Glycosylated Flavones as Selective Inhibitors of Topoisomerase IV

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/97/$04.0010

May 1997, p. 992–998 Vol. 41, No. 5

Copyright © 1997, American Society for Microbiology

Glycosylated Flavones as Selective Inhibitors of Topoisomerase IVFRANCOIS-XAVIER BERNARD,1 SERGE SABLE,2 BEATRICE CAMERON,3 JEAN PROVOST,4

JEAN-FRANCOIS DESNOTTES,1 JOEL CROUZET,3 AND FRANCIS BLANCHE3*

Antibacterial Program1 and Departments of Structural Analysis,2 Molecular Microbiology,3 andNatural Products,4 Rhone-Poulenc Rorer S.A., 94403 Vitry-sur-Seine Cedex, France

Received 3 October 1996/Returned for modification 16 December 1996/Accepted 4 March 1997

Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolatedfrom cottonseed flour and identified as quercetin 3-O-b-D-glucose-[1,6]-O-a-L-rhamnose (rutin), quercetin3-O-b-D-galactose-[1,6]-O-a-L-rhamnose, and quercetin 3-O-b-D-glucose (isoquercitrin). The most active one(rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64mg/ml) and induced the SOS response of a permeable E. coli strain. Derivatives of quercetin glycosylated atposition C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped byDNA sequence analysis to the gene encoding resistance to tetracycline. Cleavage at these sites was hardlydetectable in cleavage reactions with quercetin or fluoroquinolones. None of the three flavonoids isolated fromcottonseeds had any stimulatory activity on E. coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV. These results show that selec-tive inhibitors of topoisomerase IV can be derived from the flavone structure. This is the first report on a DNAtopoisomerase inhibitor specific for topoisomerase IV.

Two type II DNA topoisomerases have been identified inEscherichia coli, namely DNA gyrase and topoisomerase IV.These two enzymes show considerable amino acid sequencesimilarity, yet the two enzymes have distinctive topoisomeriza-tion and biological activities. Gyrase can negatively supercoilrelaxed or positively constrained DNA generated ahead of thereplication and transcription machineries (34), whereas topo-isomerase IV can unlike the catenated daughter chromosomesduring the terminal stages of the replication process (1, 24).

Both gyrase and topoisomerase IV are targets for the quin-olones (17, 25, 32), a family of synthetic antibiotics which exerttheir bactericidal effect by stabilizing the covalent topoisomer-ase-DNA reaction intermediate, known as the cleavable com-plex. By trapping this transient complex, these drugs, oftenreferred to as topoisomerase poisons, introduce latent lesionsinto intracellular DNA. Ultimately, these lesions can be con-verted into irreversible DNA breaks upon DNA replication orother cellular processes involving DNA. Cleavable complexescan be detected in vitro by the addition of a detergent, whichinduces irreversible cleavage of double-stranded DNA at spe-cific sites. During the last 20 years, thousands of quinolonederivatives have been synthesized, leading to the most active ofquinolones, the fluoroquinolones. However, the relatively highcytotoxicity of these drugs and, above all, the spread of quin-olone resistance increasingly limit the use of quinolones andrevive the interest in new series of topoisomerase poisons thatcould escape these limitations. Besides the large family ofquinolones, several natural compounds such as novobiocin(35), clerocidin (19), saintopin (7), flavones (23), and the pep-tides microcin B17 (33) and CcdB (6) have been shown totarget DNA gyrase. However, these compounds except for thetwo peptides are toxic for eukaryotic cells and/or exhibit noselectivity for the bacterial enzyme and target eukaryotic topo-isomerases as well (7, 9), thereby precluding their use as anti-bacterial therapeutic agents.

E. coli topoisomerase IV is less sensitive to quinolones and

other type II topoisomerase poisons than is gyrase in vitro (15,25), and substantial evidence indicates that topoisomerase IVis secondary to gyrase as a target of quinolone drugs in vivo(18). Yet, in E. coli resistance to quinolone antibiotics arisesvia mutations in the gyrase genes (14). By contrast, geneticstudies (10, 11, 21) have shown that in Staphylococcus aureus,topoisomerase IV is the primary target of commonly usedfluoroquinolones such as ciprofloxacin or sparfloxacin, and bio-chemical studies have shown that topoisomerase IV is moresensitive than gyrase to topoisomerase II-targeted drugs (7). Inthese two microorganisms however, compounds inhibiting gy-rase also inhibit topoisomerase IV and vice versa, and at leastin E. coli, in which this has been studied in detail, there is apositive correlation between the inhibitory activities of quino-lones on DNA gyrase supercoiling and topoisomerase IV de-catenation (15).

