-
Antibiotics (Basel, Switzerland) Jul 2023The extensive use of fluoroquinolones has been consequently accompanied by the emergence of bacterial resistance, which triggers the necessity to discover new compounds.... (Review)
Review
The extensive use of fluoroquinolones has been consequently accompanied by the emergence of bacterial resistance, which triggers the necessity to discover new compounds. Delafloxacin is a brand-new anionic non-zwitterionic fluoroquinolone with some structural particularities that give it attractive proprieties: high activity under acidic conditions, greater in vitro activity against Gram-positive bacteria-even those showing resistance to currently-used fluoroquinolones-and nearly equivalent affinity for both type-II topoisomerases (i.e., DNA gyrase and topoisomerase IV). During phases II and III clinical trials, delafloxacin showed non-inferiority compared to standard-of-care therapy in the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia, which resulted in its approval in 2017 by the Food and Drug Administration for indications. Thanks to its overall good tolerance, its broad-spectrum in vitro activity, and its ease of use, it could represent a promising molecule for the treatment of bacterial infections.
PubMed: 37627661
DOI: 10.3390/antibiotics12081241 -
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 -
Heliyon Oct 2023Chalcones and their derivatives have been widely studied due to their versatile pharmacological and biological activities, such as anti-inflammatory, antibacterial,... (Review)
Review
Chalcones and their derivatives have been widely studied due to their versatile pharmacological and biological activities, such as anti-inflammatory, antibacterial, antiviral, and antitumor effects. These compounds have shown suitable antiviral effects through the selective targeting of a variety of viral enzymes, including lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fumarate reductase, protein tyrosine phosphatase, topoisomerase-II, protein kinases, integrase/protease, and lactate/isocitrate dehydrogenase, among others. Chalcones and their derivatives have displayed excellent potential for combating pathogenic bacteria and fungi (especially, multidrug-resistant bacteria). However, relevant mechanisms should be further explored, focusing on inhibitory effects against DNA gyrase B, UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), and efflux pumps (e.g. NorA), among others. In addition, the antifungal and antiparasitic activities of these compounds (e.g., antitrypanosomal and antileishmanial properties) have prompted additional explorations. Nonetheless, systematic analysis of the relevant mechanisms, biosafety issues, and pharmacological properties, as well as clinical translation studies, are vital for practical applications. Herein, recent advancements pertaining to the antibacterial, antiviral, antiparasitic, and antifungal activities of chalcones and their derivatives are deliberated, focusing on the relevant mechanisms of action, crucial challenges, and future prospects. Furthermore, due to the great importance of greener and more sustainable synthesis of these valuable compounds, especially on an industrial scale, the progress made in this field has been briefly discussed. Hopefully, this review can serve as a catalyst for researchers to delve deeper into the exploration and designing of novel chalcone compounds with medicinal properties, especially against pathogenic viruses and multidrug-resistant bacteria as major causes of concern for human health.
PubMed: 37810815
DOI: 10.1016/j.heliyon.2023.e20428 -
EMBO Reports Jul 2023The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for...
The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.
Topics: Animals; Mice; Staphylococcus aureus; Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; Peptides
PubMed: 37166011
DOI: 10.15252/embr.202255338 -
The Journal of Biological Chemistry Dec 2023Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic...
Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic reactions. Studying these reactions in vitro, using agents such as polyetthylene glycols (PEGs) and polyvinyl alcohols (PVAs) to mimic intracellular crowding conditions, is essential due to the notable differences from enzyme behaviors observed in diluted aqueous solutions. In this article, we studied Mycobacterium tuberculosis (Mtb) DNA gyrase under macromolecular crowding conditions by incorporating PEGs and PVAs into the DNA supercoiling reactions. We discovered that high concentrations of potassium glutamate, glycine betaine, PEGs, and PVA substantially stimulated the DNA supercoiling activity of Mtb DNA gyrase. Steady-state kinetic studies showed that glycine betaine and PEG400 significantly reduced the K of Mtb DNA gyrase and simultaneously increased the V or k of Mtb DNA gyrase for ATP and the plasmid DNA molecule. Molecular dynamics simulation studies demonstrated that PEG molecules kept the ATP lid of DNA gyrase subunit B in a closed or semiclosed conformation, which prevented ATP molecules from leaving the ATP-binding pocket of DNA gyrase subunit B. The stimulation of the DNA supercoiling activity of Mtb DNA gyrase by these molecular crowding agents likely results from a decrease in water activity and an increase in excluded volume.
