title: Understanding the effect of different assay formats on agonist parameters: a study using the µ-opioid receptor.
abstract: The correct interpretation of data is fundamental to the study of G-protein-coupled receptor pharmacology. Often, new assay technologies are assimilated into the drug discovery environment without full consideration of the data generated. In this study, the authors look at µ-opioid receptor agonists in three different assays: (1) [(35)S]GTPγS binding, (2) inhibition of forskolin-stimulated cAMP production, and (3) β-arrestin recruitment. Agonist-concentration effect curves were performed before and after treatment with the irreversible antagonist β-funaltrexamine, and where appropriate, these data were fitted to the operational model of agonism. The Z' value was highest in the β-arrestin assay, followed by the [(35)S]GTPγS and cAMP assays. The cAMP data fitted well to the operational model, as did the [(35)S]GTPγS data, but the [(35)S]GTPγS assay led to an apparent overestimation of K(A) values. However, in the β-arrestin assay, data did not fit the operational model, as treatment with β-funaltrexamine reduced the Emax proportionally to receptor number, with no change in EC(50). In addition, the EC(50) values generated correlated well with affinity values. In conclusion, the β-arrestin recruitment assay does not fit with traditional pharmacological theory but is of great utility as the EC(50) value generated is a good approximation of affinity.
authors.affiliation: Discovery Biology, Pfizer Inc., Sandwich, Kent, UK. sarah.nickolls@pfizer.com
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Nickolls
authors.forename: Alison
authors.initials: A
authors.lastname: Waterfield
authors.forename: Rachael E
authors.initials: RE
authors.lastname: Williams
authors.forename: Ross A
authors.initials: RA
authors.lastname: Kinloch
mesh_terms: D019393:Arrestin / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D019393:Arrestin; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009271:Naltrexone; C024524:beta-funaltrexamine; D000242:Cyclic AMP