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The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein G i . Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free G i . DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR–G i complex to previously determined structures of other GPCRs bound to the stimulatory G protein G s reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the G i protein-coupling specificity of the µOR. A cryo-electron structure of the µ-opioid receptor in complex with the peptide agonist DAMGO and the inhibitory G protein G i reveals structural determinants of its G protein-binding specificity.

The class A adenosine A 1 receptor (A 1 R) is a G-protein-coupled receptor that preferentially couples to inhibitory G i/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A 1 R in complex with adenosine and heterotrimeric G i2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A 1 R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A 1 R primarily via amino acids in the C terminus of the Gα i α5-helix, concomitant with a 10.5 Å outward movement of the A 1 R transmembrane domain 6. Comparison with the agonist-bound β 2 adrenergic receptor–G s -protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A 1 R structure provides molecular insights into receptor and G-protein selectivity. The cryo-electron microscopy structure of the human adenosine A 1 receptor in complex with adenosine and heterotrimeric G i2 protein provides molecular insights into receptor and G-protein selectivity.

Type-A γ-aminobutyric (GABA A ) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABA A receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABA A receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABA A receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABA A receptor modulators. Cryo-electron microscopy structures are reported in which the full-length human α1β3γ2L GABA A receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA, and the benzodiazepines alprazolam and diazepam.

The σ 2 receptor has attracted intense interest in cancer imaging 1 , psychiatric disease 2 , neuropathic pain 3 – 5 and other areas of biology 6 , 7 . Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone 2 and the tool compound PB28 8 . These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ 1 receptor. Crystal structures of two ligands bound to the σ 2 receptor confirmed the docked poses. To investigate the contribution of the σ 2 receptor in pain, two potent σ 2 -selective ligands and one potent σ 1 /σ 2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model 9 , suggesting that the σ 2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology. Crystal structures of the σ 2 receptor are determined and used to perform a docking screen of nearly 500 million molecules, identifying σ 2 -selective ligands and providing insight into the role of σ 2 in neuropathic pain.

Cytokines are secreted signalling proteins that play essential roles in the initiation, maintenance and resolution of immune responses. Although the unique ability of cytokines to control immune function has garnered clinical interest in the context of cancer, autoimmunity and infectious disease, the use of cytokine-based therapeutics has been limited. This is due, in part, to the ability of cytokines to act on many cell types and impact diverse biological functions, resulting in dose-limiting toxicity or lack of efficacy. Recent studies combining structural biology, protein engineering and receptor pharmacology have unlocked new insights into the mechanisms of cytokine receptor activation, demonstrating that many aspects of cytokine function are highly tunable. Here, we discuss the pharmacological principles underlying these efforts to overcome cytokine pleiotropy and enhance the therapeutic potential of this important class of signalling molecules.The exploitation of cytokines for therapeutic use has been limited by their pleiotropic activity, which has contributed to dose-limiting toxicity and lack of efficacy. Here, Garcia and colleagues discuss how recent insights from structural biology, protein engineering and receptor pharmacology have unveiled strategies to overcome cytokine pleiotropy and enable the design of new and improved cytokine-based therapeutics.

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT 2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT 2A -Gq/11 and β-arrestin2 transducers, making their respective roles unclear. To elucidate this, we develop a series of 5-HT 2A -selective ligands with varying Gq efficacies, including β-arrestin-biased ligands. We show that 5-HT 2A -Gq but not 5-HT 2A -β-arrestin2 recruitment efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT 2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT 2A Gq-efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT 2A agonists. We also demonstrate that β-arrestin-biased 5-HT 2A receptor agonists block psychedelic effects and induce receptor downregulation and tachyphylaxis. Overall, 5-HT 2A receptor Gq-signaling can be fine-tuned to generate ligands distinct from classical psychedelics. Serotonin 5-HT 2A receptor signaling mechanisms associated with predicting psychedelic potential remain elusive. Using 5-HT 2A -selective β-arrestin-biased ligands, here the authors show that a threshold level of 5-HT 2A -Gq efficacy and not β-arrestin recruitment is associated with psychedelic potential.

G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and some of the most common drug targets. It is now well established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, GPCR kinases and β-arrestins. While these signalling pathways can be activated or blocked by 'balanced' agonists or antagonists, they can also be selectively activated in a 'biased' response. Biased responses can be induced by biased ligands, biased receptors or system bias, any of which can result in preferential signalling through G proteins or β-arrestins. At many GPCRs, signalling events mediated by G proteins and β-arrestins have been shown to have distinct biochemical and physiological actions from one another, and an accurate evaluation of biased signalling from pharmacology through physiology is crucial for preclinical drug development. Recent structural studies have provided snapshots of GPCR-transducer complexes, which should aid in the structure-based design of novel biased therapies. Our understanding of GPCRs has evolved from that of two-state, on-and-off switches to that of multistate allosteric microprocessors, in which biased ligands transmit distinct structural information that is processed into distinct biological outputs. The development of biased ligands as therapeutics heralds an era of increased drug efficacy with reduced drug side effects.

The adenosine A 1 receptor (A 1 R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain 1 , 2 . However, development of analgesic orthosteric A 1 R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects 3 . Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A 1 R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A 1 R co-bound to adenosine, MIPS521 and a G i2 heterotrimer, revealing an extrahelical lipid–detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine–receptor–G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts. MIPS521, a positive allosteric modulator of the adenosine A 1 receptor, has analgesic properties in a rat model of neuropathic pain through a mechanism by which MIPS521 stabilizes the complex between adenosine, receptor and G protein.

The authors report a 2.6 Å resolution crystal structure of the human CB1 cannabinoid receptor trapped in the inactive conformation and bound to the antagonist taranabant. CB1 cannabinoid receptor structure The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the responses to endocannabinoids and the plant cannabinoid Δ 9 -tetrahydrocannabinol (THC). They are important drug discovery targets because of the therapeutic potential of receptor modulators for controlling disorders such as pain, epilepsy and obesity. Daniel Rosenbaum and colleagues determine a crystal structure of the human CB1 receptor bound to the inhibitor taranabant. The extracellular surface of the receptor is distinct from other lipid-activated GPCRs and forms a critical part of the ligand-binding pocket. Docking studies demonstrate how this pocket might accommodate tetrahydrocannabinol. The structure should aid drug discovery efforts for novel cannabinoid system modulators as potential therapeutics. The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) and to the widely consumed plant phytocannabinoid Δ 9 -tetrahydrocannabinol (THC) 1 . The cannabinoid receptors have been the targets of intensive drug discovery efforts, because modulation of these receptors has therapeutic potential to control pain 2 , epilepsy 3 , obesity 4 , and other disorders. Although much progress in understanding the biophysical properties of GPCRs has recently been made, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. Here we report the use of GPCR engineering and lipidic cubic phase crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6-Å resolution. We found that the extracellular surface of CB1, including the highly conserved membrane-proximal N-terminal region, is distinct from those of other lipid-activated GPCRs, forming a critical part of the ligand-binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function and will aid the design and optimization of therapeutic modulators of the endocannabinoid system.

Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways 1 , 2 . Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human β-arrestin 1 (βarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with βarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin–arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin–receptor interactions. A cryo-electron microscopy structure of the neurotensin receptor 1 in complex with β-arrestin 1 is reported.