Multiplexing Label-Free and Fluorescence-Based Methods for Pharmacological Characterization of GPCR Ligands

Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Cell-based assays are essential to drug discovery and biomedical research. Most cell-based assays have been targeting specific components of signaling pathways with considerable mechanistic significance. G protein-coupled receptors (GPCRs) represent a major class of drug targets. In addition to G protein-dependent pathways, G protein-independent signaling mechanisms such as β-arrestin pathways, allosteric modulation of receptor function, and receptor oligomerization are among currently active research areas. High-throughput calcium- and membrane potential-based assays have been widely used as efficient screening platforms. The Hamamatsu FDSS7000 instrument is capable of simultaneous compound addition and fluorescence monitoring in 96- or 384-wells for fluorescence-based calcium or membrane potential assays. However, phenotypic or holistic cellular measurements of combined effects contributed by multiple signaling pathways may also be required to complement label-based assays that target specific signaling events. For this purpose, the effects mediated by GPCRs may be measured by dynamic mass redistribution (DMR) through Resonant Waveguide Grating (RWG) biosensors embedded in 384-well plates using a Corning EPIC BT label-free assay system. Herein, we describe experimental protocol for profiling ADX88178, a potent and selective positive allosteric modulator (PAM) of the metabotropic glutamate 4 receptor (mGluR4) using both Ca2+ and DMR phenotypic readouts, and discuss the complementary features of each assay type. Further, we demonstrate, for the first time, a unique integrated procedure multiplexing FDSS calcium mobilization and EPIC label-free assays using a single set of cell/compound plates.

Key words

Dynamic mass redistribution Functional drug screening system G protein-coupled receptor Label-free Resonant waveguide grating 



The authors thank Drs. Shouming Du, Yiwen Wang, Alex Sanchez, and Al McGrath of Hamamatsu Corporation, and Drs. Hung Cuong Louie Tran and Ye Fang of Corning Incorprated for technical assistance. We are also grateful to Mr. Michael Collins and Noel J. Boyle for technical support.


  1. 1.
    Flower DR (1999) Modelling G-protein-coupled receptors for drug design. Biochim Biophys Acta 1422:207–234. doi: 10.1016/S0304-4157(99)00006-4 CrossRefPubMedGoogle Scholar
  2. 2.
    Lagerstrom MC, Schioth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7:339–357. doi: 10.1038/nrd2518 CrossRefPubMedGoogle Scholar
  3. 3.
    Marinissen MJ, Gutkind JS (2001) G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci 22:368–376. doi: 10.1016/S0165-6147(00)01678-3 CrossRefPubMedGoogle Scholar
  4. 4.
    Lefkowitz RJ, Whalen EJ (2004) Beta-arrestins: traffic cops of cell signaling. Curr Opin Cell Biol 16:162–168. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  5. 5.
    Tian X, Kang DS, Benovic JL (2014) Beta-arrestins and G protein-coupled receptor trafficking. Handb Exp Pharmacol 219:173–186. doi: 10.1007/978-3-642-41199-1_9 CrossRefPubMedGoogle Scholar
  6. 6.
    Walther C, Ferguson SS (2013) Arrestins: role in the desensitization, sequestration, and vesicular trafficking of G protein-coupled receptors. Prog Mol Biol Transl Sci 118:93–113. doi: 10.1016/B978-0-12-394440-5.00004-8 CrossRefPubMedGoogle Scholar
  7. 7.
    Dewire SM, Ahn S, Lefkowitz RJ et al (2007) Beta-arrestins and cell signaling. Annu Rev Physiol 69:483–510. doi: 10.1146/annurev.physiol.69.022405.154749 CrossRefPubMedGoogle Scholar
  8. 8.
    Luttrell LM, Gesty-Palmer D (2010) Beyond desensitization: physiological relevance of arrestin-dependent signaling. Pharmacol Rev 62:305–330. doi: 10.1124/pr.109.002436 CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Kenakin T (2007) Functional selectivity through protean and biased agonism: who steers the ship? Mol Pharmacol 72:1393–1401. doi: 10.1124/mol.107.040352 CrossRefPubMedGoogle Scholar
  10. 10.
