The DiscoveRx PathHunter β-arrestin assay; A measure of agonist affinity?

Alision Waterfield, Ross Kinloch, Rachael Williams, Tim Young, Sarah Nickolls. Pfizer, Sandwich, United Kingdom.

The PathHunter β-arrestin assay uses enzyme complementation to measure the interaction of receptors with β-arrestin. This is typically referred to as a functional assay in the literature, and regarded as a good alternative to common G protein-mediated functional assays, such as measurement of cAMP levels.

We have evaluated the use of the PathHunter β-arrestin assay for the MOP receptor, and compared these results to data generated using the same cells in an inhibition of forskolin-stimulated cAMP functional assay. In both assays, U20S cells expressing the MOP receptor were incubated with 13 different agonists (+ 20μM forskolin in the cAMP assays) for 90 minutes at 37oC. Either β-arrestin recruitment or cAMP levels were then determined using the PathHunter or Cisbio HTRF cAMP detection kits, respectively. In some cases, cells had been previously treated with the irreversible antagonist β-funaltrexamine (1 or 10nM for 30 minutes) to reduce receptor number.

For all 13 compounds, the EC50 values generated in the β-arrestin assay were very similar to KA values determined by fitting cAMP data generated before and after treatment of cells with β-funaltrexamine, to the operational model of agonism (r2 = 0.89) (Black and Leff, 1983). In addition, the Emax in the β-arrestin assay was determined to be directly proportional to receptor number (a 44% decrease in Bmax gave a 44% decrease in Emax, and a 73% reduction in Bmax gave a 74% reduction in Emax). Furthermore, in the β-arrestin assay, there was no change in the EC50 values of compounds when receptor number was depleted, whereas in the cAMP assay both EC50 and Emax values were significantly affected by depleting receptor number.

These data lead us to the conclusion that a linear relationship exists between occupancy and response in the PathHunter β-arrestin assay, therefore indicating that this assay is a direct measure of agonist affinity.

Black JW, Leff P (1983). Operational models of pharmacological agonism. Proc Royal Soc Lon 220: 141-162.