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Cellular and Molecular Life Sciences :... Jun 2010Topoisomerases are vital enzymes specialized in controlling DNA topology, in particular supercoiling and decatenation, to properly handle nucleic acid packing and cell... (Review)
Review
Topoisomerases are vital enzymes specialized in controlling DNA topology, in particular supercoiling and decatenation, to properly handle nucleic acid packing and cell dynamics. The type IIA enzymes act by cleaving both strands of a double helix and having another strand from the same or another molecule cross the DNA gate before a re-sealing event completes the catalytic cycle. Here, we will consider the two types of IIA prokaryotic topoisomerases, DNA Gyrase and Topoisomerase IV, as crucial regulators of bacterial cell cycle progression. Their synergistic action allows control of chromosome packing and grants occurrence of functional transcription and replication processes. In addition to displaying a fascinating molecular mechanism of action, which transduces chemical energy into mechanical energy by means of large conformational changes, these enzymes represent attractive pharmacological targets for antibacterial chemotherapy.
Topics: Bacteria; Biophysical Phenomena; Catalysis; Chromosomes; DNA; DNA Gyrase; DNA Replication; DNA Topoisomerase IV; Isomerases
PubMed: 20165898
DOI: 10.1007/s00018-010-0299-5 -
Nature Communications Apr 2015Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM)...
Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro. Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections.
Topics: Animals; Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerases; Drug Resistance, Bacterial; Escherichia coli; Female; Fluoroquinolones; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mutagenesis, Site-Directed; Quinolones; Specific Pathogen-Free Organisms; Staphylococcal Infections; Staphylococcus aureus
PubMed: 25907309
DOI: 10.1038/ncomms7947 -
Microbial Drug Resistance (Larchmont,... Oct 2021WQ-3810 has strong inhibitory activity against and other fluoroquinolone-resistant pathogens. The unique potentiality of this is attributed to...
Effectiveness of Fluoroquinolones with Difluoropyridine Derivatives as R1 Groups on the DNA Gyrase in the Presence and Absence of Plasmid-Encoded Quinolone Resistance Protein QnrB19.
WQ-3810 has strong inhibitory activity against and other fluoroquinolone-resistant pathogens. The unique potentiality of this is attributed to 6-amino-3,5-difluoropyridine-2-yl at R1 group. The aim of this study was to examine WQ-3810 and its derivatives WQ-3334 and WQ-4065 as the new drug candidate for wild-type and that carrying QnrB19. The half maximal inhibitory concentrations (ICs) of WQ-3810, WQ-3334 (Br atom in place of methyl group at R8), and WQ-4065 (6-ethylamino-3,5-difluoropyridine-2-yl in place of 6-amino-3,5-difluoropyridine-2-yl group at R1) in the presence or absence of QnrB19 were assessed by DNA supercoiling assay utilizing recombinant DNA gyrase and QnrB19. ICs of WQ-3810, WQ-3334, and WQ-4065 against DNA gyrase were 0.031 ± 0.003, 0.068 ± 0.016, and 0.72 ± 0.39 μg/mL, respectively, while QnrB19 increased ICs of WQ-3810, WQ-3334, and WQ-4065 to 0.44 ± 0.05, 0.92 ± 0.34, and 9.16 ± 2.21 μg/mL, respectively. WQ-3810 and WQ-3334 showed stronger inhibitory activity against Typhimurium DNA gyrases than WQ-4065 even in the presence of QnrB19. The results suggest that 6-amino-3,5-difluoropyridine-2-yl group at R1 is playing an important role and WQ-3810 and WQ-3334 to be good candidates for carrying QnrB19.
Topics: Anti-Bacterial Agents; DNA Gyrase; Drug Resistance, Bacterial; Fluoroquinolones; Genes, Bacterial; Microbial Sensitivity Tests; Plasmids; Quinolones; Salmonella
PubMed: 33835868
DOI: 10.1089/mdr.2020.0455 -
Molecules (Basel, Switzerland) Feb 2021Gyrase is a bacterial type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme is essential in bacteria and is a validated drug target in the... (Review)
Review
Gyrase is a bacterial type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme is essential in bacteria and is a validated drug target in the treatment of bacterial infections. Inhibition of gyrase activity is achieved by competitive inhibitors that interfere with ATP- or DNA-binding, or by gyrase poisons that stabilize cleavage complexes of gyrase covalently bound to the DNA, leading to double-strand breaks and cell death. Many of the current inhibitors suffer from severe side effects, while others rapidly lose their antibiotic activity due to resistance mutations, generating an unmet medical need for novel, improved gyrase inhibitors. DNA supercoiling by gyrase is associated with a series of nucleotide- and DNA-induced conformational changes, yet the full potential of interfering with these conformational changes as a strategy to identify novel, improved gyrase inhibitors has not been explored so far. This review highlights recent insights into the mechanism of DNA supercoiling by gyrase and illustrates the implications for the identification and development of conformation-sensitive and allosteric inhibitors.