    Violin JD, Lefkowitz RJ (2007) Beta-arrestin-biased ligands at seven-transmembrane receptors. Trends Pharmacol Sci 28:416–422. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  11. 11.
    Michel MC, Seifert R, Bond RA (2014) Dynamic bias and its implications for GPCR drug discovery. Nat Rev Drug Discov 13:869. doi: 10.1038/nrd3954-c3 CrossRefPubMedGoogle Scholar
  12. 12.
    Chen L, Jin L, Zhou N (2012) An update of novel screening methods for GPCR in drug discovery. Exp Opin Drug Discov 7:791–806. doi: 10.1517/17460441.2012.699036 CrossRefGoogle Scholar
  13. 13.
    Changeux JP, Edelstein SJ (2005) Allosteric mechanisms of signal transduction. Science 308:1424–1428. doi: 10.1126/science.1108595 CrossRefPubMedGoogle Scholar
  14. 14.
    Kenakin T (2007) Collateral efficacy in drug discovery: taking advantage of the good (allosteric) nature of 7TM receptors. Trends Pharmacol Sci 28:407–415. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  15. 15.
    Gao ZG, Jacobson KA (2006) Keynote review: allosterism in membrane receptors. Drug Discov Today 11:191–202. doi: 10.1016/S1359-6446(05)03689-5 CrossRefPubMedGoogle Scholar
  16. 16.
    Conn PJ, Christopoulos A, Lindsley CW (2009) Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nat Rev Drug Discov 8:41–54. doi: 10.1038/nrd2760 CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Keov P, Sexton PM, Christopoulos A (2011) Allosteric modulation of G protein-coupled receptors: a pharmacological perspective. Neuropharmacology 60:24–35. doi: 10.1016/j.neuropharm.2010.07.010 CrossRefPubMedGoogle Scholar
  18. 18.
    Arneric SP, Holladay M, Williams M (2007) Neuronal nicotinic receptors: a perspective on two decades of drug discovery research. Biochem Pharmacol 74:1092–1101. doi: 10.1016/j.bcp.2007.06.033 CrossRefPubMedGoogle Scholar
  19. 19.
    Pandya A, Yakel JL (2011) Allosteric modulators of the α4β2 subtype of neuronal nicotinic acetylcholine receptors. Biochem Pharmacol 82:952–958. doi: 10.1016/j.bcp.2011.04.020 CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Wallace TL, Porter RH (2011) Targeting the nicotinic alpha7 acetylcholine receptor to enhance cognition in disease. Biochem Pharmacol 82:891–903. doi: 10.1016/j.bcp.2011.06.034 CrossRefPubMedGoogle Scholar
  21. 21.
    Zhong H, Haddjeri N, Sanchez C (2012) Escitalopram, an antidepressant with an allosteric effect at the serotonin transporter-a review of current understanding of its mechanism of action. Psychopharmacology (Berl) 219:1–13. doi: 10.1007/s00213-011-2463-5 CrossRefGoogle Scholar
  22. 22.
    Zhong H, Sanchez C, Caron MG (2012) Consideration of allosterism and interacting proteins in the physiological functions of the serotonin transporter. Biochem Pharmacol 83:435–442. doi: 10.1016/j.bcp.2011.09.020 CrossRefPubMedGoogle Scholar
  23. 23.
    Kenakin T (2013) Allosteric drugs and seven transmembrane receptors. Curr Top Med Chem 13:5–13. doi: 10.2174/1568026611313010003 CrossRefPubMedGoogle Scholar
  24. 24.
    Kenakin TP (2012) Biased signalling and allosteric machines: new vistas and challenges for drug discovery. Br J Pharmacol 165:1659–1669. doi: 10.1111/j.1476-5381.2011.01749.x CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Conn PJ, Lindsley CW, Meiler J et al (2014) Opportunities and challenges in the discovery of allosteric modulators of GPCRs for treating CNS disorders. Nat Rev Drug Discov 13:692–708. doi: 10.1038/nrd4308 CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Thomsen W, Frazer J, Unett D (2005) Functional assays for screening GPCR targets. Curr Opin Biotechnol 16:655–665. doi: 10.1016/j.copbio.2005.10.008 PubMedGoogle Scholar
  27. 27.