From the screening of natural products for type II topoisom-erase inhibitors, we discovered that glycosylated flavones pres-ent in cottonseed flour were selective poisons of E. coli topo-isomerase IV. The most active compound was purified andidentifiedasrutin(quercetin3-O-b-D-glucose-[1,6]-O-a-L-rham-nose). In this report, we describe the inhibitory properties ofrutin and the relative contributions of the aglycone and of thesugar moieties on the selectivity towards topoisomerase IVinhibition.

MATERIALS AND METHODS

Isolation from cottonseed flour of flavones which stimulate topoisomeraseIV-mediated DNA cleavage. Cottonseed flour (200 g) was first extracted withchloroform, and the extract was discarded. It was extracted again with methanol.The methanol extract was concentrated, and the oily residue (14 g) was dissolvedin 5 ml of methanol, diluted with 150 ml of water, and then applied to a columnof LiChroprep RP18 (20 3 300 mm; Merck) which was washed with 10%aqueous methanol. Fractions which stimulated topoisomerase IV-dependentDNA cleavage (eluted with 40% aqueous methanol) were pooled and evapo-rated to dryness under reduced pressure to give 370 mg of a yellow powder. Thismaterial was dissolved in 5% aqueous methanol and further purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Nucleosil 7C18 column (250 3 10 mm; Macherey-Nagel) eluted with a 5-to-100% gradientof methanol in water. The active fractions were pooled and concentrated todryness, to give 110 mg of active material which was further chromatographed in5-mg aliquots on the above-mentioned HPLC column eluted with a 15-to-25%gradient of tetrahydrofuran in 2% aqueous acetic acid over a period of 38 min at

* Corresponding author. Phone: (33) 1 55 71 80 93. Fax: (33) 1 55 7180 58. E-mail: [email protected].

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a flow rate of 3.0 ml/min. The latter HPLC system resolved the activity into threewell-separated species, initially named A (23.3 mg), B (9.4 mg), and C (8.2 mg),with retention times of 28.0, 30.0, and 35.2 min, respectively. The other flavonederivatives used in this study were from Sigma.

Enzymes and enzyme assays. Topoisomerase II was purified from calf thymusas described previously (30). E. coli DNA gyrase (6,000 U/mg) was reconstitutedfrom the GyrA and GyrB subunits purified to homogeneity from E. coli JMtacA(JM109[pPH3]) and JMtacB(JM109[pAG111]), respectively (12). E. coli topo-isomerase IV was purified to homogeneity as described previously (25), to aspecific activity of 20,000 U/mg (7), in decatenation reactions. Decatenationreactions (with kinetoplast DNA as the substrate) and topoisomerase-mediatedDNA cleavage reactions (with pBR322 DNA that was 39 end labeled EcoRIlinearized) were carried out as described previously (7). DNA quantification wasachieved by scanning densitometry (7), and each result obtained was expressedas the drug concentration at which 50% of the linear plasmid substrate wascleaved at least once (CC50).

Induction of the SOS response. The SOS response elicited by flavones wasdetected in a qualitative top-agar assay with E. coli G4420 [D(lacIPOZYA) X74galU galK strAr hsdR envA1 zac-3051/Tn10 dinD1::MudI1734(Kmr lac)] a per-meable E. coli strain. This strain was obtained by P1-mediated cotransduction ofthe envA1 allele from strain D22 (22) with a Tn10 insertion as a marker. Theinduction of an SOS response appeared as b-galactosidase activity (28). Antimi-crobial activity was detected as a circular growth inhibition area. MICs for E. coliD22 and S. aureus FDA 209-P were determined according to the NationalCommittee for Clinical Laboratory Standards protocol (20).

