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Antimicrobial Agents and Chemotherapy Apr 2022Described here is a series of spiropyrimidinetrione (SPT) compounds with activity against Mycobacterium tuberculosis through inhibition of DNA gyrase. The SPT class...
Described here is a series of spiropyrimidinetrione (SPT) compounds with activity against Mycobacterium tuberculosis through inhibition of DNA gyrase. The SPT class operates via a novel mode of inhibition, which involves Mg-independent stabilization of the DNA cleavage complex with DNA gyrase and is thereby not cross-resistant with other DNA gyrase-inhibiting antibacterials, including fluoroquinolones. Compound 22 from the series was profiled broadly and showed cidality as well as intracellular activity against M. tuberculosis in macrophages. Evidence for the DNA gyrase mode of action was supported by inhibition of the target in a DNA supercoiling assay and elicitation of an SOS response seen in a reporter strain of M. tuberculosis. Pharmacokinetic properties of 22 supported evaluation of efficacy in an acute model of M. tuberculosis infection, where modest reduction in CFU numbers was seen. This work offers promise for deriving a novel drug class of tuberculosis agent without preexisting clinical resistance.
Topics: DNA Gyrase; Fluoroquinolones; Humans; Mycobacterium tuberculosis; Topoisomerase II Inhibitors; Tuberculosis
PubMed: 35266826
DOI: 10.1128/aac.02192-21 -
Extremophiles : Life Under Extreme... Dec 2023Topoisomerases are crucial enzymes in genome maintenance that modulate the topological changes during DNA metabolism. Deinococcus radiodurans, a Gram-positive bacterium...
Topoisomerases are crucial enzymes in genome maintenance that modulate the topological changes during DNA metabolism. Deinococcus radiodurans, a Gram-positive bacterium is characterized by its resistance to many abiotic stresses including gamma radiation. Its multipartite genome encodes both type I and type II topoisomerases. Time-lapse studies using fluorescently tagged topoisomerase IB (drTopoIB-RFP) and DNA gyrase (GyrA-RFP) were performed to check the dynamics and localization with respect to DNA repair and cell division under normal and post-irradiation growth conditions. Results suggested that TopoIB and DNA gyrase are mostly found on nucleoid, highly dynamic, and show growth phase-dependent subcellular localization. The drTopoIB-RFP was also present at peripheral and septum regions but does not co-localize with the cell division protein, drFtsZ. On the other hand, DNA gyrase co-localizes with PprA a pleiotropic protein involved in radioresistance, on the nucleoid during the post-irradiation recovery (PIR). The topoIB mutant was found to be sensitive to hydroxyurea treatment, and showed more accumulation of single-stranded DNA during the PIR, compared to the wild type suggesting its role in DNA replication stress. Together, these results suggest differential localization of drTopoIB-RFP and GyrA-RFP in D. radiodurans and their interaction with PprA protein, emphasizing the functional significance and role in radioresistance.
Topics: DNA Gyrase; Deinococcus; Bacterial Proteins; DNA Damage; DNA Repair
PubMed: 38062175
DOI: 10.1007/s00792-023-01323-1 -
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 -
The Journal of Biological Chemistry May 2023DNA gyrase is an essential nucleoprotein motor present in all bacteria and is a major target for antibiotic treatment of Mycobacterium tuberculosis (MTB) infection....
DNA gyrase is an essential nucleoprotein motor present in all bacteria and is a major target for antibiotic treatment of Mycobacterium tuberculosis (MTB) infection. Gyrase hydrolyzes ATP to add negative supercoils to DNA using a strand passage mechanism that has been investigated using biophysical and biochemical approaches. To analyze the dynamics of substeps leading to strand passage, single-molecule rotor bead tracking (RBT) has been used previously to follow real-time supercoiling and conformational transitions in Escherichia coli (EC) gyrase. However, RBT has not yet been applied to gyrase from other pathogenically relevant bacteria, and it is not known whether substeps are conserved across evolutionarily distant species. Here, we compare gyrase supercoiling dynamics between two evolutionarily distant bacterial species, MTB and EC. We used RBT to measure supercoiling rates, processivities, and the geometries and transition kinetics of conformational states of purified gyrase proteins in complex with DNA. Our results show that E. coli and MTB gyrases are both processive, with the MTB enzyme displaying velocities ∼5.5× slower than the EC enzyme. Compared with EC gyrase, MTB gyrase also more readily populates an intermediate state with DNA chirally wrapped around the enzyme, in both the presence and absence of ATP. Our substep measurements reveal common features in conformational states of EC and MTB gyrases interacting with DNA but also suggest differences in populations and transition rates that may reflect distinct cellular needs between these two species.
