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A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe

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Abstract

High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N′-phenyloctandiamide.

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References

  1. West AC, Johnstone RW (2014) New and emerging HDAC inhibitors for cancer treatment. J Clin Investig 124(1):30–39

    Article  CAS  Google Scholar 

  2. Andrews KT, Haque A, Jones MK (2011) HDAC inhibitors in parasitic diseases. Immunol Cell Biol 90(1):66–77

    Article  Google Scholar 

  3. Eom GH, Kook H (2014) Posttranslational modifications of histone deacetylases: Implications for cardiovascular diseases. Pharmacol Ther. doi:10.1016/j.pharmthera.2014.02.012

    Google Scholar 

  4. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406(6799):959–964. doi:10.1038/35023079

    Article  CAS  Google Scholar 

  5. Large PJ (1992) Enzymes and pathways of polyamine breakdown in microorganisms. FEMS Microbiol Rev 8(3–4):249–262

    Article  CAS  Google Scholar 

  6. Patel CN, Wortham BW, Lines JL, Fetherston JD, Perry RD, Oliveira MA (2006) Polyamines are essential for the formation of plague biofilm. J Bacteriol 188(7):2355–2363

    Article  CAS  Google Scholar 

  7. Karatan E, Duncan TR, Watnick PI (2005) NspS, a predicted polyamine sensor, mediates activation of vibrio cholerae biofilm formation by norspermidine. J Bacteriol 187(21):7434–7443. doi:10.1128/jb.187.21.7434-7443.2005

    Article  CAS  Google Scholar 

  8. Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73

    Article  Google Scholar 

  9. Chen B-H, Wang C-C, Lu L-Y, Hung K-S, Yang Y-S (2013) Fluorescence assay for protein post-translational tyrosine sulfation. Anal Bioanal Chem 405(4):1425–1429

    Article  CAS  Google Scholar 

  10. Geng X, Zhang D, Wang H, Zhao Q (2013) Screening interaction between ochratoxin A and aptamers by fluorescence anisotropy approach. Anal Bioanal Chem 405(8):2443–2449

    Article  CAS  Google Scholar 

  11. Riester D, Hildmann C, Schwienhorst A, Meyer-Almes FJ (2007) Histone deacetylase inhibitor assay based on fluorescence resonance energy transfer. Anal Biochem 362(1):136–141. doi:10.1016/j.ab.2006.12.019

    Article  CAS  Google Scholar 

  12. Li Y, Xie W, Fang G (2008) Fluorescence detection techniques for protein kinase assay. Anal Bioanal Chem 390(8):2049–2057

    Article  CAS  Google Scholar 

  13. Diamandis EP, Christopoulos TK (1990) Europium chelate labels in time-resolved fluorescence immunoassays and DNA hybridization assays. Anal Chem 62(22):1149A–1157A

    Article  CAS  Google Scholar 

  14. Pritz S, Meder G, Doering K, Drueckes P, Woelcke J, Mayr LM, Hassiepen U (2011) A fluorescence lifetime-based assay for Abelson kinase. J Biomol Screen 16(1):65–72

    Article  CAS  Google Scholar 

  15. Riester D, Hildmann C, Haus P, Galetovic A, Schober A, Schwienhorst A, Meyer-Almes FJ (2009) Non-isotopic dual parameter competition assay suitable for high-throughput screening of histone deacetylases. Bioorg Med Chem Lett 19(13):3651–3656. doi:10.1016/j.bmcl.2009.04.102

    Article  CAS  Google Scholar 

  16. Lebakken CS, Kang HC, Vogel KW (2007) A fluorescence lifetime-based binding assay to characterize kinase inhibitors. J Biomol Screen 12(6):828–841

    Article  CAS  Google Scholar 

  17. Comley J (2003) Assay interference. Drug Discovery:91

  18. Maltman BA, Dunsmore CJ, Couturier SC, Tirnaveanu AE, Delbederi Z, McMordie RAS, Naredo G, Ramage R, Cotton G (2010) 9-Aminoacridine peptide derivatives as versatile reporter systems for use in fluorescence lifetime assays. Chem Commun 46(37):6929–6931

    Article  CAS  Google Scholar 

  19. Chou HT, Kwon DH, Hegazy M, Lu CD (2008) Transcriptome analysis of agmatine and putrescine catabolism in Pseudomonas aeruginosa PAO1. J Bacteriol 190(6):1966–1975

    Article  CAS  Google Scholar 

  20. Riester D, Wegener D, Hildmann C, Schwienhorst A (2004) Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates. Biochem Biophys Res Commun 324(3):1116–1123. doi:10.1016/j.bbrc.2004.09.155

