Triple‐Addition Assay Protocols for Detecting and Characterizing Modulators of Seven‐Transmembrane Receptors

C. David Weaver1

1 Vanderbilt University School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110060
Online Posting Date:  September, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The detection and characterization of seven‐transmembrane‐receptor modulators (orthosteric binding site agonists, antagonists, and more recently allosteric modulators) is an area of intense interest for both drug discovery and basic research. Traditionally, assays used to detect and characterize these different modes of modulation have been executed as separate, discrete protocols focused on a particular mode of action (e.g., agonism). In recent years, investigators have begun to combine aspects of these separate protocols to produce methods that detect multiple modes of modulation simultaneously. The power of such approaches is revealed not only in conservation of time and resources, but more importantly in a superior ability to discover and characterize novel modulators of the targets of interest. The protocols in this article describe a general procedure for developing, validating, and utilizing triple‐addition assays to enable the simultaneous detection and characterization of multiple modes of seven‐transmembrane‐receptor modulation. Curr. Protoc. Chem. Biol. 3:119‐140 © 2011 by John Wiley & Sons, Inc.

Keywords: seven‐transmembrane receptor; G‐protein‐coupled receptor; agonist; potentiator; antagonist; allosteric; fluorescence; high‐throughput screening

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Measurement of Seven‐Transmembrane Receptor Activity Using a Triple‐Addition Assay
  • Basic Protocol 2: Signal Uniformity Validation for High‐Throughput Screening for Modulators of Seven‐Transmembrane Receptors Using a Triple‐Addition Assay
  • Basic Protocol 3: High‐Throughput Screening for Modulators of Seven‐Transmembrane Receptors Using a Triple‐Addition Assay
  • Basic Protocol 4: Measurement of Concentration‐Response Relationships for Modulators of Seven‐Transmembrane Receptors Using a Triple‐Addition Assay
  • Alternate Protocols
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Measurement of Seven‐Transmembrane Receptor Activity Using a Triple‐Addition Assay

  • Cells expressing the seven‐transmembrane receptor of interest (e.g., mGlu5‐expressing HEK‐293 cells), e.g., in a T175 tissue culture flask
  • Cell plating medium (see recipe)
  • Dye loading solution (see recipe)
  • Seven‐transmembrane‐receptor agonist (e.g., l‐glutamic acid, Tocris, cat. no. 0218; see recipe)
  • Assay buffer (e.g., 20 mM HEPES‐buffered HBSS, pH 7.3; see recipe)
  • Dimethyl sulfoxide (DMSO)
  • 10‐ml Pasteur pipets
  • Black‐walled, clear‐bottom, poly‐D‐lysine‐coated 384‐well plates (e.g., Becton Dickinson, cat. no. 356936)
  • Multichannel electronic pipettor (e.g., Biohit, cat. no. 73036X; optional)
  • Humidified, 37°C, 5% CO 2 cell culture incubator
  • 384‐well polypropylene compound plates (e.g., Greiner, cat. no. 781280)
  • Kinetic imaging plate reader (e.g., Hamamatsu FDSS 6000)
  • Spreadsheet software (e.g., Excel 2010, Microsoft, with XLfit add‐in, IDBS)
  • Additional reagents and equipment for counting cells with a hemacytometer and trypan blue exclusion (e.g., Phelan et al., )
NOTE: All steps are performed at room temperature with medium that has been prewarmed to room temperature.

Basic Protocol 2: Signal Uniformity Validation for High‐Throughput Screening for Modulators of Seven‐Transmembrane Receptors Using a Triple‐Addition Assay

  • Control compounds, if available (e.g., mGluR5 potentiator VU 1545, Tocris, cat. no. 3325; mGluR5 antagonist MPEP hydrochloride, Tocris, cat. no. 1212)
  • Collection of compounds to be screened
  • Means of transferring test compounds from source plates to daughter plates: e.g., a contact‐based liquid handler (e.g., Bravo, Agilent) equipped with a pin tool (VP Scientific) or an acoustic liquid transfer device (e.g., Echo 550, Labcyte)
PDF or HTML at Wiley Online Library



