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Methods in Molecular Biology (Clifton,... 2018The quinolones are potent antibacterials that act by forming complexes with DNA and either gyrase or topoisomerase IV. These ternary complexes, called cleaved complexes...
The quinolones are potent antibacterials that act by forming complexes with DNA and either gyrase or topoisomerase IV. These ternary complexes, called cleaved complexes because the DNA moiety is broken, block replication, transcription, and bacterial growth. Cleaved complexes readily form in vitro when gyrase, plasmid DNA, and quinolone are combined and incubated; complexes are detected by the linearization of plasmid DNA, generally assayed by gel electrophoresis. The stability of the complexes can be assessed by treatment with EDTA, high temperature, or dilution to dissociate the complexes and reseal the DNA moiety. Properties of the complexes are sensitive to quinolone structure and to topoisomerase amino acid substitutions associated with quinolone resistance. Consequently, studies of cleaved complexes can be used to identify improvements in quinolone structure and to understand the biochemical basis of target-based resistance. Cleaved complexes can also be detected in quinolone-treated bacterial cells by their ability to rapidly block DNA replication and to cause chromosome fragmentation; they can even be recovered from lysed cells following CsCl density-gradient centrifugation. Thus, in vivo and cell-fractionation tests are available for assessing the biological relevance of work with purified components.
Topics: DNA; DNA Gyrase; DNA Replication; Edetic Acid; Escherichia coli; Fluoroquinolones; Hot Temperature; Plasmids; Structure-Activity Relationship
PubMed: 29177748
DOI: 10.1007/978-1-4939-7459-7_19 -
EcoSal Plus 2015DNA topoisomerases are enzymes that control the topology of DNA in all cells. There are two types, I and II, classified according to whether they make transient single-... (Review)
Review
DNA topoisomerases are enzymes that control the topology of DNA in all cells. There are two types, I and II, classified according to whether they make transient single- or double-stranded breaks in DNA. Their reactions generally involve the passage of a single- or double-strand segment of DNA through this transient break, stabilized by DNA-protein covalent bonds. All topoisomerases can relax DNA, but DNA gyrase, present in all bacteria, can also introduce supercoils into DNA. Because of their essentiality in all cells and the fact that their reactions proceed via DNA breaks, topoisomerases have become important drug targets; the bacterial enzymes are key targets for antibacterial agents. This article discusses the structure and mechanism of topoisomerases and their roles in the bacterial cell. Targeting of the bacterial topoisomerases by inhibitors, including antibiotics in clinical use, is also discussed.
Topics: Anti-Bacterial Agents; Bacteria; Bacteriocins; DNA Gyrase; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA, Bacterial; DNA, Superhelical; Models, Molecular; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors
PubMed: 26435256
DOI: 10.1128/ecosalplus.ESP-0010-2014 -
Microbiology Spectrum Oct 2014Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for... (Review)
Review
Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6')-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.
Topics: Acetylation; Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Biological Transport; DNA Gyrase; DNA Topoisomerase IV; Drug Resistance, Bacterial; Inactivation, Metabolic; Plasmids; Quinolones
PubMed: 25584197
DOI: 10.1128/microbiolspec.PLAS-0006-2013 -
The Journal of Antimicrobial... Aug 2014Loop B is important for low-level quinolone resistance conferred by Qnr proteins. The role of individual amino acids within QnrS1 loop B in quinolone resistance and...
OBJECTIVES
Loop B is important for low-level quinolone resistance conferred by Qnr proteins. The role of individual amino acids within QnrS1 loop B in quinolone resistance and gyrase protection was assessed.
METHODS
qnrS1 and 11 qnrS1 alleles with site-directed Ala mutations in loop B were expressed in Escherichia coli BL21(DE3) and proteins were purified by affinity chromatography. Ciprofloxacin MICs were determined with and without IPTG. Gyrase DNA supercoiling was measured with and without ciprofloxacin IC50 and with various concentrations of QnrS1 proteins.
RESULTS
Wild-type QnrS1 and QnrS1 with Asn-110→Ala and Arg-111→Ala substitutions increased the ciprofloxacin MIC 12-fold in BL21(DE3), although QnrS1 with Gln-107→Ala replacement increased it 2-fold more than wild-type did. However, QnrS1 with Ala substitutions at His-106, Val-108, Ser-109, Met-112, Tyr-113, Phe-114, Cys-115 and Ser-116 increased ciprofloxacin MIC 1.4- to 8-fold less than wild-type QnrS1. Induction by 10-1000 μM IPTG increased ciprofloxacin MICs for all mutants, reaching values similar to those for wild-type. Purified wild-type and mutated proteins differed in protection of gyrase from ciprofloxacin action. Wild-type QnrS1 produced complete protection of gyrase supercoiling from ciprofloxacin (1.8 μM) action at 0.05 nM and half protection at 0.5 pM, whereas QnrS1 with Ala replacements that conferred the least increase in ciprofloxacin MICs also required the highest QnrS1 concentrations for protection.
