Inhibitors Of Bacterial DNA Gyrase Research Articles

A New-Class Antibacterial-Almost. Lessons in Drug Discovery and Development: A Critical Analysis of More than 50 Years of Effort toward ATPase Inhibitors of DNA Gyrase and Topoisomerase IV.

The introduction into clinical practice of an ATPase inhibitor of bacterial DNA gyrase and topoisomerase IV (topo IV) would represent a new-class agent for the treatment of resistant bacterial infections. Novobiocin, the only historical member of this class, established the clinical proof of concept for this novel mechanism during the late 1950s, but its use declined rapidly and it was eventually withdrawn from the market. Despite significant and prolonged effort across the biopharmaceutical industry to develop other agents of this class, novobiocin remains the only ATPase inhibitor of gyrase and topo IV ever to progress beyond Phase I. In this review, we analyze the historical attempts to discover and develop agents within this class and highlight factors that might have hindered those efforts. Within the last 15 years, however, our technical understanding of the molecular details of the inhibition of the gyrase and topo IV ATPases, the factors governing resistance development to such inhibitors, and our knowledge of the physical properties required for robust clinical drug candidates have all matured to the point wherein the industry may now address this mechanism of action with greater confidence. The antibacterial spectrum within this class has recently been extended to begin to include serious Gram negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. In spite of this recent technical progress, adverse economics associated with antibacterial R&D over the last 20 years has diminished industry's ability to commit the resources and perseverance needed to bring new-class agents to launch. Consequently, a number of recent efforts in the ATPase class have been derailed by organizational rather than scientific factors. Nevertheless, within this context we discuss the unique opportunity for the development of ATPase inhibitors of gyrase and topo IV as new-class antibacterial agents with broad spectrum potential.

Pyridine-3-carboxamide-6-yl-ureas as novel inhibitors of bacterial DNA gyrase: Structure based design, synthesis, SAR and antimicrobial activity

The development of antibacterial drugs based on novel chemotypes is essential to the future management of serious drug resistant infections. We herein report the design, synthesis and SAR of a novel series of N-ethylurea inhibitors based on a pyridine-3-carboxamide scaffold targeting the ATPase sub-unit of DNA gyrase. Consideration of structural aspects of the GyrB ATPase site has aided the development of this series resulting in derivatives that demonstrate excellent enzyme inhibitory activity coupled to potent Gram positive antibacterial efficacy.

Novel quinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV

A structurally novel set of inhibitors of bacterial type II topoisomerases with potent in vitro and in vivo antibacterial activity was developed. Dual-targeting ability, hERG inhibition, and pharmacokinetic properties were also assessed.

Aminocoumarins inhibit osteoclast differentiation and bone resorption via downregulation of nuclear factor of activated T cells c1

Aminocoumarins, such as coumermycin A1 and novobiocin, are natural products of streptomycetes. They are potent inhibitors of bacterial DNA gyrase and are used to suppress the growth of bacteria in inflammatory diseases. However, their effect in osteoclastogenesis has not been investigated. In this study, using mouse bone-marrow-derived macrophages (BMMs), we showed that coumermycin A1 and novobiocin suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. The inhibitory effect of coumermycin A1 was associated with impaired activation of multiple signaling events downstream of RANK, including extracellular signal-regulated kinase, p38, and c-Jun terminal kinase phosphorylation, followed by decreased c-Fos and nuclear factor of activated T cells (NFAT)c1 expression. Ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of coumermycin A1, suggesting that the anti-osteoclastogenic effect of coumermycin A1 was mainly attributable to reduction in NFATc1 expression. Coumermycin A1 also abrogated RANKL-induced expression of interleukin-1β, tumor necrosis factor-α, and inducible nitric oxide synthase in mouse BMMs. Consistent with the in vitro anti-osteoclastogenic effect, the aminocoumarin suppressed lipopolysaccharide-induced osteoclast formation and bone loss in in vivo mouse experiments. Taken together, our data demonstrate that aminocoumarins inhibit osteoclast formation and bone resorption, and comprise a potential therapeutic strategy for treating bone destructive diseases.

Allosteric inhibition of the DNA-dependent ATPase activity of Escherichia coli DNA gyrase by a representative of a novel class of inhibitors

A novel class of bacterial DNA gyrase inhibitors has been shown previously to form a ternary complex with DNA and gyrase in a site distinct from the fluoroquinolone and ATP binding sites and does not cause double-strand-cleaved complex stabilization like fluoroquinolones. We show that, unlike fluoroquinolones, a representative compound inhibits DNA-dependent ATP hydrolysis by Escherichia coli gyrase and also blocks cleaved complex stabilization by ciprofloxacin. Conversely, ciprofloxacin blocks ATPase inhibition by the novel compound. We conclude that the compound acts allosterically to inhibit ATP binding or hydrolysis and interferes with the gyrase catalytic cycle at a different point than ciprofloxacin.

