Effect of forskolin concentration on the pharmacological profiling of dopamine D2 agonists using the DiscoveRx HitHunter cAMP IITM assay

Vicki Mason*, Christine Williams†, David McLoughlin† and Philip G. Strange* *School of Pharmacy, University of Reading, Whiteknights, Reading, Berkshire †Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent.

The production of cAMP from ATP on activation of adenylyl cyclase presents a reliable platform for the high-throughput-screening of G-protein coupled receptors. To screen for agonist activity at Gi-GPCRs it is necessary to activate adenylyl cyclase with forskolin and measure the agonist-induced inhibition of cAMP production. The assay type and cell line being used can affect the EC50 value for forskolin (Williams, 2004). In this study the effect of forskolin ECvalue on the pharmacological profile of a range of agonists acting at the human dopamine D2short receptor stably expressed in CHO-K1 cells (Wilson et al., 2001) was investigated in the DiscoveRx HitHunter cAMP II ä assay.

Assays were performed in triplicate in a 384 well format; total assay volume was 15μl. Forskolin concentration response curves were produced by performing 1:2 serial dilutions in PBS. Forskolin (5μl, final concentration 250μM – 15.26nM) was placed in the assay with 5μl PBS containing DTT and DMSO (final concentration 100μM and 0.5% respectively), and CHO-K1 cells stably expressing the D2S dopamine receptor (Wilson et al., 2001) (5μl DMEM, 20,000 cells/well). Agonist concentration response curves were obtained by performing 1:5 serial dilutions in PBS containing DTT and DMSO (final concentration 100μM and 0.5% respectively). Agonist (5μl, final concentration 50μM – 8fM) was placed in the assay with 5μl forskolin (final concentration 10μM, 30μM or 100μM) and CHO-K1D2S cells (5 m l DMEM, 20,000 cells/well). The assay was incubated for 1.5h at 37 ° C in a humidified atmosphere of 5% CO2.

Forskolin had an EC50 value of 14μM (pEC50 = 4.85 ± 0.05, n =11). Subsequent agonist concentration response curves were performed at 10μM, 30μM and 100μM (EC41±3 ; EC71±3 , EC90±2 respectively). p-tyramine displayed no detectable agonism, this is in contrast to previous experiments performed in the same cell line (10μM forskolin) measuring the inhibition of [3H]-cAMP accumulation where it behaved as a full agonist (99.8 ± 3.6%, n =3; Payne et al., 2002). All the other ligands behaved as full agonists at all forskolin concentrations with the exception of (-)-3PPP, which behaved as a full agonist at 10μM forskolin and as a partial agonist at 30μM and 100μM forskolin, displaying a significant decrease in relative efficacy with increasing forskolin concentration (one-way ANOVA, p < 0.05). The rank order of potency was maintained with increasing forskolin concentration (dopamine > (-)-3PPP = (+)-3PPP > m-tyramine). There was a decrease in potency for dopamine, m-tyramine and (+)-3PPP (one way ANOVA, p < 0.05) with increasing forskolin concentration, whilst no change in potency for (-)-3PPP was observed with increasing forskolin concentration (one way ANOVA, p > 0.05).

This study has shown how assay configuration and assay type can affect the pharmacological profiling of ligands. It demonstrates the need to firmly establish the potency of forskolin in the system being examined, and to exercise caution when comparing parameters obtained in different cell lines or assay types.

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