CONCLUSIONS
Key individual residues in QnrS1 loop B affect ciprofloxacin resistance and gyrase protection from ciprofloxacin action, supporting the concept that loop B is key for interaction with gyrase necessary for quinolone resistance.
Topics: Amino Acid Substitution; Anti-Bacterial Agents; Ciprofloxacin; DNA Gyrase; DNA, Superhelical; Drug Resistance, Bacterial; Escherichia coli; Escherichia coli Proteins; Isopropyl Thiogalactoside; Microbial Sensitivity Tests; Mutation; Protein Structure, Tertiary; Structure-Activity Relationship
PubMed: 24729602
DOI: 10.1093/jac/dku102 -
ELife Jun 2024DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active...
DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (-1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface 'swapping' (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed 'swivelling' mechanism for DNA gyrase (Gubaev et al., 2016).
Topics: DNA Gyrase; Protein Multimerization; DNA, Bacterial; Escherichia coli; DNA
PubMed: 38856655
DOI: 10.7554/eLife.86722 -
Biomedicine & Pharmacotherapy =... Jul 2018DNA gyrase is classified as topoisomerase II, an ATP-dependent enzyme that is vital in the transcription, replication of DNA and chromosome segregation processes. It... (Review)
Review
DNA gyrase is classified as topoisomerase II, an ATP-dependent enzyme that is vital in the transcription, replication of DNA and chromosome segregation processes. It plays a crucial role in all bacteria except higher eukaryotes and this makes it a desirable and viable therapeutic target for development of new antibacterial agents. Fluoroquinolones are commonly used effective antibacterial agents that target DNA gyrase, however the spectrum of side-effects and emerging bacterial resistance with no new drugs in the antibacterial pipeline has fuelled intensive research in this area. New chemical entities with varied scaffolds possessing DNA gyrase inhibiting properties have been determined by screening chemical libraries that could serve as good leads for antibacterial drug development. A wide range of natural products and protein-based compounds have been identified and studied as DNA gyrase inhibitors and this adds a huge amount of structural diversity that can be exploited and harnessed in the discovery of new antibacterial agents. The development of new chemical compounds with DNA gyrase inhibitory activity (from natural sources, random screens or rational design) will further validate/corroborate the potential of this enzyme as a useful target. This review presents an overview of the DNA gyrase inhibitors obtained from natural and synthetic sources, their syntheses schemes and spectrum of biological activity of a variety of scaffolds and their analogues. The authors hope to provide focused direction for development of new chemical entities, synthetic routes for analogue synthesis, structure activity relationships and biological activity. The most potent ones can be used as templates to design novel compounds targeting DNA gyrase and are effective against resistant bacterial strains and biofilms.
Topics: Anti-Bacterial Agents; DNA Gyrase; Gram-Negative Bacteria; Gram-Positive Bacteria; Microbial Sensitivity Tests; Models, Molecular; Molecular Structure; Topoisomerase II Inhibitors
PubMed: 29710509
DOI: 10.1016/j.biopha.2018.04.021 -
Expert Opinion on Therapeutic Patents Mar 2019The bacterial topoisomerases DNA gyrase and topoisomerase IV are validated targets for development of novel antibacterial agents. Fluoroquinolones inhibit the catalytic... (Review)
Review
INTRODUCTION
The bacterial topoisomerases DNA gyrase and topoisomerase IV are validated targets for development of novel antibacterial agents. Fluoroquinolones inhibit the catalytic GyrA and/or ParC(GrlA) subunit and have been commonly used, although these have toxicity liabilities that restrict their use. The ATPase GyrB and ParE(GrlB) subunits have been much less explored and after withdrawal of novobiocin, there are no further marketed inhibitors . ATP-competitive inhibitors of GyrB and/or ParE(GrlB) are of special interest, as this target has been validated, and it is expected that many of the problems associated with fluoroquinolones can be avoided.
AREAS COVERED
This review summarises the development of ATP-competitive inhibitors of GyrB and/or ParE(GrlB) as novel antibacterial agents over the last 10 years. Structural features of the new inhibitors and their optimisation strategies are highlighted.
