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PLoS Genetics Oct 2020DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing...
DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing activities of topoisomerase I, which relaxes DNA, and DNA gyrase, which compacts DNA. These enzymes are widely conserved, sharing >91% amino acid identity between the closely related species Escherichia coli and Salmonella enterica serovar Typhimurium. Why, then, do E. coli and Salmonella exhibit different DNA supercoiling when experiencing the same conditions? We now report that this surprising difference reflects disparate activation of their DNA gyrases by the polyamine spermidine and its precursor putrescine. In vitro, Salmonella DNA gyrase activity was sensitive to changes in putrescine concentration within the physiological range, whereas activity of the E. coli enzyme was not. In vivo, putrescine activated the Salmonella DNA gyrase and spermidine the E. coli enzyme. High extracellular Mg2+ decreased DNA supercoiling exclusively in Salmonella by reducing the putrescine concentration. Our results establish the basis for the differences in global DNA supercoiling between E. coli and Salmonella, define a signal transduction pathway regulating DNA supercoiling, and identify potential targets for antibacterial agents.
Topics: DNA Gyrase; DNA Topoisomerases, Type I; DNA, Superhelical; Escherichia coli; Magnesium; Putrescine; Salmonella typhimurium; Spermidine
PubMed: 33125364
DOI: 10.1371/journal.pgen.1009085 -
Molecules (Basel, Switzerland) May 2022The increased use of polyphenols nowadays poses the need for identification of their new pharmacological targets. Recently, structure similarity-based virtual screening...
The increased use of polyphenols nowadays poses the need for identification of their new pharmacological targets. Recently, structure similarity-based virtual screening of DrugBank outlined pseudopurpurin, a hydroxyanthraquinone from spp., as similar to gatifloxacin, a synthetic antibacterial agent. This suggested the bacterial DNA gyrase and DNA topoisomerase IV as potential pharmacological targets of pseudopurpurin. In this study, estimation of structural similarity to referent antibacterial agents and molecular docking in the DNA gyrase and DNA topoisomerase IV complexes were performed for a homologous series of four hydroxyanthraquinones. Estimation of shape- and chemical feature-based similarity with (S)-gatifloxacin, a DNA gyrase inhibitor, and (S)-levofloxacin, a DNA topoisomerase IV inhibitor, outlined pseudopurpurin and munjistin as the most similar structures. The docking simulations supported the hypothesis for a plausible antibacterial activity of hydroxyanthraquinones. The predicted docking poses were grouped into 13 binding modes based on spatial similarities in the active site. The simultaneous presence of 1-OH and 3-COOH substituents in the anthraquinone scaffold were emphasized as relevant features for the binding modes' variability and ability of the compounds to strongly bind in the DNA-enzyme complexes. The results reveal new potential pharmacological targets of the studied polyphenols and help in their prioritization as drug candidates and dietary supplements.
Topics: Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; Gatifloxacin; Molecular Docking Simulation; Polyphenols; Rubia
PubMed: 35630751
DOI: 10.3390/molecules27103274 -
Antimicrobial Agents and Chemotherapy Apr 2022Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans. Fluoroquinolone (FQ), used in the treatment of...
Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans. Fluoroquinolone (FQ), used in the treatment of this disease, has been known to act by inhibiting the enzymatic activities of DNA gyrase. However, the detailed molecular basis of these characteristics and the FQ resistance mechanisms in M. ulcerans remains unknown. This study investigated the detailed molecular mechanism of M. ulcerans DNA gyrase and the contribution of FQ resistance using recombinant proteins from the M. ulcerans subsp. shinshuense and Agy99 strains with reduced sensitivity to FQs. The IC of FQs against Ala91Val and Asp95Gly mutants of M. ulcerans shinshuense and Agy99 GyrA subunits were 3.7- to 42.0-fold higher than those against wild-type (WT) enzyme. Similarly, the quinolone concentrations required to induce 25% of the maximum DNA cleavage (CC) was 10- to 210-fold higher than those for the WT enzyme. Furthermore, the interaction between the amino acid residues of the WT/mutant M. ulcerans DNA gyrase and FQ side chains were assessed by molecular docking studies. This was the first elaborative study demonstrating the contribution of mutations in M. ulcerans DNA GyrA subunit to FQ resistance .
