A Series of Spiropyrimidinetriones that Enhances DNA Cleavage Mediated by Mycobacterium tuberculosis Gyrase

The rise in drug-resistant tuberculosis has necessitated the search for alternative antibacterial treatments. Spiropyrimidinetriones (SPTs) represent an important new class of compounds that work through gyrase, the cytotoxic target of fluoroquinolone antibacterials. The present study analyzed the effects of a novel series of SPTs on the DNA cleavage activity of Mycobacterium tuberculosis gyrase. H3D-005722 and related SPTs displayed high activity against gyrase and increased levels of enzyme-mediated double-stranded DNA breaks. The activities of these compounds were similar to those of the fluoroquinolones, moxifloxacin, and ciprofloxacin and greater than that of zoliflodacin, the most clinically advanced SPT. All the SPTs overcame the most common mutations in gyrase associated with fluoroquinolone resistance and, in most cases, were more active against the mutant enzymes than wild-type gyrase. Finally, the compounds displayed low activity against human topoisomerase IIα. These findings support the potential of novel SPT analogues as antitubercular drugs.

G yrase and topoisomerase IV are the targets for fluoroquinolones, which are among the most heavily prescribed broad-spectrum antibacterials worldwide. 1−6 These enzymes modulate the superhelical density of DNA and resolve knots and tangles in the bacterial chromosome. 4,7,8 Fluoroquinolones inhibit the overall catalytic activities of gyrase and topoisomerase IV and stabilize covalent enzyme-cleaved DNA complexes (i.e., cleavage complexes) that are transient intermediates in the catalytic cycles of these enzymes. 1−6 The actions of fluoroquinolones rob bacterial cells of the critical catalytic functions of gyrase and topoisomerase IV and induce enzyme-generated DNA strand breaks that trigger the SOS response and lead to cell death. 2,[4][5][6]9 The World Health Organization lists fluoroquinolones among the five "highest priority" and "critically important" antimicrobial classes. 10 An important use of fluoroquinolones is the treatment of tuberculosis. 10,11 This lung infection, which is caused by the bacterium Mycobacterium tuberculosis, is one of the leading causes of global mortality. 10 The 1.5 million fatalities attributed to tuberculosis in 2020 ranked second only to COVID-19 for deaths caused by a single infectious agent. 12 Although fluoroquinolones are used as second-line treatment for tuberculosis, members of this drug class (primarily moxifloxacin and levofloxacin) are becoming increasingly more important as a treatment for patients who have multidrugresistant tuberculosis or are intolerant of first-line therapies. 10,11 Unfortunately, the use of these drugs in the treatment of tuberculosis is being threatened by the rise of fluoroquinolone resistance mutations in M. tuberculosis gyrase, which is the only type II topoisomerase encoded by this organism. 11, 13,14 Fluoroquinolones interact with bacterial type II enzymes primarily through a water-metal ion bridge formed by a divalent metal ion that is chelated by the C3/C4 keto acid of the drug and stabilized by four water molecules. 1,15−17 Two of these water molecules are coordinated by a highly conserved serine (originally identified as Ser83 in the GyrA subunit of Escherichia coli gyrase) and an acidic residue (located four positions downstream). Mutations in the residues that anchor the bridge are the most prevalent cause of fluoroquinolone resistance. 1,5,6,18,19 In contrast to gyrase from most bacteria, M. tuberculosis gyrase contains an alanine (A90) in the place of the conserved serine. 20 However, mutations in this residue and the acidic residue (D94) disrupt interactions with the water−metal ion bridge, which diminishes drug binding and causes fluoroquinolone resistance. 16 To address the issue of fluoroquinolone resistance, two new classes of gyrase/topoisomerase IV-targeted antibacterials have been developed, novel bacterial topoisomerase inhibitors (NBTIs) and spiropyrimidinetriones (SPTs). 5,18,21 The most clinically advanced NBTI, gepotidacin, is in phase III clinical trials for the treatment of uncomplicated urinary tract infections and uncomplicated urogenital gonorrhea. 22−25 The most clinically advanced SPT, zoliflodacin, is in phase III clinical trials for the treatment of uncomplicated gonorrhea. 26−28 Although far more is known about drug−enzyme interactions and drug mechanism for NBTIs than for SPTs, both classes of antibacterials appear to interact with gyrase and topoisomerase IV through residues that are not used to bind fluoroquinolones. 15,29−31 Consequently, NBTIs and SPTs that overcome fluoroquinolone resistance have been reported. 4,18,32−35 Furthermore, novel subsets of NBTIs and SPTs have been shown to interact with wild-type and resistant M. tuberculosis gyrase and overcome fluoroquinolone resistance in cultures and mouse infection models. 4,32,34,35 Members of the novel SPT series inhibit the DNA supercoiling reaction catalyzed by wild-type M. tuberculosis gyrase. 34,35 However, virtually nothing is known regarding the effects of these SPTs on the critical DNA cleavage reaction of gyrase from this organism.
