(R1694) Use of Novel ebBRET Biosensors for Comprehensive Signaling Profiling of One Hundred Therapeutically Relevant Human GPCRs

Arturo Mancini (Domain Therapeutics)| Charlotte Avet (Institute for Research in Immunology and Cancer, Université de Montréal)| Billy Breton (Institute for Research in Immunology and Cancer, Université de Montréal)| Christian Le Gouill (Institute for Research in Immunology and Cancer, Université de Montréal)| Alexander Hauser (Department of Drug Design and Pharmacology, University of Copenhagen)| Claire Normand (Domain Therapeutics)| Florence Gross (Domain Therapeutics)| Viktoriya Lukasheva (Institute for Research in Immunology and Cancer, Université de Montréal)| Mireille Hogue (Institute for Research in Immunology and Cancer, Université de Montréal)| Sandra Morissette (Domain Therapeutics)| Eric Fauman (Pfizer Global R&D)| Jean-Philippe Fortin (Pfizer Global R&D)| Stephan Schann (Domain Therapeutics)| Xavier Leroy (Domain Therapeutics)| David Gloriam (Department of Drug Design and Pharmacology, University of Copenhagen)| Michel Bouvier (Institute for Research in Immunology and Cancer, Université de Montréal)

Functional selectivity is the ability of a given GPCR to engage multiple signaling pathways, with distinct ligands of the given receptor displaying different efficacies in engaging receptor-coupled pathways. Full exploitation of functional selectivity in drug development will require an exhaustive description of the effectors that can be engaged by a given receptor, thus revealing receptor- and ligand-specific signaling signatures. Here, we describe the signaling profiles of 100 therapeutically relevant human GPCRs in response to their endogenous (or prototypical) ligands. Profiling was performed with 15 pathway-selective enhanced bystander bioluminescence resonance energy transfer (ebBRET) biosensors monitoring the activation of specific Gα proteins and βarrestins 1 and 2. The G protein biosensors represent a new generation of BRET-based sensors that measure the translocation of G protein effectors to the plasma membrane with no need for modifying the G proteins or the receptors. Over 1,500 dose‑response curves were generated, revealing a great diversity in GPCR coupling selectivity. Our data highlight that the Gi family displayed the highest general coupling while Gs and G12/13 families were less frequently engaged by the receptors tested. Certain GPCRs (17%) showed greater selectivity, with coupling restricted to a single G protein subtype or members of the same G protein family. Others showed broader activation profiles; specifically, 39%, 35% and 9% of GPCRs tested coupled to members of two, three and all four G protein families, respectively. Somewhat surprisingly, some receptors showed G protein subtype selectivity among the members of the same family. Of note, over 50 novel GPCR/G protein couplings were uncovered. In addition to highlighting receptor G protein coupling preferences, our data revealed that 78% of GPCRs recruited βarrestin 1 and/or 2. For certain GPCRs, co‑expression of GRK2 augmented (and even exposed) βarrestin engagement. Finally, in addition to the signaling profiling application, we demonstrated the versatility and usefulness of our ebBRET biosensor platform to study constitutive GPCR activity/inverse agonism, ligand- and SNP-induced biased signaling and cross-talk systems pharmacology. Overall, this work complements and enriches the current body of data on GPCR effector coupling. Moreover, it presents innovative tools allowing to further explore novel GPCR pharmacology. The resources provided in this study, combined to other signaling profiling and omic-scale datasets, will help deconvolute the complexities of GPCR biology and pharmacology and lead to innovative therapeutic exploitation of GPCRs.