Understanding the effect of different assay formats on agonist parameters: a study using the µ-opioid receptor.

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 mu-opioid receptor agonists in three different assays: (1) [(35)S]GTP gamma S binding, (2) inhibition of forskolin-stimulated cAMP production, and (3) beta-arrestin recruitment. Agonist-concentration effect curves were performed before and after treatment with the irreversible antagonist beta-funaltrexamine, and where appropriate, these data were fitted to the operational model of agonism. The Z' value was highest in the beta-arrestin assay, followed by the [(35)S]GTP gamma S and cAMP assays. The cAMP data fitted well to the operational model, as did the [(35)S]GTP gamma S data, but the [(35)S]GTP gamma S assay led to an apparent overestimation of K A values. However, in the beta-arrestin assay, data did not fit the operational model, as treatment with beta-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 beta-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. (Journal of Biomolecular Screening 2011; 16: 706-716)