Therefore, the effects of five novel SPTs 34 on DNA cleavage mediated by wild-type and fluoroquinolone-resistant M. tuberculosis gyrase were assessed and compared to those of moxifloxacin, ciprofloxacin, and zoliflodacin. These SPTs displayed activities that were comparable to those of the fluoroquinolones and higher than that of zoliflodacin against the wild-type enzyme. Moreover, the SPTs maintained high activity against three of the most common fluoroquinoloneresistant mutant gyrase enzymes. These results, together with the previous cellular and in vivo data, 34,35 suggest that SPTs may be suitable alternatives to fluoroquinolones for the treatment of tuberculosis, especially strains that carry fluoroquinolone resistance mutations in gyrase.

Enhancement of M. tuberculosis
Gyrase-Mediated DNA Cleavage by Novel SPTs. SPTs are an emerging class of antibacterials that target gyrase and topoisomerase IV. Current SPT drug development efforts have focused on Neisseria gonorrhoeae infections. 23,26−28 A recent study determined that novel SPTs also display activity against M. tuberculosis in cultures and mouse infection models. One of the most potent of these compounds was H3D-005722 ( Figure 1, listed as compound 23 in the work of Govender et al.). 34 H3D-005722 differs from zoliflodacin by the replacement of the 5-methyloxazolidinone group at the R 2 position with a valerolactam with a 2-atom bridge to the benzisoxazole scaffold ( Figure 1). Although members of the SPT series inhibited gyrase-catalyzed DNA supercoiling, they were not critically evaluated for their ability to enhance DNA cleavage mediated by the M. tuberculosis enzyme. Therefore, the effects of H3D-005722 and related SPTs (Figure 1) on the critical DNA cleavage reaction of M. tuberculosis gyrase were assessed ( Figure 2). Results were compared to those of the fluoroquinolones moxifloxacin (which is used in the treatment of tuberculosis) and ciprofloxacin (which is heavily prescribed as a broad-spectrum antibacterial) and zoliflodacin.
The ability of H3D-005722 to induce gyrase-mediated double-stranded DNA cleavage was similar to that of moxifloxacin and ciprofloxacin and somewhat higher than that of zoliflodacin ( Figure 2, left panel). The related SPTs, H3D-004882 and H3D-005709 (which differ at the R 2 position), also induced gyrase-mediated DNA cleavage. Although their activities were slightly lower than that of H3D-005722, they were comparable to or greater than that of zoliflodacin. The activities of two additional compounds, H3D-  004912 and H3D-005867, were also examined. These SPTs are analogues of H3D-00488 and H3D-005722, respectively, and replace the oxygen at R 1 with an amino-cyano group. In both cases, the presence of the amino-cyano group enhanced the activity of the parent compound ( Figure 2, right panel; see arrows).
As seen in the gel in Figure 2 (top left), H3D-005722 (and other SPTs�not shown) induced almost exclusively doublestranded DNA breaks monitored by the conversion of negatively supercoiled plasmid to linear DNA. Virtually no increase in single-stranded DNA breaks (monitored by the generation of nicked DNA molecules) was observed.