Topics: DNA Gyrase; Drug Resistance, Bacterial; Fluoroquinolones; Microbial Sensitivity Tests; Molecular Docking Simulation; Mutation; Mycobacterium ulcerans; Quinolones
PubMed: 35041504
DOI: 10.1128/AAC.01902-21 -
Bioorganic & Medicinal Chemistry Nov 2022A series of ciprofloxacin-uracil conjugates 5a-t were synthesized and identified by H NMR, C NMR, mass spectroscopy and elemental analyses. The antibacterial results...
A series of ciprofloxacin-uracil conjugates 5a-t were synthesized and identified by H NMR, C NMR, mass spectroscopy and elemental analyses. The antibacterial results revealed that the new derivatives exhibited better activity against Gram-positive than the Gram-negative strains; most of the target compounds exhibited good activities against S. aureus ATCC 6538. Compounds 5b and 5g possess the highest activities with MICs of 1.25 and 2.37 µM, respectively, which are more potent than the parent drug ciprofloxacin, MIC, 7.58 µM. In addition, they also exhibited potent activities against MRSA AUMC 261 with MICs, 0.031 and 0.046 µM respectively, higher than ciprofloxacin with MIC, 0.57 µM. Moreover, compounds 5b and 5g showed potent inhibitory activities against DNA gyrase (IC = 1.72 and 5.72 µM) and topoisomerase IV (4.36 and 7.77 µM) compared to ciprofloxacin with IC values 0.66 and 8.16 µM, respectively. The molecular docking study revealed that compounds 5b and 5g may formed stable interaction with the active sites of DNA gyrase and topoisomerase IV similar to ciprofloxacin. Hence, 5b and 5g are considered promising antibacterial candidated against MRSA AUMC 261 strains that requires further optimization.
Topics: Anti-Bacterial Agents; Ciprofloxacin; DNA Gyrase; DNA Topoisomerase IV; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Molecular Docking Simulation; Staphylococcus aureus; Topoisomerase II Inhibitors; Uracil
PubMed: 36148773
DOI: 10.1016/j.bmc.2022.117004 -
Pharmaceuticals (Basel, Switzerland) Aug 2021DNA gyrase is an important target for the development of novel antibiotics. Although ATP-competitive DNA gyrase (GyrB) inhibitors are a well-studied class of...
DNA gyrase is an important target for the development of novel antibiotics. Although ATP-competitive DNA gyrase (GyrB) inhibitors are a well-studied class of antibacterial agents, there is currently no representative used in therapy, largely due to unwanted off-target activities. Selectivity of GyrB inhibitors against closely related human ATP-binding enzymes should be evaluated early in development to avoid off-target binding to homologous binding domains. To address this challenge, we developed selective 3D-pharmacophore models for GyrB, human topoisomerase IIα (TopoII), and the Hsp90 N-terminal domain (NTD) to be used in in silico activity profiling paradigms to identify molecules selective for GyrB over TopoII and Hsp90, as starting points for hit expansion and lead optimization. The models were used to profile highly active GyrB, TopoII, and Hsp90 inhibitors. Selected compounds were tested in in vitro assays. GyrB inhibitors and were inactive against TopoII and Hsp90, while and , potent Hsp90 inhibitors, displayed no inhibition of GyrB and TopoII, and TopoII inhibitors and were inactive at GyrB and Hsp90. The results provide a proof of concept for the use of target activity profiling methods to identify selective starting points for hit and lead identification.
PubMed: 34451886
DOI: 10.3390/ph14080789 -
Structure (London, England : 1993) May 2020Most biological processes involve formation of transient complexes where binding of a ligand allosterically modulates function. The ccd toxin-antitoxin system is...
