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Journal of Molecular Biology Aug 2019Type II topoisomerases regulate DNA topology by making a double-stranded break in one DNA duplex, transporting another DNA segment through this break and then resealing... (Review)
Review
Type II topoisomerases regulate DNA topology by making a double-stranded break in one DNA duplex, transporting another DNA segment through this break and then resealing it. Bacterial type IIA topoisomerase inhibitors, such as fluoroquinolones and novel bacterial topoisomerase inhibitors, can trap DNA cleavage complexes with double- or single-stranded cleaved DNA. To study the mode of action of such compounds, 21 crystal structures of a "gyrase" fusion truncate of Staphyloccocus aureus DNA gyrase complexed with DNA and diverse inhibitors have been published, as well as 4 structures lacking inhibitors. These structures have the DNA in various cleavage states and appear to track trajectories along the catalytic paths of the DNA cleavage/religation steps. The various conformations sampled by these multiple "gyrase" structures show rigid body movements of the catalytic GyrA WHD and GyrB TOPRIM domains across the dimer interface. Conformational changes common to all compound-bound structures suggest common mechanisms for DNA cleavage-stabilizing compounds. The structures suggest that S. aureus gyrase uses a single moving-metal ion for cleavage and that the central four base pairs need to be stretched between the two catalytic sites, in order for a scissile phosphate to attract a metal ion to the A-site to catalyze cleavage, after which it is "stored" in another coordination configuration (B-site) in the vicinity. We present a simplified model for the catalytic cycle in which capture of the transported DNA segment causes conformational changes in the ATPase domain that push the DNA gate open, resulting in stretching and cleaving the gate-DNA in two steps.
Topics: Anti-Bacterial Agents; Catalytic Domain; DNA; DNA Cleavage; DNA Gyrase; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Fluoroquinolones; Metals; Models, Molecular; Protein Conformation; Quinolones; Staphylococcus aureus; Topoisomerase II Inhibitors; Topoisomerase Inhibitors
PubMed: 31301408
DOI: 10.1016/j.jmb.2019.07.008 -
Antimicrobial Agents and Chemotherapy Jul 2021New, more-effective drugs for the treatment of lung disease caused by nontuberculous mycobacteria (NTM) are needed. Among NTM opportunistic pathogens, Mycobacterium...
New, more-effective drugs for the treatment of lung disease caused by nontuberculous mycobacteria (NTM) are needed. Among NTM opportunistic pathogens, Mycobacterium abscessus is the most difficult to cure and intrinsically multidrug resistant. In a whole-cell screen of a compound collection active against Mycobacterium tuberculosis, we previously identified the piperidine-4-carboxamide (P4C) MMV688844 (844) as a hit against M. abscessus. Here, we identified a more potent analog of 844 and showed that both the parent and improved analog retain activity against strains representing all three subspecies of the M. abscessus complex. Furthermore, P4Cs showed bactericidal and antibiofilm activity. Spontaneous resistance against the P4Cs emerged at a frequency of 10/CFU and mapped to and encoding the subunits of DNA gyrase. Biochemical studies with recombinant M. abscessus DNA gyrase showed that P4Cs inhibit the wild-type enzyme but not the P4C-resistant mutant. P4C-resistant strains showed limited cross-resistance to the fluoroquinolone moxifloxacin, which is in clinical use for the treatment of macrolide-resistant M. abscessus disease, and no cross-resistance to the benzimidazole SPR719, a novel DNA gyrase inhibitor in clinical development for the treatment of mycobacterial diseases. Analyses of P4Cs in promoter-based DNA damage reporter strains showed induction of promoter activity in the wild type but not in the P4C-resistant mutant background. This indicates that P4Cs, similar to fluoroquinolones, cause DNA gyrase-mediated DNA damage. Together, our results show that P4Cs present a novel class of mycobacterial DNA gyrase inhibitors with attractive antimicrobial activities against the M. abscessus complex.
Topics: Anti-Bacterial Agents; DNA Gyrase; Humans; Microbial Sensitivity Tests; Mycobacterium Infections, Nontuberculous; Mycobacterium abscessus; Nontuberculous Mycobacteria; Piperidines
PubMed: 34001512
DOI: 10.1128/AAC.00676-21 -
Antimicrobial Agents and Chemotherapy Dec 2022CUO246, a novel DNA gyrase/topoisomerase IV inhibitor, is active against a broad range of Gram-positive, fastidious Gram-negative, and atypical bacterial pathogens and...
