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Bioorganic & Medicinal Chemistry May 2020Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections and target bacterial type-II topoisomerases (DNA...
Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections and target bacterial type-II topoisomerases (DNA gyrase and topoisomerase IV). Fluoroquinolones, however potent, are susceptible to bacterial resistance with prolonged use, which limits their use in the clinic. Quinazoline-2,4-diones also target bacterial type-II topoisomerases and are not susceptible to bacterial resistance similar to fluoroquinolones, however, their potency pales in comparison to fluoroquinolones. To meet the increasing demand for antibacterial development, nine modified quinazoline-2,4-diones were developed to probe quinazoline-2,4-dione structure modification for possible new binding contacts with the bacterial type-II topoisomerase, DNA gyrase. Evaluation of compounds for inhibition of the supercoiling activity of DNA gyrase revealed a novel ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative as a modest inhibitor of DNA gyrase, having an IC of 3.5 μM. However, this ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate does not trap the catalytic intermediate like fluoroquinolones or typical quinazoline-2,4-diones do. Thus, the ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative discovered in this work acts as a catalytic inhibitor of DNA gyrase and therefore represents a new structural type of catalytic inhibitor of DNA gyrase.
Topics: Biocatalysis; DNA Gyrase; Dose-Response Relationship, Drug; Escherichia coli; Molecular Structure; Structure-Activity Relationship; Topoisomerase II Inhibitors
PubMed: 32234278
DOI: 10.1016/j.bmc.2020.115439 -
Journal of Enzyme Inhibition and... Dec 2022Emerging drug resistance is generating an urgent need for novel and effective antibiotics. A promising target that has not yet been addressed by approved antibiotics is...
Emerging drug resistance is generating an urgent need for novel and effective antibiotics. A promising target that has not yet been addressed by approved antibiotics is the bacterial DNA gyrase subunit B (GyrB), and GyrB inhibitors could be effective against drug-resistant bacteria, such as methicillin-resistant (MRSA). Here, we used the 4-hydroxy-2-quinolone fragment to search the Specs database of purchasable compounds for potential inhibitors of GyrB and identified or , as a novel and potent inhibitor of the target protein (IC: 1.21 µM). Structural modification was used to further identify two more potent GyrB inhibitors: (IC: 0.31 µM) and (IC: 0.28 µM). Additional experiments indicated that compound is more potent than the others in terms of antibacterial activity against MRSA (MICs: 4-8 µg/mL), non-toxic to HUVEC and HepG2 (CC: approximately 50 µM), and metabolically stable (t: > 372.8 min for plasma; 24.5 min for liver microsomes). In summary, this study showed that the discovered N-quinazolinone-4-hydroxy-2-quinolone-3-carboxamides are novel GyrB-targeted antibacterial agents; compound is promising for further development.
Topics: DNA Gyrase; Anti-Bacterial Agents; Topoisomerase II Inhibitors; Methicillin-Resistant Staphylococcus aureus; Quinazolinones; DNA, Bacterial; Microbial Sensitivity Tests; Bacteria
PubMed: 36278813
DOI: 10.1080/14756366.2022.2084088 -
The Journal of Biological Chemistry May 2014DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is...
DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.
Topics: Anti-Bacterial Agents; Ciprofloxacin; Crystallography, X-Ray; DNA Gyrase; DNA Topoisomerases, Type II; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Fluoroquinolones; Macromolecular Substances; Microbial Sensitivity Tests; Models, Molecular; Molecular Structure; Mutation; Mycobacterium smegmatis; Nucleic Acid Conformation; Protein Binding; Protein Structure, Tertiary; Topoisomerase II Inhibitors
PubMed: 24497635
DOI: 10.1074/jbc.M113.529164 -
Antimicrobial Agents and Chemotherapy May 2007Mycobacterium leprae, the causative agent of leprosy, is noncultivable in vitro; therefore, evaluation of antibiotic activity against M. leprae relies mainly upon the...
Mycobacterium leprae, the causative agent of leprosy, is noncultivable in vitro; therefore, evaluation of antibiotic activity against M. leprae relies mainly upon the mouse footpad system, which requires at least 12 months before the results become available. We have developed an in vitro assay for studying the activities of quinolones against the DNA gyrase of M. leprae. We overexpressed in Escherichia coli the M. leprae GyrA and GyrB subunits separately as His-tagged proteins by using a pET plasmid carrying the gyrA and gyrB genes. The soluble 97.5-kDa GyrA and 74.5-kDa GyrB subunits were purified by nickel chelate chromatography and were reconstituted as an enzyme with DNA supercoiling activity. Based on the drug concentrations that inhibited DNA supercoiling by 50% or that induced DNA cleavage by 25%, the 13 quinolones tested clustered into three groups. Analysis of the quinolone structure-activity relationship demonstrates that the most active quinolones against M. leprae DNA gyrase share the following structural features: a substituted carbon at position 8, a cyclopropyl substituent at N-1, a fluorine at C-6, and a substituent ring at C-7. We conclude that the assays based on DNA supercoiling inhibition and drug-induced DNA cleavage on purified M. leprae DNA gyrase are rapid, efficient, and safe methods for the screening of quinolone derivatives with potential in vivo activities against M. leprae.
