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Compounds with antibacterial activity that enhance DNA cleavage by bacterial DNA topoisomerase I

Compounds with antibacterial activity that enhance DNA cleavage by bacterial DNA topoisomerase I Abstract Objectives DNA topoisomerases utilize a covalent complex formed after DNA cleavage as an intermediate in the interconversion of topological forms via DNA cleavage and religation. Many anticancer and antibacterial therapeutic agents are effective because they stabilize or increase the level of the covalent topoisomerase–DNA complex formed by type IIA or type IB topoisomerases. Our goal is to identify small molecules that can enhance DNA cleavage by type IA DNA topoisomerase. Compounds that act in this mechanism against type IA topoisomerase have not been identified previously and could be leads for development of a new class of antibacterial agents. Methods High throughput screening was carried out to select small molecules that induce the SOS response of Escherichia coli, overexpressing recombinant Yersinia pestis topoisomerase I. The initial hit compounds were further tested for inhibition of bacterial growth and bacterial topoisomerase I activity. Results Three compounds with antibacterial activity that enhance the cleavage activity of bacterial topoisomerase I were identified. Conclusions Small molecules that can enhance the DNA cleavage activity of type IA DNA topoisomerase can be identified and may provide leads for development of novel antibacterial compounds. antibiotics, high throughput screening, Y. pestis Introduction All organisms characterized so far have at least one type I and one type II DNA topoisomerase in their genome. These enzymes are required for the regulation of DNA supercoiling and overcoming topological barriers during replication, transcription, recombination or repair.1–4 Type I topoisomerases break and rejoin one strand of DNA, whereas type II topoisomerases break and rejoin a double strand of DNA simultaneously during catalysis. The cleaved DNA is covalently linked to the topoisomerase protein via the active site tyrosine hydroxyl, and the covalent complex represents an intermediate for the cleavage and religation of DNA. Topoisomerases are further divided into subfamilies.1,2 A large number of anticancer5–7 and antibacterial8–10 therapeutic agents are effective because they stabilize the covalent intermediate formed by type IIA or type IB topoisomerases. These drugs are referred to as ‘topoisomerase poisons’.11–13 All DNA topoisomerases utilize an active site tyrosine for the formation of the phosphotyrosine linkage in the covalent complex, so it should be possible to target type IA DNA topoisomerase by a similar mechanism. However, small molecules that can act as poisons for type IA DNA topoisomerases remain to be identified. In recent years, the rapid emergence of bacterial pathogens resistant to commonly used antibacterial drugs has become a serious public health problem.14–16 There is an urgent need to identify novel antibacterial agents against new cellular targets in order to avoid the problem of cross-resistance. Every bacterium has at least one type IA topoisomerase so that the enzyme activity is available for resolving topological blocks involving DNA single strands.2,17 The transesterification domains of the type IA topoisomerases are well conserved in evolution.18 It is a logical route to search for novel antibiotics by trying to identify small molecules that can trap the covalent complex formed by bacterial type IA topoisomerases. The bactericidal action of such ‘poisons’ would not depend on the catalytic activity of the type IA topoisomerase being essential, as long as the topoisomerase is present and bound to the genomic DNA. Although Escherichia coli and Salmonella typhimurium mutants lacking topoisomerase I activity (encoded by the topA gene) can be isolated,19–22 these topA mutants have another type IA topoisomerase, topoisomerase III (encoded by the topB gene). Previous studies have shown that E. coli is not viable if both type IA topoisomerase activities are absent due to mutations in topA and topB.17 Many fluoroquinolones can act as poisons against both bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV.23,24 Therefore, it should also be possible to identify small molecules that can act as poisons against both bacterial topoisomerase I and topoisomerase III. Furthermore, loss of topoisomerase I function in E. coli has been shown to result in drastic decrease in survival rates when the organism is challenged with high temperature, oxidative stress and extreme low pH.25–28 Hence, if the bacterial pathogen gains resistance against topoisomerase I targeting poisons by loss of topA gene activity, such drug-resistant mutants are not expected to be viable in the host environment. Recently, we have isolated a mutant in the strictly conserved TOPRIM region of recombinant Y. pestis topoisomerase I that accumulates the covalent complex in E. coli by screening for Y. pestis topoisomerase I mutant proteins that induce the SOS response of E. coli.29 When the expression level is elevated, this mutant Y. pestis topoisomerase I and similar mutant E. coli topoisomerase I cause > 4 logs loss of bacterial cell viability.29 This verified that accumulation of the cleavage complex formed by bacterial topoisomerase I can lead to extensive bacterial cell killing. We report here for the first time identification of small molecules that can enhance the accumulation of cleavage complex formed by bacterial topoisomerase I. Materials and methods Strains and plasmids E. coli strain RFM44328 and Bacillus subtilis (ATCC 6633) were used as wild-type test strains for antibacterial activity. The E. coli strain BAS302330,31 has the Imp4213 mutation,31 conferring permeability to small molecules. The gene segment encoding Y. pestis topoisomerase I was excised from plasmid p-YTOP29 with restriction enzymes PmeI and SphI and then cloned into plasmid pACYC184 (from New England BioLab) digested with NruI and SphI to generate a low copy plasmid pAYTOP with chloramphenicol resistance selection. The plasmid pAYTOP-128 was constructed similarly starting with the plasmid pYTOP-128, which has the SOS-inducing mutations in the Y. pestis topoisomerase I coding sequence.29 To construct the luciferase reporter plasmid for SOS induction in E. coli, the dinD1 promoter region was amplified by PCR with primers (forward 5′-GGAATTCTGGAAAACAACCGTCTGG-3′, reverse 5′-CATTGAGCTCAATTGCAAAGAAAGTAAATCTG-3′) using genomic DNA from E. coli MG1655 as template. The PCR product was digested with the restriction enzymes EcoRI and SacI; then ligated to the luxCDABE reporter plasmid pDEW20732 digested with the same restriction enzymes to replace the grpE promoter sequence on the plasmid with the dinD1 promoter sequence. The resulting reporter plasmid was named pDinlux. Luciferase assay E. coli strain BAS3023 transformed with plasmids pDinlux and pAYTOP or its derivatives was maintained and grown overnight in LB medium with 2% glucose, 100 mg/L ampicillin and 30 mg/L chloramphenicol. Glucose was included to suppress the expression of the recombinant Y. pestis topoisomerase under the control of the BAD promoter.29 The overnight culture was diluted 1:100 into fresh LB medium with antibiotics and grown at 37°C until OD600 reached 0.4. Arabinose was added at a concentration of 0.001% for induction of recombinant topoisomerase for 90 min. The culture was then dispersed into flat bottom white microtitre plates (100 µL in each well of 96-well plate). One microlitre of test or screening compounds at various concentrations dissolved in DMSO was added to each well. Light production was measured on a Dynex ML-3000 luminometer at 37°C in 10 min cycles.32 High throughput screening High throughput screening was carried out at the Institute of Chemistry and Cell Biology (ICCB), Harvard Medical School. E. coli strain BAS3023 transformed with plasmids pDinlux and pAYTOP was cultured and induced with arabinose for 1 h, as described earlier. The induced culture was dispersed into 384-well white microtitre plates (30 µL in each well). After pin transfer of 100 nL of each compound (at 5 mg/mL in DMSO) in the libraries available at ICCB (purchased from ChemDiv2, ChemDiv3, Biomol-TimTec1, Maybridge4, Enamine1, Bionet2), the microtitre plates were incubated at 37°C for 1 h with shaking before luciferase readings were recorded with the PerkinElmer EnVision Multilabel microplate reader. The plates were agitated on a microtitre plate shaker in the incubator when they are not being read on the plate reader. After completion of one round of reading, the entire set of plates was read at least one more time. Bacterial topoisomerase I