Development of a High-Throughput Screening–Compatible Cell-Based Functional Assay to Identify Small Molecule Probes of the Galanin 3 Receptor (GalR3)

Abstract

The galanin 3 receptor (GalR3) belongs to the large G protein-coupled receptor (GPCR) family of proteins. GalR3 and two other closely related receptors, GalR1 and GalR2, together with their endogenous ligand galanin, are involved in a variety of physiological and pathophysiological processes. GalR3 in particular has been strongly implicated in addiction and mood-related disorders such as anxiety and depression. It has been the target of many drug discovery programs within the pharmaceutical industry, but despite the significant resources and effort devoted to discovery of galanin receptor subtype selective small molecule modulators, there have been very few reports for the discovery of such molecules. GalR3 has proven difficult to enable in cell-based functional assays due to its apparent poor cell surface expression in recombinant systems. Here, we describe the generation of a modified GalR3 that facilitates its cell surface expression while maintaining wild-type receptor pharmacology. The modified GalR3 has been used to develop a high-throughput screening-compatible, cell-based, cAMP biosensor assay to detect selective small molecule modulators of GalR3. The performance of the assay has been validated by challenging it against a test library of small molecules with known pharmacological activities (LOPAC; Sigma Aldrich). This approach will enable identification of GalR3 selective modulators (chemical probes) that will facilitate dissection of the biological role(s) that GalR3 plays in normal physiological processes as well as in disease states.

Physiology, Signaling, and Pharmacology of Galanin Peptides and Receptors: Three Decades of Emerging Diversity

“…A possible reason for this is that the different cells used do not express the appropriate G proteins or other receptors to allow correct GAL 3 trafficking and/or signaling. Consistent with this hypothesis, GAL 3 overexpression in these cells has been shown to generate insoluble inclusion bodies which prevent the receptor being trafficked to and expressed on the cell surface (Robinson et al, 2013;B. Brodowicz, unpublished data).…”

“…There are several GAL 3 -transfected cell lines available (Lang et al, 2005;Lu et al, 2005b), but although they express GAL 3 mRNA, they are not able to produce sufficient GAL 3 protein or GAL 3 in an appropriate form on the plasma membrane to allow galanin binding and stable signaling experiments to be performed (Robinson et al, 2013;R. Lang and A. Lang, personal communication).…”

“…Brodowicz, unpublished data). Robinson et al (2013) reported that the GAL 3 carboxy tail has multiple overlapping motifs that target expression to the endoplasmic reticulum, inhibiting receptor transport and insertion at the cell 130 membranes. Those authors showed that a modified GAL 3 construct (the C-terminal part of GAL 3 was replaced with that of GAL 1 ) facilitates cell surface expression while maintaining wild-type receptor pharmacology.…”

“…Those authors showed that a modified GAL 3 construct (the C-terminal part of GAL 3 was replaced with that of GAL 1 ) facilitates cell surface expression while maintaining wild-type receptor pharmacology. This finding (Robinson et al, 2013) should allow the GAL 3 field to move forward, because the chimeric cell line can be used to study G protein coupling, the downstream signaling pathways, and to undertake high-throughput screening to identify novel GAL 3 ligands.…”

Development of Functionally Selective, Small Molecule Agonists at Kappa Opioid Receptors

“…On the 2nd day, cells were serum-starved for 1 h followed by treatment with saponin (50 g/ml, Sigma) for 4 min to briefly permeabilize cells (44 (45,46). Maximal changes in impedance were extracted and plotted in dose-response curves.…”