fluoroquinolones inhibit dna gyrase

Biomolecules. Epub 2019 Oct 26. 1994 Jun;47(6):872-901. doi: 10.2165/00003495-199447060-00003. 2014 Dec 19;9(12):2895-904. doi: 10.1021/cb500629k. Properties of the complexes are sensitive to quinolone structure and to topoisomerase amino acid substitutions associated with quinolone resistance. Antimicrob. At specified time points, mice were killed and blood was collected, centrifuged and the serum was frozen at 80C until analysis. 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. Fluoroquinolones (FQs) were introduced into the clinic in the early 1980s and since then have become one of the most widely prescribed classes of antibiotics1,2,3. However, no colonies were observed on co-treatment (resistance frequency <1.0 1010). However, DNM showed excellent activity against the FQR NRS3 (Minimum Inhibitory Concentration, MIC=0.03gml1, Fig 1b,c). Other gyrase inhibitors include ribosomally synthesized proteinaceous poisons like microcin B17, CcdB and cyclic peptide cyclothialidines. Deoxynybomycins inhibit mutant DNA gyrase and rescue mice infected with fluoroquinolone-resistant bacteria. 38, 24772479 (1994). The .gov means its official. 2019 Dec;26(34):34830-34853. doi: 10.1007/s11356-019-06482-3. Federal government websites often end in .gov or .mil. Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria. . Data shown are from three independent replicatess.e.m. Drugs. information contained herein is in every respect accurate or complete. Would you like email updates of new search results? Agents 35, 119125 (2010). Drugs. Federal government websites often end in .gov or .mil. Reaction mixtures containing gyrase, plasmid DNA, and derivatives of ciprofloxacin were incubated for 45 min at 37 C followed by an additional 5-min incubation at the indicated temperature. Half-lives range from 3 h The antibacterial activity of the preparation was determined as described previously.31. Int. CAS Our results (Table 2) show the ability of etoposide to trap the gyraseDNA complex. 8a). Vernel-Pauillac, F., Hogan, T. R., Tapsall, J. W. & Goarant, C. Quinolone resistance in Neisseria gonorrhoeae: rapid genotyping of quinolone resistance-determining regions in gyrA and parC genes by melting curve analysis predicts susceptibility. until A600 reached 0.40.6. Blais J, Dean CR, Lapointe G, Leeds JA, Ma S, Morris L, Moser HE, Osborne CS, Prosen KR, Richie D, Skepper C, Thompson K, Vo J, Yue Q, Rivkin A. Antimicrob Agents Chemother. This is followed by passage of another duplex through this gate and finally resealing of the break. joint infections by multidrug resistant strains All animals were housed in a pathogen-free environment and received sterile food and water. M. smegmatis SN2 cells were used for purification of DNA gyrase. 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. GSK299423 is a recently discovered DNA gyrase inhibitor that is hypothesized to stabilize the DNAenzyme complex, either pre-cleavage or after the formation of a single-strand break resulting in a buildup of OC DNA38. sharing sensitive information, make sure youre on a federal This data combined with previously published data showing that DNM is non-toxic to normal (that is, non-cancerous) cell lines41 suggests that these compounds would probably be well tolerated in-vivo. Blackwell Science Ltd, Oxford. The therapeutic use of DNA gyrase inhibitors, mainly quinolone antibacterials, has proven to be a tremendous success story in the treatment of bacterial infections. 1b). Cleaved complexes readily form in vitro when gyrase, plasmid DNA, and quinolone are combined and incubated; complexes are detected by the linearization of plasmid DNA, generally assayed by gel electrophoresis. 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. and the National Institutes of Health (NIH) Grant R01HL090699 (G.W.L.). DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. Therefore, a thorough evaluation of the effects of a range of inhibitors on DNA gyrase from a Gram-positive bacterium is imperative. Supplementary Figures 1-10, Supplementary Tables 1-7, Supplementary Note 1, Supplementary Methods and Supplementary References (PDF 4516 kb), This work is licensed under a Creative Commons Attribution 4.0 International License. Biol. Although different substitution patterns were found for MRSA ParC, all sequenced strains have the same mutation in GyrA (S84L), consistent with the notion that this mutation sensitizes bacteria to DNM. 269, 22606-22614). However, the molecular factors that subsequently generate DNA double-strand breaks from the irreversible complexes and that probably initiate cell death have yet to be defined. All lesions were characterized, recorded and scored for severity (minimal=1, mild=2, moderate=3, marked=4, and severe=5). For full access to this pdf, sign in to an existing account, or purchase an annual subscription. 