cleavage assay Recombinant Y. pestis and E. coli topoisomerase I proteins were purified as described in the previous work.29 The substrate for the oligonucleotide cleavage assay was a 59 base oligonucleotide 5′-GCCCTGAAAGATTATGCAATGCGCTTTGGGCAAACCAAGAGAGCATAATCTTTCAGGGC-3′ labelled at the 5′-end using T4 polynucleotide kinase and [γ-32P]ATP. It has a preferred cleavage site CAAT↓GC for E. coli DNA topoisomerase I.33 The cleavage reaction was carried out with 0.5 pmol of DNA substrate, 100 ng of enzyme in 5 µL of 10 mM Tris–HCl, 1 mM EDTA pH 8.0 at 37°C for 30 min before the addition of 5 µL of stop solution (79% formamide, 0.2 M NaOH, 0.04% Bromophenol Blue). The reaction products were separated by electrophoresis in a 15% sequencing gel. The gel was analysed with Phosphorimager Storm 860. Vaccinia topoisomerase I cleavage assay Recombinant vaccinia topoisomerase I enzyme was purified as described previously.34 The cleavage substrate is a PCR product generated by plasmid pBAD/Thio (Invitrogen), using the pBAD forward and reverse primers. The pBAD reverse primer was labelled with 32P at the 5′-end. The 635 bp PCR product has a vaccinia topoisomerase I cleavage site.35 The cleavage reaction was carried out with 0.4 pmol of DNA substrate, 100 ng of vaccinia topoisomerase I in 5 µL of 100 mM Tris–HCl, pH 7.4 at 37°C for 10 min before the addition of SDS to 0.5% and 10 µg of protease K. After 60 min of digestion, the reaction mixtures were mixed with equal volume of loading buffer for sequencing gel (79% formamide and 0.04% Bromophenol Blue) and analysed by electrophoresis in a 6% sequencing gel. E. coli topoisomerase I relaxation assay Relaxation of negatively supercoiled plasmid DNA by E. coli topoisomerase I was assayed as described previously.29 The compound being tested in 1 µL of DMSO was mixed with the DNA before the addition of 20 ng of enzyme. After incubation at 37°C for 30 min, the reaction products were analysed by agarose gel electrophoresis.29 MIC measurements For MIC measurements, E. coli strain BAS3023 transformed with plasmid pAYTOP or its active site mutant derivative (Y325A) was grown to OD600 = 0.4 in LB medium with chloramphenicol. Arabinose (0.001%) was added to induce the expression of recombinant Y. pestis topoisomerase I for 2 h before the culture was diluted into fresh LB medium with chloramphenicol. In addition, wild-type E. coli strain RFM443 and B. subtilis strain 6633 were used to access the effect of the compounds on bacterial growth. Cells were grown to OD600 = 1 in LB medium, then diluted into fresh medium. After overnight growth in a 37°C shaker, the concentration of compound at which no visible growth could be detected was recorded as the MIC. Results The dinD1::luxCADBE luciferase reporter system can detect accumulation of DNA cleavage complex formed by recombinant Y. pestis topoisomerase I in E. coli Accumulation of the DNA cleavage complex formed by E. coli G116S and Y. pestis G122S topoisomerase I mutant enzymes led to the induction of the SOS response in E. coli, as indicated by transcription from the dinD1 promoter.29 We set up an assay system with a dinD1::luxCADBE fusion plasmid as the reporter for SOS induction. We chose Y. pestis topoisomerase I as the initial target for this assay system. An E. coli host strain with the Imp4213 mutation known to enhance entry of small molecules into E. coli31 was utilized in preparation for high throughput screening of small molecules library for compounds that can enhance the accumulation of the cleavage complex formed by wild-type Y. pestis topoisomerase I. After wild-type recombinant Y. pestis topoisomerase I was induced from pAYTOP by arabinose treatment, the luciferase signal from E. coli BAS3023/pDinlux stayed at a reasonably low level (Figure 1a). Induction of pAYOP-128 results in the synthesis of a mutant topoisomerase I that accumulates the cleaved DNA complex due to a mutation in the TOPRIM domain.29 This leads to SOS induction,29 and a robust luciferase signal was produced from BAS3023/pDinlux (Figure 1b) in a dose-dependent manner corresponding to the concentration of arabinose regulating the synthesis of the mutant topoisomerase molecules. This validates the assay for its ability to generate a positive readout from accumulation of the cleavage complex formed by the recombinant bacterial type IA topoisomerase in E. coli. Figure 1. Open in new tabDownload slide Luciferase induction from dinD1::luxCADBE after the addition of arabinose to E. coli BAS3023 with (a) wild-type pAYTOP or (b) SOS-inducing mutant pAYTOP-128. The luciferase readings were normalized as response ratio by dividing by luciferase activity reading of culture with no arabinose added. Cells were grown to early exponential phase and arabinose was added at time zero. Figure 1. Open in new tabDownload slide Luciferase induction from dinD1::luxCADBE after the addition of arabinose to E. coli BAS3023 with (a) wild-type pAYTOP or (b) SOS-inducing mutant pAYTOP-128. The luciferase readings were normalized as response ratio by dividing by luciferase activity reading of culture with no arabinose added. Cells were grown to early exponential phase and arabinose was added at time zero. Hit compounds identified by the assay include small molecules that can enhance the DNA cleavage by bacterial type IA DNA topoisomerases High throughput assay was carried out in the facilities of ICCB, Harvard Medical School. Screening of a total of 49 268 compounds was conducted over a 3 day period. Each compound was assayed at a concentration of 16.7 mg/L in duplicate and the luciferase signal had to be at least 60% above the plate median in both assays of the compound for the compound to be classified as a positive hit. One microlitre of 5 mg/mL DMSO solution of 150 hit compounds was made available by ICCB for secondary assays. These compounds were tested in DNA oligonucleotide cleavage assays using purified Y. pestis and E. coli topoisomerase I. Three of the compounds (compounds 1–3, Figure 2) from ChemDiv were found to enhance the formation of DNA cleavage products by the bacterial type IA topoisomerases (Figure 3). No effect from these three compounds was observed for DNA cleavage by the type IB vaccinia topoisomerase I (Figure 3), so this action is specific for type IA topoisomerase. Phenanthrene and fluorene, which shared common structural features with compounds 1 and 2, were found to have no effect on DNA cleavage by E. coli DNA topoisomerase I (Figure 3). Quantification of the cleavage data from three experiments on E. coli DNA topoisomerase I by phosphorimager showed that the maximal enhancement of the cleavage product formation was 107 ± 5% for lead compound 1; 80 ± 20% for lead compound 2 and 150 ± 12% for lead compound 3. Figure 2. Open in new tabDownload slide Structures of lead compounds 1–3 identified in this study along with structures of phenanthrene and fluorene for comparison. Figure 2. Open in new tabDownload slide Structures of lead compounds 1–3 identified in this study along with structures of phenanthrene and fluorene for comparison. Figure 3. Open in new tabDownload slide Effect of compounds on the accumulation of DNA cleavage products by the type IA bacterial topoisomerases (Y. pestis and E. coli) and type IB topoisomerase (vaccinia virus). Compounds were present at a concentration of 20 mg/L. The arrows point to the cleavage products. C, control with no enzyme added; E, DMSO control; 1, lead compound 1; 2, lead compound 2; 3, lead compound 3; -, other initial SOS-inducing hits from high throughput assay; P, phenanthrene; F, fluorene. Figure 3. Open in new tabDownload slide Effect of compounds on the accumulation of DNA cleavage products by the type IA bacterial topoisomerases (Y. pestis and E. coli) and type IB topoisomerase (vaccinia virus). Compounds were present at a concentration of 20 mg/L. The arrows point to the cleavage products. C, control with no enzyme added; E, DMSO control; 1, lead compound 1; 2, lead compound 2; 3, lead compound 3; -, other initial SOS-inducing hits from high throughput assay; P, phenanthrene; F, fluorene. Further characterization of lead compounds Lead compounds 1–3 were purchased from ChemDiv for further analysis. Lead compound 1 has been previously characterized as the natural product stephenanthrine, a phenanthrene alkaloid.36 Phenanthrene and fluorene were also included in the experiments for comparison. SOS induction of luciferase activity from dinD1::luxCADBE in BAS3023 strain expressing recombinant Y. pestis topoisomerase I was measured for both wild-type Y. pestis topoisomerase I and a mutant derivative with its active site Tyr-325 changed to alanine. This active site mutant of Y. pestis topoisomerase I would not be able to form the covalent cleavage