A series of control experiments was carried out to ensure that the enhancement of DNA cleavage seen in the presence of SPTs was mediated by gyrase, as opposed to a chemical reaction by the compounds (Figure 3). H3D-005722 was the SPT that was chosen for these experiments. Three separate results indicate that the double-stranded DNA breaks generated in the presence of H3D-005722 were generated by M. tuberculosis gyrase. First, no DNA cleavage was seen when 200 μM H3D-005722 was incubated with the plasmid substrate in the absence of gyrase ( Figure 3, lane 2, 200). Second, because DNA cleavage products generated by gyrase are covalently attached to the protein, 16,18 reaction mixtures must be treated with Proteinase K to digest the enzyme for cleaved DNA to run as a unique linear band. If it is not digested, protein-linked cleaved products are observed as a high molecular weight smear on the gel. As seen in lane 5 (ProK), in the absence of protease, the linear DNA cleavage product of the gyrase-containing reaction was replaced by a high-molecular-weight smear. Third, gyrase requires two active-site divalent metal ions to cleave DNA, which, in this case, is Mg 2+ . 16,18 EDTA is able to chelate the Mg 2+ and remove it from the enzyme only when the DNA is ligated. Consequently, treatment of gyrase-mediated reactions with EDTA prior to termination decreases the level of cleaved products over time. As seen in lanes 6−8 (EDTA-5, 10, and 20), treatment of reaction mixtures with the chelator diminished the presence of linear DNA cleavage products substantially over a time course of 5−20 min. This reversibility is inconsistent with a chemically induced DNA cleavage reaction. Taken together, these results provide strong evidence that the DNA scission observed in the presence H3D-005722 is mediated by M. tuberculosis gyrase.
Compounds that raise levels of gyrase−DNA cleavage complexes can act by increasing the forward rate of DNA scission or diminishing the rate of ligation. To determine the effects of SPTs on the lifetime of cleavage complexes, a DNA ligation assay was carried out in the presence of H3D-005722. After reaction mixtures were allowed to come to cleavage− ligation equilibrium at 37°C, ligation was induced by a shift to 75°C (a temperature that allows DNA ligation but not cleavage). 18,36 As seen in Figure 4, the half-life of the cleavage complex formed in the presence of H3D-005722 (∼80 s) was ∼4 times longer than that observed in the absence of the compound (∼21 s). Therefore, cleavage complexes formed in the presence of the SPT appear to be more stable than those formed in the presence of moxifloxacin or ciprofloxacin ( Figure  4).  Effects of ATP on SPT Activity. The DNA cleavage reactions shown above were carried out in the absence of ATP, which is the high energy co-factor that is required for gyrase to carry out its complete catalytic DNA supercoiling reaction. 1−6 In some cases, the inclusion of ATP has been shown to enhance the ability of drugs to induce enzyme-mediated DNA cleavage, while in others, it has diminished scission. 19,30 The effects of ATP on the stimulation of gyrase-mediated DNA cleavage by H3D-005722 are shown in Figure 5. In the presence of ATP, the activity of the SPT was ∼1.5 to 2-fold higher at every concentration examined. Furthermore, the activities of the novel SPTs (H3D-004882, H3D-004912, H3D-005709, and H3D-005867) against the M. tuberculosis enzyme were also increased modestly by ATP at 50 μM SPT, although little difference was observed at 200 μM SPT ( Figure  6). These findings contrast with results with zoliflodacin and moxifloxacin whose abilities to enhance DNA cleavage decreased slightly in the presence of ATP ( Figure 6). Thus, in the bacterial cell, which contains (on average) millimolar concentrations of ATP, 37 H3D-005722 is likely to be more active than zoliflodacin or moxifloxacin.
H3D-005722 Interacts with DNA in the Cleavage− Ligation Active Site of M. tuberculosis Gyrase. Structural studies provide strong evidence that fluoroquinolones interact with DNA in the cleavage−ligation active site of M. tuberculosis gyrase. 17 These drugs are situated between the newly generated 3′-OH and 5′-PO 4 terminal moieties of the two scissile bonds. Because SPTs, such as fluoroquinolones, induce gyrase-mediated double-stranded DNA breaks, it is presumed that all members of this class also interact at the two scissile bonds of M. tuberculosis gyrase. However, there is no direct evidence to confirm this assumption. At the present time, there is a crystal structure of a cleavage complex formed with QPT-1, which is a progenitor and smaller member of the SPT drug class, 29 and a second structure with zoliflodacin. 38 Both structures were generated with gyrase from Staphylococcus aureus).