EXPERT OPINION
The development of novel ATP-competitive inhibitors of GyrB and/or ParE(GrlB) is ongoing in industrial and academical research. Development of resistance is one of the most problematic issues, but GyrB/ParE(GrlB) inhibitors do not show cross-resistance with fluoroquinolones. Other common issues, such as low solubility, high protein binding, development of off-target resistance, are related to the structures of the inhibitors themselves, which is thus a main focus of design strategies. With some now in early clinical development, there is reasonable expectation that novel ATP-competitive inhibitors of GyrB/ParE(GrlB) will reach the market in the near future.
Topics: Adenosine Triphosphate; Animals; Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; Drug Design; Fluoroquinolones; Humans; Patents as Topic; Topoisomerase II Inhibitors
PubMed: 30686070
DOI: 10.1080/13543776.2019.1575362 -
The Journal of Biological Chemistry Dec 2023Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic...
Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic reactions. Studying these reactions in vitro, using agents such as polyetthylene glycols (PEGs) and polyvinyl alcohols (PVAs) to mimic intracellular crowding conditions, is essential due to the notable differences from enzyme behaviors observed in diluted aqueous solutions. In this article, we studied Mycobacterium tuberculosis (Mtb) DNA gyrase under macromolecular crowding conditions by incorporating PEGs and PVAs into the DNA supercoiling reactions. We discovered that high concentrations of potassium glutamate, glycine betaine, PEGs, and PVA substantially stimulated the DNA supercoiling activity of Mtb DNA gyrase. Steady-state kinetic studies showed that glycine betaine and PEG400 significantly reduced the K of Mtb DNA gyrase and simultaneously increased the V or k of Mtb DNA gyrase for ATP and the plasmid DNA molecule. Molecular dynamics simulation studies demonstrated that PEG molecules kept the ATP lid of DNA gyrase subunit B in a closed or semiclosed conformation, which prevented ATP molecules from leaving the ATP-binding pocket of DNA gyrase subunit B. The stimulation of the DNA supercoiling activity of Mtb DNA gyrase by these molecular crowding agents likely results from a decrease in water activity and an increase in excluded volume.
Topics: DNA Gyrase; Mycobacterium tuberculosis; Betaine; Kinetics; Adenosine Triphosphate; DNA; DNA, Superhelical
PubMed: 37944619
DOI: 10.1016/j.jbc.2023.105439 -
Infectious Disorders Drug Targets 2020The newly emerging infectious organisms, the global crisis in antibiotic resistance, and the threat of bioterrorism create an urgent need to discover novel antimicrobial... (Review)
Review
The newly emerging infectious organisms, the global crisis in antibiotic resistance, and the threat of bioterrorism create an urgent need to discover novel antimicrobial agents. In order to develop novel antimicrobial agents, the mechanism of infectious disease must be better understood. DNA Gyrase is a bacterial enzyme that plays an important role in the replication of DNA and transcription process. It is not present in higher eukaryotes making it a perfect target for developing new antibacterial agents. This review describes the role of DNA gyrase inhibitors in preventing various diseases. In this review, we outline the synthesis and pharmacological action of various novel DNA gyrase inhibitors. DNA gyrase inhibitors were used to treat tuberculosis, bacterial, fungal infections and malaria. DNA gyrase inhibitors mainly act by preventing the supercoiling of DNA strands..
Topics: Anti-Bacterial Agents; Bacteria; DNA Gyrase; Drug Resistance, Microbial; Humans; Topoisomerase II Inhibitors
PubMed: 33109068
DOI: 10.2174/1871526520666200102110235 -
Journal of Medicinal Chemistry May 2022New antibiotics with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The...
New antibiotics with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The present study profiles new spiropyrimidinetriones (SPTs), DNA gyrase inhibitors having activity against drug-resistant (), the causative agent of TB. While the clinical candidate zoliflodacin has progressed to phase 3 trials for the treatment of gonorrhea, compounds herein demonstrated higher inhibitory potency against DNA gyrase (e.g., compound with IC = 2.0) and lower minimum inhibitor concentrations (0.49 μM for ). Notably, and analogues showed selective activity relative to representative Gram-positive and Gram-negative bacteria. DNA gyrase inhibition was shown to involve stabilization of double-cleaved DNA, while on-target activity was supported by hypersensitivity against a gyrA hypomorph. Finally, a docking model for SPTs with DNA gyrase was developed, and a structural hypothesis was built for structure-activity relationship expansion.
Topics: Anti-Bacterial Agents; Antitubercular Agents; DNA Gyrase; Gram-Negative Bacteria; Gram-Positive Bacteria; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Topoisomerase II Inhibitors
PubMed: 35500229
DOI: 10.1021/acs.jmedchem.2c00266