complex. Significant levels of SOS induction were also observed when either wild-type or the Y325A active site mutant Y. pestis topoisomerase I was expressed for all three compounds (Figure 4). The luciferase activity induced by lead compound 3 was higher when wild-type Y. pestis topoisomerase I was expressed. Analysis of dose–response showed that for lead compounds 1 and 3, high concentrations of the compounds led to a decrease in luciferase induction, possibly due to the low level of cellular ATP from loss of viability (Figure 4). Phenanthrene and fluorene treatment did not induce comparable levels of luciferase activity (data not shown). Figure 4. Open in new tabDownload slide Induction of luciferase activity from dinD1::luxCADBE in E. coli strain BAS3023 by the three lead compounds. The reporter strain was treated with arabinose at time zero to induce the expression of either wild-type recombinant Y. pestis topoisomerase I (wtYTOP, filled symbols) or a mutant with the active site tyrosine converted to alanine (AlaYTOP, open symbols). The luciferase induction ratio was calculated by normalizing the luciferase reading of the cultures treated with the compounds against the luciferase readings from control cultures treated with DMSO measured at 290 min after addition of compounds. Figure 4. Open in new tabDownload slide Induction of luciferase activity from dinD1::luxCADBE in E. coli strain BAS3023 by the three lead compounds. The reporter strain was treated with arabinose at time zero to induce the expression of either wild-type recombinant Y. pestis topoisomerase I (wtYTOP, filled symbols) or a mutant with the active site tyrosine converted to alanine (AlaYTOP, open symbols). The luciferase induction ratio was calculated by normalizing the luciferase reading of the cultures treated with the compounds against the luciferase readings from control cultures treated with DMSO measured at 290 min after addition of compounds. The effects of the lead compounds present at different concentrations on the relaxation activity of E. coli DNA topoisomerase I were determined (Figure 5). Addition of compounds 1, 2 and 3 was shown to result in complete inhibition of the formation of relaxed DNA products. Phenanthrene and fluorene at similar concentrations had no effect on the relaxation activity of E. coli DNA topoisomerase I. Compound 2 was less effective than compounds 1 and 3 in inhibition of relaxation. This correlates with the IC50 for increase in DNA cleavage products observed for these compounds in the DNA cleavage experiments (5 µM for lead compounds 1 and 3 and 10 µM for lead compound 2). Figure 5. Open in new tabDownload slide Inhibition of E. coli topoisomerase I relaxation activity. Lead compounds 1, 2 and 3, phenanthrene (P) and fluorene (F) were present at concentrations of 100, 20 or 4 µM in the reaction mixture. C, control reaction with no enzyme added; E, enzyme only; S, supercoiled DNA; R, relaxed and nicked DNA. Figure 5. Open in new tabDownload slide Inhibition of E. coli topoisomerase I relaxation activity. Lead compounds 1, 2 and 3, phenanthrene (P) and fluorene (F) were present at concentrations of 100, 20 or 4 µM in the reaction mixture. C, control reaction with no enzyme added; E, enzyme only; S, supercoiled DNA; R, relaxed and nicked DNA. Growth inhibition of E. coli Imp4213 mutant strain BAS3023, wild-type E. coli RFM443 and B. subtilis 6633 by these lead compounds was measured. The compounds had little or no effect on growth of wild-type E. coli RFM443 probably due to failure to enter the cell. Growth of E. coli BAS3023 with the Imp4213 mutation that enhanced permeability, as well as the Gram-positive B. subtilis, could be inhibited by these compounds, but not by phenanthrene and fluorene present at similar concentrations (Table 1). E. coli strain BAS3023 transformed with wild-type pAYTOP showed slightly greater sensitivity to lead compound 3 when compared with the same strain transformed with plasmid expressing the active site mutant of YTOP, in agreement with the luciferase induction data shown in Figure 4. This suggests that overexpression of wild-type Y. pestis topoisomerase I enhanced the susceptibility to this compound. Table 1. Inhibition of bacterial growth by lead compounds Compound . MIC (μM) . E. coli IMP4213 . B. subtilis 6633 . YTOP . YTOPala . Compound 1 100 100 60 Compound 2 80 80 80 Compound 3 40 60 40 Phenanthrene >800 >800 >200 Fluorene >800 >800 >200 Compound . MIC (μM) . E. coli IMP4213 . B. subtilis 6633 . YTOP . YTOPala . Compound 1 100 100 60 Compound 2 80 80 80 Compound 3 40 60 40 Phenanthrene >800 >800 >200 Fluorene >800 >800 >200 E. coli IMP4213 strain BAS3023 was induced for expression of recombinant Y. pestis topoisomerase I YTOP or its mutant derivative YTOPala (with active site tyrosine changed to alanine) for 2 h prior to dilution into fresh medium containing the compounds. Open in new tab Table 1. Inhibition of bacterial growth by lead compounds Compound . MIC (μM) . E. coli IMP4213 . B. subtilis 6633 . YTOP . YTOPala . Compound 1 100 100 60 Compound 2 80 80 80 Compound 3 40 60 40 Phenanthrene >800 >800 >200 Fluorene >800 >800 >200 Compound . MIC (μM) . E. coli IMP4213 . B. subtilis 6633 . YTOP . YTOPala . Compound 1 100 100 60 Compound 2 80 80 80 Compound 3 40 60 40 Phenanthrene >800 >800 >200 Fluorene >800 >800 >200 E. coli IMP4213 strain BAS3023 was induced for expression of recombinant Y. pestis topoisomerase I YTOP or its mutant derivative YTOPala (with active site tyrosine changed to alanine) for 2 h prior to dilution into fresh medium containing the compounds. Open in new tab Discussion The luciferase screening system utilizing the dinD1::luxCADBE plasmid was shown here to respond to the accumulation of covalent DNA cleavage complex formed by the recombinant Y. pestis topoisomerase I G122S TOPRIM mutant expressed in E. coli. After screening 49268 compounds, three small molecules that can enhance the level of DNA cleavage product of bacterial type IA topoisomerases were identified from 150 initial positive hit compounds. These three compounds enhanced the level of DNA cleavage product formed by both E. coli and Y. pestis topoisomerase I, as expected from the high degree of identity (85%) between the two enzyme sequences. The type IB vaccinia topoisomerase I does not share any homology with these bacterial enzymes. DNA cleavage by vaccinia topoisomerase I was not enhanced by the three lead compounds identified here. Induction of SOS by these three compounds did not depend strictly on the overexpression of recombinant Y. pestis topoisomerase I. However, there are two type IA topoisomerases, topoisomerase I and III, encoded by the topA and topB genes present in E. coli. These type IA topoisomerases share significant homology in the transesterification domain. E. coli mutants lacking both topA and topB gene functions are not viable.17 Interaction of the compounds with either or both of these E. coli type IA topoisomerases might be sufficient to induce the SOS response. A large number of compounds that target type IB and type IIA topoisomerases are known to interact with DNA and this interaction influences the stability of the covalent complex formed by topoisomerases after DNA cleavage.37–40 It is therefore possible that certain poisons of type IA bacterial topoisomerases may also induce the E. coli SOS response via a second mechanism independent of topoisomerase IA cleavage activity. The modes of action of the compounds found here to enhance the bacterial topoisomerase I cleavage will be further investigated with future experiments by selecting for mutant strains resistant to the action of these compounds. The antibacterial activities of the lead compounds identified here are not potent, especially against Gram-negative E. coli. It would be desirable to identify either analogues of these lead compounds or other novel compounds that have stronger broad spectrum antibacterial activities. Nevertheless, this is the first time that the cleavage–religation equilibrium of this class of topoisomerase has been shown to be shifted towards DNA cleavage by the action of a small molecule. It is hoped that the continued high throughput screening of compound libraries and follow-up efforts can yield a candidate for development of new antibacterial agents targeting type IA DNA topoisomerases. Acknowledgements We thank Professor Thomas Silhavy (Princeton University) for providing strain BAS3023. Facilities and compound libraries for high throughput screening were made available through the NSRB/NERCE programme. We thank members of the NSRB and ICCB-Longwood (Harvard Medical School) for their ongoing advice. This work was funded by grants from the National Institutes of Health (R03 NS050782 and R01 AI 069313) and