Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays.
References
[1]
Jacoby, E.; Bouhelal, R.; Gerspacher, M.; Seuwen, K. The 7TM G-Protein-coupled receptor target family. Chem. Med. Chem. 2006, 1, 760–782.
[2]
Heilker, R.; Wolff, M.; Tautermann, C.S.; Bieler, M. G-Protein-coupled receptor-focused drug discovery using a target class platform approach. Drug Discov. Today 2009, 14, 231–240.
[3]
Zwier, J.M.; Roux, T.; Cottet, M.; Durroux, T.; Douzon, S.; Bdioui, S.; Gregor, N.; Bourrier, E.; Oueslati, N.; Nicolas, L.; et al. A fluorescent ligand-binding alternative using Tag-lite？ technology. J. Biomol. Screen. 2010, 15, 1248–1259, doi:10.1177/1087057110384611.
[4]
Chambers, C.; Smith, F.; Williams, C.; Marcos, S.; Liu, Z.H.; Hayter, P.; Ciaramella, G.; Keighley, W.; Gribbon, P.; Sewing, A. Measuring intracellular calcium fluxes in high throughput mode. Comb. Chem. High Throughput Screen. 2003, 6, 355–362.
[5]
Thomsen, W.; Frazer, J.; Unett, D. Functional assays for screening GPCR targets. Curr. Opin. Biotechnol. 2005, 16, 655–665.
[6]
Taouji, S.; Dahan, S.; Bosse, R.; Chevet, E. Current screens based on the AlphaScreen technology for deciphering cell signalling pathways. Curr. Genomics 2009, 10, 93–101, doi:10.2174/138920209787847041.
[7]
Cooper, M.A. Biosensor profiling of molecular interactions in pharmacology. Curr. Opin. Pharmacol. 2003, 3, 557–562, doi:10.1016/j.coph.2003.05.003.
[8]
Xi, B.; Yu, N.; Wang, X.; Xu, X.; Abassi, Y. The application of cell-based label-free technology in drug discovery. Biotechnol. J. 2008, 3, 484–495.
[9]
Scott, C.W.; Peters, M.F. Label-free whole-cell assays: Expanding the scope of GPCR screening. Drug Discov. Today 2010, 15, 704–716, doi:10.1016/j.drudis.2010.06.008.
[10]
Fang, Y. Label-free receptor assays. Drug Discov. Today Technol. 2010, 7, 5–11, doi:10.1016/j.ddtec.2010.05.001.
Jerman, J.C.; Brown, J.; Rocheville, M. Cellular screening for 7TM receptors using label-free detection. In Label-Free Technologies: For Drug Discovery; Cooper, M.A., Mayr, L.M., Eds.; John Wiley & Sons, Ltd.: Hoboken, NJ, USA, 2011; pp. 205–222.
[13]
Nirschl, M.; Reuter, F.; Voros, J. Review of transducer principles for label-free biomolecular interaction analysis. Biosensors 2011, 1, 70–92.
[14]
Li, G.; Ferrie, A.M.; Fang, Y. Label-free profiling of ligands for endogenous GPCRs using a cell-based high-throughput screening technology. JALA 2006, 11, 181–187.
[15]
Fang, Y. Label-free cell-based assays with optical biosensors in drug discovery. Assay Drug Dev. Technol. 2006, 4, 583–595, doi:10.1089/adt.2006.4.583.
[16]
Cooper, M.A. Optical biosensors: Where next and how soon? Drug Discov. Today 2006, 11, 1061–1067.
[17]
Fang, Y.; Fang, J.; Tran, E.; Xinying, X.; Hallstrom, M.; Frutos, A.G. High-throughput analysis of biomolecular interactions and cellular responses with resonant waveguide grating biosensors. In Label-Free Biosensors: Techniques and Applications; Cooper, M.A., Ed.; Cambridge University Press: Cambridge, UK, 2009; pp. 206–222.
[18]
Daniels, J.S.; Pourmand, N. Label-free impedance biosensors: Opportunities and challenges. Electroanalysis 2007, 19, 1239–1257, doi:10.1002/elan.200603855.
[19]
Abassi, Y.A.; Sieler, A.; Watzele, M.; Wang, X.; Xu, X. Dynamic and label-free cell-based assays using the xCELLigence system. In Label-Free Technologies: For Drug Discovery; Cooper, M.A., Mayr, L.M., Eds.; John Wiley & Sons, Ltd.: Hoboken, NJ, USA, 2011; pp. 71–81.
[20]
Giaever, I.; Keese, C.R. Use of electric fields to monitor the dynamical aspect of cell behavior in tissue culture. IEEE Trans. Biomed. Eng. 1986, 33, 242–247.
[21]
Giaever, I.; Keese, C.R. A morphological biosensor for mammalian cells. Nature 1993, 366, 591–592.
[22]
Solly, K.; Wang, X.; Xu, X.; Strulovici, B.; Zheng, W. Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays. Assay Drug Dev. Technol. 2004, 2, 363–372, doi:10.1089/adt.2004.2.363.