    Eglen RM (2005) Functional G protein-coupled receptor assays for primary and secondary screening. Comb Chem High Throughput Screen 8:311–318. doi: 10.2174/1386207054020813 CrossRefPubMedGoogle Scholar
  28. 28.
    Fang Y (2014) Label-free drug discovery. Front Pharmacol 5:52. doi: 10.3389/fphar.2014.00052 CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Rask-Andersen M, Almen MS, Schioth HB (2011) Trends in the exploitation of novel drug targets. Nat Rev Drug Discov 10:579–590. doi: 10.1038/nrd3478 CrossRefPubMedGoogle Scholar
  30. 30.
    Halai R, Cooper MA (2012) Using label-free screening technology to improve efficiency in drug discovery. Exp Opin Drug Discov 7:123–131. doi: 10.1517/17460441.2012.651121 CrossRefGoogle Scholar
  31. 31.
    Citartan M, Gopinath SC, Tominaga J et al (2013) Label-free methods of reporting biomolecular interactions by optical biosensors. Analyst 138:3576–3592. doi: 10.1039/C3AN36828A CrossRefPubMedGoogle Scholar
  32. 32.
    Lunn CA (2010) Label-free screening assays: a strategy for finding better drug candidates. Future Med Chem 2:1703–1716. doi: 10.4155/fmc.10.246 CrossRefPubMedGoogle Scholar
  33. 33.
    Wong JW, Cagney G (2010) An overview of label-free quantitation methods in proteomics by mass spectrometry. Methods Mol Biol 604:273–283. doi: 10.1007/978-1-60761-444-9_18 CrossRefPubMedGoogle Scholar
  34. 34.
    Eggert US (2013) The why and how of phenotypic small-molecule screens. Nat Chem Biol 9:206–209. doi: 10.1038/nchembio.1206 CrossRefPubMedGoogle Scholar
  35. 35.
    Lee JA, Uhlik MT, Moxham CM et al (2012) Modern phenotypic drug discovery is a viable, neoclassic pharma strategy. J Med Chem 55:4527–4538. doi: 10.1021/jm201649s CrossRefPubMedGoogle Scholar
  36. 36.
    Fang Y (2013) Troubleshooting and deconvoluting label-free cell phenotypic assays in drug discovery. J Pharmacol Toxicol Methods 67:69–81. doi: 10.1016/j.vascn.2013.01.004 CrossRefPubMedGoogle Scholar
  37. 37.
    Fang Y, Ferrie AM, Fontaine NH et al (2006) Resonant waveguide grating biosensor for living cell sensing. Biophys J 91:1925–1940. doi: 10.1529/biophysj.105.077818 CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    Fang Y, Ferrie AM, Li G (2005) Probing cytoskeleton modulation by optical biosensors. FEBS Lett 579:4175–4180. doi: 10.1016/j.febslet.2005.06.050 CrossRefPubMedGoogle Scholar
  39. 39.
    Tan CM, Brady AE, Nickols HH et al (2004) Membrane trafficking of G protein-coupled receptors. Annu Rev Pharmacol Toxicol 44:559–609. doi: 10.1146/annurev.pharmtox.44.101802.121558 CrossRefPubMedGoogle Scholar
  40. 40.
    Wehrman TS, Casipit CL, Gewertz NM et al (2005) Enzymatic detection of protein translocation. Nat Methods 2:521–527. doi: 10.1038/nmeth771 CrossRefPubMedGoogle Scholar
  41. 41.
    Garbison KE, Heinz BA, Lajiness ME et al (2004) Impedance-based technologies. In: Sittampalam GS, Gal-Edd N, Arkin M, Auld D, Austin C, Bejcek B, Glicksman M, Inglese J, Lemmon V, Li Z, McGee J, McManus O, Minor L, Napper A, Riss T, Trask OJ, Weidner J (eds) Assay guidance manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda, MDGoogle Scholar
  42. 42.