a grant from the New York State Department of Health (C-020219) administered by the Northeast Biodefense Center. Transparency declarations None to declare. References 1 Champoux JJ . DNA topoisomerases: structure, function, and mechanism , Annu Rev Biochem , 2001 , vol. 70 (pg. 369 - 413 ) Google Scholar Crossref Search ADS PubMed WorldCat 2 Wang JC . Cellular roles of DNA topoisomerases: a molecular perspective , Nat Rev Mol Cell Biol , 2002 , vol. 3 (pg. 430 - 40 ) Google Scholar Crossref Search ADS PubMed WorldCat 3 Drlica K . Control of bacterial DNA supercoiling , Mol Microbiol , 1992 , vol. 6 (pg. 425 - 33 ) Google Scholar Crossref Search ADS PubMed WorldCat 4 Luttinger A . The twisted ‘life’ of DNA in the cell: bacterial topoisomerases , Mol Microbiol , 1995 , vol. 15 (pg. 601 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat 5 Nitiss JL . DNA topoisomerases in cancer chemotherapy: using enzymes to generate selective DNA damage , Curr Opin Investig Drugs , 2002 , vol. 3 (pg. 1512 - 6 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 6 Denny WA . Emerging DNA topoisomerase inhibitors as anticancer drugs , Expert Opin Emerg Drugs , 2004 , vol. 9 (pg. 105 - 33 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 7 Capranico G , Zagotto G , Palumbo M . Development of DNA topoisomerase-related therapeutics: a short perspective of new challenges , Curr Med Chem Anti-canc Agents , 2004 , vol. 4 (pg. 335 - 45 ) Google Scholar Crossref Search ADS WorldCat 8 Anderson VE , Osheroff N . Type II topoisomerases as targets for quinolone antibacterials: turning Dr. Jekyll into Mr. Hyde , Curr Pharm Des , 2001 , vol. 7 (pg. 337 - 53 ) Google Scholar Crossref Search ADS PubMed WorldCat 9 Hooper DC . Mechanisms of action of antimicrobials: focus on fluoroquinolones , Clin Infect Dis , 2001 , vol. 32 (pg. S9 - 15 ) Google Scholar Crossref Search ADS PubMed WorldCat 10 Drilica K , Malik M . Fluoroquinolones: action and resistance , Curr Top Med Chem , 2003 , vol. 3 (pg. 249 - 82 ) Google Scholar Crossref Search ADS PubMed WorldCat 11 Liu LF . DNA topoisomerase poisons as antitumor drugs , Annu Rev Biochem , 1989 , vol. 58 (pg. 351 - 75 ) Google Scholar Crossref Search ADS PubMed WorldCat 12 Drlica K , Franco RJ . Inhibitors of DNA topoisomerases , Biochemistry , 1988 , vol. 27 (pg. 2252 - 9 ) Google Scholar Crossref Search ADS WorldCat 13 Smith PJ . DNA topoisomerase dysfunction: a new goal for antitumor chemotherapy , Bioessay , 1990 , vol. 12 (pg. 167 - 72 ) Google Scholar Crossref Search ADS WorldCat 14 Canton R , Coque TM , Baquero F . Multi-resistant gram-negative bacilli: from epidemics to endemics , Curr Opin Infect Dis , 2003 , vol. 16 (pg. 315 - 25 ) Google Scholar Crossref Search ADS PubMed WorldCat 15 Stevens DL . Community-acquired Staphylococcus aureus infections: increasing virulence and emerging methicillin resistance in the new millennium , Curr Opin Infect Dis , 2003 , vol. 16 (pg. 189 - 91 ) Google Scholar Crossref Search ADS PubMed WorldCat 16 Levy SB , Marshall B . Antibacterial resistance worldwide: causes, challenges and responses , Nat Med , 2004 , vol. 10 (pg. S122 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat 17 Zhu Q , Pongpech P , DiGate RJ . Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli. , Proc Natl Acad Sci USA , 2001 , vol. 98 (pg. 9766 - 71 ) Google Scholar Crossref Search ADS PubMed WorldCat 18 Caron PR . Compendium of DNA topoisomerase sequences , Methods Mol Biol , 1999 , vol. 94 (pg. 279 - 316 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 19 Stupina VA , Wang JC . On the viability of Escherichia coli topA mutants lacking DNA topoisomerase I , J Biol Chem , 2005 , vol. 280 (pg. 355 - 60 ) Google Scholar Crossref Search ADS PubMed WorldCat 20 Richardson SM , Higgins CF , Lilley DM . The genetic control of DNA supercoiling in Salmonella typhimurium. , EMBO J , 1984 , vol. 3 (pg. 1745 - 52 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 21 Pruss GJ , Manes SH , Drlica K . Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes , Cell , 1982 , vol. 31 (pg. 35 - 42 ) Google Scholar Crossref Search ADS PubMed WorldCat 22 DiNardo S , Voelkel KA , Sternglanz R , et al. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes , Cell , 1982 , vol. 31 (pg. 43 - 51 ) Google Scholar Crossref Search ADS PubMed WorldCat 23 Takel M , Fukuda H , Kishii R , et al. Target preference of 15 quinolones against Staphyloccus aureus, based on antibacterial activities and target inhibition , Antimicrob Agents Chemother , 2001 , vol. 45 (pg. 3544 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat 24 Hooper DC . Mechanisms of action and resistance of older and newer fluoroquinolones , Clin Infect Dis , 2000 , vol. 31 (pg. S24 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat 25 Qi H , Menzel R , Tse-Dinh YC . Effect of the deletion of the σ32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance , Mol Microbiol , 1996 , vol. 21 (pg. 703 - 11 ) Google Scholar Crossref Search ADS PubMed WorldCat 26 Qi H , Menzel R , Tse-Dinh YC . Increased sensitivity to thermal challenges associated with topA deletion and promoter mutations in Escherichia coli. , FEMS Microbiol Lett , 1999 , vol. 178 (pg. 141 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat 27 Tse-Dinh YC . Increased sensitivity to oxidative challenges associated with topA deletion in Escherichia coli. , J Bacteriol , 2000 , vol. 182 (pg. 829 - 32 ) Google Scholar Crossref Search ADS PubMed WorldCat 28 Stewart N , Feng J , Liu X , et al. Loss of topoisomerase I function affects the RpoS-dependent and GAD systems of acid resistance in Escherichia coli. , Microbiology , 2005 , vol. 151 (pg. 2783 - 91 ) Google Scholar Crossref Search ADS PubMed WorldCat 29 Cheng B , Shukla S , Vasunilashorn S , et al. Bacterial cell killing mediated by topoisomerase I DNA cleavage activity , J Biol Chem , 2005 , vol. 280 (pg. 38489 - 95 ) Google Scholar Crossref Search ADS PubMed WorldCat 30 Braun M , Silhavy TJ . Imp/OstA is required for cell envelope biogenesis in Escherichia coli. , Mol Microbiol , 2002 , vol. 45 (pg. 1298 - 302 ) Google Scholar Crossref Search ADS WorldCat 31 Sampson BA , Misra R , Benson SA . Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability , Genetics , 1989 , vol. 122 (pg. 491 - 501 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 32 Van Dyk TK , Ayers BL , Morgan RW , et al. Constricted flux through the branched-chain amino acid biosynthetic enzyme acetolactate synthase triggers elevated expression of genes regulated by rpoS and internal acidification , J Bacteriol , 1998 , vol. 180 (pg. 785 - 92 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 33 Kirkegaard K , Pflugfelder G , Wang JC . The cleavage of DNA by type I-DNA topoisomerases , Cold Spring Harb Symp Quant Biol , 1984 , vol. 49 (pg. 411 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat 34 Shuman S , Golder M , Moss B . Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. , J Biol Chem , 1988 , vol. 263 (pg. 16401 - 7 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 35 Shuman S , Prescott J . Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I , J Biol Chem , 1990 , vol. 265 (pg. 17826 - 36 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 36 Lopez-Martin J , Anam EM , Boira H , et al. Chromone and phenanthrene alkaloids from Dennettia tripietala. , Chem Pharm Bull , 2002 , vol. 50 (pg. 1613 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat 37 Pommier Y , Minford JK , Schwartz RE , et al. Effects of the DNA intercalators 4′-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage , Biochemistry , 1985 , vol. 24 (pg. 6410 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat 38 Sim SP , Gatto B , Yu C , et al. Differential poisoning of topoisomerases by menogaril and nogalamycin dictated by the minor groove-binding nogalose sugar , Biochemistry , 1997 , vol. 36 (pg. 13285 - 91 ) Google Scholar Crossref Search ADS PubMed WorldCat 39 Bailly C , Colson P , Houssier C , et al. Recognition of specific sequences in DNA by a topoisomerase I inhibitor derived from the antitumor drug rebeccamycin , Mol Pharmacol , 1998 , vol. 53 (pg. 77 - 87 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 40 McClendon AK , Osheroff N . The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs , Biochemistry , 2006 , vol. 45 (pg. 3040 - 50 ) Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org © The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Compounds with antibacterial activity that enhance DNA cleavage by bacterial DNA topoisomerase I

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Oxford University Press
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Copyright © 2022 British Society for Antimicrobial Chemotherapy
