ReviewKeynoteLabel-free whole-cell assays: expanding the scope of GPCR screening
Introduction
Cell-based assays have an important role in drug discovery. Designed appropriately, these in vitro tests can help predict the effect of chemical agents in vivo and can provide relevant biochemical and pharmacological insight that is not possible in a whole animal study. Many detection methods used in cell-based assays have been miniaturized and automated to enable high-throughput screening (HTS) of chemical libraries to seek compounds that induce desired changes in a particular cellular function. Assays for G-protein-coupled receptors (GPCRs) often use functional outputs (e.g. changes in second messenger levels) that can discriminate different pharmacological classes of ligands. These assays have suitable precision to define the structure–activity relationship (SAR) of synthetic ligands generated in a drug hunting campaign. Despite success with these assays, in vitro pharmacologists are constantly looking for improvements in cell-based GPCR detection methods to provide greater biological insight and new and broader applications, or those that can be applied to more native environments.
Label-free technologies with the potential to substantially change some aspects of whole-cell assays, including GPCR screening, have emerged within the past few years (reviewed in Refs. 1, 2, 3, 4, 5). These technologies detect changes in cellular features including adhesion and morphology, complex endpoints that are modulated by many different receptors, ion channels and signal transduction pathways. Some label-free instruments have gained particular prominence in measuring GPCR function because they can detect the activation of Gs, Gi and Gq signal transduction pathways 6, 7, 8, 9, 10, 11. These readouts are achieved in real-time and have shown exquisite sensitivity to enable detection of endogenous receptor function with output that can be quantified with high precision. In addition, these label-free assays do not require the addition of detection reagents to the cells or expression strategies involving forced G-protein coupling or promiscuous G proteins, thus offering the potential for investigating a more physiological state.
Depending on the cell type and the activating ligand, GPCRs can stimulate different or multiple signal transduction pathways. Initial studies with label-free instruments have demonstrated the potential for detecting and exploring the diversity of these signaling events in a manner not previously possible. Here, we review these findings and label-free GPCR drug discovery applications that have been validated to date. We particularly focus on assay features required to enable SAR studies with label-free instruments and to detect different classes of pharmacological agents. Current limitations and areas not yet investigated in this rapidly evolving field of study are also highlighted.
Section snippets
Detection principles and instruments
Currently available label-free instruments use either an impedance-based biosensor or an optical-based biosensor to detect changes in cell behavior (Fig. 1). The underlying concepts of these biosensors are described below. Detailed information on the technology used in these biosensors has been described previously 12, 13, 14, 15.
Comparison with traditional technologies
To validate label-free detection applications for drug discovery, the pharmacology derived from these instruments should align with more traditional measures of GPCR signaling. Ligands that span large potency and efficacy ranges should be used to determine whether (a) the instrument has sufficient dynamic range and sensitivity to discriminate high and low affinity, as well as varying efficacy; (b) the potency values show a similar rank order to that seen with other functional readouts (i.e.
Detecting a spectrum of pharmacology
Whole-cell label-free assays have been validated for routine agonist and antagonist SAR-driving assays, but for maximal impact, broader applications are required. As described below, other pharmacological profiles can also be detected and quantified with these biosensors.
Applications for drug discovery
The advantages of label-free technology can be exploited for different purposes in a drug discovery environment. Because these instruments can detect a functional response from receptors that activate Gs, Gi or Gq signaling pathways, a label-free biosensor can be used as a platform technology to support GPCR drug hunting projects. Some of the more prominent applications are listed below.
HTS. The sensitivity, precision, ease of assay development (once familiar with the technology) and throughput
Current unknowns, limitations and future opportunities
The past five years can be viewed as a period of substantial exploration and growth using label-free instruments to measure aspects of cellular biology. In particular, studying GPCR signal transduction with label-free biosensors has provided both insight and practical applications. By applying appropriate biochemical tools and cell biology perspectives, several areas of unexplored biology might reveal additional applications for these instruments. Some examples include the following.
Biochemical
Concluding remarks
Impedance- and optical-based instruments introduce two fundamental advances – label-free detection and unparalleled sensitivity. Combining these in a single instrument is at the heart of their exceptional power and versatility. Although the potential extends to a wide range of in vitro applications, the impact on GPCR drug discovery is particularly great. Specifically, this versatility and power enables detecting GPCR coupling through different classes of G proteins even when expressed at
Disclosure statement
The authors were employees of AstraZeneca Pharmaceuticals Inc. before and while performing the experiments described in this article.
Acknowledgements
We thank Robert Shaw for developing the statistical tool used to define assay precision (Fig. 4) and Lois Ann Lazor for improving the grammatical content of this manuscript.
Clay Scott is an associate director within the CNS/Pain Research Area at AstraZeneca Pharmaceuticals, providing in vitro pharmacology support for projects in early phase drug discovery. Clay has been a drug discovery project leader in several CNS and respiratory disease areas. He has a particular expertise in Lead Generation as a discipline and in implementing new methods, from technologies to process improvements, to enhance in vitro pharmacology screening. He received a Ph.D. in pharmacology
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Clay Scott is an associate director within the CNS/Pain Research Area at AstraZeneca Pharmaceuticals, providing in vitro pharmacology support for projects in early phase drug discovery. Clay has been a drug discovery project leader in several CNS and respiratory disease areas. He has a particular expertise in Lead Generation as a discipline and in implementing new methods, from technologies to process improvements, to enhance in vitro pharmacology screening. He received a Ph.D. in pharmacology from the University of Texas Southwestern Medical Center.
Matt Peters is a principle scientist and Drug Discovery Project Leader with AstraZeneca Pharmaceuticals, focusing on GPCR targets and developing capabilities to enhance the assessment of in vitro pharmacology for GPCRs. Matt received a Ph.D. in cellular and molecular physiology from the University of North Carolina followed by a postdoctoral fellowship at Johns Hopkins University.
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Both authors contributed equally to this publication.