Labeling of DNA fragments for topoisomerase IV-dependent cleavage siteanalysis. pBR322 DNA was linearized by digestion with SalI, dephosphorylatedwith alkaline phosphatase, and 59 32P end labeled with T4 polynucleotide kinase(31). After purification, the labeled linear (SalI) plasmid was incubated withtopoisomerase IV and rutin under conditions similar to those used for cleavagereactions with EcoRI-linearized pBR322 DNA.

DNA sequencing and analysis. The DNA sequence analysis from the SalIrestriction site of pBR322 was performed by the dideoxy chain terminationmethod using Sequenase (USB), [a-32P]dATP, and primer 59-TCGACCGATGCCCTTGAGAGCCTTCAA-39 (located from position 652 to 678 in the se-quence of pBR322 [31]). Sequencing reaction mixtures along with 32P-labeledDNA fragments generated in topoisomerase IV-mediated cleavage reactionsinduced by rutin were electrophoresed through a 6% polyacrylamide sequencinggel (31).

Structural analysis of flavones. All nuclear magnetic resonance (NMR) spec-tra were obtained in dmso-d6 at 383 K with a Brucker DMX 600 spectrometeroperating at 600 and 150 MHz for 1H and 13C, respectively (dmso dH, 2.54 for1H; dmso dC, 77.5 for 13C). Electrospray mass spectra were recorded on a SciexAPI-III spectrometer. Samples dissolved in water-methanol-acetic acid (50:50:1,vol/vol/vol) (final concentration, 1 to 100 pmol/ml) were introduced at a flow rateof 5 ml/min into the ion source (5,000 V) via a 100-mm-inside-diameter silicacapillary tube.

RESULTS

Isolation and structure elucidation of active metabolites.From the screening of natural products for bacterial type IItopoisomerase inhibitors, we discovered that cottonseed flourpresent in some culture media used for growing microorgan-isms contained metabolites which stimulated topoisomeraseIV-mediated DNA cleavage. Following solvent extraction andRP column chromatography, a fraction containing the majorpart of activity was obtained. This chromatographic fractionwas shown to be made up of three structurally related com-pounds, originally named A, B, and C, which were separatedfrom each other by HPLC. Compounds A, B, and C exhibitedvery similar UV-visible spectra in 2% aqueous acetic acid, withlmax at 256.8 and 357.6 nm (compound A), 256.8 and 359.5 nm(compound B), and 256.8 and 358.6 nm (compound C). Com-pound A had an extinction coefficient of 16,000 M21 cm21 at357.6 nm.

Mass spectra of compound A showed a molecular ion at m/z611[M1H]1, and this molecular weight together with 13C NMRanalysis led to the empirical molecular formula C27H30O16.Compound A was shown by mass spectrometry-mass spec-trometry to contain two hexose units (m/z 5 300) which wereidentified as b-D-glucose and a-L-rhamnose by a combinationof COSY (29), TOCSY (3), NOESY (16), HMQC (4), andheteronuclear multiple-bond connectivity (5) experimentswhile the aglycone moiety was identified as quercetin by a

combination of two-dimensional NMR experiments. 1H-13Clong-range correlations showed that the hexose units wereattached to position 3 of quercetin. The structure of compoundA was therefore quercetin 3-O-b-D-glucose-[1,6]-O-a-L-rham-nose, also known as rutin (13).

1H NMR (dmso-d6 with a few drops of methanol-d4) (d inparts per million): 1.05 (d, J 5 6.5 Hz, 3H: CH3 in 6 ofa-rhamnose), 3.10 (t, J 5 9 Hz, 1H: H-4 of b-glucose), 3.12 (t,J 5 9 Hz, 1H: H-4 of a-rhamnose), 3.34 (m, 3H: H-2, H-3, andH-5 of b-glucose), 3.38 and 3.76 (2bd, Jab511 Hz, 1H each:CH2O in 6 of b-glucose), 3.39 (m, 2H: H-3 and H-5 of a-rham-nose), 3.46 (m, J very small, 1H: H-2 of a-rhamnose), 4.48 (bs,1H: H-1 of a-rhamnose), 5.32 (d, J58 Hz, 1H: H-1 of b-glu-cose), 6.23 and 6.41 (2 bs, 1H each: H-6 and H-8), 6.89 (d,J58.5 Hz, 1H: H-59), 7.56 (dd, J 5 8.5 and 1.5 Hz, 1H: H-69),7.60 (d, J 5 1.5 Hz, 1H: H-29).