Topics: Anti-Infective Agents; DNA; DNA Gyrase; DNA, Superhelical; Enzyme Inhibitors; Mycobacterium leprae; Quinolones; Topoisomerase II Inhibitors
PubMed: 17325221
DOI: 10.1128/AAC.01282-06 -
Molecular Microbiology Aug 2006When DNA gyrase is trapped on bacterial chromosomes by quinolone antibacterials, reversible complexes form that contain DNA ends constrained by protein. Two subsequent...
When DNA gyrase is trapped on bacterial chromosomes by quinolone antibacterials, reversible complexes form that contain DNA ends constrained by protein. Two subsequent processes lead to rapid cell death. One requires ongoing protein synthesis; the other does not. The prototype quinolone, nalidixic acid, kills wild-type Escherichia coli only by the first pathway; fluoroquinolones kill by both. Both lethal processes correlated with irreversible chromosome fragmentation, detected by sedimentation and viscosity of DNA from quinolone-treated cells. However, only fluoroquinolones fragmented purified nucleoids when incubated with gyrase purified from wild-type cells. A GyrA amino acid substitution (A67S) expected to perturb a GyrA-GyrA dimer interface allowed nalidixic acid to fragment chromosomes and kill cells in the absence of protein synthesis; moreover, it made a non-inducible lexA mutant hypersusceptible to nalidixic acid, a property restricted to fluoroquinolones with wild-type cells. The GyrA variation also facilitated immunoprecipitation of DNA fragments by GyrA antiserum following nalidixic acid treatment of cells. The ability of changes in both gyrase and quinolone structure to enhance protein synthesis-independent lethality and chromosome fragmentation is explained by drug-mediated destabilization of gyrase-DNA complexes. Instability of type II topoisomerase-DNA complexes may be a general phenomenon that can be exploited to kill cells.
Topics: Amino Acid Substitution; Chromosomes, Bacterial; DNA Fragmentation; DNA Gyrase; DNA, Bacterial; Dimerization; Escherichia coli; Fluoroquinolones; Gatifloxacin; Microbial Sensitivity Tests; Mutation; Nalidixic Acid; Quinolones
PubMed: 16803589
DOI: 10.1111/j.1365-2958.2006.05275.x -
Journal of Medicinal Chemistry Jun 2020The emergence of bacterial resistance against life-saving medicines has forced the scientific community and pharmaceutical industry to take actions in the quest for... (Review)
Review
The emergence of bacterial resistance against life-saving medicines has forced the scientific community and pharmaceutical industry to take actions in the quest for novel antibacterials. These should not only overcome the existing bacterial resistance but also provide at least interim effective protection against emerging bacterial infections. Research into DNA gyrase and topoisomerase IV inhibitors has become a particular focus, with the description of a new class of bacterial topoisomerase type II inhibitors known as "novel bacterial topoisomerase inhibitors", NBTIs. Elucidation of the key structural modifications incorporated into these inhibitors and the impact these can have on their general physicochemical properties are detailed in this review. This defines novel bacterial topoisomerase inhibitors with promising antibacterial activities and potencies, which thus represent one potential example of the future "drugs for bad bugs", as identified by the World Health Organization.
Topics: Anti-Bacterial Agents; Bacteria; Binding Sites; Catalytic Domain; DNA Gyrase; DNA Topoisomerase IV; Molecular Dynamics Simulation; Structure-Activity Relationship; Topoisomerase Inhibitors
PubMed: 32027491
DOI: 10.1021/acs.jmedchem.9b01738 -
The Journal of Biological Chemistry Nov 2013Nucleoid-associated proteins play an important role in condensing chromosomal DNA and regulating gene expression. We report here the characterization of the...
