G protein-coupled receptors (GPCRs) are essential mediators of cellular signaling and important targets of drug action. Of the approximately 350 non-olfactory human GPCRs, more than 100 are still considered “orphans” as their endogenous ligand(s) remain unknown. Here, we describe a unique open-source resource that provides the capacity to interrogate the druggable human GPCR-ome via a G protein-independent β-arrestin recruitment assay. We validate this unique platform at more than 120 non-orphan human GPCR targets, demonstrate its utility for discovering new ligands for orphan human GPCRs, and describe a method (PRESTO-TANGO; Parallel Receptor-ome Expression and Screening via Transcriptional Output - TANGO) for the simultaneous and parallel interrogation of the entire human GPCR-ome.

SUMMARY The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B. The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD’s key diethylamide moiety. LSD dissociates exceptionally slowly from both 5-HT2BR and 5-HT2AR -- a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD’s slow binding kinetics may be due to a “lid” formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD’s binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD, illuminates key features of its kinetics, stereochemistry, and signaling, and provides a molecular explanation for LSD’s actions at human serotonin receptors.

The primate-exclusive MRGPRX2 G protein-coupled receptor (GPCR) has been suggested to modulate pain and itch. Despite putative peptide and small molecule MRGPRX2 agonists, selective nanomolar potency probes have not yet been reported. To identify a MRGPRX2 probe, we first screened 5,695 small molecules and found many opioid compounds activated MRGPRX2, including (−)- and (+)-morphine, hydrocodone, sinomenine, dextromethorphan and the prodynorphin-derived peptides, dynorphin A, dynorphin B, and α- and β-neoendorphin. We used these to select for mutagenesis-validated homology models and docked almost 4 million small molecules. From this docking, we predicted ZINC-3573, which represents a potent MRGPRX2-selective agonist, showing little activity against 315 other GPCRs and 97 representative kinases, and an essentially inactive enantiomer. ZINC-3573 activates endogenous MRGPRX2 in a human mast cell line inducing degranulation and calcium release. MRGPRX2 is a unique atypical opioid-like receptor important for modulating mast cell degranulation, which can now be specifically modulated with ZINC-3573.

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.

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