Literature Cited

   Bird, M.K. and Lawrence, A.J. 2009. The promiscuous mGlu5 receptor—a range of partners for therapeutic possibilities? Trends Pharmacol. Sci. 30:617‐623.
   Brideau, C., Gunter, B., Pikounis, B., and Liaw, A. 2003. Improved statistical methods for hit selection in high‐throughput screening. J. Biomol. Screen. 8:634‐647.
   Chen, J., Lake, M.R., Sabet, R.S., Niforatos, W., Pratt, S.D., Cassar, S.C., Xu, J., Gopalakrishnan, S., Pereda‐Lopez, A., Gopalakrishnan, M., Holzman, T.F., Moreland, R.B., Walter, K.A., Faltynek, C.R., Warrior, U., and Scott, V.E. 2007. Utility of large‐scale transiently transfected cells for cell‐based high‐throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists. J. Biomol. Screen. 12:61‐69.
   Christopoulos, A. and Kenakin, T. 2002. G protein‐coupled receptor allosterism and complexing. Pharmacol. Rev. 54:323‐374.
   Cleva, R.M. and Olive, M.F. 2011. Positive allosteric modulators of type 5 metabotropic glutamate receptors (mGluR5) and their therapeutic potential for the treatment of CNS disorders. Molecules 16:2097‐2106.
   Conklin, B.R., Farfel, Z., Lustig, K.D., Julius, D., and Bourne, H.R. 1993. Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature 363:274‐276.
   Conn, P.J., Christopoulos, A., and Lindsley, C.W. 2009. Allosteric modulators of GPCRs: A novel approach for the treatment of CNS disorders. Nat. Rev. Drug Discov. 8:41‐54.
   Coward, P., Chan, S.D., Wada, H.G., Humphries, G.M., and Conklin, B.R. 1999. Chimeric G proteins allow a high‐throughput signaling assay of Gi‐coupled receptors. Anal. Biochem. 270:242‐248.
   De Amici, M., Dallanoce, C., Holzgrabe, U., Tränkle, C., and Mohr, K. 2010. Allosteric ligands for G protein‐coupled receptors: A novel strategy with attractive therapeutic opportunities. Med. Res. Rev. 30:463‐549.
   Digan, M.E., Pou, C., Niu, H., and Zhang, J.H. 2005. Evaluation of division‐arrested cells for cell‐based high‐throughput screening and profiling. J. Biomol. Screen. 10:615‐623.
   Di Virgilio, F., Fasolato, C., and Steinberg, T.H. 1988. Inhibitors of membrane transport system for organic anions block fura‐2 excretion from PC12 and N2A cells. Biochem. J. 256:959‐963.
   Drake, M.T., Violin, J.D., Whalen, E.J., Wisler, J.W., Shenoy, S.K., and Lefkowitz, R.J. 2008. Beta‐arrestin‐biased agonism at the beta2‐adrenergic receptor. J. Biol. Chem. 283:5669‐5676.
   Evans, B.A., Broxton, N., Merlin, J., Sato, M., Hutchinson, D.S., Christopoulos, A., and Summers, R.J. 2011. Quantification of functional selectivity at the human α(1A)‐adrenoceptor. Mol. Pharmacol. 79:298‐307.
   Gasparini, F., Bilbe, G., Gomez‐Mancilla, B., and Spooren, W. 2008. mGluR5 antagonists: Discovery, characterization and drug development. Curr. Opin. Drug Discov. Devel. 11:655‐665.
   Hoare, S.R. 2007. Allosteric modulators of class B G‐protein‐coupled receptors. Curr. Neuropharmacol. 5:168‐179.
   Im, W.B., Chio, C.L., Alberts, G.L., and Dinh, D.M. 2003. Positive allosteric modulator of the human 5‐HT2C receptor. Mol. Pharmacol. 64:78‐84.
   Jacoby, E., Bouhelal, R., Gerspacher, M., and Seuwen, K. 2006. The 7 TM G‐protein‐coupled receptor target family. ChemMedChem 8:761‐782.
   Kost, T.A., Condreay, J.P., Ames, R.S., Rees, S., and Romanos, M.A. 2007. Implementation of BacMam virus gene delivery technology in a drug discovery setting. Drug Discov. Today 12:396‐403.
   Kunapuli, P., Zheng, W., Weber, M., Solly, K., Mull, R., Platchek, M., Cong, M., Zhong, Z., and Strulovici, B. 2005. Application of division arrest technology to cell‐based HTS: Comparison with frozen and fresh cells. Assay Drug Dev. Technol. 3:17‐26.
   Leach, K., Sexton, P.M., and Christopoulos, A. 2007. Allosteric GPCR modulators: Taking advantage of permissive receptor pharmacology. Trends Pharmacol. Sci. 28:382‐389.
   Lindsley, C.W. and Emmitte, K.A. 2009. Recent progress in the discovery and development of negative allosteric modulators of mGluR5. Curr. Opin. Drug Discov. Devel. 12:446‐457.
   Marino, M.J. and Conn, P.J. 2006. Glutamate‐based therapeutic approaches: Allosteric modulators of metabotropic glutamate receptors. Curr. Opin. Pharmacol. 6:98‐102.
   Marino, M.J., Williams, D.L. Jr., O'Brien, J.A., Valenti, O., McDonald, T.P., Clements, M.K., Wang, R., DiLella, A.G., Hess, J.F., Kinney, G.G., and Conn, P.J. 2003. Allosteric modulation of group III metabotropic glutamate receptor 4: A potential approach to Parkinson's disease treatment. Proc. Natl. Acad. Sci. U.S.A. 100:13668‐13673.