CUO246, a novel DNA gyrase/topoisomerase IV inhibitor, is active against a broad range of Gram-positive, fastidious Gram-negative, and atypical bacterial pathogens and retains activity against quinolone-resistant strains in circulation. The frequency of selection for single step mutants of wild-type S. aureus with reduced susceptibility to CUO246 was <4.64 × 10 at 4× and 8× MIC and remained low when using an isogenic QRDR mutant (<5.24 × 10 at 4× and 8× MIC). Biochemical assays indicated that CUO246 had potent inhibitory activity against both DNA gyrase (GyrAB) and topoisomerase IV (ParCE). Furthermore, CUO246 showed rapid bactericidal activity in time-kill assays and potent efficacy against S. aureus in a neutropenic murine thigh infection model. These results suggest that CUO246 may be useful in treating infections by various causative agents of acute skin and skin structure infections, respiratory tract infections, and sexually transmitted infections.
Topics: Animals; Mice; DNA Gyrase; DNA Topoisomerase IV; Topoisomerase II Inhibitors; DNA, Bacterial; Staphylococcus aureus; Microbial Sensitivity Tests; Anti-Bacterial Agents
PubMed: 36448795
DOI: 10.1128/aac.00921-22 -
Nature Chemical Biology Jun 2018Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of > 100 base pairs confers directionality...
Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of > 100 base pairs confers directionality on topoisomerization. To understand the mechanism of this nucleoprotein machine, global structural transitions must be mapped onto the nucleotide cycle of ATP binding, hydrolysis and product release. Here we investigate coupling mechanisms using single-molecule tracking of DNA rotation and contraction during Escherichia coli gyrase activity under varying nucleotide conditions. We find that ADP must be exchanged for ATP to drive the rate-limiting remodeling transition that generates the chiral wrap. ATP hydrolysis accelerates subsequent duplex strand passage and is required for resetting the enzyme and recapturing transiently released DNA. Our measurements suggest how gyrase coordinates DNA rearrangements with the dynamics of its ATP-driven protein gate, how the motor minimizes futile cycles of ATP hydrolysis and how gyrase may respond to changing cellular energy levels to link gene expression with metabolism.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; DNA; DNA Gyrase; Escherichia coli; Gene Expression Profiling; Gene Expression Regulation; Hydrolysis; Kinetics; Models, Molecular; Nucleic Acid Conformation; Nucleotides; Protein Binding; Protein Conformation; Stereoisomerism
PubMed: 29662209
DOI: 10.1038/s41589-018-0037-0 -
Molecular Diversity Aug 2023A series of N-4 piperazinyl ciprofloxacin derivatives as urea-tethered ciprofloxacin-chalcone hybrids 2a-j and thioacetyl-linked ciprofloxacin-pyrimidine hybrids 5a-i...
A series of N-4 piperazinyl ciprofloxacin derivatives as urea-tethered ciprofloxacin-chalcone hybrids 2a-j and thioacetyl-linked ciprofloxacin-pyrimidine hybrids 5a-i were synthesized. The target compounds were investigated for their antibacterial activity against S. aureus, P. aeruginosa, E. coli, and C. albicans strains, respectively. Ciprofloxacin derivatives 2a-j and 5a-i revealed broad antibacterial activity against either Gram positive or Gram negative strains, with MIC range of 0.06-42.23 µg/mL compared to ciprofloxacin with an MIC range of 0.15-3.25 µg/mL. Among the tested compounds, hybrids 2b, 2c, 5a, 5b, 5h, and 5i exhibited remarkable antibacterial activity with MIC range of 0.06-1.53 µg/mL against the tested bacterial strains. On the other hand, compounds 2c, 2e, 5c, and 5e showed comparable antifungal activity to ketoconazole against candida albicans with MIC range of 2.03-3.89 µg/mL and 2.6 µg/mL, respectively. Further investigations showed that some ciprofloxacin hybrids have inhibitory activity against DNA gyrase as potential molecular target compared to ciprofloxacin with IC range of 0.231 ± 0.01-7.592 ± 0.40 µM and 0.323 ± 0.02 µM, respectively. Docking studies of compounds 2b, 2c, 5b, 5c, 5e, 5h, and 5i on the active site of DNA gyrase (PDB: 2XCT) confirmed their ability to form stable complex with the target enzyme like that of ciprofloxacin.