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0305-7453
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10.1093/jac/dkl556
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17317696
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Abstract

Abstract Objectives DNA topoisomerases utilize a covalent complex formed after DNA cleavage as an intermediate in the interconversion of topological forms via DNA cleavage and religation. Many anticancer and antibacterial therapeutic agents are effective because they stabilize or increase the level of the covalent topoisomerase–DNA complex formed by type IIA or type IB topoisomerases. Our goal is to identify small molecules that can enhance DNA cleavage by type IA DNA topoisomerase. Compounds that act in this mechanism against type IA topoisomerase have not been identified previously and could be leads for development of a new class of antibacterial agents. Methods High throughput screening was carried out to select small molecules that induce the SOS response of Escherichia coli, overexpressing recombinant Yersinia pestis topoisomerase I. The initial hit compounds were further tested for inhibition of bacterial growth and bacterial topoisomerase I activity. Results Three compounds with antibacterial activity that enhance the cleavage activity of bacterial topoisomerase I were identified. Conclusions Small molecules that can enhance the DNA cleavage activity of type IA DNA topoisomerase can be identified and may provide leads for development of novel antibacterial compounds. antibiotics, high throughput screening, Y. pestis Introduction All organisms characterized so far have at least one type I and one type II DNA topoisomerase in their genome. These enzymes are required for the regulation of DNA supercoiling and overcoming topological barriers during replication, transcription, recombination or repair.1–4 Type I topoisomerases break and rejoin one strand of DNA, whereas type II topoisomerases break and rejoin a double strand of DNA simultaneously during catalysis. The cleaved DNA is covalently linked to the topoisomerase protein via the active site tyrosine hydroxyl, and the covalent complex represents an intermediate for the cleavage and religation of DNA. Topoisomerases are further divided into subfamilies.1,2 A large number of anticancer5–7 and antibacterial8–10 therapeutic agents are effective because they stabilize the covalent intermediate formed by type IIA or type IB topoisomerases. These drugs are referred to as ‘topoisomerase poisons’.11–13 All DNA topoisomerases utilize an active site tyrosine for the formation of the phosphotyrosine linkage in the covalent complex, so it should be possible to target type IA DNA topoisomerase by a similar mechanism. However, small molecules that can act as poisons for type IA DNA topoisomerases remain to be identified. In recent years, the rapid emergence of bacterial pathogens resistant to commonly used antibacterial drugs has become a serious public health problem.14–16 There is an urgent need to identify novel antibacterial agents against new cellular targets in order to avoid the problem of cross-resistance. Every bacterium has at least one type IA topoisomerase so that the enzyme activity is available for resolving topological blocks involving DNA single strands.2,17 The transesterification domains of the type IA topoisomerases are well conserved in evolution.18 It is a logical route to search for novel antibiotics by trying to identify small molecules that can trap the covalent complex formed by bacterial type IA topoisomerases. The bactericidal action of such ‘poisons’ would not depend on the catalytic activity of the type IA topoisomerase being essential, as long as the topoisomerase is present and bound to the genomic DNA. Although Escherichia coli and Salmonella typhimurium mutants lacking topoisomerase I activity (encoded by the topA gene) can be isolated,19–22 these topA mutants have another type IA topoisomerase, topoisomerase III (encoded by the topB gene). Previous studies have shown that E. coli is not viable if both type IA topoisomerase activities are absent due to mutations in topA and topB.17 Many fluoroquinolones can act as poisons against both bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV.23,24 Therefore, it should also be possible to identify small molecules that can act as poisons against both bacterial topoisomerase I and topoisomerase III. Furthermore, loss of topoisomerase I function in E. coli has been shown to result in drastic decrease in survival rates when the organism is challenged with high temperature, oxidative stress and extreme low pH.25–28 Hence, if the bacterial pathogen gains resistance against topoisomerase I targeting poisons by loss of topA gene activity, such drug-resistant mutants are not expected to be viable in the host environment. Recently, we have isolated a mutant in the strictly conserved TOPRIM region of recombinant Y. pestis topoisomerase I that accumulates the covalent complex in E. coli by screening for Y. pestis topoisomerase I mutant proteins that induce the SOS response of E. coli.29 When the expression level is elevated, this mutant Y. pestis topoisomerase I and similar mutant E. coli topoisomerase I cause > 4 logs loss of bacterial cell viability.29 This verified that accumulation of the cleavage complex formed by bacterial topoisomerase I can lead to extensive bacterial cell killing. We report here for the first time identification of small molecules that can enhance the accumulation of cleavage complex formed by bacterial topoisomerase I. Materials and methods Strains and plasmids E. coli strain RFM44328 and Bacillus subtilis (ATCC 6633) were used as wild-type test strains for antibacterial activity. The E. coli strain BAS302330,31 has the Imp4213 mutation,31 conferring permeability to small molecules. The gene segment encoding Y. pestis topoisomerase I was excised from plasmid p-YTOP29 with restriction enzymes PmeI and SphI and then cloned into plasmid pACYC184 (from New England BioLab) digested with NruI and SphI to generate a low copy plasmid pAYTOP with chloramphenicol resistance selection. The plasmid pAYTOP-128 was constructed similarly starting with the plasmid pYTOP-128, which has the SOS-inducing mutations in the Y. pestis topoisomerase I coding sequence.29 To construct the luciferase reporter plasmid for SOS induction in E. coli, the dinD1 promoter region was amplified by PCR with primers (forward 5′-GGAATTCTGGAAAACAACCGTCTGG-3′, reverse 5′-CATTGAGCTCAATTGCAAAGAAAGTAAATCTG-3′) using genomic DNA from E. coli MG1655 as template. The PCR product was digested with the restriction enzymes EcoRI and SacI; then ligated to the luxCDABE reporter plasmid pDEW20732 digested with the same restriction enzymes to replace the grpE promoter sequence on the plasmid with the dinD1 promoter sequence. The resulting reporter plasmid was named pDinlux. Luciferase assay E. coli strain BAS3023 transformed with plasmids pDinlux and pAYTOP or its derivatives was maintained and grown overnight in LB medium with 2% glucose, 100 mg/L ampicillin and 30 mg/L chloramphenicol. Glucose was included to suppress the expression of the recombinant Y. pestis topoisomerase under the control of the BAD promoter.29 The overnight culture was diluted 1:100 into fresh LB medium with antibiotics and grown at 37°C until OD600 reached 0.4. Arabinose was added at a concentration of 0.001% for induction of recombinant topoisomerase for 90 min. The culture was then dispersed into flat bottom white microtitre plates (100 µL in each well of 96-well plate). One microlitre of test or screening compounds at various concentrations dissolved in DMSO was added to each well. Light production was measured on a Dynex ML-3000 luminometer at 37°C in 10 min cycles.32 High throughput screening High throughput screening was carried out at the Institute of Chemistry and Cell Biology (ICCB), Harvard Medical School. E. coli strain BAS3023 transformed with plasmids pDinlux and pAYTOP was cultured and induced with arabinose for 1 h, as described earlier. The induced culture was dispersed into 384-well white microtitre plates (30 µL in each well). After pin transfer of 100 nL of each compound (at 5 mg/mL in DMSO) in the libraries available at ICCB (purchased from ChemDiv2, ChemDiv3, Biomol-TimTec1, Maybridge4, Enamine1, Bionet2), the microtitre plates were incubated at 37°C for 1 h with shaking before luciferase readings were recorded with the PerkinElmer EnVision Multilabel microplate reader. The plates were agitated on a microtitre plate shaker in the incubator when they are not being read on the plate reader. After completion of one round of reading, the entire set of plates was read at least one more time. Bacterial topoisomerase