Therefore, we carried out modeling studies to help elucidate the interactions of SPTs in the active site of M. tuberculosis gyrase. The model shown in Figure 7 with H3D-005722 bound to the enzyme was constructed based on coordinates from the crystal structures of QPT-1 in a cleavage complex with S. aureus gyrase 29 and moxifloxacin in a cleavage complex with M. tuberculosis gyrase, 17 as had been carried out previously for H3D-005687. 34 Note that the only difference between H3D-005722 and H3D-005687 is the replacement of the R 1 morpholine oxygen of the former with the amino-cyano group of the latter. Modeling studies suggest that H3D-005722 stabilizes double-stranded DNA breaks mediated by M. tuberculosis gyrase by interacting at the two scissile bonds cleaved by the enzyme (Figure 7A). This presumes the binding of two SPT molecules, one at each scissile bond as seen in the crystal structures of QPT-1 and zoliflodacin bound to the S. aureus gyrase. 29,38 The region surrounding the H3D-005722 R 2 valerolactam side chain includes the GyrB residues Arg482 and Lys441 that appear to be conformationally mobile ( Figure 7B). Equivalent residues in S. aureus gyrase (Arg 458 and Lys 417) tend to have poor electron density and vary in their positions depending on the bound inhibitor. Hence, it is likely that the conformation of the R 1 valerolactam is ambiguous. Figure 7C shows two possible poses for the side chain using the superposition of the model of H3D-05772 and that previously published for H3D-005687 (compound 42 in the publication). 34 The conformational mobility in the region probably accounts for the wide range of R 2 benzisoxazole substituents ( Figure 1) that are acceptable for gyrase inhibitory potency. 26,34,35 However, the differences in the positioning of the valerolactam between the two structures could also contribute to the enhanced activity of H3D-005687 as compared to H3D-05772.
To more directly determine whether H3D-005722 functions in the DNA cleavage−ligation active site of the enzyme, competition studies were carried out using GSK000, which is an NBTI derivative with high activity against M. tuberculosis gyrase. 18 In contrast to SPTs, only a single NBTI molecule binds in the cleavage complex. The left-hand side of the NBTI sits in a pocket in the DNA on the twofold axis of the complex,  midway between the two DNA cleavage sites, and the righthand side sits in a pocket on the twofold axis between the two GyrA subunits. Competition studies have demonstrated that NBTIs and fluoroquinolones that interact at the scissile bonds cannot coexist in the active site of gyrase. 18,30 The actions of NBTIs and SPTs can be distinguished because GSK000 induces only gyrase-mediated single-stranded DNA breaks, 18 while H3D-005722 induces primarily doublestranded breaks (Figure 2). This difference was used as the basis for a competition assay to determine whether the NBTI and H3D-005722 can simultaneously act on M. tuberculosis gyrase. In the assay, cleavage complexes were formed in the presence of a mixture of 200 μM H3D-005722 and increasing concentrations of GSK000 (0−100 μM). Competition was monitored by the loss of double-stranded DNA breaks, which only could have been induced by the SPT. As seen in Figure 8, levels of double-stranded breaks dropped ∼80% in the presence of 100 μM GSK000, which indicates competition between the NBTI and SPT.
