Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development - Progress in Molecular Biology and Translational Science - page 275

5.3 Multiplexing: Simultaneous analysis of a compound effect in
several signaling pathways 306

5.4 Toward further miniaturization 307

6. Conclusion 308

References 308

Abstract
High-throughput screening requires easy-to-monitor, rapid, robust, reliable, and minia-
turized methods to test thousands of compounds on a target in a short period, in order
to find active drugs. Only a few methods have been proved to fulfill all these require-
ments. New screening approaches based on fluorescence and especially on the prin-
ciple of resonance energy transfer are being developed to study one of the main
targets in the pharmaceutical industry, namely, the G protein-coupled receptors
(GPCRs). Two types of approaches are clearly defined: generic approaches that are im-
mediately applicable to a lot of targets such as second messenger kits or kinase kits;
target-specific approaches that sense the receptor itself such as fluorescent ligands
or fluorescent partners. This chapter focuses on sensors and approaches using the
time-resolved Förster resonance energy transfer and homogeneous time-resolved
fluorescence principle, their use, and their prospective applications for screening drugs
acting on GPCRs.

1. INTRODUCTION
Drug discovery remains a challenge in the pharmaceutical industry. To
be validated as a future medicine, a compound must fulfill certain criteria to
ensure its specificity for the target, its mode of action, and limited side effects.
To assess whether it fulfills all these criteria, the molecules are tested in various
assays starting fromhit identification by screening large libraries of compounds
using rapid and accurate tests, and followed by a precise analysis of the prop-
erties of a reduced number of molecules using animal models and clinical tests.
While the duration of the later part of the process is rather incompress-
ible, efforts have been made to reduce the experimental time spent on the
identificationofleadcompoundsandalsotoreachgoodpharmacological
characterization before qualifying a compound for further tests. Accord-
ingly, several different assays have been developed to rapidly test a large
number of molecules during high-throughput screening (HTS) cam-
paigns. Over the past decades, the assays used for HTS have constantly
evolved, together with the new findings and concepts arising from basic
research on the target (identification of new mechanisms, new signaling