediated by gyrase (left) is shown in the absence of the compound (black, open circle) or in the initial presence of 10 μM gepotidacin (blue, closed circle) or 50 μM ciprofloxacin (orange, open circle).Gyrase and plasmid concentrations in initial assay mixtures were 500 and 50 nM, respectively.The persistence of DNA cleavage complexes mediated by topoisomerase IV (right panel) is shown in the absence of compound (black, open circle) or in the initial presence of 0.1 μM gepotidacin (blue, closed circle) or 10 μM ciprofloxacin (orange, open circle).Topoisomerase IV and plasmid concentrations in initial assay mixtures were 100 and 50 nM, respectively.Initial reaction mixtures contained 5 mM MgCl 2 .Assays were initiated by the 20-fold dilution of reaction mixtures into buffer that lacked divalent cation.Double-stranded DNA breaks generated in the absence of a compound or in the presence of ciprofloxacin are shown as open circles, while single-stranded breaks generated in the presence of gepotidacin are shown as closed circles.DNA cleavage at time 0 was set to 100%.Error bars represent the SD of at least three independent experiments.

Figure 7 .
Figure 7. Gepotidacin competes against ciprofloxacin for the DNA cleavage/ligation active site of WT E. coli gyrase and topoisomerase IV.Assays with gyrase (left) contained 50 μM ciprofloxacin and 0−100 μM gepotidacin.Assays with topoisomerase IV (right panel) contained 10 μM ciprofloxacin and 0−25 μM gepotidacin.The disappearance of ciprofloxacin-induced double-stranded breaks (DS, open circles) and the appearance of gepotidacin-induced single-stranded breaks (SS, closed circles) are shown.Levels of ciprofloxacin-induced doublestranded breaks in the absence of gepotidacin were set to 100%, as were maximal levels of gepotidacin-induced single-stranded breaks.Error bars represent the SD of at least three independent experiments.The gels shown at the top are representative DNA cleavage competition assays with gepotidacin.The mobilities of nicked (Nick), linear (Lin), and negatively supercoiled [(−)SC] are indicated.

Figure 8 .
Figure 8. Effects of gepotidacin and ciprofloxacin on DNA supercoiling catalyzed by WT E. coli gyrase, the fluoroquinolone-resistant mutant GyrA S83L enzyme, and the mutant GyrA P35L enzyme.The effects of gepotidacin (left panel, closed circles) and ciprofloxacin (right panel, open circles) on DNA supercoiling mediated by WT (black), fluoroquinolone-resistant GyrA S83L (red), and mutant GyrA P35L (purple) gyrases are shown.Error bars represent the SD of at least three independent experiments.

Figure 9 .
Figure 9. Effects of gepotidacin and ciprofloxacin on DNA cleavage mediated by WT E. coli gyrase, the fluoroquinolone-resistant mutant GyrA S83L enzyme and the mutant GyrA P35L enzyme.The enhancement of single-stranded DNA breaks in the presence of gepotidacin (left panel, closed circles) and double-stranded breaks in the presence of ciprofloxacin (right panel, open circles) mediated by WT gyrase (black), fluoroquinolone-resistant GyrA S83L (red), and mutant GyrA P35L (purple) are shown.Error bars represent the SD of at least three independent experiments.

Figure 10 .
Figure 10.Effects of gepotidacin and ciprofloxacin on DNA decatenation catalyzed by WT topoisomerase IV, the fluoroquinolone-resistant ParC S80L enzyme, and the mutant ParC D79G enzyme.The effects of gepotidacin (left panel, closed circles) and ciprofloxacin (right panel, open circles) on DNA decatenation mediated by WT topoisomerase IV (black), fluoroquinolone-resistant ParC S80L (red), and ParC D79G (purple) are shown.Error bars represent the SD of at least three independent experiments.

Figure 11 .
Figure 11.Effects of gepotidacin and ciprofloxacin on DNA cleavage mediated by E. coli topoisomerase IV, the fluoroquinolone-resistant mutant ParC S80L enzyme, and the mutant ParC D79G enzyme.The enhancement of single-stranded DNA breaks in the presence of gepotidacin (left panel, closed circles) and double-stranded breaks in the presence of ciprofloxacin (right panel, open circles) mediated by WT topoisomerase IV (black), fluoroquinolone-resistant ParC S80L (red), and ParC D79G (purple) is shown.Error bars represent the SD of at least three independent experiments.

Table 1 .
Gepotidacin Displays Dual-Targeting of Gyrase and Topoisomerase IV in E. coli Cells a

Table 2 .
Summary of IC 50 and CC50Values for Gepotidacin against WT E. coli Gyrase and Topoisomerase IV, Mutant Gyrase Enzymes that Carry the GyrA S83L (S83L) or GyrAP35L