The effect of receptor expression level on positive allosteric modulation (PAM) of human Dopamine D1 receptor

H. M. Saunders, E. McIver, J. C. Jerman. MRC Technology, Stevenage, UNITED KINGDOM.

Introduction: The ability of researchers to genetically engineer host cells to overexpress exogenous GPCRs has facilitated pharmacology and drug discovery. The development of high-throughput functional assays to support plate-based compound profiling is now commonplace. The desire for assay robustness, improved signal:background and high sensitivity tends to drive receptor expression and/or coupling efficiency upward. However, this can lead to gross amplification of potency/efficacy and physiologically unrepresentative agonist pharmacology. The use of titratable expression systems has enabled control and configuration of assays for a particular purpose e.g. high sensitivity versus physiological predictability. The present study reports the effect of receptor expression on allosteric potentiation of dopamine D1 receptors (D1R) using BacMam (Life Technologies).

Method: CHO cells were transduced with 1-15% (v/v) D1R BacMam (15K cells/well, 24hr, 37°C). On the day of assay, cells were pre-incubated with PAM (10pM-100nM) for 30min prior to exposure to agonist (100pM-10µM) for 30mins. After assay termination, cAMP activity was measured using Homogeneous Time Resolved Fluorescence (Cisbio). Responses were generally expressed relative to the dopamine max and iteratively fitted to a four-parameter logistic model (GraphPad Prism Software). To generate log-alpha factors, PAM data were analysed according to the Ternary Complex Model (1).

Results: Increasing viral load increased potency of full agonists dopamine and SKF83822 without effect on apparent efficacy. Conversely, the potency of partial agonist SKF83959 remained constant with increasing viral load but was accompanied by an increase in apparent efficacy (relative to dopamine within that condition).

Using a highly selective D1 PAM in leftward-shift experiments, at 1, 5, 10 and 15% (v/v) D1R BacMam, yielded Log-alpha factors of 1.34, 1.23, 1.32 and 1.43, respectively.

Conclusions: In the absence of Bmax values, increased receptor expression and/or coupling efficacy is highly inferred by the agonist pharmacology in each condition. Of note is the increase in potency of the full agonists and the increase in intrinsic activity of the partial agonist, each consistent with receptor theory. In this study the use of the partial agonist SKF83959 was particularly useful in ‘benchmarking’ experimental conditions relative to physiologically relevant assays and systems. Despite clear amplification of agonist pharmacology, the effect of the PAM was unaltered by receptor expression/coupling. This is also consistent with theory and is a useful empirical confirmation important to those configuring assays focussing on PAMs.

References: 1. Christopoulos A and Kenakin T (2002). Pharmacol Rev 54: 323-374.