Functional assays for screening GPCR targets.

G-protein-coupled receptors (GPCRs) are valuable molecular targets for drug discovery. An important aspect of the early drug discovery process is the design and implementation of high-throughput GPCR functional assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates. Several functional assay kits based on fluorescence and/or chemiluminescence detection are commercially available for convenient screen development, each having advantages and disadvantages. In addition, new GPCR biosensors and high-content imaging technologies have recently been developed that hold promise for the development of functional GPCR screens in living cells.

[1]  Michel Bouvier,et al.  Methods to monitor the quaternary structure of G protein‐coupled receptors , 2005, The FEBS journal.

[2]  L. Luttrell,et al.  Signaling in time and space: G protein-coupled receptors and mitogen-activated protein kinases. , 2003, Assay and drug development technologies.

[3]  M. Fidock,et al.  Identification of orphan G protein-coupled receptor ligands using FLIPR assays. , 2005, Methods in molecular biology.

[4]  Vincent J. Dupriez,et al.  Adaptation of Aequorin Functional Assay to High Throughput Screening , 2002, Journal of biomolecular screening.

[5]  E. Scolnick,et al.  Scintillation proximity assay of inositol phosphates in cell extracts: high-throughput measurement of G-protein-coupled receptor activation. , 2003, Analytical biochemistry.

[6]  Anita Preininger,et al.  Insights into G protein structure, function, and regulation. , 2003, Endocrine reviews.

[7]  M. Rosenkilde,et al.  High content screening for G protein-coupled receptors using cell-based protein translocation assays. , 2005, Combinatorial chemistry & high throughput screening.

[8]  Sadashiva S Karnik,et al.  Multiple Signaling States of G-Protein-Coupled Receptors , 2005, Pharmacological Reviews.

[9]  K. Beaumont,et al.  Identification of signal transduction pathways used by orphan g protein-coupled receptors. , 2003, Assay and drug development technologies.

[10]  S. Rees,et al.  Chimaeric G alpha proteins: their potential use in drug discovery. , 1999, Trends in pharmacological sciences.

[11]  Michel Bouvier,et al.  Real-time monitoring of receptor and G-protein interactions in living cells , 2005, Nature Methods.

[12]  J. Way,et al.  Use of constitutive G protein-coupled receptor activity for drug discovery. , 2000, Molecular pharmacology.

[13]  O. Civelli,et al.  High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide-gated channels. , 2003, European journal of pharmacology.

[14]  Martin J. Lohse,et al.  β-Arrestin Binding to the β2-Adrenergic Receptor Requires Both Receptor Phosphorylation and Receptor Activation* , 2005, Journal of Biological Chemistry.

[15]  P. Marin,et al.  GPCR interacting proteins (GIP). , 2004, Pharmacology & therapeutics.

[16]  R. Lefkowitz,et al.  Multifaceted roles of β-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling , 2003 .

[17]  Christine Williams,et al.  cAMP detection methods in HTS: selecting the best from the rest , 2004, Nature Reviews Drug Discovery.

[18]  N. Delapp.  The antibody-capture [(35)S]GTPgammaS scintillation proximity assay: a powerful emerging technique for analysis of GPCR pharmacology. , 2004, Trends in pharmacological sciences.

[19]  Emmanuel Hermans,et al.  Biochemical and pharmacological control of the multiplicity of coupling at G-protein-coupled receptors. , 2003, Pharmacology & therapeutics.

[20]  P. Devreotes,et al.  Receptor-Mediated Activation of Heterotrimeric G-Proteins in Living Cells , 2001, Science.

[21]  A. Hanyaloglu,et al.  Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells , 2002, Trends in Endocrinology & Metabolism.

[22]  R. Eglen,et al.  Beta galactosidase enzyme fragment complementation as a novel technology for high throughput screening. , 2003, Combinatorial chemistry & high throughput screening.

[23]  Pascale G. Charest,et al.  Palmitoylation of the V2 Vasopressin Receptor Carboxyl Tail Enhances β-Arrestin Recruitment Leading to Efficient Receptor Endocytosis and ERK1/2 Activation* , 2003, Journal of Biological Chemistry.

[24]  J. Dunlop,et al.  Evaluation of FLIPR Calcium 3 Assay Kit—A New No-Wash Fluorescence Calcium Indicator Reagent , 2003, Journal of biomolecular screening.

[25]  A. Närvänen,et al.  Assessing activation of the human neuropeptide FF2 receptor with a non-radioactive GTP binding assay , 2004, Peptides.

[26]  Alan Wise,et al.  The identification of ligands at orphan G-protein coupled receptors. , 2004, Annual review of pharmacology and toxicology.

[27]  Graeme Milligan,et al.  High-content assays for ligand regulation of G-protein-coupled receptors. , 2003, Drug discovery today.

[28]  Keith A Houck,et al.  High-content screening assay for activators of the Wnt/Fzd pathway in primary human cells. , 2005, Assay and drug development technologies.

[29]  E. Kostenis.  POTENTIATION OF GPCR-SIGNALING VIA MEMBRANE TARGETING OF G PROTEIN α SUBUNITS , 2002 .

[30]  E. Kostenis.  Is Gα16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors? , 2001 .

[31]  G. Bird,et al.  Techniques: high-throughput measurement of intracellular Ca(2+) -- back to basics. , 2005, Trends in pharmacological sciences.

