The roles of bacterial RecA in the evolution and transmission of antibiotic resistance genes make it an attractive target for inhibition by small molecules. We report two complementary fluorescence-based ATPase assays that were used to screen for inhibitors of RecA. We elected to employ the ADP-linked variation of the assay, with a Z′ factor of 0.83 in 96-well microplates, to assess whether 18 select compounds could inhibit ATP hydrolyis by RecA. The compounds represented five sets of related inhibitor scaffolds, each of which had the potential to cross-inhibit RecA. Although nucleotide analogs, known inhibitors of GHL ATPases, and known protein kinase inhibitors were not active against RecA, we found that three suramin-like agents substantially inhibited RecA's ATPase activity

Singleton, Scott F.

Wigle, Tim J.

PubMed

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Directed Molecular Screening for RecA ATPase InhibitorsTim J. Wigle and Scott F. Singleton**School of Pharmacy, Division of Medicinal Chemistry & Natural Products, The University of North Carolinaat Chapel Hill, CB #7360, Chapel Hill, NC 27599-7360, USAAbstractThe roles of bacterial RecA in the evolution and transmission of antibiotic resistance genes make itan attractive target for inhibition by small molecules. We report two complementary fluorescence-based ATPase assays that were used to screen for inhibitors of RecA. We elected to employ the ADP-linked variation of the assay, with a Z′ factor of 0.83 in 96-well microplates, to assess whether 18select compounds could inhibit ATP hydrolyis by RecA. The compounds represented five sets ofrelated inhibitor scaffolds, each of which had the potential to cross-inhibit RecA. Although nucleotideanalogs, known inhibitors of GHL ATPases, and known protein kinase inhibitors were not activeagainst RecA, we found that three suramin-like agents substantially inhibited RecA's ATPaseactivity.Drug resistance is an ever-increasing problem in the chemotherapy of bacterial infectiousdiseases. The de novo development and clonal spread of drug-resistant bacteria, and thehorizontal transfer of resistance factors among bacteria have resulted in a dramatic increase inthe incidence of drug-resistant infections. One strategy to improve the efficacy of existingantibacterial drugs involves countering bacterial mechanisms of drug resistance. In this context,RecA has emerged as a potential target because its activities allow bacteria to overcome themetabolic stress induced by a range of antibacterial agents, and promote the de novodevelopment and transmission of antibiotic resistance genes.1-5 Although potent and selectiveinhibitors of RecA could be used to modulate its activities in the development of antibioticresistance, no small-molecule natural product inhibitor of RecA's activities has been reported.Herein, we report two rapid, microvolume molecular screening assays and theirimplementation in the directed screening of prospective inhibitors of RecA's ATPase activity.We have previously demonstrated that select NDP and NTP analogs inhibit RecA ATPhydrolysis.6,7 Because nucleotide analogs are largely unsuited for use in cell-based assays, wescreened a small, focused set of commercially available compounds to discover non-nucleotideinhibitors of RecA. The compounds we elected to study can be ordered in five groups (Figure1). The first group comprises vanillin,8,9 cinnamaldehyde,9 curcumin,10 and the soy-derivedcompounds genistin and genistein,11 all of which may inhibit RecA based on their activitiesin microbiological assays. The second group includes adenosine nucleotide-likecompounds12,13 that may extend upon our previous success with ADP analogs. The thirdgroup is composed of inhibitors of the gyrase-Hsp90-like (GHL) family of ATPases.14,15 Thefourth group includes adenine-like inhibitors of protein kinases.16 The fifth group comprisescompounds related to the non-nucleotide inhibitors of purine nucleotide receptors, suramin andPPADS.17,18Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptBioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.Published in final edited form as:Bioorg Med Chem Lett. 2007 June 15; 17(12): 3249–3253.