2016 Apr 20;44(7):3304-16. doi: 10.1093/nar/gkw161. In addition, minimal change was observed with 5M CIP at up to 1.5h (Fig. However, the FQ stabilization of a single-strand break causes an even faster second cleavage event that is also stabilized by FQs, thus explaining the rapid buildup of linear DNA37. DNM derivatives were evaluated against both FQS S. aureus (ATCC 29213) and FQR MRSA (NRS3), and their MIC values are listed in Fig. PK parameters were determined using GraphPad Prism Version 5.00 for Windows. However, P. aeruginosa is similar to VRE in that the Thr is mutated to an Ile in FQR strains. PMC Alternatively, when either DNM or DNM-2 was incubated with WT DNA gyrase, neither showed similar increases in L or OC DNA, suggesting that these compounds are poor inhibitors of WT DNA gyrase (Fig. Rinehart, K. L. & Renfroe, H. B. Quinolones and the Clinical Laboratory | HAI | CDC Microbiol. } Thank you for visiting nature.com. of Pseudomonas and Enterobacter. Using a synthetic route building on a palladium-catalysed mixed cross-coupling and a methylene bridge insertion, hundreds of milligrams of the natural-product DNM were prepared as described herein. The authors thank J. C. Wang and A. Maxwell for overexpressing constructs of E. coli topoisomerase I and DNA gyrase, respectively. Drlica K, Mustaev A, Towle TR, Luan G, Kerns RJ, Berger JM. Forty millilitres of an overnight bacterial culture was centrifuged at 3,000 g for 10min and resuspended in 0.4ml of sterile PBS. An update of their pharmacology and therapeutic use. Redgrave, L. S., Sutton, S. B., Webber, M. A. The calibration curve (measured by ultraviolet absorbance) was linear over this range. Inhibition of DNA gyrase blocks The column was washed with TGEM and the holoenzyme was eluted with 5 M urea. Epub 2019 Feb 11. Get the most important science stories of the day, free in your inbox. Whilst active against both Gram-negative and -positive bacteria, the emergence of resistance has affected the clinical effectiveness of fluoroquinolones in both groups. Chem. Please enable it to take advantage of the complete set of features! (c) Doseresponse curves for FQS S. aureus (ATCC 29213) and FQR S. aureus (NRS3) treated with DNM. A problem with novel antibacterials is that bacterial resistance typically necessitates the development of a new drug to treat those drug-resistant pathogens. Agents Chemother. Chen, S. H., Chan, N. L. & Hsieh, T. S. New mechanistic and functional insights into DNA topoisomerases. A brief overview of the DNA gyrase structure is provided. Mermer A, Faiz O, Demirbas A, Demirbas N, Alagumuthu M, Arumugam S. Bioorg Chem. Am. Mutations are selected first in the more susceptible target: DNA gyrase, in gram-negative bacteria, or topoisomerase IV, in gram-positive bacteria. & Howells, A. J. The supernatant was batch loaded onto 1ml of 1:1 Ni-NTA agarose (Qiagen) at 4C for 30min with inversion. Similarly, E. coli DNA gyrase was susceptible to microcin B17 whereas the M. smegmatis enzyme was resistant (data not shown). Introduction Quinolones are potent antibacterial drugs that are used in the treatment of a wide range of bacterial infections. National Library of Medicine MeSH Dual small-molecule targeting of procaspase-3 dramatically enhances zymogen activation and anticancer activity. gram negative cocci and bacilli: Enterobacteriaceae, Pseudomonas, With increasing amounts of CcdB, there was a concentration-dependent increase in the cleaved product with E. coli DNA gyrase (Figure 3a and Table 2). The https:// ensures that you are connecting to the In general, compounds with a single methyl addition retained good activity against NRS3 (25). J. Clin. Drugs. S, supercoiled DNA; OC, open-circular DNA; L, linear pBR322 DNA. 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. Am. