The CellKey (MDS Sciex, South San Francisco, CA) system enables comprehensive pharmacological evaluation of cell surface receptors, including G-protein coupled receptors (GPCRs) and tyrosine kinase receptors, using adherent and suspension cell lines and primary cells. A unique application enabled by the ability of the CellKey system to reliably quantify activation of endogenous receptors is receptor panning. This application allows investigators to easily screen disease-relevant cell types for functionally active target receptors by treating cells with a panel of receptor-specific ligands. Receptor panning of multiple cell types including Chinese hamster ovary, human embryonic kidney 293, HeLa, U-937, U-2 OS, and TE671 cells resulted in the identification of many functionally active, differently coupled endogenous GPCRs, some of which have not been previously documented in the literature. Upon detecting GPCR activation in live cells, unique cellular dielectric spectroscopy (CDS) response profiles are generated within minutes that reflect the signaling pathways utilized and have been shown to be characteristic of Gs, Gq, and Gi GPCRs. The fact that the CDS response profiles are predictive of the G-protein coupling mechanism of the receptor was demonstrated by using examples of subtype-selective agonists/antagonists to identify the subtypes of the endogenous histamine and beta-adrenergic receptors expressed in U-2 OS cells. A direct correlation is shown between receptor subtype G-protein coupling and CDS response profile. In addition, complex pharmacology, including detection of partial agonism and Schild analysis for endogenous receptors, is presented. The CellKey system allows investigators to conduct studies using endogenously expressed receptors to generate data that are physiologically relevant and in disease context.

The past decade has seen a number of significant changes in identifying higher quality lead compounds earlier in the drug discovery process. Cell-based assay technologies yielding high-content information have emerged to achieve this goal. Although most of these systems are based on fluorescence detection, this article describes the development and application of an innovative cellular assay technology based on radio frequency spectrometry and bioimpedance measurements. Using this technique, the authors have discovered a link between cellular bioimpedance changes and receptor-mediated signal transduction events. By performing dielectric spectroscopy of cells across a spectrum of frequencies (1 KHz to 110 MHz), a series of receptorspecific, frequency-dependent impedance patterns is collected. These raw data patterns are used to determine the identity of the cellular receptor-signaling pathway being tested and to quantify stimulation endpoints and kinetics. The authors describe the application of this technology to the analysis of ligand-induced cellular responses mediated by the 3 major classes of Gprotein-coupled receptors (GPCRs) and protein tyrosine kinase receptors. This single assay platform can be used with ease to monitor G s , G i , and G q GPCRs without the need for chimeric or promiscuous G-proteins, fluorophors, or tagged proteins. In contrast to other methods of monitoring cellular signal transduction, this approach provides high information content in a sim-

C ellular dielectric spectroscopy (CDS) provides realtime, label-free, universal measurements, enabling comprehensive pharmacological evaluation of cell surface receptors in living cells. The sensitivity of the measurement allows monitoring of ligand-mediated activation of endogenous receptors, therefore generating physiologically relevant data. Activation of receptors results in CDS response profiles that are characteristic of main subsets of G-protein coupled receptors (GPCRs) within a cell line. This allows cluster analysis of response profiles that may be used in several important applications, which include identification of the G-protein coupling of orphan GPCRs and the cataloging of active endogenous receptors in cells. In this study, CDS technology is used in the pharmacological evaluation of multiple receptors in many cell types, including primary cells. Specifically, data is presented demonstrating hit confirmation, receptor selectivity analysis, ligand potency, and Schild analysis of receptor-selective antagonists. CDS results compare favorably to other cell-based assays, and the robustness and reproducibility of CDS assays are reflected by low assay coefficient of variation (CVs) and reliable Z'-scores of the data. Because CDS requires no stable or transiently transfected cells or special reagents, assay development and data acquisition is simple and fast.The ease of use, universality, and label-free nature of the CDS-based platform make it well suited to secondary screening applications in drug discovery.