Topics: Adenosine Triphosphate; DNA; DNA Gyrase; DNA, Superhelical; Escherichia coli; Mycobacterium tuberculosis; Molecular Dynamics Simulation
PubMed: 36775125
DOI: 10.1016/j.jbc.2023.103003 -
Scientific Reports Jul 2019Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in...
Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25-30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.
Topics: Cell Culture Techniques; DNA Gyrase; DNA Replication; DNA, Bacterial; Leprosy; Mycobacterium leprae
PubMed: 31346236
DOI: 10.1038/s41598-019-47364-5 -
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 -
Drug Development Research Sep 2023An efficient one-pot reaction utilizing readily available chemical reagents was used to prepare novel 2-amino-1,5-diaryl-1H-pyrrole-3-carbonitrile derivatives and the...
An efficient one-pot reaction utilizing readily available chemical reagents was used to prepare novel 2-amino-1,5-diaryl-1H-pyrrole-3-carbonitrile derivatives and the structures of these compounds were validated by spectroscopic data and elemental analyses. All the synthetic compounds were evaluated for their antimicrobial activities (MZI assay). The tested compounds proved high activities on Staphylococcus aureus (Gram-positive bacteria) and Candida albicans (Pathogenic fungi). However, they did not show any activity on Escherichia coli (Gram-negative bacteria). The most effective compounds in MZI assay 7c, 9a, 9b, 11a, and 11b were selected to determine their MIC on S. aureus and C. albicans. Furthermore, DNA gyrase and 14-α demethylase inhibitory assays were performed to study the inhibitory activities of 7c, 9a, 9b, 11a, and 11b. The results illustrated that compound 9b was the most DNA gyrase inhibitor (IC of 0.0236 ± 0.45 µM, which was 1.3- fold higher than gentamicin reference IC values of 0.0323 ± 0.81 µM). In addition, compound 9b demonstrated the highest 14-α demethylase inhibitory effect with IC of 0.0013 ± 0.02 µM, compared to ketoconazole (IC of 0.0008 ± 0.03 µM) and fluconazole (IC of 0.00073 ± 0.01 µM), as antifungal reference drugs. Lastly, docking studies were performed to rationalize the dual inhibitory activities of the highly active compounds on both DNA gyrase and 14-α demethylase enzymes.
Topics: Molecular Docking Simulation; 14-alpha Demethylase Inhibitors; DNA Gyrase; Staphylococcus aureus; Anti-Bacterial Agents; Pyrroles; Antifungal Agents; Escherichia coli; Microbial Sensitivity Tests; Molecular Structure; Structure-Activity Relationship
PubMed: 37165799
DOI: 10.1002/ddr.22080 -
Molecules (Basel, Switzerland) Mar 2024There is an urgent need to discover and develop novel antibacterial agents. Accordingly, we synthesised 2-(piperazin-1-yl)naphtho[2,3-d]thiazole-4,9-dione (PNT), which...
There is an urgent need to discover and develop novel antibacterial agents. Accordingly, we synthesised 2-(piperazin-1-yl)naphtho[2,3-d]thiazole-4,9-dione (PNT), which exhibits antimicrobial activity. The aim of this study was to characterise PNT as an effective antimicrobial agent. Fluorescence microscopy was used to measure PNT's uptake into microbial cells (strains of , , and methicillin-resistant (MRSA)), transmission electron microscopy (TEM) was used to investigate the influence of PNT on the configuration of microbial cells, and a DNA gyrase supercoiling assay was used to investigate whether PNT inhibits DNA gyrase. PNT was taken up by more than 50% of microbial cells within 30 min. Using TEM, hollowed-out bacterial cytoplasms were observed in the specimen treated with PNT, although there was no disintegration of the bacterial membrane. In the DNA gyrase supercoiling assay, a dose-dependent reduction in fluorescence intensity was observed as the concentration of PNT increased. This suggests that PNT is taken up by microbial cells, resulting in cell disruption, and it reveals that one of the mechanisms underlying the antimicrobial activity of PNT is the inhibition of DNA gyrase.
Topics: Staphylococcus; Thiazoles; Methicillin-Resistant Staphylococcus aureus; DNA Gyrase; Anti-Bacterial Agents; Microbial Sensitivity Tests
PubMed: 38542913
DOI: 10.3390/molecules29061277 -
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