4a and the complete list of resistant strains generated and the sequences of their QRDR is shown in Supplementary Fig. Quinolones, coumarins, cyclothialidines, CcdB and microcin B17 inhibit DNA gyrase. 23, 10361040 (2000). The pellet was washed three times with sterile saline (0.9% NaCl in water) by repeated gentle suspension and centrifugation. Three independent replicates of each compound were performed. Blackwell Science Ltd, Oxford. All other author declare no competing financial interests. Concentrations were 0.01, 0.04, 0.017, 0.68, 2.7 and 10.8M, except for DNM, which was 8.9M for the highest concentration. Fluoroquinolone antibiotics. How many fluoroquinolones are approved for human use? J. Vet. MeSH Furthermore, no homologue of topoisomerase IV has been identified in the M. tuberculosis and Mycobacterium leprae genomes, indicating that the sole target of the quinolone family of inhibitors in these organisms is DNA gyrase. Chen, S. H., Chan, N. L. & Hsieh, T. S. New mechanistic and functional insights into DNA topoisomerases. Wang, S., Wang, Y., Shen, J., Wu, Y. In addition, the N-terminal half of GyrB hydrolyses ATP, and the C-terminal half is involved in binding to GyrA and DNA. Chemotherapy 57, 363371 (2011). Similarly, Bacillus anthracis, E. coli and A. baumannii also have the analogous serine mutated to leucine43,44,45. . At specified time points, mice were killed and blood was collected, centrifuged and the serum was frozen at 80C until analysis. The reaction mixtures were resolved on a 0.8% agarose gel in 40 mM Trisacetate buffer containing 1 mM EDTA. The animal studies (PK, in-vivo toxicity and in-vivo efficacy) were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. As representatives of cyclothialidines, the compound Ro 09-1437 and its derivative Ro 48-2865 were tested for their ability to inhibit the enzyme activity (Figure 1). DNA gyrase cleavage assays were performed as previously described with minor changes12,37,54. } Parkinson, E. I., Bair, J. S., Cismesia, M. & Hergenrother, P. J. Gyrase Docking 1. However, in mycobacteria, DNA gyrase is the only type II topoisomerase identified so far. When the effect of ciprofloxacin was tested on the supercoiling reaction, detectable inhibition (MIC) required 1.3 M of the drug, in agreement with earlier reports.38 In contrast, cleavage was doubled at concentrations (CC2) as low as 0.17 M (Figure 2 and Table 2). designed and executed the biological experiments including MIC determinations, sequencing of the QRDR of clinical isolates, in vitro analysis of DNA gyrase inhibition and development of resistant mutants. Biol. DNA gyrase, or simply gyrase, is an enzyme within the class of topoisomerase and is a subclass of Type II topoisomerases that reduces topological strain in an ATP dependent manner while double-stranded DNA is being unwound by elongating RNA-polymerase or by helicase in front of the progressing replication fork. (1998) J. Biol. Finally, we discovered a strong correlation between the nimB-promoter variant and a Thr82Ile mutation in DNA Gyrase A (GyrA), which confers fluoroquinolone resistance and has been linked to the . Mechanisms of Action and Resistance of Older and Newer Fluoroquinolones Expression of WT E. coli gyrase A and gyrase B was performed as previously described53. Extended spectrum of quinolone resistance, even to a potential latter third-generation agent, as a result of a minimum of two GrlA and two GyrA alterations in quinolone-resistant Staphylococcus aureus. DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. The site is secure. Jacoby, G. A. Mechanisms of resistance to quinolones. Relative cleavage is the amount of cleavage product seen in the presence of CcdB normalized to the intrinsic cleavage produced by the enzyme alone. 38, 24772479 (1994). Bair, J. S., Palchaudhuri, R. & Hergenrother, P. J. Chemistry and biology of deoxynyboquinone, a potent inducer of cancer cell death. Fluoroquinolones inhibit the ability of the enzymes to ligate cleaved DNA and result in single-and double-stranded DNA breaks. Ciprofloxacin (Cipro)/ofloxacin (Floxin): gonococcal infection. Inhibition of DNA synthesis by quinolones requires the targeted topoisomerase to have DNA cleavage capability, and collisions of the replication fork with reversible quinolone-DNA-topoisomerase complexes convert them to an irreversible form. A. they are proteins B. they have turnover numbers that can be in the thousands and more C. they form 68, 27662772 (2013). 3b. Further modifications of these compounds would be needed to enhance their potency. Deoxynybomycins inhibit mutant DNA gyrase and rescue mice infected with fluoroquinolone-resistant bacteria. 2022 Dec 20;66(12):e0092122. 