13C NMR (d in parts per million): 17.5 (C-6 of a-rhamnose),67 (C-6 of b-glucose), 68.2 (C-5 of a-rhamnose), 70 (C-3 ofa-rhamnose), 70.3 (C-4 of b-glucose), 70.5 (C-2 of a-rham-nose), 71 (C-4 of a-rhamnose), 74 (C-2 of b-glucose), 75.9 and76.4 (C-3 and C-5 of b-glucose), 93 (C-8), 98 (C-6), 101 (C-1 ofa-rhamnose), 102 (C-1 of b-glucose), 104 (C-4a), 115 (C-59),116 (C-29), 1.21 (C-19 and C-69), 133 (C-3), 144 (C-39), 148(C-49), 156 (C-2 and C-8a), 161 (C-5), 163.5 (C-7), 177 (C-4).

Compound B was shown to be closely related to compoundA when analyzed by mass spectrometry and NMR. The onlydifference observed was an inversion of configuration at posi-tion 4 in the glucose unit. Therefore, the structure of thiscompound was quercetin 3-O-b-D-galactose-[1,6]-O-a-L-rham-nose.

1H NMR (d in parts per million): 1.13 (d, J 5 6.5 Hz, 3H:CH3 in 6 of a-rhamnose), 3.19 (t, J 5 9 Hz, 1H: H-4 ofa-rhamnose), 3.37 and 3.69 (2 dd, J 5 10.5 and 6 Hz, 1H each:CH2O in 6 of b-galactose), 3.41 (dd, J 5 9 and 3 Hz, 1H: H-3of a-rhamnose), 3.43 (m, 1H: H-5 of a-rhamnose), 3.46 (dd,J 5 9 and 3.5 Hz, 1H: H-3 of b-galactose), 3.52 (m, J very small,1H: H-2 of a-rhamnose), 3.58 (bt, J 5 6 Hz, 1H: H-5 ofb-galactose), 3.64 (dd, J 5 9 and 8 Hz: H-2 of b-galactose),3.69 (bd, J 5 3.5 Hz, 1H: H-4 of b-galactose), 4.51 (bs, 1H: H-1of a-rhamnose), 5.29 (d, J 5 8 Hz, 1H: H-1 of b-galactose),6.23 and 6.43 (2 bs, 1H each: H-6 and H-8), 6.88 (d, J 5 8.5 Hz,1H: H-59), 7.57 (d, J 5 1.5 Hz: H-29), 7.36 (dd, J 5 8.5 and 1.5Hz, 1H: H-69).

13C NMR (d in parts per million): 17 (C-6 of a-rhamnose),65 (C-6 of b-galactose), 68 (C-5 of a-rhamnose and C-4b ofb-galactose), 70 (C-2 of a-rhamnose), 70.5 (C-3 of a-rham-nose), 71 (C-2 of b-galactose), 72 (C-4 of a-rhamnose), 73 (C-3of b-galactose), 73.5 (C-5 of b-galactose), 93 (C-8), 98 (C-6),100 (C-1 of a-rhamnose), 102 (C-1 of b-galactose), 104 (C-4a),115 (C-59), 116 (C-29), 1.21 (C-19 and C-69), 133 (C-3), 144(C-39), 148 (C-49), 156 (C-2 and C-8a), 161 (C-5), 163.5 (C-7),177 (C-4).

Compound C, which showed a molecular ion at m/z 5 463,compatible with a monoglycoside derivative of quercetin, wasidentified by the same combination of NMR techniques as thatused to identify rutin. Compound C was shown to be quercetin3-O-b-D-glucose, also known as isoquercitrin.