Inhibition of DNA gyrase and DNA topoisomerase IV of Staphylococcus aureus and Escherichia coli by aminocoumarin antibiotics

Aminocoumarin antibiotics are potent inhibitors of bacterial DNA gyrase. We investigated the inhibitory and antibacterial activity of naturally occurring aminocoumarin antibiotics and six structural analogues (novclobiocins) against DNA gyrase and DNA topoisomerase IV from Escherichia coli and Staphylococcus aureus as well as the effect of potassium and sodium glutamate on the activity of these enzymes. The inhibitory concentrations of the aminocoumarins were determined in gyrase supercoiling assays and topoisomerase IV decatenation assays. Both subunits of S. aureus topoisomerase IV were purified as His-Tag proteins in E. coli. The MIC was tested in vivo for the control organisms E. coli ATCC 25922 and S. aureus ATCC 29213. DNA gyrase is the primary target in vitro of all investigated aminocoumarins. With the exception of simocyclinone D8, all other aminocoumarins inhibited S. aureus gyrase on average 6-fold more effectively than E. coli gyrase. Potassium glutamate is essential for the activity of S. aureus gyrase and increases the sensitivity of E. coli gyrase to aminocoumarins ≥ 10-fold. The antibacterial activity of the tested compounds mirrored their relative activities against topoisomerases. The study provides insights about the substituents that are important for the inhibitory activity of aminocoumarins against the target enzymes, which will facilitate the rational design of improved antibiotics.

N-Benzyl-3-sulfonamidopyrrolidines Are a New Class of Bacterial DNA Gyrase Inhibitors

This paper characterizes N-benzyl-3-sulfonamidopyrrolidines (gyramides) as DNA gyrase inhibitors. Gyramide A was previously shown to exhibit antimicrobial activity that suggested it inhibited bacterial cell division. In this study, we conducted target identification studies and identified DNA gyrase as the primary target of gyramide A. The gyramide A resistance-determining region in DNA gyrase is adjacent to the DNA cleavage gate and is a new site for inhibitor design. We studied the antibiotic effects of gyramides A-C in combination with the Gram-negative efflux pump inhibitor MC-207,110 (60 μM). The gyramides had a minimum inhibitory concentration of 10-40 μM against Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, and Streptococcus pneumoniae; the compounds were ineffective against Enterococcus faecalis. The IC(50) of gyramides A-C against E. coli DNA gyrase was 0.7- 3.3 μM. The N-benzyl-3-sulfonamidopyrrolidines described in this manuscript represent a starting point for development of antibiotics that bind a new site in DNA gyrase.

Metabolic stress-induced programmed cell death in Xanthomonas

Xanthomonas campestris pv. glycines (Xcg), an etiological agent of the bacterial pustule disease of soybean, displayed nutritionally regulated caspase-dependent programmed cell death (PCD). Experiments showed that Xcg was under metabolic stress during PCD, as evident from the intracellular accumulation of NADH and ATP. Further, the accumulation of reactive oxygen species (ROS), as confirmed by 2',7'-dichlorofluorescein diacetate labeling, electron spin resonance spectroscopy, and scopoletin assay, was also observed along with the activation of caspase-3. ROS scavengers such as dimethylsulfoxide, glutathione, n-propyl gallate, and catalase significantly inhibited caspase biosynthesis as well as its activity, eventually leading to the inhibition of PCD. The presence of a sublethal concentration of an electron transport chain uncoupler, 2,4-dinitrophenol, was found to reduce the ROS generation and the increase in the cell survival. These results indicated that Xcg cells grown in a protein-rich medium experienced metabolic stress due to electron leakage from the electron transport chain, leading to the generation of ROS and the expression as well as the activation of caspase-3, and resulting in PCD. A bacterial DNA gyrase inhibitor, nalidixic acid, was also found to inhibit PCD. Gyrase, which regulates DNA superhelicity, and consequently DNA replication and cell multiplication, appears to be involved in the process.

