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Methods in Molecular Biology (Clifton,... 2018Most bacterial cells have a motor enzyme termed DNA gyrase, which is a type-2 topoisomerase that reduces the linking number (Lk) of DNA. The supercoiling energy...
Most bacterial cells have a motor enzyme termed DNA gyrase, which is a type-2 topoisomerase that reduces the linking number (Lk) of DNA. The supercoiling energy generated by gyrase is essential to maintain the bacterial chromosome architecture and regulate its DNA transactions. This chapter describes the use of agarose-gel electrophoresis to detect the unconstrained supercoiling of DNA generated by gyrase or other gyrase-like activities. Particular emphasis is made on the preparation of a relaxed plasmid as initial DNA substrate, on the distinction of constrained and unconstrained DNA supercoils, and on the measurement of the DNA supercoiling density achieved by gyrase activity.
Topics: Animals; Cattle; DNA Gyrase; DNA, Superhelical; Electrophoresis, Agar Gel; Humans; Substrate Specificity
PubMed: 29971724
DOI: 10.1007/978-1-4939-8556-2_15 -
Journal of Applied Microbiology Sep 2022This study was conducted to evaluate 35 natural flavonoids for their in vitro susceptibility against E. coli (ATCC 25922), Ps. aeruginosa (ATCC 27853), B. subtilis (ATCC...
AIMS
This study was conducted to evaluate 35 natural flavonoids for their in vitro susceptibility against E. coli (ATCC 25922), Ps. aeruginosa (ATCC 27853), B. subtilis (ATCC 530) and Staph. aureus (ATCC 6538) in search of a potential broad-spectrum antibiotic.
METHODS AND RESULTS
Glabridin, a natural isoflavonoid isolated from Glycyrrhiza glabra L., was identified to be highly active with a MIC of 8-16 μg ml against Staph. aureus, B. subtilis and E. coli. By the results of the docking simulation, we located the potential targets of glabridin as DNA gyrase and dihydrofolate reductase (DHFR). The subsequent DNA gyrase inhibition assays (glabridin: IC = 0.8516 μmol L , ciprofloxacin: IC = 0.04697 μmol L ), DHFR inhibition assays (glabridin: inhibition ratio = 29%, methotrexate: inhibition ratio = 45% under 100 μmol L treatment) and TUNEL confirmed that glabridin acted as DNA gyrase inhibitor and DHFR mild inhibitor, exerting bactericidal activity by blocking bacterial nucleic acid synthesis. CCK-8 and in silico calculations were also conducted to verify the low cytotoxicity and acceptable druggability of glabridin.
CONCLUSION
These findings suggest that glabridin represents the prototypical member of an exciting structural class of natural antimicrobial agents.
SIGNIFICANCE AND IMPACT OF THE STUDY
This study reports a novel mechanism of bactericidal activity of glabridin against Staph. aureus.
Topics: Anti-Bacterial Agents; DNA Gyrase; Escherichia coli; Flavonoids; Glycyrrhiza; Microbial Sensitivity Tests; Staphylococcus aureus
PubMed: 35801665
DOI: 10.1111/jam.15705 -
Future Medicinal Chemistry Feb 2020DNA gyrase and topoisomerase IV are essential bacterial enzymes, and in the fight against bacterial resistance, they are important targets for the development of novel...
DNA gyrase and topoisomerase IV are essential bacterial enzymes, and in the fight against bacterial resistance, they are important targets for the development of novel antibacterial drugs. Building from our first generation of 4,5,6,7-tetrahydrobenzo[]thiazole-based DNA gyrase inhibitors, we designed and prepared an optimized series of analogs that show improved inhibition of DNA gyrase and topoisomerase IV from and , with IC values in the nanomolar range. Importantly, these inhibitors also show improved antibacterial activity against Gram-positive strains. The most promising inhibitor, , is active against , and wild-type and resistant strains, with minimum inhibitory concentrations between 4 and 8 μg/ml, which represents good starting point for development of novel antibacterials.
Topics: Anti-Bacterial Agents; Benzothiazoles; DNA Gyrase; Dose-Response Relationship, Drug; Gram-Positive Bacteria; Humans; Microbial Sensitivity Tests; Models, Molecular; Molecular Structure; Structure-Activity Relationship; Topoisomerase II Inhibitors
PubMed: 32043377
DOI: 10.4155/fmc-2019-0127 -
Biomedicine & Pharmacotherapy =... Feb 2021DNA gyrase and Topoisomerase IV are promising antibacterial drug targets as they regulate bacterial DNA replication and topology. In a quest for novel DNA topoisomerase...
DNA gyrase and Topoisomerase IV are promising antibacterial drug targets as they regulate bacterial DNA replication and topology. In a quest for novel DNA topoisomerase inhibitors, a multidisciplinary approach was adopted that involves computational prediction of binding sites and molecular modelling followed by green synthesis and biological evaluation of antibacterial activity of spirobenzimidazo quinazolines derivatives. Using basic quantum chemistry principles, we evaluated spirobenzimidazo quinazolines derivatives with their pharmacokinetic profiles. Based on the results of the aforesaid in-silico studies, we synthesized a series of titled compounds using green synthetic methodology that were validated as potential antimicrobial agents. Quantum chemoinformatics based predicted activity for the synthesized compounds 9b, 9c, and 9j was concomitant with biological evaluation of broadspectrum antibacterial activity. Biological evaluation revealed that inhibition of biofilm formation was due to their potential antibacterial activity. We believe that the novel spirobenzimidazo quinazolines have the potential to be alternatives to aminocoumarins and classical quinazolines upon detailed target specific biological studies.
