Bacterial DNA Gyrase Research Articles

Structure-Based Discovery of Substituted 4,5′-Bithiazoles as Novel DNA Gyrase Inhibitors

Bacterial DNA gyrase is a well-established and validated target for the development of novel antibacterials. Starting from the available structural information about the binding of the natural product inhibitor, clorobiocin, we identified a novel series of 4'-methyl-N(2)-phenyl-[4,5'-bithiazole]-2,2'-diamine inhibitors of gyrase B with a low micromolar inhibitory activity by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques (differential scanning fluorimetry, surface plasmon resonance, and microscale thermophoresis). The binding mode of the potent inhibitor 18 was revealed by X-ray crystallography, confirming our initial in silico binding model. Furthermore, the high resolution of the complex structure allowed for the placement of the Gly97-Ser108 flexible loop, thus revealing its role in binding of this class of compounds. The crystal structure of the complex protein G24 and inhibitor 18 provides valuable information for further optimization of this novel class of DNA gyrase B inhibitors.

Evolution of antibiotic resistance, catabolic pathways and niche colonization

Evolution of antibiotic resistance, catabolic pathways and niche colonization

In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).

Development of a copper(II)-catalyzed three-component tandem synthesis of isoindolinone derivatives

Isoindolinone derivatives are important pharmaceutical building blocks in medicinal chemistry. Isoindolo[2,1-a]quinolines are a class of interesting compounds that possess protective effects against N2-induced hypoxia and inhibitory activities against human topoisomerase II and bacterial DNA-gyrase. The conventional methods for synthesizing these compounds are unsatisfactory because of their harsh reaction conditions, complex starting materials, and multistaged purification procedures. This synthesis of the propargyl-isoindolinone core features an aldehyde–alkyne–amine (A3) coupling-based tandem strategy using methyl 2-formylbenzoate, primary aryl amine, and terminal alkyne under Cu(OTf)2 catalysis.

Inhibition of DNA Gyrase by Levofloxacin and Related Fluorine-Containing Heterocyclic Compounds

Fluoroquinolones are an important class of modern and efficient antibacterial drugs with a broad spectrum of activity. Levofloxacin (the optically active form of ofloxacin) is one of the most promising fluoroquinolone drugs, and its antibacterial activity is substantially higher than the activity of other drugs of the fluoroquinolone family. Earlier, in the Postovsky Institute of Organic Synthesis, UB RAS, an original method of levofloxacin synthesis was developed, and now the pilot batch of the drug is being prepared. Bacterial DNA gyrase is a specific target of fluoroquinolones; hence, the study of the enzyme-drug interaction is of theoretical and practical importance. Moreover, the parameters of DNA gyrase inhibition may serve as a criterion for drug quality. Here, we present the results of studying the interaction of DNA gyrase with a number of fluoroquinolones and their analogs: intermediates and semi-products of the levofloxacin synthesis, and also samples from the pilot batches of this drug. The importance of two structural elements of the levofloxacin molecule for the efficiency of the inhibition is revealed. The data obtained may be useful for the design of new drugs derived from levofloxacin.

Identifikasi Pseudomonas aeruginosa dan tes sensitivitas siprofloksasin pada abses periodontal Identification of Pseudomonas aeruginosa and sensitivity test of ciprofloxacin on periodontal abcess

Periodontitis is a common type of periodontal disease caused by expansion of the early stages of gingivalinflammation. Expansion of inflammation to the tissue structures supporting the teeth can be modified by thepathogenic ability of plaque or host resistance factors. A total of 200 different bacteria have been identified on theplaque. Resistance to antimicrobials can be natural because the microbes develop mechanisms to defendthemselves. Ciprofloxacin is a synthetic drug of the second generation quinolones derivatives. Mechanism of itsaction is to inhibit the activity of bacterial DNA gyrase, which is bactericidal with a broad spectrum against Grampositiveor negative. This observational study identified P. aeruginosa and sensitivity test was performed tociprofloxacin in periodontal abscesses. Study conducted in 23 patients with periodontal abscess. Of those,Pseudomonas was acquired for 8 samples and 4 of them was resistant to ciprofloxacin.