I cleavage assay Recombinant Y. pestis and E. coli topoisomerase I proteins were purified as described in the previous work.29 The substrate for the oligonucleotide cleavage assay was a 59 base oligonucleotide 5′-GCCCTGAAAGATTATGCAATGCGCTTTGGGCAAACCAAGAGAGCATAATCTTTCAGGGC-3′ labelled at the 5′-end using T4 polynucleotide kinase and [γ-32P]ATP. It has a preferred cleavage site CAAT↓GC for E. coli DNA topoisomerase I.33 The cleavage reaction was carried out with 0.5 pmol of DNA substrate, 100 ng of enzyme in 5 µL of 10 mM Tris–HCl, 1 mM EDTA pH 8.0 at 37°C for 30 min before the addition of 5 µL of stop solution (79% formamide, 0.2 M NaOH, 0.04% Bromophenol Blue). The reaction products were separated by electrophoresis in a 15% sequencing gel. The gel was analysed with Phosphorimager Storm 860. Vaccinia topoisomerase I cleavage assay Recombinant vaccinia topoisomerase I enzyme was purified as described previously.34 The cleavage substrate is a PCR product generated by plasmid pBAD/Thio (Invitrogen), using the pBAD forward and reverse primers. The pBAD reverse primer was labelled with 32P at the 5′-end. The 635 bp PCR product has a vaccinia topoisomerase I cleavage site.35 The cleavage reaction was carried out with 0.4 pmol of DNA substrate, 100 ng of vaccinia topoisomerase I in 5 µL of 100 mM Tris–HCl, pH 7.4 at 37°C for 10 min before the addition of SDS to 0.5% and 10 µg of protease K. After 60 min of digestion, the reaction mixtures were mixed with equal volume of loading buffer for sequencing gel (79% formamide and 0.04% Bromophenol Blue) and analysed by electrophoresis in a 6% sequencing gel. E. coli topoisomerase I relaxation assay Relaxation of negatively supercoiled plasmid DNA by E. coli topoisomerase I was assayed as described previously.29 The compound being tested in 1 µL of DMSO was mixed with the DNA before the addition of 20 ng of enzyme. After incubation at 37°C for 30 min, the reaction products were analysed by agarose gel electrophoresis.29 MIC measurements For MIC measurements, E. coli strain BAS3023 transformed with plasmid pAYTOP or its active site mutant derivative (Y325A) was grown to OD600 = 0.4 in LB medium with chloramphenicol. Arabinose (0.001%) was added to induce the expression of recombinant Y. pestis topoisomerase I for 2 h before the culture was diluted into fresh LB medium with chloramphenicol. In addition, wild-type E. coli strain RFM443 and B. subtilis strain 6633 were used to access the effect of the compounds on bacterial growth. Cells were grown to OD600 = 1 in LB medium, then diluted into fresh medium. After overnight growth in a 37°C shaker, the concentration of compound at which no visible growth could be detected was recorded as the MIC. Results The dinD1::luxCADBE luciferase reporter system can detect accumulation of DNA cleavage complex formed by recombinant Y. pestis topoisomerase I in E. coli Accumulation of the DNA cleavage complex formed by E. coli G116S and Y. pestis G122S topoisomerase I mutant enzymes led to the induction of the SOS response in E. coli, as indicated by transcription from the dinD1 promoter.29 We set up an assay system with a dinD1::luxCADBE fusion plasmid as the reporter for SOS induction. We chose Y. pestis topoisomerase I as the initial target for this assay system. An E. coli host strain with the Imp4213 mutation known to enhance entry of small molecules into E. coli31 was utilized in preparation for high throughput screening of small molecules library for compounds that can enhance the accumulation of the cleavage complex formed by wild-type Y. pestis topoisomerase I. After wild-type recombinant Y. pestis topoisomerase I was induced from pAYTOP by arabinose treatment, the luciferase signal from E. coli BAS3023/pDinlux stayed at a reasonably low level (Figure 1a). Induction of pAYOP-128 results in the synthesis of a mutant topoisomerase I that accumulates the cleaved DNA complex due to a mutation in the TOPRIM domain.29 This leads to SOS induction,29 and a robust luciferase signal was produced from BAS3023/pDinlux (Figure 1b) in a dose-dependent manner corresponding to the concentration of arabinose regulating the synthesis of the mutant topoisomerase molecules. This validates the assay for its ability to generate a positive readout from accumulation of the cleavage complex formed by the recombinant bacterial type IA topoisomerase in E. coli. Figure 1. Open in new tabDownload slide Luciferase induction from dinD1::luxCADBE after the addition of arabinose to E. coli BAS3023 with (a) wild-type pAYTOP or (b) SOS-inducing mutant pAYTOP-128. The luciferase readings were normalized as response ratio by dividing by luciferase activity reading of culture with no arabinose added. Cells were grown to early exponential phase and arabinose was added at time zero. Figure 1. Open in new tabDownload slide Luciferase induction from dinD1::luxCADBE after the addition of arabinose to E. coli BAS3023 with (a) wild-type pAYTOP or (b) SOS-inducing mutant pAYTOP-128. The luciferase readings were normalized as response ratio by dividing by luciferase activity reading of culture with no arabinose added. Cells were grown to early exponential phase and arabinose was added at time zero. Hit compounds identified by the assay include small molecules that can enhance the DNA cleavage by bacterial type IA DNA topoisomerases High throughput assay was carried out in the facilities of ICCB, Harvard Medical School. Screening of a total of 49 268 compounds was conducted over a 3 day period. Each compound was assayed at a concentration of 16.7 mg/L in duplicate and the luciferase signal had to be at least 60% above the plate median in both assays of the compound for the compound to be classified as a positive hit. One microlitre of 5 mg/mL DMSO solution of 150 hit compounds was made available by ICCB for secondary assays. These compounds were tested in DNA oligonucleotide cleavage assays using purified Y. pestis and E. coli topoisomerase I. Three of the compounds (compounds 1–3, Figure 2) from ChemDiv were found to enhance the formation of DNA cleavage products by the bacterial type IA topoisomerases (Figure 3). No effect from these three compounds was observed for DNA cleavage by the type IB vaccinia topoisomerase I (Figure 3), so this action is specific for type IA topoisomerase. Phenanthrene and fluorene, which shared common structural features with compounds 1 and 2, were found to have no effect on DNA cleavage by E. coli DNA topoisomerase I (Figure 3). Quantification of the cleavage data from three experiments on E. coli DNA topoisomerase I by phosphorimager showed that the maximal enhancement of the cleavage product formation was 107 ± 5% for lead compound 1; 80 ± 20% for lead compound 2 and 150 ± 12% for lead compound 3. Figure 2. Open in new tabDownload slide Structures of lead compounds 1–3 identified in this study along with structures of phenanthrene and fluorene for comparison. Figure 2. Open in new tabDownload slide Structures of lead compounds 1–3 identified in this study along with structures of phenanthrene and fluorene for comparison. Figure 3. Open in new tabDownload slide Effect of compounds on the accumulation of DNA cleavage products by the type IA bacterial topoisomerases (Y. pestis and E. coli) and type IB topoisomerase (vaccinia virus). Compounds were present at a concentration of 20 mg/L. The arrows point to the cleavage products. C, control with no enzyme added; E, DMSO control; 1, lead compound 1; 2, lead compound 2; 3, lead compound 3; -, other initial SOS-inducing hits from high throughput assay; P, phenanthrene; F, fluorene. Figure 3. Open in new tabDownload slide Effect of compounds on the accumulation of DNA cleavage products by the type IA bacterial topoisomerases (Y. pestis and E. coli) and type IB topoisomerase (vaccinia virus). Compounds were present at a concentration of 20 mg/L. The arrows point to the cleavage products. C, control with no enzyme added; E, DMSO control; 1, lead compound 1; 2, lead compound 2; 3, lead compound 3; -, other initial SOS-inducing hits from high throughput assay; P, phenanthrene; F, fluorene. Further characterization of lead compounds Lead compounds 1–3 were purchased from ChemDiv for further analysis. Lead compound 1 has been previously characterized as the natural product stephenanthrine, a phenanthrene alkaloid.36 Phenanthrene and fluorene were also included in the experiments for comparison. SOS induction of luciferase activity from dinD1::luxCADBE in BAS3023 strain expressing recombinant Y. pestis topoisomerase I was measured for both wild-type Y. pestis topoisomerase I and a mutant derivative with its active site Tyr-325 changed to alanine. This active site mutant of Y. pestis topoisomerase I would not be able to form the covalent cleavage