    Article  CAS  Google Scholar 

  21. Lombardi PM, Angell HD, Whittington DA, Flynn EF, Rajashankar KR, Christianson DW (2011) Structure of prokaryotic polyamine deacetylase reveals evolutionary functional relationships with eukaryotic histone deacetylases. Biochemistry 50(11):1808–1817. doi:10.1021/bi101859k

    Article  CAS  Google Scholar 

  22. Wessig P, Wawrzinek R, Möllnitz K, Feldbusch E, Schilde U (2011) A new class of fluorescent dyes based on 1,3-benzodioxole and [1,3]-dioxolo[4.5-f]benzodioxole. Tetrahedron Lett 52(46):6192–6195

    Article  CAS  Google Scholar 

  23. Henkes LM, Haus P, Jager F, Ludwig J, Meyer-Almes FJ (2012) Synthesis and biochemical analysis of 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoro-N-hydroxy-octanediamides as inhibitors of human histone deacetylases. Bioorg Med Chem 20(2):985–995. doi:10.1016/j.bmc.2011.11.041

    Article  CAS  Google Scholar 

  24. Boivin J, Kaim LE, Zard SZ (1995) A new and efficient synthesis of trifluoromethyl ketones from carboxylic acids. Part I. Tetrahedron 51(9):2573–2584

    Article  CAS  Google Scholar 

  25. Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, Garcia KC, Bogyo M (2009) Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag. PLoS One 4(12):e8119. doi:10.1371/journal.pone.0008119

    Article  Google Scholar 

  26. Hochuli E, Bannwarth W, Dobeli H, Gentz R, Stuber D (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nat Biotechnol 6(11):1321–1325

    Article  CAS  Google Scholar 

  27. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85

    Article  CAS  Google Scholar 

  28. Wegener D, Hildmann C, Riester D, Schober A, Meyer-almes F-J, Deubzer HE, Oehme I, Witt O, Lang S, Jaensch M, Makarov V, Lange C, Busse B, Schwienhorst A (2008) Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay. Biochem J 413(1):143–150. doi:10.1042/bj20080536

    Article  CAS  Google Scholar 

  29. Volund A (1978) Application of the four-parameter logistic model to bioassay: comparison with slope ratio and parallel line models. Biometrics 34(3):357–365

    Article  CAS  Google Scholar 

  30. Wawrzinek R, Wessig P, Mollnitz K, Nikolaus J, Schwarzer R, Muller P, Herrmann A (2012) DBD dyes as fluorescent probes for sensing lipophilic environments. Bioorg Med Chem Lett 22(17):5367–5371. doi:10.1016/j.bmcl.2012.07.056

    Article  CAS  Google Scholar 

  31. Wawrzinek R, Ziomkowska J, Heuveling J, Mertens M, Herrmann A, Schneider E, Wessig P (2013) DBD dyes as fluorescence lifetime probes to study conformational changes in proteins. Chemistry 19(51):17349–17357. doi:10.1002/chem.201302368

    Article  CAS  Google Scholar 

  32. Beher D, Wu J, Cumine S, Kim KW, Lu SC, Atangan L, Wang M (2009) Resveratrol is not a direct activator of SIRT1 enzyme activity. Chem Biol Drug Des 74(6):619–624. doi:10.1111/j.1747-0285.2009.00901.x

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The work has been supported by a grant of the Deutsche Forschungsgemeinschaft (DFG). The excellent technical support of Michael Schröder is greatfully acknowledged.

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Authors and Affiliations

  1. Department of Chemical Engineering and Biotechnology, University of Applied Sciences Darmstadt, Schnittspahnstr. 12, 64287, Darmstadt, Germany

    Christian Meyners, Andreas Krämer, Steffen Hinz & Franz-Josef Meyer-Almes

  2. Institut für Chemie, Universität Potsdam, 14476, Potsdam, Germany

    Robert Wawrzinek & Pablo Wessig

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  1. Christian Meyners
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  2. Robert Wawrzinek
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  3. Andreas Krämer
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  4. Steffen Hinz
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  6. Franz-Josef Meyer-Almes
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Correspondence to Franz-Josef Meyer-Almes.

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Meyners, C., Wawrzinek, R., Krämer, A. et al. A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe. Anal Bioanal Chem 406, 4889–4897 (2014). https://doi.org/10.1007/s00216-014-7886-5

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  • DOI: https://doi.org/10.1007/s00216-014-7886-5

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