Toxicity of the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin to photoautotrophic aquatic organisms

The present study investigated the growth inhibition effect of the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin on four photoautotrophic aquatic species: the freshwater microalga Desmodesmus subspicatus, the cyanobacterium Anabaena flos-aquae, the monocotyledonous macrophyte Lemna minor, and the dicotyledonous macrophyte Myriophyllum spicatum. Both antibiotics, which act by inhibiting the bacterial DNA gyrase, demonstrated high toxicity to A. flos-aquae and L. minor and moderate to slight toxicity to D. subspicatus and M. spicatum. The cyanobacterium was the most sensitive species with median effective concentration (EC50) values of 173 and 10.2 µg/L for enrofloxacin and ciprofloxacin, respectively. Lemna minor proved to be similarly sensitive, with EC50 values of 107 and 62.5 µg/L for enrofloxacin and ciprofloxacin, respectively. While enrofloxacin was more toxic to green algae, ciprofloxacin was more toxic to cyanobacteria. Calculated EC50s for D. subspicatus were 5,568 µg/L and >8,042 µg/L for enrofloxacin and ciprofloxacin, respectively. These data, as well as effect data from the literature, were compared with predicted and reported environmental concentrations. For two of the four species, a risk was identified at ciprofloxacin concentrations found in surface waters, sewage treatment plant influents and effluents, as well as in hospital effluents. For ciprofloxacin the results of the present study indicate a risk even at the predicted environmental concentration. In contrast, for enrofloxacin no risk was identified at predicted and measured concentrations.

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme.

A fluoroquinolone-resistant Acinetobacter baumannii without the quinolone resistance-determining region mutations

Sir, Acinetobacter baumannii is an emerging Gram-negative multidrug-resistant (MDR) nosocomial pathogen responsible for causing lung, bloodstream, urinary tract and skin infections. Additionally, A. baumannii has emerged as a significant threat in injured US military personnel returning for medical treatment from Afghanistan and Iraq. Because an increasing number of A. baumannii isolates are resistant to the most commonly clinically utilized antibiotics, it is imperative to understand the development of antibiotic resistance in A. baumannii at the molecular level. Fluoroquinolones are broad-spectrum bactericidal agents used to treat diverse bacterial infections. Fluoroquinolones are emerging as viable alternatives for treating A. baumannii infections, but the clinical incidence of fluoroquinolone resistance continues to increase. The most extensively described mechanism of resistance to fluoroquinolones is mediated by mutations in the quinolone resistance-determining regions (QRDRs) of gyrA that encodes the DNA gyrase A subunit and parC that encodes the topoisomerase IV subunit. Additionally, resistance can also be mediated by efflux pumps such as AdeABC and AdeM. Recently, plasmid-mediated quinolone resistance (PMQR) genes have been reported in Gram-negative bacteria, including Escherichia coli and Pseudomonas spp., and include the qnr, qep and oqx systems. The qnr system encodes a pentapeptide repeat protein that blocks the action of ciprofloxacin on bacterial DNA gyrase and topoisomerase IV and was first described in Klebsiella pneumoniae. In contrast, the qep and oqx systems encode plasmid-mediated efflux pumps. However, the PMQR genes have not been demonstrated in A. baumannii. The prevalent theory is that the fluoroquinolone resistance is chiefly mediated by chromosomal mutation, whereas the acquisition of the PMQR genes results in enhancement of the resistance because the PMQR genes by themselves result in a low level of fluoroquinolone resistance. To address the progression of antibiotic resistance in A. baumannii ATCC 17978, we attempted to isolate A. baumannii ATCC 17978 ciprofloxacin-resistant mutants by plating 10– 10 cfu on Mueller–Hinton agar plates containing 2×–16× the MIC of ciprofloxacin. Among the mutants analysed, we isolated a mutant named AB-7, which exhibited a ciprofloxacin MIC of 16 mg/L (Table 1). The CLSI breakpoints for ciprofloxacin are ≤1 mg/L, susceptible; and ≥4 mg/L, resistant. Additionally, the AB-7 strain also exhibited resistance to other fluoroquinolones tested, thus demonstrating class-wide resistance; however, AB-7 was not resistant to the unrelated antibiotic polymyxin B (Table 1). Surprisingly, however, DNA sequencing of the QRDRs did not exhibit any mutations in gyrA, gyrB, parC and parE when compared with the parental strain (Table 1). A. baumannii ATCC 17978 has been whole-genome sequenced and the data have been deposited at the National Center for Biotechnology Information (NCBI; GenBank accession number CP000521.1). The DNA sequence was then analysed for existence of the PMQR genes, namely qnr, qep and oqx. None of the PMQR genes was identified, as has been reported by multiple investigators. – 9 It has been speculated that a number of clinical bacterial isolates that express phenotypic resistance do not exhibit co-relatable genotypic mutations. This phenomenon has recently been reported in the HUE1 E. coli clinical isolate from Japan. In comparison with the HUE1 E. coli isolate, no mutation was detected in the QRDRs as well as no PMQR genes in AB-7. However, other mechanisms may be involved in fluoroquinolone resistance and they are being investigated further. This finding demonstrates a clear exception to the well-accepted mechanism of resistance for fluoroquinolones. The results also underscore the importance of undertaking systems-level studies to re-learn mechanisms of action and evolution of resistance for conventional antibiotics. In conclusion, we present the first report of fluoroquinolone resistance in A. baumannii that is independent of mutations in QRDRs and in the absence of PMQR.