Topics: Ciprofloxacin; Topoisomerase II Inhibitors; Molecular Docking Simulation; DNA Gyrase; Escherichia coli; Staphylococcus aureus; Anti-Infective Agents; Anti-Bacterial Agents; Microbial Sensitivity Tests; Structure-Activity Relationship; Molecular Structure
PubMed: 36152132
DOI: 10.1007/s11030-022-10528-z -
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 -
Science (New York, N.Y.) Apr 2024DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules,...
DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.
Topics: Cryoelectron Microscopy; DNA; DNA Gyrase; DNA, Superhelical; Escherichia coli; Escherichia coli Proteins; Protein Domains
PubMed: 38603484
DOI: 10.1126/science.adl5899 -
Antimicrobial Agents and Chemotherapy Sep 2021Malaria persists as a major health problem due to the spread of drug resistance and the lack of effective vaccines. DNA gyrase is a well-validated and extremely...
Malaria persists as a major health problem due to the spread of drug resistance and the lack of effective vaccines. DNA gyrase is a well-validated and extremely effective therapeutic target in bacteria, and it is also known to be present in the apicoplast of malarial species, including Plasmodium falciparum. This raises the possibility that it could be a useful target for novel antimalarials. To date, characterization and screening of this gyrase have been hampered by difficulties in cloning and purification of the GyrA subunit, which is necessary together with GyrB for reconstitution of the holoenzyme. To overcome this, we employed a library of compounds with specificity for P. falciparum GyrB and assessed them in activity tests utilizing P. falciparum GyrB together with Escherichia coli GyrA to reconstitute a functional hybrid enzyme. Two inhibitory compounds were identified that preferentially inhibited the supercoiling activity of the hybrid enzyme over the E. coli enzyme. Of these, purpurogallin (PPG) was found to disrupt DNA binding to the hybrid gyrase complex and thus reduce the DNA-induced ATP hydrolysis of the enzyme. Binding studies indicated that PPG showed higher-affinity binding to P. falciparum GyrB than to the E. coli protein. We suggest that PPG achieves its inhibitory effect on gyrase through interaction with P. falciparum GyrB leading to disruption of DNA binding and, consequently, reduction of DNA-induced ATPase activity. The compound also showed an inhibitory effect against the malaria parasite and may be of interest for further development as an antimalarial agent.
Topics: Apicoplasts; DNA Gyrase; Escherichia coli; Humans; Malaria, Falciparum; Plasmodium falciparum
PubMed: 34339271
DOI: 10.1128/AAC.00267-21 -
ACS Chemical Biology Jun 2014Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new...
Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Anti-Bacterial Agents; Chromosomes, Bacterial; DNA Gyrase; DNA Replication; DNA, Bacterial; Drug Resistance, Bacterial; Escherichia coli; Flow Cytometry; Molecular Structure; Mutation; Pyrrolidines; Topoisomerase II Inhibitors
PubMed: 24712739
DOI: 10.1021/cb500154m -
Proceedings of the National Academy of... Aug 2011DNA gyrase introduces negative supercoils into DNA in an ATP-dependent reaction. DNA supercoiling is catalyzed by a strand-passage mechanism, in which a T-segment of DNA...
DNA gyrase introduces negative supercoils into DNA in an ATP-dependent reaction. DNA supercoiling is catalyzed by a strand-passage mechanism, in which a T-segment of DNA is passed through the gap in a transiently cleaved G-segment. Strand passage requires the coordinated closing and opening of three protein interfaces in gyrase, the N-gate, DNA-gate, and C-gate. We show here that DNA binding to the DNA-gate of gyrase and wrapping of DNA around the C-terminal domains of GyrA induces a narrowing of the N-gate. This half-closed state prepares capture of a T-segment in the upper cavity of gyrase. Subsequent N-gate closure upon binding of ATP then poises the reaction toward strand passage. The N-gate reopens after ATP hydrolysis, allowing for further catalytic cycles. DNA binding, cleavage, and wrapping and N-gate narrowing are intimately linked events that coordinate conformational changes at the DNA and the N-gate.
Topics: Adenosine Triphosphate; Bacillus subtilis; DNA Gyrase; DNA, Superhelical; Hydrolysis; Models, Biological; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Recombinant Fusion Proteins
PubMed: 21817063
DOI: 10.1073/pnas.1102100108