In vitro inhibition of bacterial DNA gyrase by cinodine, a glycocinnamoylspermidine antibiotic

Abstract

Cinodine, a broad-spectrum glycocinnamoylspermidine antibiotic, binds to DNA and irreversibly inhibits bacterial and phage DNA synthesis. Cinodine was found to inhibit the activity of Micrococcus luteus DNA gyrase in vitro, but it did not inhibit the activities of two other DNA-binding enzymes, namely, topoisomerase I and BamHI. Although we cannot yet conclude that DNA gyrase is an intracellular target of the drug, in vitro inhibition of the enzyme by cinodine appears to be specific.Bacterial topoisomerase II (DNA gyrase) is an essential enzyme that is known to be the target of two classes of antibiotics. The coumarins, natural products which include coumermycin Al and novobiocin, inhibit gyrase probably by competing with ATP for binding to the B subunit of the enzyme (6, 11). The quinolones, synthetic products typified by nalidixic and oxolinic acids, target the A subunit of the enzyme and probably act by interfering with the DNArejoining step of the gyrase-mediated, DNA strand-passing reaction (1, 4, 5, 12; for recent reviews of topoisomerase inhibitors, see references 2 and 13).The broad-spectrum antibiotic cinodine, which is produced by a Nocardia species (3, 9), represents a class of antibiotics referred to as the glycocinnamoylspermidines.The three components of cinodine, ,B, -Yl, and Y2, are distinguished by the structure of the terminal pentose of the trisaccharide (Fig. 1). In previous studies of its mechanism of action (7), cinodine was shown to cause an immediate and irreversible cessation of DNA synthesis in growing cultures of Escherichia coli, whereas RNA synthesis remained unaffected for at least 30 min. Furthermore, equilibrium dialysis studies showed that cinodine was able to bind physically to DNA (7). In the present study, we investigated the mechanism of action of cinodine further, and we found that the drug inhibits bacterial DNA gyrase in vitro.DNA gyrase activity of Micrococcus luteus is inhibited by cinodine. The conversion of relaxed plasmid pBR322 DNA to the supercoiled form by M. luteus DNA gyrase was tested in the presence and absence of cinodine. Purified DNA gyrase from M. luteus was obtained from Bethesda Research Laboratories, Inc., Gaithersburg, Md. DNA gyrase was assayed in vitro for the ability to convert relaxed covalently closed circular DNA to the supercoiled form in the presence of ATP by the standard electrophoretic assay method of Otter and Cozzarelli (10). The relaxed substrate (0.5 ,ug of plasmid pBR322) was prepared by treating supercoiled plasmid DNA with topoisomerase I isolated from calf thymus (Bethesda Research Laboratories), according to the directions of the manufacturer, followed by three phenol-chloroform extractions (to remove the enzyme) and ethanol precipitation of the DNA. Gyrase assays were carried out in a total volume of 20 ,ul. Enzyme (1 U) was added to the assay mixture as the last step, to initiate the reaction; the mixture was then incubated at 30°C for 1 h and applied directly to a 0.8% agarose gel. Samples were electrophoresed at 150 * Correspondi...

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives

“…4 ) (Martin et al 1978 ; Tresner et al 1978 ); three different forms, β, γ 1 and γ 2 have been identified. It has been shown to inhibit bacterial DNA synthesis (Greenstein et al 1981 ) and DNA supercoiling by Micrococcus luteus gyrase in vitro (Osburne et al 1990 ). Cinodine-resistant mutants have been found to map to the gyrA gene (Osburne 1995 ), supporting the idea that gyrase is the target of cinodine in vivo.…”

Mode of action of GR122222X, a novel inhibitor of bacterial DNA gyrase

“…A 24-kDa N-terminal fragment of GyrB has been cloned and shown to contain the coumarin-drugbinding site (9). Other inhibitors, which target DNA gyrase but which are not members of the quinolone and coumarin families, include cinodine (11,21), microcin B17 (26), clerocidin (17), and cyclothialidine (10,20).…”

Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates

“…The enzyme has proven to be a good target for antimicrobial agents; the actions of the A subunits are inhibited by quinolones such as nalidixic acid and ciprofloxacin, while those of the B subunits are inhibited by coumarins such as coumermycin Al and novobiocin. The peptide antibiotic microcin B17 and the glycocinnamoylspermidine agent cinodine have also been shown to be inhibitors of DNA gyrase function (22,33). The contiguous gyrB-gyrA locus has been cloned and sequenced in Bacillus subtilis (19), Staphylococcus aureus (10), Mycoplasma pneumoniae (1), and Haloferax spp.…”