complex. Significant levels of SOS induction were also observed when either wild-type or the Y325A active site mutant Y. pestis topoisomerase I was expressed for all three compounds (Figure 4). The luciferase activity induced by lead compound 3 was higher when wild-type Y. pestis topoisomerase I was expressed. Analysis of dose–response showed that for lead compounds 1 and 3, high concentrations of the compounds led to a decrease in luciferase induction, possibly due to the low level of cellular ATP from loss of viability (Figure 4). Phenanthrene and fluorene treatment did not induce comparable levels of luciferase activity (data not shown). Figure 4. Open in new tabDownload slide Induction of luciferase activity from dinD1::luxCADBE in E. coli strain BAS3023 by the three lead compounds. The reporter strain was treated with arabinose at time zero to induce the expression of either wild-type recombinant Y. pestis topoisomerase I (wtYTOP, filled symbols) or a mutant with the active site tyrosine converted to alanine (AlaYTOP, open symbols). The luciferase induction ratio was calculated by normalizing the luciferase reading of the cultures treated with the compounds against the luciferase readings from control cultures treated with DMSO measured at 290 min after addition of compounds. Figure 4. Open in new tabDownload slide Induction of luciferase activity from dinD1::luxCADBE in E. coli strain BAS3023 by the three lead compounds. The reporter strain was treated with arabinose at time zero to induce the expression of either wild-type recombinant Y. pestis topoisomerase I (wtYTOP, filled symbols) or a mutant with the active site tyrosine converted to alanine (AlaYTOP, open symbols). The luciferase induction ratio was calculated by normalizing the luciferase reading of the cultures treated with the compounds against the luciferase readings from control cultures treated with DMSO measured at 290 min after addition of compounds. The effects of the lead compounds present at different concentrations on the relaxation activity of E. coli DNA topoisomerase I were determined (Figure 5). Addition of compounds 1, 2 and 3 was shown to result in complete inhibition of the formation of relaxed DNA products. Phenanthrene and fluorene at similar concentrations had no effect on the relaxation activity of E. coli DNA topoisomerase I. Compound 2 was less effective than compounds 1 and 3 in inhibition of relaxation. This correlates with the IC50 for increase in DNA cleavage products observed for these compounds in the DNA cleavage experiments (5 µM for lead compounds 1 and 3 and 10 µM for lead compound 2). Figure 5. Open in new tabDownload slide Inhibition of E. coli topoisomerase I relaxation activity. Lead compounds 1, 2 and 3, phenanthrene (P) and fluorene (F) were present at concentrations of 100, 20 or 4 µM in the reaction mixture. C, control reaction with no enzyme added; E, enzyme only; S, supercoiled DNA; R, relaxed and nicked DNA. Figure 5. Open in new tabDownload slide Inhibition of E. coli topoisomerase I relaxation activity. Lead compounds 1, 2 and 3, phenanthrene (P) and fluorene (F) were present at concentrations of 100, 20 or 4 µM in the reaction mixture. C, control reaction with no enzyme added; E, enzyme only; S, supercoiled DNA; R, relaxed and nicked DNA. Growth inhibition of E. coli Imp4213 mutant strain BAS3023, wild-type E. coli RFM443 and B. subtilis 6633 by these lead compounds was measured. The compounds had little or no effect on growth of wild-type E. coli RFM443 probably due to failure to enter the cell. Growth of E. coli BAS3023 with the Imp4213 mutation that enhanced permeability, as well as the Gram-positive B. subtilis, could be inhibited by these compounds, but not by phenanthrene and fluorene present at similar concentrations (Table 1). E. coli strain BAS3023 transformed with wild-type pAYTOP showed slightly greater sensitivity to lead compound 3 when compared with the same strain transformed with plasmid expressing the active site mutant of YTOP, in agreement with the luciferase induction data shown in Figure 4. This suggests that overexpression of wild-type Y. pestis topoisomerase I enhanced the susceptibility to this compound. Table 1. Inhibition of bacterial growth by lead compounds Compound . MIC (μM) . E. coli IMP4213 . B. subtilis 6633 . YTOP . YTOPala . Compound 1 100 100 60 Compound 2 80 80 80 Compound 3 40 60 40 Phenanthrene >800 >800 >200 Fluorene >800 >800 >200 Compound . MIC (μM) . E. coli IMP4213 . B. subtilis 6633 . YTOP . YTOPala . Compound 1 100 100 60 Compound 2 80 80 80 Compound 3 40 60 40 Phenanthrene >800 >800 >200 Fluorene >800 >800 >200 E. coli IMP4213 strain BAS3023 was induced for expression of recombinant Y. pestis topoisomerase I YTOP or its mutant derivative YTOPala (with active site tyrosine changed to alanine) for 2 h prior to dilution into fresh medium containing the compounds. Open in new tab Table 1. Inhibition of bacterial growth by lead compounds Compound . MIC (μM) . E. coli IMP4213 . B. subtilis 6633 . YTOP . YTOPala . Compound 1 100 100 60 Compound 2 80 80 80 Compound 3 40 60 40 Phenanthrene >800 >800 >200 Fluorene >800 >800 >200 Compound . MIC (μM) . E. coli IMP4213 . B. subtilis 6633 . YTOP . YTOPala . Compound 1 100 100 60 Compound 2 80 80 80 Compound 3 40 60 40 Phenanthrene >800 >800 >200 Fluorene >800 >800 >200 E. coli IMP4213 strain BAS3023 was induced for expression of recombinant Y. pestis topoisomerase I YTOP or its mutant derivative YTOPala (with active site tyrosine changed to alanine) for 2 h prior to dilution into fresh medium containing the compounds. Open in new tab Discussion The luciferase screening system utilizing the dinD1::luxCADBE plasmid was shown here to respond to the accumulation of covalent DNA cleavage complex formed by the recombinant Y. pestis topoisomerase I G122S TOPRIM mutant expressed in E. coli. After screening 49268 compounds, three small molecules that can enhance the level of DNA cleavage product of bacterial type IA topoisomerases were identified from 150 initial positive hit compounds. These three compounds enhanced the level of DNA cleavage product formed by both E. coli and Y. pestis topoisomerase I, as expected from the high degree of identity (85%) between the two enzyme sequences. The type IB vaccinia topoisomerase I does not share any homology with these bacterial enzymes. DNA cleavage by vaccinia topoisomerase I was not enhanced by the three lead compounds identified here. Induction of SOS by these three compounds did not depend strictly on the overexpression of recombinant Y. pestis topoisomerase I. However, there are two type IA topoisomerases, topoisomerase I and III, encoded by the topA and topB genes present in E. coli. These type IA topoisomerases share significant homology in the transesterification domain. E. coli mutants lacking both topA and topB gene functions are not viable.17 Interaction of the compounds with either or both of these E. coli type IA topoisomerases might be sufficient to induce the SOS response. A large number of compounds that target type IB and type IIA topoisomerases are known to interact with DNA and this interaction influences the stability of the covalent complex formed by topoisomerases after DNA cleavage.37–40 It is therefore possible that certain poisons of type IA bacterial topoisomerases may also induce the E. coli SOS response via a second mechanism independent of topoisomerase IA cleavage activity. The modes of action of the compounds found here to enhance the bacterial topoisomerase I cleavage will be further investigated with future experiments by selecting for mutant strains resistant to the action of these compounds. The antibacterial activities of the lead compounds identified here are not potent, especially against Gram-negative E. coli. It would be desirable to identify either analogues of these lead compounds or other novel compounds that have stronger broad spectrum antibacterial activities. Nevertheless, this is the first time that the cleavage–religation equilibrium of this class of topoisomerase has been shown to be shifted towards DNA cleavage by the action of a small molecule. It is hoped that the continued high throughput screening of compound libraries and follow-up efforts can yield a candidate for development of new antibacterial agents targeting type IA DNA topoisomerases. Acknowledgements We thank Professor Thomas Silhavy (Princeton University) for providing strain BAS3023. Facilities and compound libraries for high throughput screening were made available through the NSRB/NERCE programme. We thank members of the NSRB and ICCB-Longwood (Harvard Medical School) for their ongoing advice. This work was funded by grants from the National Institutes of Health (R03 NS050782 and R01 AI 