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). The use of first-generation quinolones (such as nalidixic acid and pipemidic acid) was limited due to their narrow spectrum activity and low serum levels. Doxorubicin was used as a positive control. In addition to vehicle controls, wells lacking either DNA or EtBr were also used to ensure that these did not have an effect on fluorescence. This enhanced its activity against bacteria, and improved its absorption, metabolism, and excretion characteristics. Bookshelf Fluoroquinolones inhibit both DNA gyrase and topoisomerase IV by interaction with GyrA and ParC, respectively. Although doxorubicin inhibited human topoisomerase II at concentrations as low as 3M, DNM-2 showed no significant inhibition at concentrations up to 30M (Supplementary Fig. Soc. 2022 Nov 17;27(22):7993. doi: 10.3390/molecules27227993. Although M. smegmatis DNA gyrase had lower affinity for etoposide, it showed higher levels of cleavage under saturating concentrations of the drug. scientific or other professionals. A low-level CIP-resistant strain (29213-C1) was used in these studies. DNM and DNM-2 induce only small increases in L DNA with S83L DNA gyrase (Fig. 2). Grohs, P., Houssaye, S., Aubert, A., Gutmann, L. & Varon, E. In vitro activities of garenoxacin (BMS-284756) against Streptococcus pneumoniae, viridans group streptococci, and Enterococcus faecalis compared to those of six other quinolones. A variety of inhibitors have been found to interfere with specific enzymic reactions of DNA gyrase, rendering it inactive.46 Two major families of compounds that inhibit E. coli DNA gyrase are quinolones and coumarins. Various inhibitors interfere with one or more of the substeps in the reaction. DNM was also evaluated against a panel of Gram-negative bacteria (Supplementary Table 2). Infection 40, 239262 (2012). Willmott, Christopher J. R . Cite this article. Consequently, studies of cleaved complexes can be used to identify improvements in quinolone structure and to understand the biochemical basis of target-based resistance. Fluoroquinolones have been used with limited success as part of a second-line chemotherapeutic regime against mycobacterial diseases. Type II topoisomerasesinhibitors, repair mechanisms and mutations Sanfilippo, C. M., Hesje, C. K., Haas, W. & Morris, T. W. Topoisomerase mutations that are associated with high-level resistance to earlier fluoroquinolones in Staphylococcus aureus have less effect on the antibacterial activity of besifloxacin. Med. and transmitted securely. sharing sensitive information, make sure youre on a federal The quinolones are potent antibacterials that act by forming complexes with DNA and either gyrase or topoisomerase IV. Full synthetic routes along with experimental details and characterization data can be found in the Supplementary Methods. Before Bax, B. D. et al. growing cartilage (not recommend for use in ADS Chem. Primers for mutagenesis were designed using QuikChange Primer Design (Agilent) and their sequences can be found in Supplementary Table 7. Quinolone Antibiotics: Resistance and Therapy. PMC The fluoroquinolone class of antibiotics promises to become as diverse and as important as -lactam agents. Antimicrob. P. aeruginosa differs in that it naturally has a Thr instead of the Ser. It was found to be well tolerated when dosed either subcutaneously, orally or by intraperitoneal injection27. Natl Acad. Epub 2019 Jul 10. . 4a). 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. Adelmann, S., Baldhoff, T., Koepcke, B. Forbis, R. M. & Rinehart, K. L. Nybomycin.4. 2023 Feb 16:rs.3.rs-2525765. Cleaved complexes; DNA gyrase; DNA topoisomerase; Fluoroquinolone; Plasmid DNA isolation; Quinolone. Gyrase Docking 1. An official website of the United States government. Recent advances in DNA gyrase-targeted antimicrobial agents patients. 52, 29993005 (2008). The mice received vehicle alone, 50mgkg1 CIP, or 50mgkg1 DNM-2 by oral gavage once daily for 10 days; n=15 for each group. 82, 139170 (2013). Linear product was revealed by addition of 0.2% SDS and 0.1gml1 proteinase K for 30min at 37C. The https:// ensures that you are connecting to the Corresponding author. (, Lewis, R. J., Tsai, F. T. F. & Wigley, D. B. Concurrent administration of theophylline and MeSH 1a)34. Agents Chemother. Antimicrob Agents Chemother 37: 126-127. n+=2) { elm=FP_getObjectByID(args[n]); if(elm) { doc.$imgSwaps[doc.