Nucleoid-associated proteins play an important role in condensing chromosomal DNA and regulating gene expression. We report here the characterization of the nucleoid-associated protein YejK, which was detected in a yeast two-hybrid screen using the ParE subunit of topoisomerase IV as bait. The purified protein likely exists in a monomer-dimer equilibrium in solution and can form tetramers. Cross-linking of the protein bound to DNA suggests that the active form could be either a dimer or tetramer. YejK, which is present at about 24,000 copies of monomer per mid-log phase cell, binds double-stranded DNA with a site size of 12-14 base pairs/monomer, does not display a significant preference for either bent compared with straight DNA or supercoiled compared with relaxed DNA, and untwists DNA somewhat as it binds. YejK binds RNA, but not single-stranded DNA, with 65% of the avidity with which it binds DNA. However, cells deleted for yejK do not show defects in either RNA or protein synthesis. YejK interacts with all the subunits of both DNA gyrase and topoisomerase IV and has measurable effects on their activities. In the presence of YejK, relaxation of negatively supercoiled DNA by topoisomerase IV becomes distributive, whereas relaxation of positively supercoiled DNA is stimulated. Relaxation of negatively supercoiled DNA by DNA gyrase is inhibited, whereas the extent of supercoiling of relaxed DNA is limited. A YejK-GFP chimera is an effective marker for the nucleoid in live cell imaging.
Topics: DNA Gyrase; DNA Topoisomerase IV; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Protein Binding; Saccharomyces cerevisiae; Two-Hybrid System Techniques
PubMed: 24043617
DOI: 10.1074/jbc.M113.494237 -
Acta Crystallographica. Section F,... Feb 2012Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the...
Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolone in the treatment of tuberculosis. The C-terminal domain (CTD) of the DNA gyrase A subunit possesses a unique feature, the ability to wrap DNA in a chiral manner, that plays an essential role during the catalytic cycle. A construct of 36 kDa corresponding to this domain has been overproduced, purified and crystallized. Diffraction data were collected to 1.55 Å resolution. Cleavage of the N-terminal His tag was crucial for obtaining crystals. The crystals belonged to space group P2(1)2(1)2(1), with one molecule in the asymmetric unit and a low solvent content (33%). This is the first report of the crystallization and preliminary X-ray diffraction studies of a DNA gyrase CTD from a species that contains one unique type II topoisomerase.
Topics: Crystallization; Crystallography, X-Ray; DNA Gyrase; Mycobacterium tuberculosis
PubMed: 22297993
DOI: 10.1107/S1744309111051888 -
Molecules (Basel, Switzerland) Dec 2023The emergence of antimicrobial resistance due to the widespread and inappropriate use of antibiotics has now become the global health challenge. Flavonoids have long...
The emergence of antimicrobial resistance due to the widespread and inappropriate use of antibiotics has now become the global health challenge. Flavonoids have long been reported to be a potent antimicrobial agent against a wide range of pathogenic microorganisms in vitro. Therefore, new antibiotics development based on flavonoid structures could be a potential strategy to fight against antibiotic-resistant infections. This research aims to screen the potency of flavonoids of the genus Erythrina as an inhibitor of bacterial ATPase DNA gyrase B. From the 378 flavonoids being screened, 49 flavonoids show potential as an inhibitor of ATPase DNA gyrase B due to their lower binding affinity compared to the inhibitor and ATP. Further screening for their toxicity, we identified 6 flavonoids from these 49 flavonoids, which are predicted to have low toxicity. Among these flavonoids, erystagallin B () is predicted to have the best pharmacokinetic properties, and therefore, could be further developed as new antibacterial agent.
Topics: Anti-Bacterial Agents; DNA Gyrase; Flavonoids; Erythrina; Adenosine Triphosphatases; Microbial Sensitivity Tests; Bacteria; Topoisomerase II Inhibitors
PubMed: 38138500
DOI: 10.3390/molecules28248010 -
Molecular Microbiology Apr 2018In bacteria, initiation of DNA replication requires the DnaA protein. Regulation of DnaA association and activity at the origin of replication, oriC, is the predominant...
In bacteria, initiation of DNA replication requires the DnaA protein. Regulation of DnaA association and activity at the origin of replication, oriC, is the predominant mechanism of replication initiation control. One key feature known to be generally important for replication is DNA topology. Although there have been some suggestions that topology may impact replication initiation, whether this mechanism regulates DnaA-mediated replication initiation is unclear. We found that the essential topoisomerase, DNA gyrase, is required for both proper binding of DnaA to oriC as well as control of initiation frequency in Bacillus subtilis. Furthermore, we found that the regulatory activity of gyrase in initiation is specific to DnaA and oriC. Cells initiating replication from a DnaA-independent origin, oriN, are largely resistant to gyrase inhibition by novobiocin, even at concentrations that compromise survival by up to four orders of magnitude in oriC cells. Furthermore, inhibition of gyrase does not impact initiation frequency in oriN cells. Additionally, deletion or overexpression of the DnaA regulator, YabA, significantly modulates sensitivity to gyrase inhibition, but only in oriC and not oriN cells. We propose that gyrase is a negative regulator of DnaA-dependent replication initiation from oriC, and that this regulatory mechanism is required for cell survival.
Topics: Bacillus subtilis; Bacterial Proteins; DNA Gyrase; DNA Replication; DNA, Bacterial; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Replication Origin
PubMed: 29396913
DOI: 10.1111/mmi.13920