Anti-proliferative effects of simocyclinone D8 (SD8), a novel catalytic inhibitor of topoisomerase II

Simocyclinone D-8 (SD8), a semi-synthetic compound derived from yeast, has been shown to decrease the proliferation of MCF-7 breast cancer cells. It has been shown to be a potent bacterial DNA gyrase inhibitor, a homologue of human topoisomerase II (hTopoII). We tested SD8 activity alone and in combination with cisplatin against malignant mesothelioma (MM) and non-small cell cancer (NSCLC) cell lines. Inhibition of hTopoII supercoiling function by SD8 and a known hTopoII poison, etoposide, were done by in vitro assay using purified hTopoII and kinetoplast DNA as the substrate. The DNA products were analyzed by agarose gel electrophoresis after treatment with increasing concentrations of each drug. Mesothelioma cell lines (H2373, H2461 and H2596) and NSCLC cell lines (H2030, H460, and H2009) grown in RPMI with 10% calf serum were used. Non-malignant mesothelial cells, LP9, were grown in 1:1 ratio of MCDB:199E medium supplemented with 15% calf serum, 0.4 microg/mL hydrocortisone, and 15 ng/mL epidermal grown factor. Cell proliferation assays were performed in 96-well plates using the CCK-8 kit (Dojindo inc.). Cells were treated for 72 h with various SD8 concentrations and controls containing equal volume of the vehicle, DMSO. Treated cells were assayed for the induction of apoptosis with poly ADP-ribose polymerase-1 (PARP) cleavage assay. Biochemical assays revealed that the IC(50) for hTopoII inhibition was 100 microM for SD8 and 400 microM for etoposide. SD8 inhibited hTopoII function without inducing DNA cleavage events. SD8 inhibited the growth of NSCLC and Mesothelioma cells with IC(50) ranging from 75-125 microM. Furthermore, SD8 was not toxic to non-transformed primary mesothelial cell line, LP9 at the IC(50) doses. SD8 induced apoptosis in all cell lines tested. SD8 inhibits hTopoII in vitro without inducing DNA strands breaks and has significant activity against NSCLC and MM cell lines. While doses required for SD8 anticancer activity are unlikely to be achieved in vivo, chemical modifications to SD8 to increase its potency could lead to improved therapies for these diseases.

Mycobacterium tuberculosis DNA Gyrase as a Target for Drug Discovery

Bacterial DNA gyrase is an important target of antibacterial agents, including fluoroquinolones. In most bacterial species, fluoroquinolones inhibit DNA gyrase and topoisomerase IV and cause bacterial cell-death. Other naturally occurring bacterial DNA gyrase inhibitors, such as novobiocin, are also known to be effective as antibacterial agents. DNA gyrase is an ATP-dependent enzyme that acts by creating a transient double-stranded DNA break. It is unique in catalyzing the negative supercoiling of DNA and is essential for efficient DNA replication, transcription, and recombination. DNA gyrase is a tetrameric A2B2 protein. The A subunit carries the breakage-reunion active site, whereas the B subunit promotes ATP hydrolysis. The M. tuberculosis genome analysis has identified a gyrB-gyrA contig in which gyrA and gyrB encode the A and B subunits, respectively. There is no evidence that M. tuberculosis has homologs of the topoisomerase IV, parC and parE genes, which are present in most other bacteria. Newer fluoroquinolones, including moxifloxacin and gatifloxacin, exhibit potent activity against M. tuberculosis, and show potential to shorten the duration for TB treatment. Resistance to fluoroquinolones remains uncommon in clinical isolates of M. tuberculosis. M. tuberculosis DNA gyrase is thus a validated target for anti-tubercular drug discovery. Inhibitors of this enzyme are also active against non-replicating mycobacteria, which might be important for the eradication of persistent organisms. A novel inhibitor of M. tuberculosis DNA gyrase would be effective against multi-drug resistant (MDR)-TB, and it could also be effective against fluoroquinolone-resistant M. tuberculosis.

Expression and characterization of the ATP-binding domain of a malarial Plasmodium vivax gene homologous to the B-subunit of the bacterial topoisomerase DNA gyrase

We have previously reported the presence of a DNA gyrase-like topoisomerase activity associated with the 35 kb apicoplast DNA in the malarial parasite Plasmodium falciparum [Weissig V, Vetro-Widenhouse TS, Rowe TC. Topoisomerase II inhibitors induce cleavage of nuclear and 35 kb plastid DNAs in the malarial parasite Plasmodium falciparum. DNA Cell Biol 1997;16:1483]. Sequences encoding polypeptides homologous to both the A and B subunits of bacterial DNA gyrase have been identified in the genome sequence of P. falciparum among data produced by the Malaria Genome Consortium and the University of Florida Malaria Gene Sequence Tag Project. Based on these findings, we have cloned and expressed a region of the Plasmodium vivax GyrB gene encoding a 43 kDa polypeptide homologous to the ATP-binding domain of Escherichia coli DNA gyrase. The 43 kDa PvGyrB polypeptide was found to have intrinsic ATPase activity with a K m of 0.27 mM and a k cat of 0.051 s −1. The PvGyrB ATPase was also sensitive to the bacterial DNA gyrase inhibitor coumermycin. The implications of these findings are discussed.