Topics: Bacteria; DNA Gyrase; Models, Molecular; Topoisomerase II Inhibitors
PubMed: 33669078
DOI: 10.3390/molecules26051234 -
Antimicrobial Agents and Chemotherapy Oct 2021SPR720 (phosphate prodrug of SPR719) is a novel aminobenzimidazole bacterial DNA gyrase (GyrB) inhibitor in development for nontuberculous mycobacterial pulmonary... (Randomized Controlled Trial)
Randomized Controlled Trial
SPR720 (phosphate prodrug of SPR719) is a novel aminobenzimidazole bacterial DNA gyrase (GyrB) inhibitor in development for nontuberculous mycobacterial pulmonary disease (NTM-PD) and pulmonary tuberculosis. SPR719 has demonstrated activity against clinically relevant mycobacteria and in murine and hollow-fiber infection models. This phase 1 randomized, double-blind, placebo-controlled, single ascending dose (SAD)/multiple ascending dose (MAD) trial evaluated the safety, tolerability, and pharmacokinetics of SPR720/SPR719. A total of 96 healthy volunteers ( = 8/cohort, 3:1 randomization) received SPR720 (or placebo) as single oral doses ranging from 100 to 2,000 mg or repeat total daily doses ranging from 500 to 1,500 mg for 7 or 14 days. SPR720 was well tolerated at daily doses of up to 1,000 mg for up to 14 days. Across SAD/MAD cohorts, the most common adverse events (AEs) were gastrointestinal (nausea, vomiting, and diarrhea) and headache, all of mild or moderate severity and dose dependent. No serious AEs were reported. The median SPR719 ranged from 2.8 to 8.0 h across cohorts, and the ranged from 2.9 to 4.5 h and was shown to be dose independent. Dosing with food decreased SPR719 plasma exposure by approximately 20%. In the MAD cohorts, SPR719 plasma exposure declined approximately 40% between days 1 and 7, suggesting induction of an elimination pathway. However, plasma AUC was comparable between days 7 and 14. The results of this first-in-human study suggest that predicted therapeutic exposures of SPR719 can be attained with a once-daily oral administration of SPR720. (This study has been registered at ClinicalTrials.gov under registration no. NCT03796910.).
Topics: Administration, Oral; Animals; Area Under Curve; DNA Gyrase; DNA, Bacterial; Dose-Response Relationship, Drug; Double-Blind Method; Humans; Mice; Mycobacterium; Mycobacterium Infections, Nontuberculous; Topoisomerase II Inhibitors
PubMed: 34491803
DOI: 10.1128/AAC.01208-21 -
Antimicrobial Agents and Chemotherapy Apr 2008The sugarcane pathogen Xanthomonas albilineans produces a family of antibiotics and phytotoxins termed albicidins, which inhibit plant and bacterial DNA gyrase...
The sugarcane pathogen Xanthomonas albilineans produces a family of antibiotics and phytotoxins termed albicidins, which inhibit plant and bacterial DNA gyrase supercoiling activity, with a 50% inhibitory concentration (50 nM) comparable to those of coumarins and quinolones. Here we show that X. albilineans has an unusual, antibiotic-resistant DNA gyrase. The X. albilineans gyrA and gyrB genes are not clustered with previously described albicidin biosynthesis and self-protection genes. The GyrA and GyrB products differ from Escherichia coli homologues through several insertions and through changes in several amino acid residues implicated in quinolone and coumarin resistance. Reconstituted X. albilineans DNA gyrase showed 20- to 25-fold-higher resistance than E. coli DNA gyrase to albicidin and ciprofloxacin and 8-fold-higher resistance to novobiocin in the supercoiling assay. The X. albilineans DNA gyrase is unusual in showing a high degree of distributive supercoiling and little DNA relaxation activity. X. albilineans GyrA (XaA) forms a functional gyrase heterotetramer with E. coli GyrB (EcB) and can account for albicidin and quinolone resistance and low levels of relaxation activity. XaB probably contributes to both coumarin resistance and the distributive supercoiling pattern. Although XaB shows fewer apparent changes relative to EcB, the EcA.XaB hybrid relaxed DNA in the presence or absence of ATP and was unable to supercoil. A fuller understanding of structural differences between albicidin-sensitive and -resistant gyrases may provide new clues into features of the enzyme amenable to interference by novel antibiotics.