1H NMR (d in parts per million): 3.16 (ddd, J 5 9, 5.5, and2.5 Hz, 1H: H-5 of b-glucose), 3.20 (t, J 5 9 Hz, 1H: H-4 ofb-glucose), from 3.25 to 3.35 (m, 2H: H-2 and H-3 of b-glu-cose), 3.43 and 3.63 (2 dd, J 5 11.5 and 5.5 Hz and J 5 11.5 and2.5 Hz, respectively, 1H each: CH2O in 6 of b-glucose), 5.39 (d,J 5 7.5 Hz, 1H: H-1 of b-glucose), 6.23 and 6.43 (2 d, J 5 1.5Hz, 1H each: H-6 and H-8, 6.89 (d, J 5 8.5 Hz, 1H: H-59), 7.58(dd, J 5 8.5 and 1.5 Hz, 1H: H-69), 7.63 (d, J 5 1.5 Hz, 1H:H-29).

VOL. 41, 1997 TOPOISOMERASE IV INHIBITION BY RUTIN 993

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13C NMR (d in parts per million): 61 (C-6 of b-glucose), 70(C-4 of b-glucose), 74 (C-2 of b-glucose), 76 (C-5 of b-glu-cose), 76.5 (C-3 of b-glucose), 93 (C-8), 98 (C-6), 101.5 (C-1 ofb-glucose), 104 (C-4a), 115 (C-59), 116 (C-29), 1.21 (C-19 andC-69), 133 (C-3), 144 (C-39), 148 (C-49), 156 (C-2 and C-8a),161 (C-5), 163.5 (C-7), 177 (C-4).

Inhibition of topoisomerase IV-dependent reaction. Amongthe three flavones, rutin was clearly the most potent in stimu-lating topoisomerase IV-dependent DNA cleavage (CC50 5 1mg/ml) (Fig. 1), followed by quercetin 3-O-b-D-galactose-[1,6]-O-a-L-rhamnose, which was fivefold less effective. Isoquerci-trin was very weakly active. Both commercially available rutinand our sample exhibited the same activity in cleavage reac-tions (data not shown). In addition to stabilizing the cleavablecomplex, rutin also blocked the catalytic activity of topoisomer-ase IV. The decatenation activity of topoisomerase IV wasinhibited by rutin (50% inhibitory concentration of 64 mg/ml)(Fig. 2).

Both quercetin and its disaccharide derivative rutin stimu-lated topoisomerase IV-associated DNA breaks (Fig. 3), sug-gesting that the presence of sugar residues at position C-3 hadno effect on inhibitory activity. By contrast, whereas quercetinwas potent in cleavage reactions with E. coli gyrase or calfthymus topoisomerase II, rutin was very weakly active withgyrase (at 50 mg/ml) and was completely inactive with topo-isomerase II. The presence of a dissacharide at the C-3 posi-tion on quercetin reduced by more than 50-fold (Table 1) thepotency of the flavone to stabilize the covalent topoisomeraseII-DNA intermediate.

Flavonoids as poisons of bacterial type II topoisomerase.The abilities of selected flavonoids to stabilize the covalenttopoisomerase-DNA complex with gyrase or topoisomerase IV

were tested (Table 1). Among the flavonoid aglycones, the close-ly related flavones quercetin and apigenin were the most po-tent compounds in poisoning DNA gyrase (CC50 5 2 mg/ml),whereas phloretin was fivefold less potent and taxifolin andgenistein were inactive at concentrations up to 50 mg/ml. Onthe other hand, quercetin was by far the most active unglyco-sylated flavonoid inhibitor of topoisomerase IV. The other fla-vonoids tested were at least 25- to 50-fold less potent (Table 1).

Derivatives of quercetin at the C-3 position were also testedas potential bacterial type II topoisomerase poisons. In allcases studied (Table 2), the presence of sugars at this positionresulted in complete loss of activity in cleavage reactions withgyrase. The presence of a sulfate group at the C-3 position hadthe same effect. By contrast, the effect on topoisomerase IV-mediated cleavage was quite dependent on the nature of the3-O-glycosidic moiety (Table 2). Introduction of one sugarresidue led to quercetin derivatives that were either inactive(e.g., glucoside or galactoside) or weakly active (e.g., rhamno-side), and interestingly, introduction of a second sugar restored

FIG. 1. Stabilization of topoisomerase IV-mediated pBR322 cleavage by thethree quercetin derivatives isolated from cottonseed flour. Lane 1, control 39-end-labeled linearized pBR322 DNA (7); lane 2, DNA and E. coli topoisomeraseIV, without drug; lanes 3 and 4, same as lane 2 but with pefloxacin (0.1 and 5mg/ml, respectively); lanes 5 to 10, rutin (0.5, 1, 5, 10, 25, and 50 mg/ml, respec-tively); lanes 11 to 15, quercetin 3-O-b-D-galactose-[1,6]-O-a-L-rhamnose (com-pound B) (1, 5, 10, 25, and 50 mg/ml, respectively); lanes 16 to 20, isoquercitrin(1, 5, 10, 25, and 50 mg/ml, respectively). C, rutin-stimulated natural cleavagesite; CC, rutin-induced cleavage site.