069313) and a grant from the New York State Department of Health (C-020219) administered by the Northeast Biodefense Center. Transparency declarations None to declare. References 1 Champoux JJ . DNA topoisomerases: structure, function, and mechanism , Annu Rev Biochem , 2001 , vol. 70 (pg. 369 - 413 ) Google Scholar Crossref Search ADS PubMed WorldCat 2 Wang JC . Cellular roles of DNA topoisomerases: a molecular perspective , Nat Rev Mol Cell Biol , 2002 , vol. 3 (pg. 430 - 40 ) Google Scholar Crossref Search ADS PubMed WorldCat 3 Drlica K . Control of bacterial DNA supercoiling , Mol Microbiol , 1992 , vol. 6 (pg. 425 - 33 ) Google Scholar Crossref Search ADS PubMed WorldCat 4 Luttinger A . The twisted ‘life’ of DNA in the cell: bacterial topoisomerases , Mol Microbiol , 1995 , vol. 15 (pg. 601 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat 5 Nitiss JL . DNA topoisomerases in cancer chemotherapy: using enzymes to generate selective DNA damage , Curr Opin Investig Drugs , 2002 , vol. 3 (pg. 1512 - 6 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 6 Denny WA . Emerging DNA topoisomerase inhibitors as anticancer drugs , Expert Opin Emerg Drugs , 2004 , vol. 9 (pg. 105 - 33 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 7 Capranico G , Zagotto G , Palumbo M . Development of DNA topoisomerase-related therapeutics: a short perspective of new challenges , Curr Med Chem Anti-canc Agents , 2004 , vol. 4 (pg. 335 - 45 ) Google Scholar Crossref Search ADS WorldCat 8 Anderson VE , Osheroff N . Type II topoisomerases as targets for quinolone antibacterials: turning Dr. Jekyll into Mr. Hyde , Curr Pharm Des , 2001 , vol. 7 (pg. 337 - 53 ) Google Scholar Crossref Search ADS PubMed WorldCat 9 Hooper DC . Mechanisms of action of antimicrobials: focus on fluoroquinolones , Clin Infect Dis , 2001 , vol. 32 (pg. S9 - 15 ) Google Scholar Crossref Search ADS PubMed WorldCat 10 Drilica K , Malik M . Fluoroquinolones: action and resistance , Curr Top Med Chem , 2003 , vol. 3 (pg. 249 - 82 ) Google Scholar Crossref Search ADS PubMed WorldCat 11 Liu LF . DNA topoisomerase poisons as antitumor drugs , Annu Rev Biochem , 1989 , vol. 58 (pg. 351 - 75 ) Google Scholar Crossref Search ADS PubMed WorldCat 12 Drlica K , Franco RJ . Inhibitors of DNA topoisomerases , Biochemistry , 1988 , vol. 27 (pg. 2252 - 9 ) Google Scholar Crossref Search ADS WorldCat 13 Smith PJ . DNA topoisomerase dysfunction: a new goal for antitumor chemotherapy , Bioessay , 1990 , vol. 12 (pg. 167 - 72 ) Google Scholar Crossref Search ADS WorldCat 14 Canton R , Coque TM , Baquero F . Multi-resistant gram-negative bacilli: from epidemics to endemics , Curr Opin Infect Dis , 2003 , vol. 16 (pg. 315 - 25 ) Google Scholar Crossref Search ADS PubMed WorldCat 15 Stevens DL . Community-acquired Staphylococcus aureus infections: increasing virulence and emerging methicillin resistance in the new millennium , Curr Opin Infect Dis , 2003 , vol. 16 (pg. 189 - 91 ) Google Scholar Crossref Search ADS PubMed WorldCat 16 Levy SB , Marshall B . Antibacterial resistance worldwide: causes, challenges and responses , Nat Med , 2004 , vol. 10 (pg. S122 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat 17 Zhu Q , Pongpech P , DiGate RJ . Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli. , Proc Natl Acad Sci USA , 2001 , vol. 98 (pg. 9766 - 71 ) Google Scholar Crossref Search ADS PubMed WorldCat 18 Caron PR . Compendium of DNA topoisomerase sequences , Methods Mol Biol , 1999 , vol. 94 (pg. 279 - 316 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 19 Stupina VA , Wang JC . On the viability of Escherichia coli topA mutants lacking DNA topoisomerase I , J Biol Chem , 2005 , vol. 280 (pg. 355 - 60 ) Google Scholar Crossref Search ADS PubMed WorldCat 20 Richardson SM , Higgins CF , Lilley DM . The genetic control of DNA supercoiling in Salmonella typhimurium. , EMBO J , 1984 , vol. 3 (pg. 1745 - 52 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 21 Pruss GJ , Manes SH , Drlica K . Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes , Cell , 1982 , vol. 31 (pg. 35 - 42 ) Google Scholar Crossref Search ADS PubMed WorldCat 22 DiNardo S , Voelkel KA , Sternglanz R , et al. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes , Cell , 1982 , vol. 31 (pg. 43 - 51 ) Google Scholar Crossref Search ADS PubMed WorldCat 23 Takel M , Fukuda H , Kishii R , et al. Target preference of 15 quinolones against Staphyloccus aureus, based on antibacterial activities and target inhibition , Antimicrob Agents Chemother , 2001 , vol. 45 (pg. 3544 - 7 ) Google Scholar Crossref Search ADS PubMed WorldCat 24 Hooper DC . Mechanisms of action and resistance of older and newer fluoroquinolones , Clin Infect Dis , 2000 , vol. 31 (pg. S24 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat 25 Qi H , Menzel R , Tse-Dinh YC . Effect of the deletion of the σ32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance , Mol Microbiol , 1996 , vol. 21 (pg. 703 - 11 ) Google Scholar Crossref Search ADS PubMed WorldCat 26 Qi H , Menzel R , Tse-Dinh YC . Increased sensitivity to thermal challenges associated with topA deletion and promoter mutations in Escherichia coli. , FEMS Microbiol Lett , 1999 , vol. 178 (pg. 141 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat 27 Tse-Dinh YC . Increased sensitivity to oxidative challenges associated with topA deletion in Escherichia coli. , J Bacteriol , 2000 , vol. 182 (pg. 829 - 32 ) Google Scholar Crossref Search ADS PubMed WorldCat 28 Stewart N , Feng J , Liu X , et al. Loss of topoisomerase I function affects the RpoS-dependent and GAD systems of acid resistance in Escherichia coli. , Microbiology , 2005 , vol. 151 (pg. 2783 - 91 ) Google Scholar Crossref Search ADS PubMed WorldCat 29 Cheng B , Shukla S , Vasunilashorn S , et al. Bacterial cell killing mediated by topoisomerase I DNA cleavage activity , J Biol Chem , 2005 , vol. 280 (pg. 38489 - 95 ) Google Scholar Crossref Search ADS PubMed WorldCat 30 Braun M , Silhavy TJ . Imp/OstA is required for cell envelope biogenesis in Escherichia coli. , Mol Microbiol , 2002 , vol. 45 (pg. 1298 - 302 ) Google Scholar Crossref Search ADS WorldCat 31 Sampson BA , Misra R , Benson SA . Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability , Genetics , 1989 , vol. 122 (pg. 491 - 501 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 32 Van Dyk TK , Ayers BL , Morgan RW , et al. Constricted flux through the branched-chain amino acid biosynthetic enzyme acetolactate synthase triggers elevated expression of genes regulated by rpoS and internal acidification , J Bacteriol , 1998 , vol. 180 (pg. 785 - 92 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 33 Kirkegaard K , Pflugfelder G , Wang JC . The cleavage of DNA by type I-DNA topoisomerases , Cold Spring Harb Symp Quant Biol , 1984 , vol. 49 (pg. 411 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat 34 Shuman S , Golder M , Moss B . Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. , J Biol Chem , 1988 , vol. 263 (pg. 16401 - 7 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 35 Shuman S , Prescott J . Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I , J Biol Chem , 1990 , vol. 265 (pg. 17826 - 36 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 36 Lopez-Martin J , Anam EM , Boira H , et al. Chromone and phenanthrene alkaloids from Dennettia tripietala. , Chem Pharm Bull , 2002 , vol. 50 (pg. 1613 - 5 ) Google Scholar Crossref Search ADS PubMed WorldCat 37 Pommier Y , Minford JK , Schwartz RE , et al. Effects of the DNA intercalators 4′-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage , Biochemistry , 1985 , vol. 24 (pg. 6410 - 6 ) Google Scholar Crossref Search ADS PubMed WorldCat 38 Sim SP , Gatto B , Yu C , et al. Differential poisoning of topoisomerases by menogaril and nogalamycin dictated by the minor groove-binding nogalose sugar , Biochemistry , 1997 , vol. 36 (pg. 13285 - 91 ) Google Scholar Crossref Search ADS PubMed WorldCat 39 Bailly C , Colson P , Houssier C , et al. Recognition of specific sequences in DNA by a topoisomerase I inhibitor derived from the antitumor drug rebeccamycin , Mol Pharmacol , 1998 , vol. 53 (pg. 77 - 87 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 40 McClendon AK , Osheroff N . The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs , Biochemistry , 2006 , vol. 45 (pg. 3040 - 50 ) Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org The online version of this article has been published under an open access model. 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Journal

Journal of Antimicrobial Chemotherapy – Oxford University Press

Published: Apr 1, 2007

Keywords: anti-bacterial agents; cytokinesis; dna, bacterial

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