Topics: Amino Acid Sequence; Anti-Bacterial Agents; Cloning, Molecular; Computational Biology; DNA Gyrase; DNA, Superhelical; Drug Resistance, Multiple, Bacterial; Microbial Sensitivity Tests; Molecular Sequence Data; Organic Chemicals; Sequence Analysis, DNA; Xanthomonas
PubMed: 18268084
DOI: 10.1128/AAC.01551-07 -
Proceedings of the National Academy of... May 2004DNA gyrase is the bacterial DNA topoisomerase (topo) that supercoils DNA by using the free energy of ATP hydrolysis. The enzyme, an A(2)B(2) tetramer encoded by the gyrA...
DNA gyrase is the bacterial DNA topoisomerase (topo) that supercoils DNA by using the free energy of ATP hydrolysis. The enzyme, an A(2)B(2) tetramer encoded by the gyrA and gyrB genes, catalyses topological changes in DNA during replication and transcription, and is the only topo that is able to introduce negative supercoils. Gyrase is essential in bacteria and apparently absent from eukaryotes and is, consequently, an important target for antibacterial agents (e.g., quinolones and coumarins). We have identified four putative gyrase genes in the model plant Arabidopsis thaliana; one gyrA and three gyrB homologues. DNA gyrase protein A (GyrA) has a dual translational initiation site targeting the mature protein to both chloroplasts and mitochondria, and there are individual targeting sequences for two of the DNA gyrase protein B (GyrB) homologues. N-terminal fusions of the organellar targeting sequences to GFPs support the hypothesis that one enzyme is targeted to the chloroplast and another to the mitochondrion, which correlates with supercoiling activity in isolated organelles. Treatment of seedlings and cultured cells with gyrase-specific drugs leads to growth inhibition. Knockout of A. thaliana gyrA is embryo-lethal whereas knockouts in the gyrB genes lead to seedling-lethal phenotypes or severely stunted growth and development. The A. thaliana genes have been cloned in Escherichia coli and found to complement gyrase temperature-sensitive strains. This report confirms the existence of DNA gyrase in eukaryotes and has important implications for drug targeting, organelle replication, and the evolution of topos in plants.
Topics: Anti-Infective Agents; Arabidopsis; Chloroplasts; Ciprofloxacin; Cytoplasm; DNA; DNA Gyrase; Mitochondria; Phylogeny; Seedlings; Sequence Analysis, DNA
PubMed: 15136745
DOI: 10.1073/pnas.0400836101 -
Nucleic Acids Research 2006We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled...
We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.
Topics: Clinical Enzyme Tests; DNA Gyrase; DNA Topoisomerase IV; DNA, Bacterial; Escherichia coli; Plasmids
PubMed: 16936317
DOI: 10.1093/nar/gkl504 -
Nature Communications Dec 2015New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by...
New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.
Topics: Anti-Bacterial Agents; Antineoplastic Agents; DNA Gyrase; DNA, Bacterial; Drug Resistance, Bacterial; Etoposide; Fluoroquinolones; Models, Molecular; Molecular Structure; Moxifloxacin; Staphylococcus aureus; Topoisomerase II Inhibitors
PubMed: 26640131
DOI: 10.1038/ncomms10048 -
The Journal of Biological Chemistry Aug 2021DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate...
DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.
Topics: Catalytic Domain; DNA Gyrase; DNA Topoisomerases, Type II; DNA, Bacterial; DNA, Superhelical; Models, Molecular; Mutagenesis, Site-Directed; Pneumococcal Infections; Protein Structural Elements; Streptococcus pneumoniae
PubMed: 34303706
DOI: 10.1016/j.jbc.2021.101000