Topics: Anti-Bacterial Agents; Bacteria; Benzimidazoles; Binding Sites; Biofilms; Computer-Aided Design; DNA Gyrase; Drug Design; Green Chemistry Technology; Microbial Sensitivity Tests; Molecular Docking Simulation; Molecular Structure; Protein Binding; Quantitative Structure-Activity Relationship; Quinazolines; Topoisomerase II Inhibitors
PubMed: 33360050
DOI: 10.1016/j.biopha.2020.111132 -
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 -
Design, synthesis and biological evaluation of novel Pseudomonas aeruginosa DNA gyrase B inhibitors.Bioorganic Chemistry Jul 2020In the present study, we attempted to develop a novel class of compounds active against Pseudomonas aeruginosa (Pa) by exploring the pharmaceutically well exploited...
In the present study, we attempted to develop a novel class of compounds active against Pseudomonas aeruginosa (Pa) by exploring the pharmaceutically well exploited enzyme targets. Since, lack of Pa gyrase B crystal structures, Thermus thermophilus gyrase B in complex with novobiocin (1KIJ) was used as template to generate model structure by performing homology modeling. Further the best model was validated and used for high-throughput virtual screening, docking and dynamics simulations using the in-house database for identification of Pa DNA gyrase B inhibitors. This study led to an identification of three lead molecules with IC values in range of 6.25-15.6 µM against Pa gyrase supercoiling assay. Lead-1 optimization and expansion resulted in 15 compounds. Among the synthesized compounds six compounds were shown good enzyme inhibition than Lead-1 (IC 6.25 µM). Compound 13 emerged as the most potential compound exhibiting inhibition of Pa gyrase supercoiling with an IC of 2.2 µM; and in-vitro Pa activity with MIC of 8 µg/mL in presence of efflux pump inhibitor; hence could be further developed as novel inhibitor for Pa gyrase B.
Topics: Anti-Bacterial Agents; Bacterial Proteins; DNA Gyrase; Drug Design; Humans; Models, Molecular; Oxazoles; Pseudomonas Infections; Pseudomonas aeruginosa; Thiazoles; Topoisomerase II Inhibitors
PubMed: 32388436
DOI: 10.1016/j.bioorg.2020.103905 -
European Journal of Medicinal Chemistry Dec 2023A new type of benzopyrone-mediated quinolones (BMQs) was rationally designed and efficiently synthesized as novel potential antibacterial molecules to overcome the...
A new type of benzopyrone-mediated quinolones (BMQs) was rationally designed and efficiently synthesized as novel potential antibacterial molecules to overcome the global increasingly serious drug resistance. Some synthesized BMQs effectively suppressed the growth of the tested strains, outperforming clinical drugs. Notably, ethylidene-derived BMQ 17a exhibited superior antibacterial potential with low MICs of 0.5-2 μg/mL to clinical drugs norfloxacin, it not only displayed rapid bactericidal performance and inhibited bacterial biofilm formation, but also showed low toxicity toward human red blood cells and normal MDA-kb2 cells. Mechanistic investigation demonstrated that BMQ 17a could effectually induce bacterial metabolic disorders and promote the enhancement of reactive oxygen species to disrupt the bacterial antioxidant defense system. It was found that the active molecule BMQ 17a could not only form supramolecular complex with lactate dehydrogenase, which disturbed the biological functions, but also effectively embed into calf thymus DNA, thus affecting the normal function of DNA and achieving cell death. This work would provide an insight into developing new molecules to reduce drug resistance and expand antibacterial spectrum.
Topics: Humans; Anti-Bacterial Agents; DNA Gyrase; Microbial Sensitivity Tests; Norfloxacin; Quinolones; Benzopyrans
PubMed: 37866337
DOI: 10.1016/j.ejmech.2023.115878 -
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 -
Proceedings of the National Academy of... May 2017A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens,...
A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.
Topics: Anti-Bacterial Agents; Crystallography, X-Ray; DNA Cleavage; DNA Gyrase; Drug Discovery; Models, Molecular; Thiophenes
PubMed: 28507124
DOI: 10.1073/pnas.1700721114 -
FEMS Microbiology Reviews Jan 2018Simocyclinones are antibiotics produced by Streptomyces and Kitasatospora species that inhibit the validated drug target DNA gyrase in a unique way, and they are thus of... (Review)
Review
Simocyclinones are antibiotics produced by Streptomyces and Kitasatospora species that inhibit the validated drug target DNA gyrase in a unique way, and they are thus of therapeutic interest. Structural approaches have revealed their mode of action, the inducible-efflux mechanism in the producing organism, and given insight into one step in their biosynthesis. The crystal structures of simocyclinones bound to their target (gyrase), the transcriptional repressor SimR and the biosynthetic enzyme SimC7 reveal fascinating insight into how molecular recognition is achieved with these three unrelated proteins.
Topics: Anti-Bacterial Agents; DNA Gyrase; Enzyme Activation; Glycosides; Ligands
PubMed: 29126195
DOI: 10.1093/femsre/fux055