A caveat to this conclusion is the fact that GSK000 suppresses double-stranded DNA breaks generated by M. tuberculosis gyrase. 18 Thus, it is possible that the NBTI and H3D-005722 interact with gyrase at separate sites and the apparent competition is due to this double-stranded DNA break suppression. If this were the case, GSK000 would decrease the actions of H3D-005722 at a concentration that reflects its binding to M. tuberculosis gyrase. In the absence of a competing compound, the concentration at which GSK000 induces one-half maximal single-stranded DNA cleavage with M. tuberculosis gyrase is ∼2.5 μM. 18 However, in the presence of the SPT, considerably higher concentrations of GSK000 were required to reduce double-stranded DNA breaks by 50% (IC 50 ≅ 17 μM; Figure 8). The reduced affinity of GSK000 for M. tuberculosis gyrase in the presence of H3D-005722 indicates that the decrease in double-stranded DNA breaks is due primarily to a competition between the SPT and the NBTI. Taken together, these findings provide evidence that GSK000 and H3D-005722 cannot co-exist in the cleavage complex,  which suggests that the SPT exerts its actions on M. tuberculosis gyrase by interacting in the active site of the enzyme.

SPTs Maintain Activity against M. tuberculosis Gyrase Enzymes Carrying Common Mutations Associated with
Fluoroquinolone Resistance. In a previous study, an SPT similar to H3D-005722 maintained activity against M. tuberculosis cells that carried the gyrase mutation GyrA A90V , which elicits fluoroquinolone resistance. 35 However, the effects of SPTs on the DNA cleavage activity of M. tuberculosis gyrase enzymes that harbor this and other common fluoroquinolone resistance mutations have not been examined. Therefore, the effects of SPTs on DNA scission mediated by gyrase enzymes that harbor the GyrA A90V , GyrA D94G , or GyrA D94H mutation, three of the most common mutations associated with fluoroquinolone-resistance in M. tuberculosis, 20 were determined.
As seen in Figure 9 (middle and right panels), the three mutant enzymes displayed resistance toward moxifloxacin and ciprofloxacin. In contrast, H3D-005722 maintained the ability to induce double-stranded DNA cleavage by the mutant gyrase enzymes (Figure 9, left panel). Although the activity of the SPT against GyrA D94H gyrase approximated that of the wildtype enzyme, H3D-005722 induced considerably higher levels (approximately three-fold) of DNA cleavage with the GyrA A90V and GyrA D94G mutant enzymes. Similar results were observed for all the SPTs utilized in the present study ( Figure 10). In all cases, the compounds displayed much higher levels of DNA cleavage with the GyrA A90V and GyrA D94G enzymes and at least wild-type activity against GyrA D94H gyrase. Modeling studies indicate that GyrA residues A90 and D94 are not proximal to the bound SPT ( Figure 7B). Thus, at the present time, there is not an obvious explanation for the enhanced activity of SPTs against fluoroquinolone-resistant mutations at these amino acid residues. However, the results of DNA cleavage assays demonstrate that H3D-005722 and related SPTs overcome the most common causes of target-mediated fluoroquinolone resistance in tuberculosis, at least at the enzyme level.
Effects of SPTs on DNA Cleavage Mediated by Human Topoisomerase IIα. Because gyrase and topoisomerase IIα are homologous enzymes, some antibacterial drug classes have the capacity to cross over into mammalian systems. 39−41 Therefore, the effects of H3D-005722 and related SPTs on the DNA cleavage activity of human topoisomerase IIα were assessed (Figures 11 and 12). At 200 μM, H3D-005722 induced a modest rise in doublestranded DNA breaks generated by topoisomerase IIα. However, virtually no increase in either double-or singlestranded cleavage was observed at any lower concentrations. Even at the highest concentration of H3D-005722 examined (200 μM), the rise in total DNA cleavage (double-and singlestranded) was dwarfed by that of etoposide, a topoisomerase II-targeted anticancer drug. At 200 μM, all the SPT and fluoroquinolone antibacterials examined displayed modest activity against human topoisomerase IIα compared to etoposide and CP-115,955, 41 which is a fluoroquinolone designed to have high activity against eukaryotic type II   topoisomerases ( Figure 12). Therefore, these SPTs maintain their potential for development as antibacterial drugs.

