Probing Adhesion GPCR Mechanisms via High Throughput Screening for Novel GPR56 Modulatory Compounds

Abstract

Adhesion G Protein-Coupled receptors (AGPCRs) are a poorly understood subset of class B GPCRs that comprises 33 members across nine subfamilies. These GPCRs are unique in their possession of large extracellular regions that include a highly conserved GPCR autoproteolysis inducing (GAIN) domain that cleaves the receptor into two fragments that remain noncovalently attached in the inactive, holoreceptor form. These fragments, deemed the extracellular N-terminal fragment (NTF) and the membrane-embedded C-terminal fragment (CTF), can dissociate from one another to expose a small stalk region on the CTF termed the tethered-peptide-agonist (tethered agonist, or TA). Exposure of the TA allows it to bind to the orthosteric site of the receptor to maximally activate G protein signaling. AGPCRs play critical roles in several cellular processes, including but not limited to the regulation of cell migration, shape, polarity, differentiation, and immune response. AGPCRs are also notable oncogenes and biomarkers for a wide array of different cancers. However, despite holding enormous therapeutic potential, most AGPCRs are classified as orphans with few molecular tools to study their activation in vivo. This has confounded the study of their activation in endogenous tissue systems, and significantly impeded the development of AGPCR-targeted therapeutics. This issue is further exacerbated by a clear lack of AGPCR structures that showcase the receptor in its active, TA-bound form. Here, we highlight the first cryo-EM structures of the activated forms of two model AGPCRs, GPR56 and LPHN3. High-resolution maps allowed for us to detail the exact mechanism of orthosteric TA activation: Following exposure to the extracellular environment, the tethered agonist bends 180° to adopt a partial helix conformation deep within the orthosteric site. Mutational analysis revealed that the TA forms critical interactions with conserved residues on TMs 1, 2, 6, 7, and extracellular loop 2. TA binding also introduces breaks in TMs 6 and 7 that open the intracellular side of the receptor to enhance G protein signaling. Following this, active and inactive forms of GPR56 were used in a modified cell-based luciferase gene reporter assay to screen for novel agonists and antagonists from a collection of over 200,000 compounds. Our agonist screen revealed that hexahydroquinoline (HHQ) derivatives are potent, selective full agonists for GPR56 that are predicted via in silico docking to bind to the orthosteric site in a manner similar to the TA. Structure activity relationship (SAR) analysis resulted in the identification of an optimized structure, Compound 36.40, with a twenty-fold improvement in potency over synthetic peptides that typically used to study AGPCRs. Additionally, our antagonist screen allowed for the early-stage characterization of two novel GPR56 inhibitors that robustly attenuated GPR56-depndent aggregation of human platelets. Taken together, we have elucidated the tethered agonist mechanism of activation for AGPCRs while also discovering novel, potent GPR56 activators and inhibitors that will prove useful as pharmacological tools, or as leads for AGPCRs-targeted therapeutics