Potentiation of Helicobacter pylori CagA Protein Virulence through Homodimerization

Chronic infection with Helicobacter pylori cagA-positive strains is associated with atrophic gastritis, peptic ulceration, and gastric carcinoma. The cagA gene product, CagA, is delivered into gastric epithelial cells via type IV secretion, where it undergoes tyrosine phosphorylation at the EPIYA motifs. Tyrosine-phosphorylated CagA binds and aberrantly activates the oncogenic tyrosine phosphatase SHP2, which mediates induction of elongated cell morphology (hummingbird phenotype) that reflects CagA virulence. CagA also binds and inhibits the polarity-regulating kinase partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) via the CagA multimerization (CM) sequence independently of tyrosine phosphorylation. Because PAR1 exists as a homodimer, two CagA proteins appear to be passively dimerized through complex formation with a PAR1 dimer in cells. Interestingly, a CagA mutant that lacks the CM sequence displays a reduced SHP2 binding activity and exhibits an attenuated ability to induce the hummingbird phenotype, indicating that the CagA-PAR1 interaction also influences the morphological transformation. Here we investigated the role of CagA dimerization in induction of the hummingbird phenotype with the use of a chemical dimerizer, coumermycin. We found that CagA dimerization markedly stabilizes the CagA-SHP2 complex and thereby potentiates SHP2 deregulation, causing an increase in the number of hummingbird cells. Protrusions of hummingbird cells induced by chemical dimerization of CagA are further elongated by simultaneous inhibition of PAR1. This study revealed a role of the CM sequence in amplifying the magnitude of SHP2 deregulation by CagA, which, in conjunction with the CM sequence-mediated inhibition of PAR1, evokes morphological transformation that reflects in vivo CagA virulence.

Cethromycin-Mediated Protection against the Plague Pathogen Yersinia pestis in a Rat Model of Infection and Comparison with Levofloxacin

The Gram-negative plague bacterium, Yersinia pestis, has historically been regarded as one of the deadliest pathogens known to mankind, having caused three major pandemics. After being transmitted by the bite of an infected flea arthropod vector, Y. pestis can cause three forms of human plague: bubonic, septicemic, and pneumonic, with the latter two having very high mortality rates. With increased threats of bioterrorism, it is likely that a multidrug-resistant Y. pestis strain would be employed, and, as such, conventional antibiotics typically used to treat Y. pestis (e.g., streptomycin, tetracycline, and gentamicin) would be ineffective. In this study, cethromycin (a ketolide antibiotic which inhibits bacterial protein synthesis and is currently in clinical trials for respiratory tract infections) was evaluated for antiplague activity in a rat model of pneumonic infection and compared with levofloxacin, which operates via inhibition of bacterial topoisomerase and DNA gyrase. Following a respiratory challenge of 24 to 30 times the 50% lethal dose of the highly virulent Y. pestis CO92 strain, 70 mg of cethromycin per kg of body weight (orally administered twice daily 24 h postinfection for a period of 7 days) provided complete protection to animals against mortality without any toxic effects. Further, no detectable plague bacilli were cultured from infected animals' blood and spleens following cethromycin treatment. The antibiotic was most effective when administered to rats 24 h postinfection, as the animals succumbed to infection if treatment was further delayed. All cethromycin-treated survivors tolerated 2 subsequent exposures to even higher lethal Y. pestis doses without further antibiotic treatment, which was related, in part, to the development of specific antibodies to the capsular and low-calcium-response V antigens of Y. pestis. These data demonstrate that cethromycin is a potent antiplague drug that can be used to treat pneumonic plague.

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.

Quercetin Diacylglycoside Analogues Showing Dual Inhibition of DNA Gyrase and Topoisomerase IV as Novel Antibacterial Agents

A structure-guided molecular design approach was used to optimize quercetin diacylglycoside analogues that inhibit bacterial DNA gyrase and topoisomerase IV and show potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. In this paper, such novel 3,7-diacylquercetin, quercetin 6''-acylgalactoside, and quercetin 2'',6''-diacylgalactoside analogues of lead compound 1 were prepared to assess their target specificities and preferences in bacteria. The significant enzymatic inhibition of both Escherichia coli DNA gyrase and Staphylococcus aureus topoIV suggest that these compounds are dual inhibitors. Most of the investigated compounds exhibited pronounced inhibition with MIC values ranging from 0.13 to 128 μg/mL toward the growth of multidrug-resistant Gram-positive methicillin-resistant S. aureus, methicillin sensitive S. aureus, vancomycin-resistant enterococci (VRE), vancomycin intermediate S. aureus, and Streptococcus pneumoniae bacterial strains. Structure-activity relationship studies revealed that the acyl moiety was absolutely essential for activity against Gram-positive organisms. The most active compound 5i was 512-fold more potent than vancomycin and 16-32-fold more potent than 1 against VRE strains. It also has realistic in situ intestinal absorption in rats and showed very low acute toxicity in mice. So far, this compound can be regarded as a leading antibacterial agent.