■ DISCUSSION
There is a need for the development of novel antitubercular agents to address rising drug resistance in this disease. Like the fluoroquinolones, SPTs act through gyrase, which is a validated drug target in M. tuberculosis. 10,11 Results of the present study indicate that H3D-005722 and related SPTs display high activity against M. tuberculosis gyrase and increase levels of enzyme-mediated double-stranded DNA cleavage. The activities of the SPTs examined were similar to those of moxifloxacin and ciprofloxacin and greater than that of zoliflodacin, a clinically advanced SPT. Finally, H3D-005722 and other novel SPTs overcome the most common mutations in gyrase that cause fluoroquinolone resistance and, in most cases, were more active against the mutant enzymes than the wild-type gyrase.
All of the compounds that were assessed in the present study inhibit the growth of cultured H37Rv M. tuberculosis cells with minimal inhibitor concentrations (MICs) in the micromolar range. 34 The order of efficacy was H3D-005867 > H3D-005722 > H3D-005709 > H3D-004882 > zoliflodacin > H3D-004912. This order is similar but not identical to the maximal levels of M. tuberculosis gyrase double-stranded DNA cleavage induced by these compounds, H3D-005867 > H3D-004912 > H3D-005722 > H3D-005709 ≈ H3D-004882 > zoliflodacin ( Figure 2). The most notable difference is that H3D-004912, which was the second most efficacious SPT with regard to DNA cleavage was the least inhibitory with regard to cell growth. The reasons that underlie this discrepancy are not known but may reflect cellular uptake, efflux, or metabolism. It is also notable that the range of MIC values is considerably lower (0.5−7.8 μM) 34 than the concentrations used in the present study. However, the relationship between concentrations of compounds used in cellular assays and the actual levels that accumulate in cells is unclear. Furthermore, the number of DNA strand breaks required to kill M. tuberculosis cells is not known.
Finally, SPTs (like fluoroquinolones and NBTIs) have two effects on M. tuberculosis gyrase. They increase levels of enzyme-mediated DNA cleavage and they inhibit overall catalytic activity. 16,18,34 It is generally assumed that drugs that enhance gyrase-mediated DNA cleavage (i.e., poisons) kill cells primarily by inducing breaks in the bacterial genome. 2,[4][5][6]9 In this case, the higher the cellular concentration of gyrase, the greater the number of DNA breaks, and the more lethal the drug. However, a recent report indicated that M. tuberculosis cells that were gyrase hypomorphs displayed hypersensitivity to SPTs. 34 This finding suggests that SPTs are also capable of killing cells by inhibiting the essential catalytic activities (removing positive DNA supercoils that accumulate ahead of replication forks and introducing negative supercoils into the genome) of gyrase. 4,7,8 Our findings, along with the previous cellular and in vitro studies, 34 suggest a model in which SPTs (and other gyrase-targeted drugs) may be able to kill M. tuberculosis cells in a bimodal fashion. At high gyrase concentrations, drugs act as "poisons" that fragment the genome. Conversely, at low gyrase concentrations, drugs act as catalytic inhibitors that rob cells of a critical enzyme activity. This "bimodal model" of cell kill may allow SPTs to act under a variety of growth conditions. Taken together, these findings further support the potential of novel SPT derivatives as antitubercular drugs.

Enzymes and Materials.
Wild-type M. tuberculosis gyrase subunits (GyrA and GyrB) and fluoroquinolone-resistant GyrA mutants (GyrA A90V , GyrA D94H , and GyrA D94G ) were expressed and purified as described previously 16 and were stored at −80°C . Human topoisomerase IIα was expressed and purified from Saccharomyces cerevisiae 42,43 and was stored in liquid N 2 .
Negatively supercoiled pBR322 DNA was prepared from E. coli using a Plasmid Mega Kit (Qiagen) as described by the manufacturer.
