G protein-independent cell-based assays for drug discovery on seven-transmembrane receptors

Antagonists of GPR35 Display High Species Ortholog Selectivity and Varying Modes of Action

“…In part, this reflects that in both academic and industrial settings GPCR agonist screens based on interactions with a ␤-arrestin have become more dominant (Hamdan et al, 2005), because of a combination of the relative ease of performing such studies and the argument that they are more "universal" (Verkaar et al, 2008). In the case of poorly characterized GPCRs this has an abiding attraction if the G protein-coupling profile of the receptor is unknown.…”

Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

“…A great deal of information is obtained from high-content assays based on imaging techniques that use fluorescent signals to yield information about receptor interaction with ␤-arrestin and subsequent movement of the receptor/␤-arrestin complex within the cytoplasm (Milligan, 2003;Lefkowitz and Whalen, 2004;Fredriksson and Schiöth, 2005). These responses can be monitored directly through observation of receptor/␤-arrestin green fluorescent protein complexes (Oakley et al, 2002;Ghosh et al, 2005;Ross et al, 2008;Hanson et al, 2009;van der Lee et al, 2009), with bioluminescence resonance energy transfer (Milligan, 2004;Hamdan et al, 2005), with enzyme fragment complementation (Olson and Eglen, 2007;Zhao et al, 2008;van der Lee et al, 2009), or with proteaseactivated transcriptional reporter genes (Barnea et al, 2008;Verkaar et al, 2008). Green fluorescent protein and immunofluorescence-based technologies can also be multiplexed to gain multiple readouts from the same cell to compare signaling pathways (Henriksen et al, 2008).…”

Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements

“…This phenomenon, also referred to as biased agonism, functional selectivity or signal trafficking, has an important impact on GPCR drug discovery because it raises the possibility to design signaling pathway-specific therapeutics. Activation of downstream signaling events of GPCRs is traditionally recorded with assays based on quantification of distinct intracellular second messengers such as Ca 2+ , IP1 and cyclic AMP 5,9-11 and/or translocation of β-arrestin proteins 5,[12][13][14][15][16] . Since GPCRs from different coupling classes typically produce one or more specific second messenger, and may additionally engage non-G protein effectors [17][18][19][20][21] , several assays are needed to obtain quantitative information about each signaling event.…”