The members of the G protein- coupled receptor (GPCR) superfamily are important contributors to many processes of development and disease [1, 2]. They consist of at least 300 nonsensory human family members that are known or predicted to be activated by endogenous ligands. GPCRs are an important drug target class for the pharmaceutical industry, and more than 20% of currently top selling drugs are GPCR related [3, 4]. However, only a small proportion of the GPCR family has been fully exploited, and a significant number of orphan GPCRs are considered potential drug targets [5].

With the implementation of high-throughput screening (HTS) and the exponential growth of compound libraries over the past two decades, it has become increasingly important to consider assay time, robustness, and costs in the design of screening campaigns for any new GPCR target. This has led to the development of a relatively small set of HTS-compatible GPCR assays that are currently used in drug discovery. These formats include, among others, real-time Ca2+ release assays for Gaq coupling, cAMP assays for Gas and Gai coupling, GTPyS assays, mainly for Gai coupling, or reporter gene assays that can be easily adapted for almost any known G protein coupling pathway [6] . This apparent simplicity of GPCR biology has led to wide implementation of drug discovery platforms that are specialized in the development of GPCR assays adapted for HTS.

The process of GPCR drug discovery has thus became fully industrialized, allowing the screening of dozens of GPCR targets per year. However, as a typical HTS campaign often leads to hit rates in excess of 1% of the total library, which may contain up to 2 million compounds, secondary assays have become increasingly important to validate hits emerging from primary screens. Nevertheless, the industrialization process of the GPCR drug discovery workflow has not fully translated into the expected flood of novel drugs, and there is much debate on the reasons of this apparent "failure" and how it can be remedied [7-9].

Besides nonmechanism-based toxicological or tolerability problems of compounds in preclinical or clinical development, there may be an additional issue with respect to the target GPCR biology and the pharmacological behavior of synthetic GPCR modulators. It is evident from the current literature that clinically successful GPCR- t argeting compounds, exemplified by dopa-mine receptor agonists and P-adrenergic agonists, do not necessarily have a straightforward pharmacological profile when they are carefully characterized

TABLE 11.1 Summary of Label-Free Technologies and Equipment

Label-Free Systems with Potential Applications in GPCR Cell Signaling Pathway Analysis

TABLE 11.1 Summary of Label-Free Technologies and Equipment

Label-Free Systems with Potential Applications in GPCR Cell Signaling Pathway Analysis



Assay Principle

Provider Home Page

Cytosensor microphysiometer

Molecular Devices Inc.

Extracellular pH

XF analyzer

Seahorse Bioscience

02 consumption/

extracellular pH

EPIC System



BIND Reader

SRU Biosystems




Optical grating-

coupled waveguide

xCELLigence system

Roche Applied Science



MDS Sciex



Applied Biophysics


Bionas 2500 analyzer


Extracellular pH, 02,

http ://www.bionas. de

and impedance

Other Label-Free Systems



Automated microscopy


Chip Man Technologies

Automated microscopy

Q-Sense El or E4



in a panel of different GPCR signaling assays [10-13] . Matters become even more complex, when considering that the pharmacology of GPCR targeting compounds may significantly depend on the cellular background or disease condition [14] . Based on these recent insights, which are translated into the setting of modern GPCR drug discovery, it is currently considered essential to investigate novel synthetic modulators in multiple signaling pathways for any given GPCR and to fully characterize the pharmacological behavior of these compounds, especially in primary cells [15]. It is thus assumed that a detailed pharmacological understanding of the compound in vitro will eventually help to design innovative compounds with potentially fewer side effects in vivo.

Today, it is common practice to consider the full spectrum of potential synthetic GPCR modulators that could emerge from a HTS campaign, including full or partial agonists or antagonists, pathway-selective (biased) agonists, inverse agonists, allosteric modulators, or compounds with the potential to specifically modulate GPCR dimers [16-18]. Consequently, the assay development and compound profiling strategy must be adequately adapted to identify suitable lead molecules and to allow a careful characterization of the target GPCR pharmacology in physiological or disease-relevant cells.

In this context, it is questionable whether the traditional screening strategy, which includes the widespread use of recombinant systems, often combined with forced coupling using chimeric Ga proteins or promiscuous Ga16 proteins, is sufficient to identify and characterize molecules with complex GPCR pharmacology. Furthermore, by using highly artificial screening assays, there is an inherent risk to find molecules that display nonphysiological pharmacology in disease-relevant target systems. It is for these reasons that the pharmaceutical industry has developed an increasing interest in label-free systems, avoiding the use of dyes and other molecular interventions to measure compound activity and to incorporate real-time kinetics. Label-free systems thus simplify assay development and offer novel opportunities to measure GPCR pharmacology in nonrecombinant or primary cells even in high- t hroughput screens. This review looks at the evolution of label-free detection methods over the past years and describes current technologies (Table 11.1) and their use in the context of GPCR pharmacology, especially in drug discovery.

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