The SPTs H3D-004882, H3D-004912, H3D-005709, H3D-005722, and H3D-005867 were synthesized as described previously by Govender et al. 34 In that paper, H3D-004882 and H3D-005722 were referred to as compounds 8 and 23, respectively. Zoliflodacin was obtained from MedChemExpress, moxifloxacin from LKT Laboratories, and ciprofloxacin and etoposide from Sigma-Aldrich (Millipore Sigma). The SPTs, moxifloxacin, etoposide, and the NBTI derivative GSK000 (gift from Monica Cacho) were stored at −20°C as 20 mM stock solutions in 100% dimethyl sulfoxide (DMSO). H3D-005722 working concentrations (5−200 μM) were not soluble in 10% DMSO but were soluble in 40% DMSO. Consequently, all drug dilutions for the SPTs and fluoroquinolones were in 40% DMSO. The final concentration of DMSO in reaction mixtures was 4%. Ciprofloxacin and the fluoroquinolone CP-115,955 (gift from Robert Kerns) were stored as 40 mM stock solutions in 0.1 N NaOH and stored at −20°C. Prior to their use in assays, these fluoroquinolones were diluted fivefold into 10 mM Tris−HCl (pH 7.9).
DNA Cleavage. DNA cleavage reactions were based on the procedure of Aldred et al. 16 Reactions were carried out in the presence or absence of SPTs or fluoroquinolones and contained 100 nM wild-type or fluoroquinolone-resistant mutant (GyrA A90V , GyrA D94H , and GyrA D94G ) gyrase (1.5:1 GyrA:GyrB ratio) and 10 nM negatively supercoiled pBR322 in a total volume of 20 μL of gyrase cleavage buffer [10 mM Tris−HCl (pH 7.5), 40 mM KCl, 6 mM MgCl 2 , 0.1 mg/mL bovine serum albumin, and 10% glycerol]. In some cases, 1.5 mM ATP was included in reaction mixtures. Unless stated otherwise, reactions were incubated at 37°C for 10 min. Enzyme−DNA cleavage complexes were trapped by adding 2 μL of 4% sodium dodecyl sulfate (SDS) followed by 1 μL of 375 mM Na 2 EDTA and 2 μL of 0.8 mg/mL Proteinase K (Sigma Aldrich). Reaction mixtures were incubated at 45°C for 30 min to digest the enzyme. Samples were mixed with 2 μL of 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 loading 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 with medium-range ultraviolet light and quantified using an Alpha Innotech digital imaging system. DNA single or doublestranded cleavage was monitored by the conversion of negatively supercoiled plasmid to nicked or linear molecules, respectively, and quantified in comparison to a control reaction in which an equal amount of DNA was digested by EcoRI (New England BioLabs).
In reactions that examined the effects of compounds on the DNA cleavage activity of human topoisomerase IIα, the procedure of Fortune and Osheroff was employed. 44 Reaction mixtures contained 0−200 μM H3D-005722 or etoposide, 110 nM topoisomerase IIα, and 10 nM negatively supercoiled pBR322 in a total volume of 20 μL of 10 mM Tris−HCl (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 5 mM MgCl 2, and 2.5% (v/v) glycerol. Assay mixtures were incubated at 37°C for 6 min. Reactions were terminated, and products were analyzed as described above. Additional experiments with topoisomerase IIα compared DNA cleavage induced by 200 μM zoliflodacin, the rest of the novel SPT series, moxifloxacin, ciprofloxacin, and CP-115,955, 41 which is a fluoroquinolone developed for its high activity against eukaryotic type II topoisomerases.
DNA Ligation. DNA ligation assays were carried out in the absence or presence of H3D-005722, moxifloxacin, or ciprofloxacin following the procedure of Gibson et al. 18 Reaction mixtures (20 μL) contained 100 nM wild-type M. tuberculosis gyrase and 10 nM negatively supercoiled pBR322 in gyrase cleavage buffer. In experiments carried out in the absence of a drug, MgCl 2 in the cleavage buffer was replaced with 6 mM CaCl 2 to increase baseline levels of DNA cleavage. DNA cleavage−religation equilibria were established at 37°C for 10 min. Ligation was initiated by shifting the temperature from 37 to 75°C. Reactions were stopped by the addition of 2 μL of 4% SDS followed by 1 μL of 375 mM EDTA (pH 8.0). Samples were digested with Proteinase K, processed, and visualized as described above. Levels of double-stranded DNA cleavage were set to 100 at time = 0 s, and ligation was assessed by the loss of the linear reaction product over time.