title: A one-day, dispense-only IP-One HTRF assay for high-throughput screening of Galphaq protein-coupled receptors: towards cells as reagents.
abstract: Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio International) for HTS in a high-density format, but furthermore the successful use of cryopreserved and suspension cells in a one-day functional cell-based assay.
authors.affiliation: Department of Screening, Lead Discovery Berlin, Bayer Schering Pharma AG, Berlin, Germany. christian.bergsdorf@bayerhealthcare.com
authors.forename: Christian
authors.initials: C
authors.lastname: Bergsdorf
authors.forename: Carmen
authors.initials: C
authors.lastname: Kropp-Goerkis
authors.forename: Irene
authors.initials: I
authors.lastname: Kaehler
authors.forename: Lars
authors.initials: L
authors.lastname: Ketscher
authors.forename: Ulf
authors.initials: U
authors.lastname: Boemer
authors.forename: Karsten
authors.initials: K
authors.lastname: Parczyk
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bader
mesh_terms: D000818:Animals; D001331:Automation; D016466:CHO Cells; D002138:Calibration; D002448:Cell Adhesion; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000187:drug effects*; D015723:Gene Library; D007202:Indicators and Reagents; D007700:Kinetics; D016014:Linear Models; D012371:Robotics
chemical_list: D007202:Indicators and Reagents; D043802:GTP-Binding Protein alpha Subunits, Gq-G11