FIG. 2. Inhibitory activity of rutin on topoisomerase IV-dependent decatena-tion of kinetoplast DNA (kDNA). Standard reaction mixtures contained 2.0 U oftopoisomerase IV and 128, 96, 80, 64, 48, 32, and 16 mg of rutin (lanes 1 to 7,respectively) per ml. m, monomeric minicircles.

FIG. 3. Stimulation by quercetin (Q) and rutin (R) of DNA cleavage cata-lyzed by E. coli gyrase, E. coli topoisomerase IV (topo IV), and calf thymustopoisomerase II (topo II). Flavone concentrations (in micrograms per milliliter)are indicated above the lanes. Lane D, control pBR322 DNA (same as in Fig. 1).C, rutin-stimulated natural cleavage site; CC, rutin-induced cleavage site.

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inhibitory activity either partially (e.g., rhamnosyl-galactosideor glucosyl-arabinoside) or completely (e.g., rhamnosyl-gluco-side). Glycosylated derivatives of phloretin or apigenin testedwere not inhibitory on gyrase (Table 1).

Induction of SOS response. Among the quercetin deriva-tives, rutin was the only compound which elicited induction ofSOS response in the SOS test (Table 2). Unlike the othercompounds tested, rutin showed a detectable antibacterial ac-tivity against the permeable E. coli D22 (MIC, 32 mg/ml). How-ever, this activity was not detected on the nonpermeable wild-type E. coli K-12. Rutin also exhibited a weak antibacterialactivity (MIC, 128 mg/ml) on S. aureus FDA 209-P.

Mapping major DNA cleavage sites induced by rutin. TheDNA cleavage patterns obtained with quercetin in the pres-ence of either topoisomerase IV or gyrase were similar to eachother (Fig. 3) and were similar to that obtained in the presenceof a large excess of enzyme without drug (data not shown). Incontrast, rutin not only strongly enhanced one natural topo-

isomerase IV-mediated DNA cleavage site (Fig. 3, fragmentC) but also promoted cleavage at a specific site, generating onemajor DNA fragment (Fig. 3, fragment CC) not observed withquercetin. Fragment CC, which migrated at a slightly lowermolecular weight than uncleaved DNA, was also induced byother glycosylated flavones such as quercetin 3-O-b-D-galac-tose-[1,6]-O-a-L-rhamnose or quercetin 3-O-b-D-glucose (Fig.1). This fragment was observed at low concentrations of rutin(#5 mg/ml) and was subsequently cleaved at higher rutin con-centrations (Fig. 3). Cleavage patterns obtained with variousrestriction fragments of linearized pBR322 used as topoisomer-ase IV substrates (data not shown) indicated that fragment CCwas located between the SalI and the BspMI sites on therestriction map of pBR322, at a position located less than 400bp downstream from the SalI site. A topoisomerase IV-depen-dent cleavage reaction was performed with a SalI-linearized59-end-labeledpBR322substrate.Twoenzyme-freelabeledfrag-ments were generated and electrophoresed through a sequenc-

TABLE 1. Stimulation activities of selected flavonoids on DNA cleavagea

Structure Compound(R in structure)

CC50 (mg/ml)

Topo IV Gyrase Topo II

Quercetin (OH) 1 2 5

Rutin (O-glucose-rhamnose) 1 .50 .50

Phloretin (OH) 50 10 NDb

Phlorizin (R 5 O-glucose) .50 .50 ND

Apigenin (OH) 25 2 ND

Apiin (O-glucose-apiose) .50 25 ND

Genistein (OH) 25 .50 ND

Daidzein (H) .50 .50 ND

Taxifolin 25 .50 ND

Control quinolone Pefloxacin 1 0.05 .50

a Activities on E. coli gyrase-, E. coli topoisomerase IV (Topo IV)-, and calf thymus topoisomerase II (Topo II)-dependent DNA cleavage were studied.b ND, not determined.