[23]
McGuinness, R.; Verdonk, E. Electrical impedance technology applied to cell-based assays. In Label-Free Biosensors: Techniques and Applications; Cooper, M.A., Ed.; Cambridge University Press: Cambridge, UK, 2009; pp. 251–277.
Szaszi, K.; Kurashima, K.; Kaibuchi, K.; Grinstein, S.; Orlowski, J. Role of the cytoskeleton in mediating cAMP-dependent protein kinase inhibition of the epithelial Na+/H+ exchanger NHE3. J. Biol. Chem. 2001, 276, 40761–40768.
[26]
Monk, P.N.; Scola, A.M.; Madala, P.; Fairlie, D.P. Function, structure and therapeutic potential of complement C5a receptors. Br. J. Pharmacol. 2007, 152, 429–448.
[27]
Ward, P.A. Role of C5 activation products in sepsis. Sci. World J. 2010, 10, 2395–2402, doi:10.1100/tsw.2010.216.
[28]
Marc, M.M.; Kristan, S.S.; Rozman, A.; Kern, I.; Flezar, M.; Kosnik, M.; Korosec, P. Complement factor C5a in acute exacerbation of chronic obstructive pulmonary disease. Scand. J. Immunol. 2010, 71, 386–391, doi:10.1111/j.1365-3083.2010.02385.x.
Yancey, K.B.; Lawley, T.J.; Dersookian, M.; Harvath, L. Analysis of the interaction of human C5a and C5a des Arg with human monocytes and neutrophils: Flow cytometric and chemotaxis studies. J. Invest. Dermatol. 1989, 92, 184–189.
[31]
Chenoweth, D.E.; Hugli, T.E. Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. USA 1978, 75, 3943–3947, doi:10.1073/pnas.75.8.3943.
[32]
Haviland, D.; McCoy, R.; Whitehead, W.; Akama, H.; Molmenti, E.; Brown, A.; Haviland, J.; Parks, W.; Perlmutter, D.; Wetsel, R. Cellular expression of the C5a anaphylatoxin receptor (C5aR): Demonstration of C5aR on nonmyeloid cells of the liver and lung. J. Immunol. 1995, 154, 1861–1869.
[33]
Larsen, G.L.; McCarthy, K.; Webster, R.O.; Henson, J.; Henson, P.M. A differential effect of C5a and C5a des Arg in the induction of pulmonary inflammation. Am. J. Pathol. 1980, 100, 179–192.
[34]
Konteatis, Z.; Siciliano, S.; van Riper, G.; Molineaux, C.; Pandya, S.; Fischer, P.; Rosen, H.; Mumford, R.; Springer, M. Development of C5a receptor antagonists. Differential loss of functional responses. J. Immunol. 1994, 153, 4200–4205.
[35]
Halili, M.A.; Andrews, M.R.; Labzin, L.I.; Schroder, K.; Matthias, G.; Cao, C.; Lovelace, E.; Reid, R.C.; Le, G.T.; Hume, D.A.; et al. Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll-like receptor 4 agonist LPS. J. Leukoc. Biol. 2010, 87, 1103–1114.
[36]
Dutertre, S.; Croker, D.; Daly, N.L.; Andersson, A.; Muttenthaler, M.; Lumsden, N.G.; Craik, D.J.; Alewood, P.F.; Guillon, G.; Lewis, R.J. Conopressin-T from Conus tulipa reveals an antagonist switch in Vasopressin-like peptides. J. Biol. Chem. 2008, 283, 7100–7108.
[37]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254.
[38]
Alouani, S. Scintillation proximity binding assay. In Chemokine Protocols; Proudfoot, A.E.I., Wells, T.N.C., Power, C.A., Eds.; Humana Press: Totowa, NJ, USA, 2000; Volume 138, pp. 135–141.
[39]
Eglen, R.M.; Reisine, T.; Roby, P.; Rouleau, N.; Illy, C.; Bosse, R.; Bielefeld, M. The use of AlphaScreen technology in HTS: Current status. Curr. Chem. Genomics 2008, 1, 2–10.
[40]
Klempner, M.S.; Gallin, J.I.; Balow, J.E.; van Kammen, D.P. The effect of hemodialysis and C5a des Arg on neutrophil subpopulations. Blood 1980, 55, 777–783.
[41]
Gerard, C.; Chenoweth, D.E.; Hugli, T.E. Response of human neutrophils to C5a: A role for the oligosaccharide moiety of human C5a des Arg-74 but not of C5a in biologic activity. J. Immunol. 1981, 127, 1978–1982.
[42]
Marder, S.R.; Chenoweth, D.E.; Goldstein, I.M.; Perez, H.D. Chemotactic responses of human peripheral blood monocytes to the complement-derived peptides C5a and C5a des Arg. J. Immunol. 1985, 134, 3325–3331.