    Bockaert J, Fagni L, Dumuis A et al (2004) GPCR interacting proteins (GIP). Pharmacol Ther 103:203–221. doi: 10.1016/j.pharmthera.2004.06.004 CrossRefPubMedGoogle Scholar
  43. 43.
    Deng H, Sun H, Fang Y (2013) Label-free cell phenotypic assessment of the biased agonism and efficacy of agonists at the endogenous muscarinic M3 receptors. J Pharmacol Toxicol Methods 68:323–333. doi: 10.1016/j.vascn.2013.07.005 CrossRefPubMedGoogle Scholar
  44. 44.
    Sun H, Wei Y, Deng H et al (2014) Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel. Sci Rep 4:4934. doi: 10.1038/srep04934 PubMedCentralPubMedGoogle Scholar
  45. 45.
    Carter RL, Grisanti LA, Yu JE, Repas AA, Woodall M, Ibetti J, Koch WJ, Jacobson MA, Tilley DG (2014) Dynamic mass redistribution analysis of endogenous β-adrenergic receptor signaling in neonatal rat cardiac fibroblasts. Pharmacol Res Perspect 2:24. doi: 10.1002/prp2.24 CrossRefGoogle Scholar
  46. 46.
    Watts AO, Scholten DJ, Heitman LH et al (2012) Label-free impedance responses of endogenous and synthetic chemokine receptor CXCR3 agonists correlate with Gi-protein pathway activation. Biochem Biophys Res Commun 419:412–418. doi: 10.1016/j.bbrc.2012.02.036 CrossRefPubMedGoogle Scholar
  47. 47.
    Verdonk E, Johnson K, Mcguinness R et al (2006) Cellular dielectric spectroscopy: a label-free comprehensive platform for functional evaluation of endogenous receptors. Assay Drug Dev Technol 4:609–619. doi: 10.1016/j.jala.2005.06.002 CrossRefPubMedGoogle Scholar
  48. 48.
    Geetha T, Langlais P, Luo M et al (2011) Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1. J Am Soc Mass Spectrom 22:457–466. doi: 10.1007/s13361-010-0051-2 CrossRefPubMedCentralPubMedGoogle Scholar
  49. 49.
    Zhu T, Fang LY, Xie X (2008) Development of a universal high-throughput calcium assay for G-protein-coupled receptors with promiscuous G-protein Galpha15/16. Acta Pharmacol Sin 29:507–516. doi: 10.1111/j.1745-7254.2008.00775.x CrossRefPubMedGoogle Scholar
  50. 50.
    Walker MW, Jones KA, Tamm J et al (2005) Use of Caenorhabditis elegans Gαq chimeras to detect G-protein-coupled receptor signals. J Biomol Screen 10:127–136. doi: 10.1177/1087057104272006 CrossRefPubMedGoogle Scholar
  51. 51.
    New DC, Wong YH (2004) Characterization of CHO cells stably expressing a G alpha 16/z chimera for high throughput screening of GPCRs. Assay Drug Dev Technol 2:269–280. doi: 10.1089/1540658041410641 CrossRefPubMedGoogle Scholar
  52. 52.
    Shirokova E, Schmiedeberg K, Bedner P et al (2005) Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants. J Biol Chem 280:11807–11815. doi: 10.1074/jbc.M411508200 CrossRefPubMedGoogle Scholar
  53. 53.
    Krueger KM, Witte DG, Ireland-Denny L et al (2005) G protein-dependent pharmacology of histamine H3 receptor ligands: evidence for heterogeneous active state receptor conformations. J Pharmacol Exp Ther 314:271–281. doi: 10.1124/jpet.104.078865 CrossRefPubMedGoogle Scholar
  54. 54.
    Niswender CM, Johnson KA, Weaver CD et al (2008) Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4. Mol Pharmacol 74:1345–1358. doi: 10.1124/mol.108.049551 CrossRefPubMedCentralPubMedGoogle Scholar
  55. 55.