[32]  Graeme Milligan,et al.  G Protein-Coupled Receptor Dimerization: Function and Ligand Pharmacology , 2004, Molecular Pharmacology.

[33]  M. Scheinin,et al.  Nonradioactive GTP binding assay to monitor activation of g protein-coupled receptors. , 2003, Assay and drug development technologies.

[34]  Gerhard Hessler,et al.  Drug Design Strategies for Targeting G‐Protein‐Coupled Receptors , 2002, Chembiochem : a European journal of chemical biology.

[35]  Mark H Ellisman,et al.  A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells , 2005, Nature Methods.

[36]  G. Zaman,et al.  High-Throughput Screening Using β-Lactamase Reporter-Gene Technology for Identification of Low-Molecular-Weight Antagonists of the Human Gonadotropin Releasing Hormone Receptor , 2005 .

[37]  J. Hepler.  RGS protein and G protein interactions: a little help from their friends. , 2003, Molecular pharmacology.

[38]  M. Ferrer,et al.  A 1536-well cAMP assay for Gs- and Gi-coupled receptors using enzyme fragmentation complementation. , 2004, Assay and drug development technologies.

[39]  M. Ferrer,et al.  A fully automated [35S]GTPgammaS scintillation proximity assay for the high-throughput screening of Gi-linked G protein-coupled receptors. , 2003, Assay and drug development technologies.

[40]  Daniela Gabriel,et al.  High throughput screening technologies for direct cyclic AMP measurement. , 2003, Assay and drug development technologies.

[41]  Terry Kenakin,et al.  Efficacy at g-protein-coupled receptors , 2002, Nature Reviews Drug Discovery.

[42]  P. Knight,et al.  Chimeric G Proteins Extend the Range of Insect Cell-Based Functional Assays for Human G Protein-Coupled Receptors , 2004, Journal of receptor and signal transduction research.

[43]  E. Kostenis.  Is Galpha16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors? , 2001, Trends in pharmacological sciences.

[44]  B. Conklin,et al.  Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. , 1999, Analytical biochemistry.

[45]  P. Devreotes,et al.  Monitoring receptor-mediated activation of heterotrimeric G-proteins by fluorescence resonance energy transfer. , 2002, Methods.

[46]  Rasmus Jorgensen,et al.  Development of a BRET2 screening assay using beta-arrestin 2 mutants. , 2004, Journal of biomolecular screening.

[47]  M. Bouvier,et al.  Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function. , 2003, Pharmacology & therapeutics.

[48]  Y. Wong,et al.  Characterization of CHO cells stably expressing a G alpha 16/z chimera for high throughput screening of GPCRs. , 2004, Assay and drug development technologies.

[49]  D. Devost,et al.  Real-time detection of interactions between the human oxytocin receptor and G protein-coupled receptor kinase-2. , 2004, Molecular endocrinology.

[50]  N. Gautam,et al.  A Fluorescence Resonance Energy Transfer-based Sensor Indicates that Receptor Access to a G Protein Is Unrestricted in a Living Mammalian Cell*[boxs] , 2004, Journal of Biological Chemistry.

[51]  Rasmus Jorgensen,et al.  Development of a BRET2 Screening Assay Using β-Arrestin 2 Mutants , 2004 .

[52]  Pascale G. Charest,et al.  Monitoring agonist‐promoted conformational changes of β‐arrestin in living cells by intramolecular BRET , 2005, EMBO reports.

[53]  Andreas Sewing,et al.  Measuring intracellular calcium fluxes in high throughput mode. , 2003, Combinatorial chemistry & high throughput screening.

[54]  James Inglese,et al.  Miniaturization of Whole Live Cell-Based GPCR Assays Using Microdispensing and Detection Systems , 2004, Journal of biomolecular screening.

[55]  J. Pelletier,et al.  High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based β-Arrestin2 Recruitment Assay , 2005, Journal of biomolecular screening.

[56]  Jay Liu,et al.  An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis. , 2003, Analytical biochemistry.

[57]  Y. Wong,et al.  Gα 16/z Chimeras Efficiently Link a Wide Range of G Protein— Coupled Receptors to Calcium Mobilization , 2003, Journal of biomolecular screening.

[58]  R. Eglen,et al.  Functional G protein-coupled receptor assays for primary and secondary screening. , 2005, Combinatorial chemistry & high throughput screening.

[59]  B. Conklin,et al.  Substitution of three amino acids switches receptor specificity of Gqα to that of Giα , 1993, Nature.

[60]  G. Milligan.  Principles: extending the utility of [35S]GTP gamma S binding assays. , 2003, Trends in pharmacological sciences.

[61]  M. Gewecke,et al.  A green-fluorescent-protein-based assay for the characterization of G-protein-coupled receptors. , 2004, Analytical biochemistry.

[62]  Christine C. Hudson,et al.  Quantitative Cell-Based High-Content Screening for Vasopressin Receptor Agonists Using Transfluor®Technology , 2005, Journal of biomolecular screening.

[63]  J. Chambers,et al.  A G Protein-coupled Receptor for UDP-glucose* , 2000, The Journal of Biological Chemistry.

[64]  Martin Oppermann,et al.  Characterization of Sequence Determinants within the Carboxyl-terminal Domain of Chemokine Receptor CCR5 That Regulate Signaling and Receptor Internalization* , 2001, The Journal of Biological Chemistry.