Development and Use of a High-Throughput Bacterial DNA Gyrase Assay to Identify Mammalian Topoisomerase II Inhibitors with Whole-Cell Anticancer Activity

A high-throughput screen (HTS) was developed and used to identify inhibitors of bacterial DNA gyrase. Among the validated hits were 53 compounds that also inhibited mammalian topoisomerase II with IC(50) values of <12.5 micro g/mL for 51 of them. Using computational methods, these compounds were subjected to cluster analysis to categorize them according to their chemical and structural properties. Nine compounds from different clusters were tested for their whole-cell inhibitory activity against 3 cancer cell lines-NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS)-at a concentration of 100 micro M. Five compounds inhibited cell growth by >50% for all 3 cell lines tested. These compounds were tested further against a panel of 53 to 57 cell lines representing leukemia, melanoma, colon, CNS, ovarian, renal, prostate, breast, and non-small cell lung cancers. In this assay, PGE-7143417 was found to be the most potent compound, which inhibited the growth of all the cell lines by 50% at a concentration range of 0.31 to 2.58 micro M, with an average of 1.21 micro M. An additional 17 compounds were also tested separately against a panel of 10 cell lines representing melanoma, colon, lung, mammary, ovarian, prostate, and renal cancers. In this assay, 4 compounds-PGE-3782569, PGE-7411516, PGE-2908955, and PGE-3521917-were found to have activity with concentrations for 50% cell growth inhibition in the 0.59 to 3.33, 22.5 to 59.1, 7.1 to >100, and 24.7 to >100 micro M range.

Clorobiocin Biosynthesis in Streptomyces: Identification of the Halogenase and Generation of Structural Analogs

Clorobiocin ( clo) and novobiocin ( nov) are potent inhibitors of bacterial DNA gyrase. The two substances differ in the substitution pattern at C-8′ of the aminocoumarin ring, carrying a chlorine atom or a methyl group, respectively. By gene inactivation, clo-hal was identified as the gene of the halogenase responsible for the introduction of the chlorine atom of clorobiocin. Inactivation of cloZ did not affect clorobiocin formation, showing that this ORF is not essential for clorobiocin biosynthesis. Expression of the methyltransferase gene novO in the clo-hal − mutant led to the very efficient formation of a hybrid antibiotic containing a methyl group instead of a chlorine atom at C-8′. Comparison of the antibacterial activity of clorobiocin analogs with -Cl, -H, or -CH 3 at C-8′ showed that chlorine leads to 8-fold higher activity than hydrogen and to 2-fold higher activity than a methyl group.

Synthesis and topoisomerase inhibitory activities of novel aza-analogues of flavones

Aseries of aza-flavones (3-hydroxy-2-phenyl-4-quinolones) were designed and synthesized as inhibitors of bacterial DNA-gyrase and mammalian topoisomerase II. Structure activity relationships of the compounds against each of the enzymes are discussed.

Genotoxic activity of four inhibitors of DNA topoisomerases in larval cells of Drosophila melanogaster as measured in the wing spot assay

Four inhibitors of DNA topoisomerases namely nalidixic acid, camptothecin, m-amsacrine and etoposide, have been evaluated for genotoxic effects in the wing spot test of Drosophila melanogaster. This assay assesses somatic recombination and mutational events. We studied nalidixic acid as an inhibitor of bacterial DNA gyrase, camptothecin as a topoisomerase I inhibitor, as well as m-amsacrine and etoposide as topoisomerse II inhibitors. The genotoxic effects were determined from the appearance of wing spots in flies trans-heterozygous for the recessive markers multiple wing hairs (mwh) and flare, as well as in flies heterozygous for mwh and the multiply inverted TM3 balancer chromosome. From our results it appears that whilst nalidixic acid and m-amsacrine were compounds that did not increase the incidence of mutant clones, camptothecin and etoposide proved to be significantly genotoxic in this test, being camptothecin more effective than etoposide. A significant proportion of the total spot induction was due to mitotic recombination, confirming previously reported data. On the other hand, the cotreatments of each topoisomerase inhibitor with the alkylating agent ethyl methanesulfonate (EMS) indicate that, while nalidixic acid, m-amsacrine and etoposide show a tendency to an antagonistic interaction, camptothecin shows an additive effect, suggesting mechanistic differences between the activity of the four inhibitors of DNA topoisomerases studied.