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptHigh-throughput screening is a useful method for the identification of novel inhibitoryscaffolds. Recently, we reported a coupled enzyme assay that was optimized for determinationof E. coli RecA's ssDNA-dependent ATPase activity, which is a useful indicator of activeRecA-DNA filament assembly.7 It was undesirable to use this assay to screen a larger, morediverse library because many of the compounds may be UV active at 360 nm. This interferingabsorbance would lead to false negatives in a high-throughput screening project.To address this concern, we developed two robust and reproducible microplate assays forRecA's ATPase activity that are suitable for screening collections of small molecules asprospective RecA inhibitors without the potential for signal interference generated by UV-active compounds (Figure 2). Each variation of the assay utilizes one product of ATPhydrolysis, either ADP or Pi, as a substrate for commercially available enzymes and, for everymolecule of ATP hydrolyzed by RecA, one molecule of amplex red is ultimately oxidized toresorufin, which has a unique fluorescence emission at 595 nm.19 In one variant of the assay,Pi and inosine serve as substrates for PNP in the production of hypoxanthine and ribose-1-phosphate. In turn, the O2-dependent oxidation of hypoxanthine by xanthine oxidase producesuric acid and H2O2, the latter of which is used by horseradish peroxidase to oxidize amplexred to resorufin. In the other assay variant, ADP and phosphoenolpyruvate serve as substratesfor the commercially available enzyme pyruvate kinase to produce ATP and pyruvate, the latterof which is a substrate for O2-dependent oxidization by pyruvate oxidase in the production ofacetylphosphate and H2O2.20 Identical to the first assay, horseradish peroxidase uses H2O2 tocatalyze the oxidation of amplex red to resorufin.To determine if these assays were suitable for high-throughput screening, we assessed theirrobustness and reproducibility using a statistical analysis.21 In our hands, the ADP-linkedATPase assay was more useful as a screening assay because the Pi-linked assay was sensitiveto variations in the residual phosphate contaminating enzyme and DNA preparations. For theADP-linked ATPase assay optimized for 96-well microplates,22 positive and negative controlexperiments were performed on three different days with 48 wells per condition to simulatethe day-to-day and well-to-well variability between assays (Figure 3). Statistical evaluation ofthe results yielded a reproducible Z′ factor of 0.83, demonstrating the excellent utility of theassay for reproducibly differentiating normal activity from inhibition. Furthermore, theinclusion of the two most potent NTP-analog inhibitors discovered in our previous work7verified that known RecA ATPase inhibitors would prevent resorufin production using thisassay system (Figure 3).With a suitable ATPase assay in hand, we assessed the abilities of the 18 compounds in ourdirected mini-library (Figure 1) to inhibit RecA's ATPase activity at 100 μM. The fractionalinhibition observed in the presence of each compound was obtained by comparing the totalfluorescence in wells containing the reaction in the presence and absence of inhibitor (Figure4). Only curcumin from Group 1 and the polysulfated naphthyl compounds suramin, CongoRed and bis-ANS, from Group 5, appeared to inhibit RecA's ATPase activity under theseconditions.Upon the identification of molecules that attenuated resorufin production, it was necessary todetermine whether these compounds were selectively inhibiting RecA or other components ofthe coupled assay system. Therefore, four compounds were evaluated in subsequent controlreactions in the presence of 500 μM ADP, but in the absence of RecA. Because all four of thesecompounds also inhibited pyruvate kinase in the control assay, we evaluated their abilities toinhibit RecA's ATPase activity using the Pi-linked fluorescence ATPase assay described above.Suramin, Congo Red, and bis-ANS, but not curcumin, inhibited ATP hydrolysis by RecA.Wigle and Singleton Page 2Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptIt is important to note that none of the compounds expected to inhibit RecA based on priorbiological activity studies (Group 1) inhibited RecA's ATPase activity in vitro. Possibleexplanations for the apparent inconsistency include the following: (1) these compounds mayinhibit RecA-associated proteins rather than RecA itself; or (2) these compounds may interferewith RNA or protein synthesis. Of additional importance is the observation that no