A Flow-Through Fluorescence Polarization Detection System for Measuring GPCR-Mediated Modulation of cAMP Production

Abstract

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z′ factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H₃ G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC₅₀ values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements. (Journal of Biomolecular Screening 2007:1074-1083)

Keywords

fluorescence polarization flow-through cAMP histamine H3 receptor GPCR

References

Carnero A. : High throughput screening in drug discovery. Clin Transl Oncol 2006;8:482-490.

Koehn FE , Carter GT : The evolving role of natural products in drug discovery. Nat Rev Drug Discov 2005;4:206-220.

Geysen HM , Schoenen F. , Wagner D. , Wagner R. : Combinatorial compound libraries for drug discovery: an ongoing challenge . Nat Rev Drug Discov 2003;2:222-230.

Sportsman JR , Daijo J. , Gaudet EA : Fluorescence polarization assays in signal transduction discovery. Comb Chem High Throughput Screen 2003; 6:195-200.

Eglen RM : Functional G protein-coupled receptor assays for primary and secondary screening . Comb Chem High Throughput Screen 2005;8:311-318.

Lundstrom K. : Latest development in drug discovery on G protein-coupled receptors. Curr Protein Pept Sci 2006;7:465-470.

Luttrell LM : Transmembrane signaling by G protein-coupled receptors. Methods Mol Biol 2006;332:3-49.

Scott JD : Compartmentalized cAMP signalling: a personal perspective. Biochem Soc Trans 2006;34:465-467.

Jacoby E. , Bouhelal R. , Gerspacher M. , Seuwen K. : The 7 TM G-protein-coupled receptor target family. ChemMedChem 2006;1:761-782.

10.

Gales C. , Rebois RV , Hogue M. , Trieu P. , Breit A. , Hebert TE , et al: Real-time monitoring of receptor and G-protein interactions in living cells. Nat Methods 2005;2:177-184.

11.

Williams C. : cAMP detection methods in HTS: selecting the best from the rest. Nat Rev Drug Discov 2004 ;3:125-135.

12.

Prystay L. , Gagne A. , Kasila P. , Yeh LA , Banks P. : Homogeneous cell-based fluorescence polarization assay for the direct detection of cAMP. J Biomol Screen 2001;6:75-82.

13.

Allen M. , Hall D. , Collins B. , Moore K. : A homogeneous high throughput nonradioactive method for measurement of functional activity of Gscoupled receptors in membranes. J Biomol Screen 2002;7:35-44.

14.

Passani MB , Lin JS , Hancock A. , Crochet S. , Blandina P. : The histamine H3 receptor as a novel therapeutic target for cognitive and sleep disorders. Trends Pharmacol Sci 2004 ;25:618-625.

15.

Bakker RA : Histamine H3-receptor isoforms. Inflamm Res 2004;53:509-516.

16.

Leurs R. , Bakker RA , Timmerman H. , de Esch IJ : The histamine H3 receptor: from gene cloning to H3 receptor drugs . Nat Rev Drug Discov 2005; 4:107-120.

17.

Nordstedt C. , Fredholm BB : A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid . Anal Biochem 1990;189:231-234.

18.

Takeda T. , Kuno T. , Shuntoh H. , Tanaka C. : A rapid filtration assay for cAMP. J Biochem ( Tokyo) 1989;105:327-329.

19.

Das R. , Esposito V. , Abu-Abed M, Anand GS, Taylor SS, Melacini G: cAMP activation of PKA defines an ancient signaling mechanism. Proc Natl Acad Sci USA 2007;104:93-98.

20.

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

21.

Kariv II , Stevens ME , Behrens DL , Oldenburg KR : High throughput quantitation of cAMP production mediated by activation of seven transmembrane domain receptors. J Biomol Screen 1999;4:27-32.

22.

Lim HD , van Rijn RM , Ling P. , Bakker RA , Thurmond RL , Leurs R. : Evaluation of histamine H1-, H2-, and H3-receptor ligands at the human histamine H4 receptor: identification of 4-methylhistamine as the first potent and selective H4 receptor agonist. J Pharmacol Exp Ther 2005; 314:1310-1321.

23.

Burke TJ , Loniello KR , Beebe JA , Ervin KM : Development and application of fluorescence polarization assays in drug discovery . Comb Chem High Throughput Screen 2003;6:183-194.

24.

Allen M. , Reeves J. , Mellor G. : High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors. J Biomol Screen 2000;5:63-69.

25.

Schopfer U. , Engeloch C. , Stanek J. , Girod M. , Schuffenhauer A. , Jacoby E. , et al: The Novartis compound archive: from concept to reality. Comb Chem High Throughput Screen 2005;8:513-519.

26.

Ellson R. , Stearns R. , Mutz M. , Brown C. , Browning B. , Harris D. , et al: In situ DMSO hydration measurements of HTS compound libraries . Comb Chem High Throughput Screen 2005;8:489-498.

27.

Suzuki S. , Ohno K. , Santa T. , Imai K. : Study on interactions of endocrine disruptors with estrogen receptor-beta using fluorescence polarization. Anal Sci 2003;19:1103-1108.

28.

van Elswijk DA , Schobel UP , Lansky EP , Irth H. , van der Greef J. : Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical detection coupled to mass spectrometry Phytochemistry 2004;65:233-241.

29.

Hesley J. , Daijo J. , Ferguson AT : Stable, sensitive, fluorescence-based method for detecting cAMP. Biotechniques 2002;33:691-694.

30.

Blommel P. , Hanson GT , Vogel KW : Multiplexing fluorescence polarization assays to increase information content per screen: applications for screening steroid hormone receptors. J Biomol Screen 2004;9:294-302.

31.

Ozers MS , Hill JJ , Ervin K. , Wood JR , Nardulli AM , Royer CA , et al: Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy. J Biol Chem 1997;272:30405-30411.

32.

Wang SY , Ahn BS , Harris R. , Nordeen SK , Shapiro DJ : Fluorescence anisotropy microplate assay for analysis of steroid receptor-DNA interactions. Biotechniques 2004;37:807-808.

33.

Kool J. , van Liempd SM , Ramautar R. , Schenk T. , Meerman JH , Irth H. , et al: Development of a novel cytochrome p450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography . J Biomol Screen 2005;10:427-436.

34.

Kool J. , Eggink M. , van Rossum H. , van Liempd SM , van Elswijk DA , Irth H. , et al: Online biochemical detection of glutathione-S-transferase P1-specific inhibitors in complex mixtures. J Biomol Screen 2007;12:396-405.

35.

Derks RJ , Hogenboom AC , van der Zwan G. , Irth H. : On-line continuous-flow, multi-protein biochemical assays for the characterization of bioaffinity compounds using electrospray quadrupole time-of-flight mass spectrometry. Anal Chem 2003;75:3376-3384.