Synthesis of 4-des-hydroxyl clorobiocin analogues as possible bacterial DNA gyrase B and human Hsp90 inhibitors

Synthesis of 4-des-hydroxyl clorobiocin analogues as possible bacterial DNA gyrase B and human Hsp90 inhibitors

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.

REVIVING AN OLD ANTIBIOTIC TARGET

A NEW ANGLE on an old molecular target could point the way to a fresh class of antibacterial agents for tackling drug-resistant bacteria, a study suggests ( Nature , DOI: 10.1038/nature09197). The study also sheds new light on how the target, a bacterial enzyme, works. Despite the looming threat of drug resistance, only two new classes of antibiotics have emerged in the past 40 years. To come up with other drug classes, researchers at GlaxoSmithKline have searched for new drug targets. But recent success came from reexamining an established one—bacterial DNA gyrase, also known as type IIA topoisomerase. This enzyme is critical in bacteria, where it cuts and reseals DNA to regulate a variety of important tasks. Quinolone antibiotics, which target the gyrase, have been available since 1962, but drug resistance to quinolones is on the rise. Now the GSK team, led by Michael N. Gwynn of the company’s infectious diseases research group, has found an ...

ChemInform Abstract: Microbial Resistance: Novel Screens for a Contemporary Problem

Historically, natural products have been the source of a large variety of antibacterial agents. In the 1980s, no additional useful antibacterial agents were discovered, leading to the belief that most useful chemotypes from natural product sources had already been discovered. At this time, advances in biotechnology made it feasible to produce sufficient enzyme to set up cell-free screens. Chemical compound libraries and combinatorial synthesis became the source of chemical diversity for the screens. In spite of these efforts, very few new antibacterial agents have been discovered in the last decade. At Small Molecule Therapeutics, Inc., we have developed phenotype-based screens that take advantage of the natural physiology and biochemistry of the target enzymes. We have developed a screen to identify bacterial DNA gyrase and topoisomerase IV poisons. The “hits” identified in this screen are being characterized further. A second screen has also been developed against bacterial topoisomerase 1 in which compounds that cause DNA damage through their interaction with bacterial topoisomerase 1 have been identified. Three of the compounds identified in the screen inhibit DNA relaxation mediated by bacterial topoisomerase 1, induce DNA cleavage, are noncytotoxic at >10μM, and have MICs of 4.0 μg/mL against Staphylococcus aureus. © 1999 John Wiley & Sons, Inc. Med Res Rev, 19, No. 6, 559–568, 1999

Fluoroquinolone Resistance in Campylobacter

Campylobacter is a commensal in poultry, and therefore, poultry and poultry products are major sources of Campylobacter infections in humans. Fluoroquinolones inhibit the growth of Campylobacter and other microorganisms by binding to bacterial DNA gyrase and DNA topoisomerase IV. These enzymes are associated with bacterial transcription, replication, and chromosome condensation and segregation. Selection pressure in the presence of fluoroquinolones rapidly leads to resistance in Campylobacter, due to the selection for mutations in DNA gyrase. Fluoroquinolone-resistant campylobacters have been found in poultry feces and carcasses, and in retail poultry meat products in most areas of the world. In addition, other food animals and the meat products from those animals have been shown contaminated with fluoroquinolone-resistant campylobacters. Even the removal of fluoroquinolones from use in treating animal diseases has not entirely eliminated the presence of resistant Campylobacter jejuni and Campylobacter coli from animals and animal products. Human exposure to Campylobacter infection could be reduced by using strategies that decrease colonization of chickens by the pathogen.

Discovery of pyrazolthiazoles as novel and potent inhibitors of bacterial gyrase

Bacterial DNA gyrase is an attractive target for the investigation of new antibacterial agents. Inhibitors of the GyrB subunit, which contains the ATP-binding site, are described in this communication. Novel, substituted 5-(1 H-pyrazol-3-yl)thiazole compounds were identified as inhibitors of bacterial gyrase. Structure-guided optimization led to greater enzymatic potency and moderate antibacterial potency. Data are presented for the demonstration of selective enzyme inhibition of Escherichia coli GyrB over Staphlococcus aureus GyrB.