    Dhanya RP, Sheffler DJ, Dahl R et al (2014) Design and synthesis of systemically active metabotropic glutamate subtype-2 and -3 (mGlu2/3) receptor positive allosteric modulators (PAMs): pharmacological characterization and assessment in a rat model of cocaine dependence. J Med Chem 57:4154–4172. doi: 10.1021/jm5000563 CrossRefPubMedCentralPubMedGoogle Scholar
  56. 56.
    Monn JA, Valli MJ, Massey SM et al (2013) Synthesis and pharmacological characterization of 4-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates: identification of new potent and selective metabotropic glutamate 2/3 receptor agonists. J Med Chem 56:4442–4455. doi: 10.1021/jm4000165 CrossRefPubMedGoogle Scholar
  57. 57.
    Wenthur CJ, Morrison RD, Daniels JS et al (2014) Synthesis and SAR of substituted pyrazolo[1,5-a]quinazolines as dual mGlu(2)/mGlu(3) NAMs. Bioorg Med Chem Lett 24:2693–2698.  doi: 10.1016/j.bmcl.2014.04.051 CrossRefPubMedGoogle Scholar
  58. 58.
    Hammond AS, Rodriguez AL, Townsend SD et al (2010) Discovery of a novel chemical class of mGlu(5) allosteric ligands with distinct modes of pharmacology. ACS Chem Neurosci 1:702–716. doi: 10.1021/cn100051m CrossRefPubMedCentralPubMedGoogle Scholar
  59. 59.
    Iacovelli L, Felicioni M, Nistico R et al (2014) Selective regulation of recombinantly expressed mGlu7 metabotropic glutamate receptors by G protein-coupled receptor kinases and arrestins. Neuropharmacology 77:303–312. doi: 10.1016/j.neuropharm.2013.10.013 CrossRefPubMedGoogle Scholar
  60. 60.
    Iacovelli L, Salvatore L, Capobianco L et al (2003) Role of G protein-coupled receptor kinase 4 and beta-arrestin 1 in agonist-stimulated metabotropic glutamate receptor 1 internalization and activation of mitogen-activated protein kinases. J Biol Chem 278:12433–12442. doi: 10.1074/jbc.M203992200 CrossRefPubMedGoogle Scholar
  61. 61.
    Le Poul E, Bolea C, Girard F et al (2012) A potent and selective metabotropic glutamate receptor 4 positive allosteric modulator improves movement in rodent models of Parkinson’s disease. J Pharmacol Exp Ther 343:167–177. doi: 10.1124/jpet.112.196063 CrossRefPubMedGoogle Scholar
  62. 62.
    Perdona E, Faggioni F, Buson A et al (2011) Pharmacological characterization of the ghrelin receptor antagonist, GSK1614343 in rat RC-4B/C cells natively expressing GHS type 1a receptors. Eur J Pharmacol 650:178–183. doi: 10.1016/j.ejphar.2010.10.042 CrossRefPubMedGoogle Scholar
  63. 63.
    Miller TR, Witte DG, Ireland LM et al (1999) Analysis of apparent noncompetitive responses to competitive H1-histamine receptor antagonists in fluorescent imaging plate reader-based calcium assays. J Biomol Screen 4:249–258. doi: 10.1177/108705719900400506 CrossRefPubMedGoogle Scholar
  64. 64.
    Arunlakshana O, Schild HO (1959) Some quantitative uses of drug antagonists. Br J Pharmacol Chemother 14:48–58CrossRefPubMedCentralPubMedGoogle Scholar
  65. 65.
    Kenakin T (2004) Principles: receptor theory in pharmacology. Trends Pharmacol Sci 25:186–192. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  66. 66.
    Schrage R, Seemann WK, Klockner J et al (2013) Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor. Br J Pharmacol 169:357–370. doi: 10.1111/bph.12003 CrossRefPubMedCentralPubMedGoogle Scholar
  67. 67.
    Deng H, Wang C, Su M et al (2012) Probing biochemical mechanisms of action of muscarinic M3 receptor antagonists with label-free whole cell assays. Anal Chem 84:8232–8239. doi: 10.1021/ac301495n CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.U-Pharm Laboratories LLCParsippanyUSA
  2. 2.Lundbeck Research USAParamusUSA

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