Chemogenomic Discovery of Allosteric Antagonists at the GPRC6A Receptor

Abstract

GPRC6A is a Family C G protein-coupled receptor recently discovered and deorphanized by our group. This study integrates chemogenomic ligand inference, homology modeling, compound synthesis, and pharmacological mechanism-of-action studies to disclose two noticeable results of methodological and pharmacological character: (1) chemogenomic lead identification through the first, to our knowledge, ligand inference between two different GPCR families, Families A and C; and (2) the discovery of the most selective GPRC6A allosteric antagonists discovered to date. The unprecedented inference of pharmacological activity across GPCR families provides proof-of-concept for in silico approaches against Family C targets based on Family A templates, greatly expanding the prospects of successful drug design and discovery. The antagonists were tested against a panel of seven Family A and C G protein-coupled receptors containing the chemogenomic binding sequence motif where some of the identified GPRC6A antagonists showed some activity. However, three compounds with at least ∼3-fold selectivity for GPRC6A were discovered, which present a significant step forward compared with the previously published GPRC6A antagonists, calindol and NPS 2143, which both display ∼30-fold selectivity for the calcium-sensing receptor compared to GPRC6A. The antagonists constitute novel research tools toward investigating the signaling mechanism of the GPRC6A receptor at the cellular level and serve as initial ligands for further optimization of potency and selectivity enabling future ex vivo/in vivo pharmacological studies.

Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

“…The lack of L-Orn response in the host cell line Flp-In-CHO strongly indicates that the response in mGPRC6A-CHO is in fact mediated by the GPRC6A receptor. This was further confirmed by the complete inhibition of L-Orn-induced IP 1 accumulation by a GPRC6A-selective antagonist, compound 1 (Gloriam et al, 2011) (Fig. 4A).…”

“…4A). This antagonist has proven to be selective toward GPRC6A when tested against a panel of receptors, including the homologous calcium-sensing receptor (Gloriam et al, 2011).…”

“…Complete protease inhibitor cocktail tablets were purchased from Roche (Basel, Switzerland), and phospho-p44/42 MAPK (ERK1/2) rabbit antibody and p44/42 MAPK (ERK1/2) mouse antibody were purchased from Cell Signaling Technology (Danvers, MA). Compound 1 (2-[N-methyl(29-morpholino-29-oxoethyl)amino]-1-(299-phenyl-1H-indol-399-yl)-ethanone) was synthesized by Henrik Johansson and Daniel S. Pedersen (University of Copenhagen, Denmark) as previously described (Gloriam et al, 2011).…”

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

“…When testing mouse GPRC6A in transient mammalian expression systems, it has been necessary to co-express a mutated G q G66D protein that facilitates G q coupling to obtain robust, functional responses (4,26,39). Likewise, no functional response of rat GPRC6A was measured in the absence of G q G66D (Fig.…”

From Three-Dimensional GPCR Structure to Rational Ligand Discovery

“…These GPCR crystal structures (Salon et al 2011;Katritch et al 2012) offer unique opportunities to push the limits of structure-based rational ligand discovery and design Salon et al 2011), and offer higher resolution templates for modeling the structures of GPCRs for which crystal structures have not yet been solved Stevens et al 2012;Rodriguez and Gutierrez-de-Teran 2013). It should however be noted that modeling of GPCRs with low homology to the currently available GPCR crystal structures (e.g., class B (Miller et al 2011;Vohra et al 2013;de Graaf et al 2011a) and class C GPCRs (Petrel et al 2004;Malherbe et al 2006;Gloriam et al 2011)) still remains a difficult task in which experimental data are of utmost importance to restrict the number of possible models (de Graaf and Rognan 2009;Roumen et al 2011). The challenges of GPCR modeling has been for example demonstrated in recent community wide competitions to predict GPCR crystal structures (GPCR DOCK 2008(Katritch et al 2010aMichino and Abola 2009), and GPCR DOCK 2010 Kufareva et al 2011)) and GPCR modeling methods have been described in several reviews (de Graaf and Rognan 2009;de Graaf et al 2011a;Cavasotto 2011;Kooistra et al 2013).…”