Topics: DNA Gyrase; Mycobacterium tuberculosis; Betaine; Kinetics; Adenosine Triphosphate; DNA; DNA, Superhelical
PubMed: 37944619
DOI: 10.1016/j.jbc.2023.105439 -
Microbiology Spectrum Jun 2023Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus...
Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix rosea (LrAgo) are active in heterologous Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin, depending on the host double-strand break repair machinery. Both pAgos are preferentially loaded with small guide DNAs (smDNAs) derived from the sites of replication termination. Ciprofloxacin increases the amounts of smDNAs from the termination region and from the sites of genomic DNA cleavage by gyrase, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin enhances asymmetry in the distribution of smDNAs around Chi sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host from ciprofloxacin. These results suggest that pAgo nucleases may help to complete replication of chromosomal DNA by promoting chromosome decatenation or participating in the processing of gyrase cleavage sites, and may switch their functional activities depending on the host species. Prokaryotic Argonautes (pAgos) are programmable nucleases with incompletely understood functions . In contrast to eukaryotic Argonautes, most studied pAgos recognize DNA targets. Recent studies suggested that pAgos can protect bacteria from invader DNA and counteract phage infection and may also have other functions including possible roles in DNA replication, repair, and gene regulation. Here, we have demonstrated that two cyanobacterial pAgos, SeAgo and LrAgo, can assist DNA replication and facilitate cell division in the presence of topoisomerase inhibitors in Escherichia coli. They are specifically loaded with small guide DNAs from the region of replication termination and protect the cells from the action of the gyrase inhibitor ciprofloxacin, suggesting that they help to complete DNA replication and/or repair gyrase-induced breaks. The results show that pAgo proteins may serve as a backup to topoisomerases under conditions unfavorable for DNA replication and may modulate the resistance of host bacterial strains to antibiotics.
Topics: Bacterial Proteins; Escherichia coli; Argonaute Proteins; Topoisomerase Inhibitors; Bacteria; Ciprofloxacin; DNA; Cell Division
PubMed: 37102866
DOI: 10.1128/spectrum.04146-22 -
Biology Jan 2024DNA gyrase is essential for the successful replication of circular chromosomes, such as those found in most bacterial species, by relieving topological stressors... (Review)
Review
DNA gyrase is essential for the successful replication of circular chromosomes, such as those found in most bacterial species, by relieving topological stressors associated with unwinding the double-stranded genetic material. This critical central role makes gyrase a valued target for antibacterial approaches, as exemplified by the highly successful fluoroquinolone class of antibiotics. It is reasonable that the activity of gyrase could be intrinsically regulated within cells, thereby helping to coordinate DNA replication with doubling times. Numerous proteins have been identified to exert inhibitory effects on DNA gyrase, although at lower doses, it can appear readily reversible and therefore may have regulatory value. Some of these, such as the small protein toxins found in plasmid-borne addiction modules, can promote cell death by inducing damage to DNA, resulting in an analogous outcome as quinolone antibiotics. Others, however, appear to transiently impact gyrase in a readily reversible and non-damaging mechanism, such as the plasmid-derived Qnr family of DNA-mimetic proteins. The current review examines the origins and known activities of protein inhibitors of gyrase and highlights opportunities to further exert control over bacterial growth by targeting this validated antibacterial target with novel molecular mechanisms. Furthermore, we are gaining new insights into fundamental regulatory strategies of gyrase that may prove important for understanding diverse growth strategies among different bacteria.