$imgSwaps.length]=elm; Supercoiling reactions were performed in the presence of various concentrations of cyclothialidine Ro 48-2865 (lanes 210) with (a) E. coli gyrase and (b) M. smegmatis gyrase. An oral drug for MRSA and VRE is a well-recognized clinical need and DNM-2 has tremendous promise in this regard. This study systematically investigated the performance of MeltPro and next-generation sequencing in the diagnosis of fluoroquinolone (FQ) resistance among multidrug-resistant tuberculosis patients and explored the relationship between nucleotide alteration and the level of phenotypic susceptibility Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Google Scholar. Correlation of antimicrobial resistance with beta-lactamases, the OmpA-like porin, and efflux pumps in clinical isolates of Acinetobacter baumannii endemic to New York City. On treatment with either CIP or DNM-2, resistant colonies were observed. 68, 27662772 (2013). Commun. The ability of CIP, DNM and DNM-2 to inhibit S83L or S83R DNA gyrase was then determined. Antimicrob. Finally, 1U of human topoisomerase (1l of 1Ul1 stock) is added to each tube for a final volume of 20l. Eur. CIP was much less effective at inhibiting either S83L or S83R DNA gyrase compared with WT, with only small increases in L DNA being observed (Fig. Epub 2019 Jan 8. Goldmeier MN, Khononov A, Belakhov V, Pieko T, Orbach N, Gilad Barzilay Y, Baasov T. J Med Chem. A single point mutation in the DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyrase-DNA complex. As shown in Fig. (3) DNM-2 is extremely well tolerated in mice, with no signs of toxicity at the dose levels tested. (norfloxacin (Noroxin) and ciprofloxacin (Cipro)) Chemotherapy 56, 153157 (2010). government site. Monit. FOIA Briefly, pTRCHisA-GyrA, pTRCHisA-GyrAS83L, pTRCHisA-GyrAS83R, or pTRCHisA-GyrB were introduced into One Shot BL21 Star (DE3) (NEB) by chemical transformation. 95, 50035013 (1973). administration. Reactions are stopped by the addition of 20l of 24:1 chloroform:isoamyl alcohol and 20l of stop dye (40% sucrose, 1mM EDTA, 100mM Tris-HCl pH 7.5, 0.5gml1 bromophenol blue). All animals were housed in a pathogen-free environment and received sterile food and water. This leads to cell death and turns out to be a very effective way of killing bacteria. & Crouzet, J. Full details of the sensitivity of each strain can be found in Supplementary Tables 3 and 4. The bioavailability of these compounds mirrors the aqueous solubility, suggesting that at least for this limited set of compounds aqueous solubility could be a reasonable predictor of oral bioavailability. High abundance and diversity of antimicrobial resistance determinants among early vancomycin-resistant Enterococcus faecium in Poland. Mycobacterium tuberculosis DNA gyrase as a target for drug - PubMed Int J Mol Sci. J. In addition, in this study we found that orally administered DNM-2 is effective in treating mice infected with MRSA, thus showing the first in-vivo efficacy for this class of compounds. DNA gyrase cleavage assays were performed as previously described with minor changes12,37,54. Guillemin, I., Sougakoff, W., Cambau, E., Revel-Viravau, V., Moreau, N. & Jarlier, V. (, Maki, S., Takiguchi, S., Miki, T. & Horiuchi, T. (, Guillemin, I., Jarlier, V. & Cambau, E. (, Anderson, V. E., Zaniewski, R. P., Kaczmarek, F. S., Gootz, T. D. & Osheroff, N. (, Oxford University Press is a department of the University of Oxford. Unauthorized use of these marks is strictly prohibited. Longer or more alkyl chains do not display similar increases in aqueous solubility probably because of the increased hydrophobicity of these compounds. 47, 35423547 (2003). Cleavage reactions were carried out with M. smegmatis gyrase in the presence of various ciprofloxacin concentrations (lanes 15). 1999 Apr;6(4):322-6 Discovery of Novel Inhibitors of Bacterial DNA Gyrase Using a QSAR National Library of Medicine The https:// ensures that you are connecting to the (1996). Evolution of resistance to a tricyclic pyrimidoindole DNA gyrase Next, after a selection round with DNM, a FQS/DNM-resistant strain was found. patients under 18 years old); however, since such Curing bacteria of antibiotic resistance: reverse antibiotics, a novel class of antibiotics in nature. Keywords: Although inhibitors like quinolones and their derivatives inhibit the overall DNA supercoiling activity of the enzyme, this inhibition and their cytotoxicity is a consequence of trapping the gyraseDNA complexes.1114 This is exemplified in the case of CcdB and microcin B17, which do not inhibit the supercoiling reaction of DNA gyrase but trap the gyraseDNA cleavage complex.20,39 Therefore, the extent of accumulation of the cleaved DNA in the presence of such inhibitors is a direct measure of the efficacy of the compound, and enables a comparison of all inhibitors that act by stabilizing the cleavage complex.
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