[43]
Barker, M.D.; Jose, P.J.; Williams, T.J.; Burton, D.R. The chemoattractant des-Arg74-C5a regulates the expression of its own receptor on a monocyte-like cell line. Biochem. J. 1986, 236, 621–624.
[44]
Senior, R.M.; Griffin, G.L.; Perez, H.D.; Webster, R.O. Human C5a and C5a des Arg exhibit chemotactic activity for fibroblasts. J. Immunol. 1988, 141, 3570–3574.
[45]
Bürgi, B.; Brunner, T.; Dahinden, C.A. The degradation product of the C5a anaphylatoxin C5a des Arg retains basophil-activating properties. Eur. J. Immunol. 1994, 24, 1583–1589.
[46]
Werfel, T.; Oppermann, M.; Butterfield, J.H.; Begemann, G.; Elsner, J.; Gotze, O.; Zwirner, J. The human mast cell line HMC-1 expresses C5a receptors and responds to C5a but not to C5a(desArg). Scand. J. Immunol. 1996, 44, 30–36.
[47]
Crass, T.; Bautsch, W.; Cain, S.A.; Pease, J.E.; Monk, P.N. Receptor activation by human C5a des Arg74 but not intact C5a is dependent on an interaction between Glu199 of the receptor and Lys68 of the ligand. Biochemistry 1999, 38, 9712–9717.
[48]
Krasel, C.; Vilardaga, J.P.; Bunemann, M.; Lohse, M.J. Kinetics of G-protein-coupled receptor signalling and desensitization. Biochem. Soc. Trans. 2004, 32, 1029–1031.
[49]
Sheth, B.; Banks, P.; Burton, D.R.; Monk, P.N. The regulation of actin polymerization in differentiating U937 cells correlates with increased membrane levels of the pertussis-toxin-sensitive G-protein Gi2. Biochem. J. 1991, 275, 809–811.
[50]
Skokowa, J.; Ali, S.R.; Felda, O.; Kumar, V.; Konrad, S.; Shushakova, N.; Schmidt, R.E.; Piekorz, R.P.; Nurnberg, B.; Spicher, K.; et al. Macrophages induce the inflammatory response in the pulmonary arthus reaction through Gαi2 activation that controls C5aR and Fc receptor cooperation. J. Immunol. 2005, 174, 3041–3050.
[51]
Oksjoki, R.; Laine, P.; Helske, S.; Vehmaan-Kreula, P.; M？yr？np？？, M.I.; Gasque, P.; Kovanen, P.T.; Pentik？inen, M.O. Receptors for the anaphylatoxins C3a and C5a are expressed in human atherosclerotic coronary plaques. Atherosclerosis 2007, 195, 90–99.
[52]
Koch, W.J.; Hawes, B.E.; Allen, L.F.; Lefkowitz, R.J. Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras. Proc. Natl. Acad. Sci. USA 1994, 91, 12706–12710.
[53]
Mochizuki, N.; Ohba, Y.; Kiyokawa, E.; Kurata, T.; Murakami, T.; Ozaki, T.; Kitabatake, A.; Nagashima, K.; Matsuda, M. Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with Gαi. Nature 1999, 400, 891–894.
[54]
Watson, J.; Collin, L.; Ho, M.; Riley, G.; Scott, C.; Selkirk, J.V.; Price, G.W. 5-HT(1A) receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems. Br. J. Pharmacol. 2000, 130, 1108–1114.
[55]
Shapira, M.; Vogel, Z.; Sarne, Y. Opioid and cannabinoid receptors share a common pool of GTP-binding proteins in cotransfected cells, but not in cells which endogenously coexpress the receptors. Cell Mol. Neurobiol. 2000, 20, 291–304.
[56]
Prinster, S.C.; Hague, C.; Hall, R.A. Heterodimerization of G protein-coupled receptors: Specificity and functional significance. Pharmacol. Rev. 2005, 57, 289–298.
Fang, Y.; Ferrie, A.M.; Fontaine, N.H.; Yuen, P.K. Characteristics of dynamic mass redistribution of epidermal growth factor receptor signaling in living cells measured with label-free optical biosensors. Anal. Chem. 2005, 77, 5720–5725.
[59]
Fang, Y. Non-invasive optical biosensor for probing cell signaling. Sensors 2007, 7, 2316–2329.
[60]
Schroder, R.; Janssen, N.; Schmidt, J.; Kebig, A.; Merten, N.; Hennen, S.; Muller, A.; Blattermann, S.; Mohr-Andra, M.; Zahn, S.; et al. Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat. Biotechnol. 2010, 28, 943–949.
[61]
Dodgson, K.; Gedge, L.; Murray, D.C.; Coldwell, M. A 100K well screen for a muscarinic receptor using the Epic label-free system-a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors. J. Recept. Signal Transduct. Res. 2009, 29, 163–172.
[62]
Schuster, D.; Laggner, C.; Langer, T. Why drugs fail-a study on side effects in new chemical entitie. Curr. Pharm. Des. 2005, 11, 3545–3559.
