Application of the BD ACT One ™ Technology for the High-Throughput Screening of G s -Coupled Receptor Antagonists

Abstract

A novel technology for monitoring the changes of 3,′5′-adenosine cyclic monophosphate (cAMP) in live cells suitable for drug screening relies on the use of cyclic nucleotide-gated channels as biosensors coexpressed with the appropriate target receptor. The technique (termed BD ACTOne™) offers measurement of cAMP-dependent calcium influx or membrane depolarization with conventional fluorescent methods both in kinetic and in endpoint modes, optimal for high-throughput and subsequent compound screening. The utility of the technique is reported here based on assay development and high-throughput screening for small-molecule antagonists of the peptide parathyroid hormone 2 receptor (PTH2R). The dual-signaling properties of the receptor were retained in the recombinant system, and the observed pharmacological profile corresponded to data from radiolabeled cAMP determination. The membrane-potential-based high-throughput assay produced reproducible actives and led to the identification of several chemical scaffolds with potential utility as PTH2R ligands. (Journal of Biomolecular Screening 2007:1068-1073)

Keywords

cyclic nucleotide-gated channel high-throughput screening cAMP G-protein-coupled receptor peptide parathyroid hormone 2 receptor

References

Wise A. , Gearing K. , Rees S. : Target validation of G-protein-coupled receptors: an overview of GPCR target validation. Drug Disc Today 2002; 7: 235-246 .

Chambers C. , Smith F. , Williams C. , Marcos S. , Liu ZH , Hayter P. , et al: Measuring intracellular calcium fluxes in high-throughput mode . Comb Chem High Throughput Screen 2003;6: 355-362 .

Williams C. : cAMP detection methods in HTS: selecting the best from the rest . Nat Rev Drug Disc 2004;3: 125-135 .

Warrior U. , Gopalakrishnan S. , Vanhauwe J. , Burns D. : High-throughput screening assays for G protein coupled receptors. In Lundstrom KH , Chiu ML (eds): G Protein-Coupled Receptors in Drug Discovery . Boca Raton, FL: CRC Press , 2006:159-189.

Kostenis E. : Is Gα16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors? Trends Pharmacol Sci 2001;22: 560-564 .

Mannion JC , Vanek PG , Yao Y. : Developing a new biosensor for cyclic AMP . Gen Eng News 2003; 23 .

Rich TC , Fagan KA , Nakata H. , Schaak J. , Cooper DM , Karpen JW : Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion . J Gen Physiol 2000;116: 147-162 .

Tang Y. , Li X. , He J. , Lu J. , Diwu Z. : Real-time and high-throughput monitoring of cAMP in live cells using a fluorescent membrane potential-sensitive dye . Assay Drug Dev Techn 2006;4: 461-471 .

Usdin TB , Bonner TI , Hoare SRJ : The parathyroid hormone 2 (PTH2) receptor. Recept Channels 2002;8: 211-218 .

10.

Derrickson BH , Mandel LJ : Parathyroid hormone inhibits Na-K-ATPase through Gq/G11 and the calcium-independent phospholipase A2 . Am J Physiol 1997;272: F781-F788 .

11.

Zhang JH , Chung TD , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high-throughput screening assays . J Biomol Screen 1999;4: 67-73 .

12.

Bánki Z. , Báthor M. , Molnár L. , Bielik A. , Keserű GM : Scheduling a flexible, open-architecture robotic workstation under LabWindows . J Assoc Lab Aut 2005;10: 149-154 .

13.

Gribbon, P. , Lyons R. , Laflin P. , Bradley J. , Chambers C. , Williams BS , et al: Evaluating real-life high-throughput screening data . J Biomol Screen 2005;10: 99-107 .

14.

Hoare SR , Usdin TB : Specificity and stability of a new PTH1 receptor antagonist, mouse TIP(7-39) . Peptides 2002;23: 989-998 .

15.

Vanhauwe JF , Fraeyman N. , Francken BJ , Luyten WH , Leysen JE : Comparison of the ligand binding and signaling properties of human dopamine D(2) and D(3) receptors in Chinese hamster ovary cells . J Pharmacol Exp Ther 1999;290: 908-916 .