Application of the BD ACTOne technology for the high-throughput screening of Gs-coupled receptor antagonists.
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.
Visegrády
András
A
Lead Discovery Unit, Gedeon Richter Plc, Budapest, Hungary. visegrady@richter.hu
Boros
András
A
Némethy
Zsolt
Z
Kiss
Béla
B
Keseru
György M
GM
D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D009479:Neuropeptides / Q000494:pharmacology; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D054815:Cyclic Nucleotide-Gated Cation Channels; D009479:Neuropeptides; D044168:Receptor, Parathyroid Hormone, Type 1; D043562:Receptors, G-Protein-Coupled; C400136:tuberoinfundibular peptide 39; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D002118:Calcium