PubMed: 38392303
DOI: 10.3390/biology13020084 -
BioRxiv : the Preprint Server For... Oct 2023Fluoroquinolones (FQs) are potent antibiotics of clinical significance, known for their unique mechanism of action as gyrase poisons, which stabilize gyrase-DNA cleavage...
Fluoroquinolones (FQs) are potent antibiotics of clinical significance, known for their unique mechanism of action as gyrase poisons, which stabilize gyrase-DNA cleavage complexes and convert gyrase into a DNA-damaging machinery. Unfortunately, FQ resistance has emerged, and these antibiotics can cause severe side effects. Therefore, discovering novel gyrase poisons with different chemical scaffolds is essential. The challenge lies in efficiently identifying them from compound libraries containing thousands or millions of drug-like compounds, as high-throughput screening (HTS) assays are currently unavailable. Here we report a novel fluorescence-based, T5 exonuclease-amplified DNA cleavage assay for gyrase poison discovery. This assay capitalizes on recent findings showing that multiple gyrase molecules can simultaneously bind to a plasmid DNA molecule, forming multiple gyrase-DNA cleavage complexes on the same plasmid. These gyrase-DNA cleavage complexes, stabilized by a gyrase poison, can be captured using sarkosyl. Proteinase K digestion results in producing small DNA fragments. T5 exonuclease, selectively digesting linear and nicked DNA, can fully digest the fragmented linear DNA molecules and, thus, "amplify" the decrease in fluorescence signal of the DNA cleavage products after SYBR Green staining. This fluorescence-based, T5 exonuclease-amplified DNA cleavage HTS assay is validated using a 50-compound library, making it suitable for screening large compound libraries.
PubMed: 37904923
DOI: 10.1101/2023.10.16.562555 -
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 -
MSphere Oct 2023is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant is an urgent public health threat. Currently, the...
is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant is an urgent public health threat. Currently, the diagnosis of infection requires expensive laboratory infrastructure, while antimicrobial susceptibility determination requires bacterial culture, both of which are infeasible in low-resource areas where the prevalence of infection is highest. Recent advances in molecular diagnostics, such as pecific igh-sensitivity nzymatic eporter uning (SHERLOCK) using CRISPR-Cas13a and isothermal amplification, have the potential to provide low-cost detection of pathogen and antimicrobial resistance. We designed and optimized RNA guides and primer sets for SHERLOCK assays capable of detecting via the A gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A (A) gene. We evaluated their performance using both synthetic DNA and purified isolates. For A we created both a fluorescence-based assay and lateral flow assay using a biotinylated fluorescein reporter. Both methods demonstrated sensitive detection of 14 . isolates and no cross-reactivity with 3 non-gonococcal isolates. For A, we created a fluorescence-based assay that correctly distinguished between 20 purified isolates with phenotypic ciprofloxacin resistance and 3 with phenotypic susceptibility. We confirmed the A genotype predictions from the fluorescence-based assay with DNA sequencing, which showed 100% concordance for the isolates studied. We report the development of Cas13a-based SHERLOCK assays that detect and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates. IMPORTANCE the cause of gonorrhea, disproportionately affects resource-limited settings. Such areas, however, lack the technical capabilities for diagnosing the infection. The consequences of poor or absent diagnostics include increased disease morbidity, which, for gonorrhea, includes an increased risk for HIV infection, infertility, and neonatal blindness, as well as an overuse of antibiotics that contributes to the emergence of antibiotic resistance. We used a novel CRISPR-based technology to develop a rapid test that does not require laboratory infrastructure for both diagnosing gonorrhea and predicting whether ciprofloxacin can be used in its treatment, a one-time oral pill. With further development, that diagnostic test may be of use in low-resource settings.
Topics: Infant, Newborn; Humans; Neisseria gonorrhoeae; Gonorrhea; HIV Infections; Drug Resistance, Bacterial; Microbial Sensitivity Tests; Anti-Bacterial Agents; Ciprofloxacin
PubMed: 37732792
DOI: 10.1128/msphere.00416-23