95, 50035013 (1973). These ternary complexes, called cleaved complexes because the DNA moiety is broken, block replication, transcription, and bacterial growth. J. These enzymes catalyse the introduction of negative supercoils and the decatenation of interlinked chromosomes, respectively6,7,8. Therefore, a thorough evaluation of the effects of a range of inhibitors on DNA gyrase from a Gram-positive bacterium is imperative. effective in treating soft-tissue, bone, and [PMC free article] . 9 and Supplementary Table 5). return null; 1998 Apr 14;95(8):4652-7 Vila, J., Ruiz, J., Goni, P., Marcos, A. The site is secure. Interestingly, M. smegmatis gyrase was refractory to the plasmid-borne proteinaceous inhibitors CcdB and microcin B17. Unlike FQs that bind within the two active sites, it binds between the active sites stabilizing either an uncleaved or a single-stranded cleaved DNA. The mice received vehicle alone, 50mgkg1 CIP, or 50mgkg1 DNM-2 by oral gavage once daily for 10 days; n=15 for each group. 83, 37293731 (1961). Why is resistance to quinolones important? Werner, G. et al. HHS Vulnerability Disclosure, Help DNM was evaluated against both FQ-sensitive S. aureus (ATCC 29213) and FQR MRSA (NRS3, which has GyrA S84L and ParC S80F). A brief overview of the DNA gyrase structure is provided. CcdB and microcin B17 are both plasmid-encoded proteinaceous inhibitors produced by Gram-negative bacteria.16,17 CcdB has been shown to bind to GyrA of E. coli and stabilize the gyraseDNA complex in a manner reminiscent of, but not identical to, the quinolones.18 In agreement with this, mutations conferring resistance to CcdB map to GyrA.19 Like CcdB, microcin B17 also acts by stabilizing the gyraseDNA covalent complex; however, the exact mode of inhibition remains to be elucidated.20 In contrast to CcdB and quinolones, the only mutation in E. coli conferring resistance to microcin B17 maps to Trp-751 in GyrB. The pellet was dissolved in and dialysed against TGEM, and loaded on to a novobiocinSepharose column. 2019 Apr;85:308-318. doi: 10.1016/j.bioorg.2019.01.009. (c) Doseresponse curves for FQS S. aureus (ATCC 29213) and FQR S. aureus (NRS3) treated with DNM. The authors thank J. C. Wang and A. Maxwell for overexpressing constructs of E. coli topoisomerase I and DNA gyrase, respectively. 2022 Feb;148(2):461-473. doi: 10.1007/s00432-021-03625-3. 6). After 30 min, the reaction was stopped with 0.6% SDS. Both E. coli and M. smegmatis gyrase were susceptible to etoposide. FOIA 4b). Expression of S83L and S83R GyrA was performed identically to expression of the WT GyrA. Would you like email updates of new search results? generally resistant. 2). The highest concentration of the inhibitor that failed to show any detectable inhibition of the supercoiling activity was termed the maximal non-effective concentration (MNEC), whereas the minimum concentration that produced complete inhibition was termed the IC100. 39, 12011203 (1995). 2). Recent progress in the discovery and development of DNA gyrase B inhibitors. Paul J. Hergenrother. Because they may affect the development of cartilage, all fluoroquinolones are contraindicated in children, adolescents, and pregnant or breast-feeding women. E. coli strains ZK4 and ZK650 were the microcin B17 susceptible and producer strains, respectively.30, Novobiocin and ciprofloxacin (Sigma, St Louis, MO, USA) were dissolved in water and 0.1 M NaOH, respectively. inhibited;Anaerobes: Site-directed mutagenesis was carried out with the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent), according to the manufacturers instructions, with the modification that NEB Turbo Competent E. coli were used as the host strain. CC2 was defined as the concentration of inhibitor required to stimulate basal cleavage by two-fold while maximum cleavage represents the fold increase in cleavage in the presence of saturating concentrations of the inhibitor. Microbiol. nausea vomiting diarrhea. Despite this similarity in binding position, the phenotype of DNM in the DNA cleavage assay (that is, the buildup of OC DNA) suggests that its overall mode of inhibition is more similar to that of GSK299423. Antimicrob Agents Chemother 37: 126-127. Dis. Fractions containing pure protein were pooled and dialysed against TDEN buffer (50mM Tris-HCl pH 7.5, 5mM dithiothreitol (DTT), 1mM EDTA, 150mM NaCl) overnight at 4C, using a Slide-A-Lyzer Dialysis Cassette, 10 000 MWCO (Thermo Scientific) and concentrated to 0.51ml, using an Amicon Ultra-15 50K Centrifugal Filter Device. The proteins were renatured by step dialysis against TGEM containing 4, 3, 2, 1 and 0 M urea. listed as inventors. National Library of Medicine doi: 10.1128/mBio.01093-21. for(m=0; m