knowninhibitor from Group 2, 3 or 4 substantially inhibited RecA's ATPase activity. The failure todiscover a RecA inhibitor among these compounds suggests that the development of a potentRecA inhibitor will not be a trivial exercise. However, the same lack of cross-inhibition ofRecA by known inhibitors of other ATP-dependent enzymes also suggests the likelihood ofultimately discovering specific inhibitors of RecA's activities.Although we did not observe inhibition of RecA by any of the molecules in Groups 1, 2, 3, or4, we did find that the polysulfated naphthyl compounds Congo Red, suramin, and bis-ANSstrongly inhibited the ATPase activity of RecA. The nature of the inhibition by thesecompounds is reputed to be promiscuous. Indeed, it has been established that, in aqueoussolution, Congo Red self-assembles into supramolecular aggregates23,24 that can result inapparent inhibition by the reversible sequestration of enzyme.25,26 In contrast, suramin, whichis also active against many different enzymes27 and is structurally similar to Congo Red, doesnot form aggregates and does not inhibit model enzymes that are sensitive to supramolecularligands.25,26 The possibility that suramin may be a structure- or mechanism-specific inhibitorof RecA is supported by the observation that PPADS does not inhibit RecA's ATPase activity,despite the fact that both compounds are potent antagonists of the P2X1 nucleotide receptors.18 Finally, it is noteworthy that select transition metal cations trap inactive RecA as insolubleaggregates,28 but no visible precipitates were formed in the presence of suramin, Congo Red,or bis-ANS.To probe this class of polysulfated naphthyl compounds further, we characterized the natureof the inhibition of RecA by suramin. While suramin inhibited RecA's ATPase activity withan IC50 of approximately 2 μM, the inhibition was not competitive with respect to ATP orssDNA binding (data not shown). We speculate that suramin modulates RecA's activity bybinding to an allosteric region of the protein and trapping the protein in its inactiveconformation (Figure 5).Importantly, we demonstrated that suramin interferes with the RecA-mediated DNA strandexchange reaction, an established RecA activity that serves as an in vitro model for itsphysiologic recombinational functions.29 It is known that RecA must hydrolyze ATP in orderto carry out the strand exchange reaction between ϕχ174 cssDNA and homologous, lineardsDNA (S) to yield a nicked circular dsDNA product (P), which migrates more slowly underelectrophoretic conditions than the substrate DNA molecules. The presence of suramin (100μM) in this reaction completely abrogated the formation of nicked circular dsDNA product,even over the course of 90 min (Figure 6).In conclusion, we have reported two new fluorescence-based assays for screening potentialinhibitors of RecA's ATPase activity. We previously developed activity assays for RecA'sATPase and filament assembly activities,6,7 and the new molecular screening assayscomplement and extend the previous assays by providing an observable parameter that is notinfluenced adversely by UV-active compounds. Moreover, the assays reported herein wereoptimized for use with RecA based on its production of either free phosphate or ADP. Wefurther reported that suramin, Congo Red, and bis-ANS strongly inhibit RecA's ATPase assayand compose a new structural class of RecA inhibitors. We expect that polysulfated naphthylcompounds such as these are likely to be of little therapeutic utility due to membrane-impermeability caused by their negative charges. Nonetheless these compounds may be usedin future rational modification procedures for the synthesis microbiological tools to tease apartWigle and Singleton Page 3Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptthe roles of RecA in various aspects of pathogenicity. We envision that such inhibitors mayultimately be developed into novel adjuvants for antibiotic chemotherapy that moderate thedevelopment and transmission of antibiotic resistance genes and increase the antibiotictherapeutic index.AcknowledgmentsThe authors gratefully acknowledge support of this project by NIH grant GM58114.References1. Beaber JW, Hochhut B, Waldor MK. Nature 2004;427:72. [PubMed: 14688795]2. Hastings PJ, Rosenberg SM, Slack A. Trends Microbiol 2004;12:401. [PubMed: 15337159]3. Hersh MN, Ponder RG, Hastings PJ, Rosenberg SM. Res. Microbiol 