   May, L.T., Leach, K., Sexton, P.M., and Christopoulos, A. 2007. Allosteric modulation of G protein‐coupled receptors. Annu. Rev. Pharmacol. Toxicol. 47:1‐51.
   Miller, M., Wu, M., Xu, J., Weaver, D., Li, M., and Zhu, M.X. 2011. High‐throughput screening of TRPC channel ligands using cell‐based assays. In TRP Channels (Methods in Signal Transduction Series, M.X. Zhu, ed.) pp. 2‐18. Taylor and Francis, Boca Raton, Fla.
   Motulsky, H. and Christopoulos, A. 2004. Fitting Models to Biological Data Using Linear and Non‐Linear Regression: A Practical Guide to Curve Fitting. Oxford University Press, New York.
   Niswender, C.M., Johnson, K.A., Weaver, C.D., Jones, C.K., Xiang, Z., Luo, Q., Rodriguez, A.L., Marlo, J.E., de Paulis, T., Thompson, A.D., Days, E.L., Nalywajko, T., Austin, C.A., Williams, M.B., Ayala, J.E., Williams, R., Lindsley, C.W., and Conn, P.J. 2008. Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4. Mol. Pharmacol. 74:1345‐1358.
   O'Brien, J.A., Lemaire, W., Chen, T.B., Chang, R.S., Jacobson, M.A., Ha, S.N., Lindsley, C.W., Schaffhauser, H.J., Sur, C., Pettibone, D.J., Conn, P.J., and Williams, D.L. Jr. 2003. A family of highly selective allosteric modulators of the metabotropic glutamate receptor subtype 5. Mol. Pharmacol. 64:731‐740.
   Offermanns, S. and Simon, M.I. 1995. G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. 270:15175‐15180.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Posner, B.A., Xi, H., and Mills, J.E. 2009. Enhanced HTS hit selection via a local hit rate analysis. J. Chem. Inf. Model. 49:2202‐2210.
   Rees, S., Morrow, D., and Kenakin, T. 2008. GPCR drug discovery through the exploitation of allosteric drug binding sites. Receptors Channels 8:261‐268.
   Rodriguez, A.L., Nong, Y., Sekaran, N.K., Alagille, D., Tamagnan, G.D., and Conn, P.J. 2005. A close structural analog of 2‐methyl‐6‐(phenylethynyl)‐pyridine acts as a neutral allosteric site ligand on metabotropic glutamate receptor subtype 5 and blocks the effects of multiple allosteric modulators. Mol. Pharmacol. 68:1793‐1802.
   Rodriguez, A.L., Grier, M.D., Jones, C.K., Herman, E.J., Kane, A.S., Smith, R.L., Williams, R., Zhou, Y., Marlo, J.E., Days, E.L., Blatt, T.N., Jadhav, S., Menon, U.N., Vinson, P.N., Rook, J.M., Stauffer, S.R., Niswender, C.M., Lindsley, C.W., Weaver, C.D., and Conn, P.J. 2010. Discovery of novel allosteric modulators of metabotropic glutamate receptor subtype 5 reveals chemical and functional diversity and in vivo activity in rat behavioral models of anxiolytic and antipsychotic activity. Mol. Pharmacol. 78:1105‐1123.
   Sharma, S., Rodriguez, A.L., Conn, P.J., and Lindsley, C.W. 2008. Synthesis and SAR of a mGluR5 allosteric partial antagonist lead: Unexpected modulation of pharmacology with slight structural modifications to a 5‐(phenylethynyl)pyrimidine scaffold. Bioorg. Med. Chem. Lett. 18:4098‐4101.
   Sharma, S., Kedrowski, J., Rook, J.M., Smith, R.L., Jones, C.K., Rodriguez, A.L., Conn, P.J., and Lindsley, C.W. 2009. Discovery of molecular switches that modulate modes of metabotropic glutamate receptor subtype 5 (mGlu5) pharmacology in vitro and in vivo within a series of functionalized, regioisomeric 2‐ and 5‐(phenylethynyl)pyrimidines. J. Med. Chem. 52:4103‐4106.
   Stables, J., Green, A., Marshall, F., Fraser, N., Knight, E., Sautel, M., Milligan, G., Lee, M., and Rees, S. 1997. A bioluminescent assay for agonist activity at potentially any G‐protein‐coupled receptor. Anal. Biochem. 252:115‐126.
   Shoichet, B.K. 2006. Screening in a spirit haunted world. Drug Discov. Today 11:607‐615.
   Urwyler, S. 2011. Allosteric modulation of family C G‐protein‐coupled receptors: From molecular insights to therapeutic perspectives. Pharmacol. Rev. 63:59‐126.
   Vaidehi, N. and Kenakin, T. 2010. The role of conformational ensembles of seven transmembrane receptors in functional selectivity. Curr. Opin. Pharmacol. 10:775‐781.
   Wang, L., Martin, B., Brenneman, R., Luttrell, L.M., and Maudsley, S. 2009. Allosteric modulators of g protein‐coupled receptors: Future therapeutics for complex physiological disorders. J. Pharmacol. Exp. Ther. 331:340‐348.
   Zhang, J.H., Chung, T.D., and Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4:67‐73.
Internet Resources
  NIH Chemical Genomics Center Assay Guidance Manual. This manual provides an overview of a wide variety of high‐throughput screening methods.
PDF or HTML at Wiley Online Library