Most biological processes involve formation of transient complexes where binding of a ligand allosterically modulates function. The ccd toxin-antitoxin system is involved in plasmid maintenance and bacterial persistence. The CcdA antitoxin accelerates dissociation of CcdB from its complex with DNA gyrase, binds and neutralizes CcdB, but the mechanistic details are unclear. Using a series of experimental and computational approaches, we demonstrate the formation of transient ternary and quaternary CcdA:CcdB:gyrase complexes and delineate the molecular steps involved in the rejuvenation process. Binding of region 61-72 of CcdA to CcdB induces the vital structural and dynamic changes required to facilitate dissociation from gyrase, region 50-60 enhances the dissociation process through additional allosteric effects, and segment 37-49 prevents gyrase rebinding. This study provides insights into molecular mechanisms responsible for recovery of CcdB-poisoned cells from a persister-like state. Similar methodology can be used to characterize other important transient, macromolecular complexes.
Topics: Bacterial Proteins; Bacterial Toxins; Binding Sites; Cysteine; DNA Gyrase; Fluorescence Resonance Energy Transfer; Models, Molecular; Multiprotein Complexes; Mutation; Surface Plasmon Resonance
PubMed: 32294467
DOI: 10.1016/j.str.2020.03.006 -
The FEBS Journal Mar 2023Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing...
Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Site-directed mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an α-helix structure, and that the E27 residue is located around the centre of the α-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases.
Topics: Escherichia coli; Bacterial Proteins; Staphylococcus aureus; DNA Gyrase; Toxin-Antitoxin Systems; Antitoxins; RNA
PubMed: 36148483
DOI: 10.1111/febs.16634 -
Research in Microbiology 2023Cells have evolved strategies to safeguard their genome integrity. We describe a mechanism to counter double strand breaks in the chromosome that involves the protection...
Cells have evolved strategies to safeguard their genome integrity. We describe a mechanism to counter double strand breaks in the chromosome that involves the protection of an essential housekeeping enzyme from external agents. YacG is a DNA gyrase inhibitory protein from Escherichia coli that protects the bacterium from the cytotoxic effects of catalytic inhibitors as well as cleavage-complex stabilizers of DNA gyrase. By virtue of blocking the primary DNA binding site of the enzyme, YacG prevents the accumulation of double strand breaks induced by gyrase poisons. It also enables the bacterium to resist the growth-inhibitory property of novobiocin. Gyrase poison-induced oxidative stress upregulates YacG production, probably as a cellular response to counter DNA damage. YacG-mediated protection of the genome is specific for gyrase targeting agents as the protection is not observed from the action of general DNA damaging agents. YacG also intensifies the transcription stress induced by rifampicin substantiating the importance of gyrase activity during transcription. Although essential for bacterial survival, DNA gyrase often gets entrapped by external inhibitors and poisons, resulting in cell death. The existence of YacG to specifically protect an essential housekeeping enzyme might be a strategy adopted by bacteria for competitive fitness advantage.
PubMed: 37343614
DOI: 10.1016/j.resmic.2023.104093 -
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 -
ACS Omega May 2023In pursuit of new antitubercular agents, we here report the antimycobacterial (HRv) and DNA gyrase inhibitory potential of daidzein and khellin natural products (NPs)....
In pursuit of new antitubercular agents, we here report the antimycobacterial (HRv) and DNA gyrase inhibitory potential of daidzein and khellin natural products (NPs). We procured a total of 16 NPs based on their pharmacophoric similarities with known antimycobacterial compounds. The HRv strain of was found to be susceptible to only two out of the 16 NPs procured; specifically, daidzein and khellin each exhibited an MIC of 25 μg/mL. Moreover, daidzein and khellin inhibited the DNA gyrase enzyme with IC values of 0.042 and 0.822 μg/mL, respectively, compared to ciprofloxacin with an IC value of 0.018 μg/mL. Daidzein and khellin were found to have lower toxicity toward the vero cell line, with IC values of 160.81 and 300.23 μg/mL, respectively. Further, molecular docking study and MD simulation of daidzein indicated that it remained stable inside the cavity of DNA GyrB domain for 100 ns.
PubMed: 37179626
DOI: 10.1021/acsomega.3c00684