Abstract
G protein-coupled receptors (GPCRs) are central targets in drug discovery given their involvement in multiple physiological processes [1]. These transmembrane receptors direct an array of intracellular pathways through the coupling of distinct partners, with the heterotrimeric G proteins being key players in this process. Despite the long-standing interest in the mechanisms underpinning GPCR signaling, a lack of understanding of their biology and function, mainly caused by their intrinsic dynamics, hampers full therapeutic exploitation. In this context, multiple strategies have been developed to stabilize GPCRs into active signaling state conformations. During previous efforts, we already successfully developed peptides mimicking the Gαs protein. As a next step and based on the highly conserved C-terminal α5 helix of Gα subunits, the same strategy was considered to identify peptide mimetics of other G proteins (Figure 1).The α5 -helix of both Gαq and mini-Gαs/q was identified as a central epitope for GPCR-G protein coupling [2]. The best-performing modification of the Gαs-mimetics, including a triazole staple to improve helicity, a Leu-to-Cha (cyclohexylalanine) substitution at the penultimate position in the helix, and a solubilizing trilysine motif, were inserted into the (mini-)Gαq sequences. In this study, the growth hormone secretagogue receptor (GHSR) was used to screen G q peptidomimetics for receptor binding and stabilization. Different fluorescence-based assays were used to investigate the allosteric effect of the peptides on the conformation of the isolated receptor at different levels, including the orthosteric ligand binding site and G protein-coupling domain. These findings altogether showcase the importance of the Gq mimetics developed in this work as allosteric tools to study GPCR signaling in addition to their valuable role in screening approaches.References1. Lagerstrom C, Schioth HB. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov. 2008; 7: 339-357.2. Mannes et al. Development of generic G protein peptidomimetics able to stabilize active state Gs protein-coupled receptors for application in drug discovery. Angew Chem Int Ed Engl. 2021; 60: 10,247-10,254.