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ing gel (Fig. 4). Also included was a dideoxy sequencing ladderobtained from an oligonucleotide whose 59 end coincided withthe position of the 59 end on the labeled DNA fragment (Fig.4). This study indicated that topoisomerase IV-mediated DNAbreaks specifically induced by rutin occurred at positions ap-proximately 788 and 955 on pBR322. Note that CC2 (position955) was the major fragment stabilized with 1 mg of rutin perml and that CC1 (position 788) and CC2 were almost equallystabilized at rutin concentrations of $10 mg/ml. The differencein length between the CC1 and CC2 fragments was so smallthat the two fragments comigrated as a single band (CC) in 1%agarose gels (Fig. 1 and 3).

DISCUSSION

A screening for compounds that target topoisomerase IVwas developed in addition to the screening for gyrase inhibi-tors. Surprisingly, positive responses in our topoisomerase IVassay were detected in several culture media for actinomycetes,all of them containing cottonseed flour. Extracts from controlseed flour were also positive in the topoisomerase IV-mediatedDNA cleavage assay, indicating that the potent compound wasindeed brought by the cottonseed flour. In addition, cultivationof microorganisms in cottonseed flour-containing media usu-ally led to reduction or complete loss of topoisomerase IV-poisoning activity (compared to the activity of the mediumprior to cultivation), which suggests that the active com-pound(s) was in most cases metabolized by the microorgan-

FIG. 4. Mapping of pBR322 specific cleavage sites induced by E. coli topoisomerase IV in the presence of rutin. (A) DNA sequencing reactions. Lanes G, A, T,and C correspond to the sequencing reactions of pBR322 from SalI; lanes 25, 10, 1, and 0 correspond to rutin concentrations (in micrograms per milliliter) used intopoisomerase IV-mediated cleavage reactions. (B) Nucleotide sequence from positions 651 to 1063 of pBR322. The positions of the two rutin-dependent cleavage sitesand CC1 and CC2 (nucleotides 59 to the breaks [boldface letters]), preceded by conserved residues (boxed sequences), are shown. (C) Position of the two rutin-inducedcleavage sites, CC1 and CC2, on the pBR322 map.

TABLE 2. Structure-activity relationships in the quercetin seriesa

Compound R in structureCC50 (mg/ml) SOS

(diam)bTopo IV Gyrase

Quercetin OH 1 2 2 (0)Quercetin-3-sulfate Sulfate .50 .50 2 (0)Quercitrin O-Rhamnose 10 .50 2 (0)Isoquercitrin O-Glucose .50 .50 2 (0)Hyperoside O-Galactose .50 .50 2 (0)Avicularin O-Arabinose .50 .50 2 (0)Rutin O-Glucose-rhamnose 1 .50 1 (10)Compound B O-Galactose-rhamnose 5 .50 2 (0)Peltatoside O-Arabinose-glucose 20 .50 2 (#3)Pefloxacin 1 0.05 1 (25)

a The effects of 3-O glycosylation of quercetin on gyrase- and topoisomeraseIV (Topo IV)-dependent DNA cleavage activity and on induction of the SOSsystem were studied.

b The plus and minus signs indicate that the compound does or does notinduce, respectively, the SOS response in the spot test. The diameter of the zoneof growth inhibition is given (in millimeters).


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isms. Three potent glycosylated derivatives of the flavone com-pound quercetin were isolated from cottonseed flour, namely,quercetin 3-O-b-D-glucose (isoquercitrin), quercetin 3-O-b-D-galactose-[1,6]-O-a-L-rhamnose, and quercetin 3-O-b-D-glu-cose-[1,6]-O-a-L-rhamnose (rutin), among which rutin was themost active compound in stabilizing topoisomerase IV-inducedDNA cleavage. Rutin also inhibited the catalytic activity oftopoisomerase IV; it was inactive in DNA gyrase-dependentDNA cleavage reactions and therefore was specific to topo-isomerase IV. Similarly, rutin promoted DNA cleavage medi-ated by S. aureus topoisomerase IV but not by S. aureus DNAgyrase (data not shown).