2004;155:352. [PubMed:15207867]4. Matic I, Taddei F, Radman M. Res. Microbiol 2004;155:337. [PubMed: 15207865]5. Foster PL. Mutat. Res 2005;569:3. [PubMed: 15603749]6. Lee AM, Ross CT, Zeng BB, Singleton SF. J. Med. Chem 2005;48:5408. [PubMed: 16107138]7. Wigle TJ, Lee AM, Singleton SF. Biochemistry 2006;45:4502. [PubMed: 16584186]8. Ohta T, Watanabe M, Shirasu Y, Inoue T. Mutat. Res 1988;201:107. [PubMed: 3047569]9. Shaughnessy DT, Schaaper RM, Umbach DM, Demarini DM. Mutat. Res 2006;602:54. [PubMed:16999979]10. Oda Y. Mutat. Res 1995;348:67. [PubMed: 7477054]11. Yang Y, Fix D. Mutat Res 2006;600:193. [PubMed: 16872640]12. Knight KL, McEntee K. J. Biol. Chem 1985;260:10177. [PubMed: 3894365]13. Scholz G, Barritt GJ, Kwok F. Eur. J. Biochem 1992;210:461. [PubMed: 1333953]14. Burlison JA, Neckers L, Smith AB, Maxwell A, Blagg BS. J. Am. Chem. Soc 2006;128:15529.[PubMed: 17132020]15. Corbett KD, Berger JM. Nucleic Acids Res 2006;34:4269. [PubMed: 16920739]16. Wang Y, Wagner MB, Kumar R, Cheng J, Joyner RW. Pflugers Arch 2003;446:485. [PubMed:12719980]17. Lambrecht G, Braun K, Damer M, Ganso M, Hildebrandt C, Ullmann H, Kassack MU, Nickel P.Curr. Pharm. Des 2002;8:2371. [PubMed: 12369951]18. Jacobson KA, Costanzi S, Ohno M, Joshi BV, Besada P, Xu B, Tchilibon S. Curr. Top. Med. Chem2004;4:805. [PubMed: 15078212]19. Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP. Anal. Biochem 1997;253:162. [PubMed:9367498]20. Zhang B, Senator D, Wilson CJ, Ng SC. Anal. Biochem 2005;345:326. [PubMed: 16125662]21. Zhang JH, Chung TD, Oldenburg KR. J. Biomol. Screen 1999;4:67. [PubMed: 10838414]22. Optimized ADP-linked assay conditions (100 μL volume, flat-bottom 96-well blackplates): 0.5 μME. coli RecA, 5 μM-nts poly(dT) ssDNA, 10 mM Mg(OAc)2, 500μM ATP, 250 μMphosphoenolpyruvate, 100 μM amplex red, 1 U/mL pyruvate kinase, 1 U/mL pyruvate oxidase, 1 U/mL horseradish peroxidase, and 25 mM Tris·HOAc, pH 7.5. After 25-min incubation at 37 °C thereaction was excited at 485 nm and emission was observed at 595 nm in a microplate reader. Positivecontrol reactions were performed in the absence of any inhibitor to simulate an unimpeded ATPasereaction and negative control reactions lacked RecA to simulate the complete inhibition of ATPhydrolysis.23. Stopa B, Konieczny L, Piekarska B, Roterman I, Rybarska J, Skowronek M. Biochimie 1997;79:23.[PubMed: 9195042]24. Konieczny L, Piekarska B, Rybarska J, Skowronek M, Stopa B, Tabor B, Dabros W, Pawlicki R,Roterman I. Folia Histochem. Cytobiol 1997;35:203. [PubMed: 9619419]25. McGovern SL, Caselli E, Grigorieff N, Shoichet BK. J. Med. Chem 2002;45:1712. [PubMed:11931626]Wigle and Singleton Page 4Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript26. McGovern SL, Helfand BT, Feng B, Shoichet BK. J. Med. Chem 2003;46:4265. [PubMed: 13678405]27. Stein CA. Cancer Res 1993;53:2239. [PubMed: 8485709]28. Lee AM, Singleton SF. J. Inorg. Biochem 2004;98:1981. [PubMed: 15522426]29. Lusetti SL, Cox MM. Annu. Rev. Biochem 2002;71:71. [PubMed: 12045091]Wigle and Singleton Page 5Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.Five classes of compounds screened for RecA inhibition.Wigle and Singleton Page 6Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2.Two fluorescent ATPase assay schemes used to monitor ATP hydrolysis by RecA .Wigle and Singleton Page 7Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 3.Interday and intercolumn precision for the ADP-linked fluorescent ATPase assay.Wigle and Singleton Page 8Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 4.Results of the directed screen of 18 selected compounds against RecA ATP hydrolysis activity.Wigle and Singleton Page 9Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 5.Cartoons depicting the inactive and active conformation of RecA filaments and the roles of thelatter in the de novo development and transmission of antibiotic resistance (AR) genes.Wigle and Singleton Page 10Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 6.Suramin inhibits the RecA-mediated DNA three strand exchange reaction.Wigle and Singleton Page 11Bioorg Med Chem Lett. Author manuscript; available in PMC 2008 June 15.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Directed molecular screening for RecA ATPase inhibitors

http://dx.doi.org/10.1016/j.bmcl.2007.04.013

Directed molecular screening for RecA ATPase inhibitors

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