Preparation and antimicrobial assessment of 2-thioether-linked quinolonyl-carbapenems.

This reports the synthesis and in vitro antimicrobial properties of a series of 2-thioether-linked quinolonyl-carbapenems. Although the title compounds exhibited broad spectrum activity, the MICs were generally higher than those observed for selected benchmark carbapenems, quinolonyl-penems, and quinolones. Enzyme assays suggested that the title compounds are potent inhibitors of penicillin binding proteins and inefficient inhibitors of bacterial DNA-gyrase. Uptake studies indicated that the new compounds are not substrates for the norA encoded quinolone efflux pump.

Synthesis of 2‐substituted 4‐quinazolone‐5‐carboxylic acids as inhibitors of DNA‐gyrase

AbstractA series of 4‐quinazolone‐5‐carboxylic acids were designed as bacterial DNA gyrase inhibitors. The syntheses of the target compounds were accomplished by reacting 3‐aminophthalimide with aroyl chlorides followed by rearrangement of the resulting 3‐acylaminophthalimides under basic conditions. The designed compounds showed moderate DNA gyrase inhibitory activity.

Mode of action of GR122222X, a novel inhibitor of bacterial DNA gyrase.

GR122222X is a potent inhibitor of the supercoiling reaction of bacterial DNA gyrase. We show that this compound binds stoichiometrically to inactivate the ATPase activity of a 43-kDa N-terminal fragment of the B subunit and competitively inhibits the binding of a radiolabelled coumarin drug to N-terminal fragments of GyrB. These and other data suggest that GR122222X has a mode of action similar, but not identical, to that of coumarin antibiotics.

Efficacies of ABT-719 and related 2-pyridones, members of a new class of antibacterial agents, against experimental bacterial infections.

The 2-pyridones are a new class of broad-spectrum orally bioavailable antibacterial agents. These compounds are potent bacterial DNA gyrase inhibitors which differ from fluoroquinolones by placement of the nitrogen atom in the ring juncture. ABT-719 is an S isomer and a representative 2-pyridone. ABT-719 administered orally or subcutaneously was 4- to 10-fold more effective than ciprofloxacin against Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes infections in normal mice. ABT-719 was equivalent in efficacy to ciprofloxacin for treatment of gram-negative bacterial infections caused by Pseudomonas aeruginosa or Escherichia coli. The racemate and R forms of ABT-719 produced similar results against gram-positive and gram-negative bacterial infections. The 50% effective doses of ABT-719 were at least threefold lower than those of ciprofloxacin for therapy of intracellular infections caused by Salmonella typhimurium or Listeria monocytogenes. In immunosuppressed mice, ABT-719 was more effective than ciprofloxacin against quinolone-sensitive S. aureus, Enterococcus faecalis, and Enterococcus faecium. The pharmacokinetic properties of ABT-719 were consistent with its relative efficacy. The 2-pyridones are potent, orally available antibacterial agents with efficacy against gram-positive and gram-negative bacterial infections in mice.

Testing Potential Gyrase Inhibitors of Bacterial DNA Gyrase: A Comparison of the Supercoiling Inhibition Assay and "Cleavable Complex" Assay

Inhibitory activity of test compounds against Escherichia coli DNA gyrase in a "cleavable complex" assay, readily observed in vitro at the enzyme level by the artificial addition of a denaturing agent, is found to be an excellent indicator of 4-quinolone inhibition of DNA gyrase, and as accurate a predictor of target enzyme inhibitory activity as the measurement of the inhibition of DNA gyrase supercoiling. This study was designed to examine the specificity of DNA gyrase inhibitors of various chemical classes in these two DNA gyrase assays, and define the use of these two assays in understanding the nature of inhibition by experimental compounds. Supercoiling inhibition was detected by determination of the 50% inhibition level, and cleavable complex inhibition measured by the determination of the drug concentration at which 50% of the maximal (of control) formation of linear, cleaved DNA was obtained. Results indicate that these two assays can serve several different functions in microbiological research, among them: (1) quantitation of enzyme inhibitory activity at the target level; and (2) distinguishing between nonspecific inhibition or artifactual inhibition of DNA gyrase and true, mechanism-based inhibition of the catalytic activity of DNA gyrase.