Naturally occurring flavonoids have been reported to inhibitreverse transcriptase, protein kinase C, and DNA topoisomer-ases (2). Specifically, quercetin has demonstrated significantactivity in DNA cleavage reactions mediated by eukaryotictopoisomerase I or topoisomerase II (2, 8, 9), and flavonesclosely related to quercetin have been shown to stabilize thecleavable complex with DNA gyrase and to inhibit its super-coiling activity (23). Quercetin glycosylation at position C-3 hasbeen shown to almost completely abolish activity of the flavoneon mammalian DNA topoisomerase II. Our study confirmedthis structure-activity relationship and extended it to DNAgyrase. In addition, we showed that some quercetin derivativeswere still moderately active on topoisomerase IV and thatrutin in particular retained full potency. Importantly, our studywith rutin demonstrated that the requirement of a hydroxylgroup at the C-3 position for DNA cleavage activity with typeII topoisomerases is not true for topoisomerase IV.

Flavones exhibit almost no antibacterial activity under stan-dard in vitro conditions on either gram-positive or gram-neg-ative organisms. However, some of them are moderately activeon permeable E. coli cells (23). During this study, rutin exhib-ited a low level of antibacterial activity which was restricted topermeable E. coli strains whereas no antibacterial activity wasdetected with the other quercetin derivatives assayed. In addi-tion, rutin was the only tested flavone which induced an SOSresponse, observed with a din::lacZ E. coli strain. Since topo-isomerase IV is essential for cell survival, our results suggestthat rutin-induced topoisomerase IV-mediated DNA cleavageleads to an SOS response and growth inhibition of E. coli cells.

Both quercetin and rutin are topoisomerase IV poisons.However, glycosylation at C-3 completely modified the DNAcleavage pattern by promoting two specific topoisomerase IV-dependent cleavage sites. Compounds that exhibit specific cleav-age patterns are in most cases intercalators or compounds thatbind selectively to specific DNA sequences, thereby trappingtopoisomerase on sites that differ from its natural cleavagesites. This mechanism, which is observed with anticancer topo-isomerase inhibitors such as anthracyclins, ellipticin, and into-plicin (27), is different from that of, e.g., quinolones. Quino-lones do not substantially modify but simply enhance thepattern of DNA cleavage by gyrase or topoisomerase IV (26),probably because these drugs come into play once the enzyme-DNA complex has been formed, as suggested by currentinhibition models (32). The reason why rutin and other3-O-glycosylated derivatives of quercetin induce specific topo-isomerase IV-mediated DNA cleavage sites is not yet clear.Peng and Marians have shown that topoisomerase IV protectsa 34-bp region (G1C content, 45%) roughly centered aboutthe cleavage site (26). During this study, we have identified tworutin-induced cleavage sites which are located around posi-tions 788 and 955 in the gene encoding tetracycline resistancein pBR322 DNA. The 34-bp regions surrounding the twocleavage sites both have a high G1C content (68%) and oneunique sequence located 14 and 20 bp upstream from the CC1

and CC2 cleavage sites, respectively. This sequence is 59-PyGCCGGCA-39, it is perfectly palindromic upstream of CC1,and the palindrome is shorter upstream of CC2. However, therelationship between the palindromic sequence and the rutin-induced cleavage site specificity is not established.

In conclusion, this study demonstrated that despite aminoacid sequence, structural, and mechanistic similarities betweenDNA gyrase and topoisomerase IV, compounds selectivelyacting as topoisomerase IV poisons and inactive on gyrase doexist. Such compounds may represent valuable tools to studythe role of topoisomerase IV in the bacterial cell cycle as wellas to validate topoisomerase IV as a potential target for findingnew bactericidal agents.

ACKNOWLEDGMENTS

We thank D. Bisch, A. Goniot, C. Lecoq, A. Le Prado, and L. Matonfor excellent technical assistance, T. Ciora for the synthesis of oligo-nucleotides, and M. Danzer for mass spectrometry analyses.

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Documents

Glycosylated Flavones as Selective Inhibitors of Topoisomerase IV