Molecular basis of opioid receptor signaling.

Opioids are used for pain management despite the side effects that contribute to the opioid crisis. The pursuit of non-addictive opioid analgesics remains unattained due to the unresolved intricacies of opioid actions, receptor signaling cascades, and neuronal plasticity. Advancements in structural, molecular, and computational tools illuminate the dynamic interplay between opioids and opioid receptors, as well as the molecular determinants of signaling pathways, which are potentially interlinked with pharmacological responses. Here, we review the molecular basis of opioid receptor signaling with a focus on the structures of opioid receptors bound to endogenous peptides or pharmacological agents. These insights unveil specific interactions that dictate ligand selectivity and likely their distinctive pharmacological profiles. Biochemical analysis further unveils molecular features governing opioid receptor signaling. Simultaneously, the synergy between computational biology and medicinal chemistry continues to expedite the discovery of novel chemotypes with the promise of yielding more efficacious and safer opioid compounds.

SnapShot: Psychedelics and serotonin receptor signaling.

Serotonin (5-hydroxytryptamine; 5HT) signaling regulates processes in every major organ system, but it is most widely known for its role as a neurotransmitter in modulating a plethora of human behaviors. Psychedelics target the 5HT2A receptor and represent potentially transformative therapeutics for neuropsychiatric disorders. To view this SnapShot, open or download the PDF.

Structures of the entire human opioid receptor family.

Opioids are effective analgesics, but their use is beset by serious side effects, including addiction and respiratory depression, which contribute to the ongoing opioid crisis. The human opioid system contains four opioid receptors (µOR, δOR, κOR, and NOPR) and a set of related endogenous opioid peptides (EOPs), which show distinct selectivity toward their respective opioid receptors (ORs). Despite being key to the development of safer analgesics, the mechanisms of molecular recognition and selectivity of EOPs to ORs remain unclear. Here, we systematically characterize the binding of EOPs to ORs and present five structures of EOP-OR-Gi complexes, including ß-endorphin- and endomorphin-bound µOR, deltorphin-bound δOR, dynorphin-bound κOR, and nociceptin-bound NOPR. These structures, supported by biochemical results, uncover the specific recognition and selectivity of opioid peptides and the conserved mechanism of opioid receptor activation. These results provide a structural framework to facilitate rational design of safer opioid drugs for pain relief.

Structure-based discovery of conformationally selective inhibitors of the serotonin transporter.

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

Structural Insights Accelerate the Discovery of Opioid Alternatives.

Opioids such as morphine and oxycodone are analgesics frequently prescribed for the treatment of moderate or severe pain. Unfortunately, these medications are associated with exceptionally high abuse potentials and often cause fatal side effects, mainly through the µ-opioid receptor (MOR). Efforts to discover novel, safer, and more efficacious analgesics targeting MOR have encountered challenges. In this review, we summarize alternative strategies and targets that could be used to develop safer nonopioid analgesics. A molecular understanding of G protein-coupled receptor activation and signaling has illuminated not only the complexities of receptor pharmacology but also the potential for pathway-selective agonists and allosteric modulators as safer medications. The availability of structures of pain-related receptors, in combination with high-throughput computational tools, has accelerated the discovery of multitarget ligands with promising pharmacological profiles. Emerging clinical evidence also supports the notion that drugs targeting peripheral opioid receptors have potential as improved analgesic agents.

Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor.

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions-both therapeutic and hallucinogenic-are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH-a prototypical hallucinogen-in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.

VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells.

Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10-3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.

Discovery of Human Signaling Systems: Pairing Peptides to G Protein-Coupled Receptors.

The peptidergic system is the most abundant network of ligand-receptor-mediated signaling in humans. However, the physiological roles remain elusive for numerous peptides and more than 100 G protein-coupled receptors (GPCRs). Here we report the pairing of cognate peptides and receptors. Integrating comparative genomics across 313 species and bioinformatics on all protein sequences and structures of human class A GPCRs, we identify universal characteristics that uncover additional potential peptidergic signaling systems. Using three orthogonal biochemical assays, we pair 17 proposed endogenous ligands with five orphan GPCRs that are associated with diseases, including genetic, neoplastic, nervous and reproductive system disorders. We also identify additional peptides for nine receptors with recognized ligands and pathophysiological roles. This integrated computational and multifaceted experimental approach expands the peptide-GPCR network and opens the way for studies to elucidate the roles of these signaling systems in human physiology and disease. VIDEO ABSTRACT.

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology.

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-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C 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-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C 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.

Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.

How Ligands Illuminate GPCR Molecular Pharmacology.

G protein-coupled receptors (GPCRs), which are modulated by a variety of endogenous and synthetic ligands, represent the largest family of druggable targets in the human genome. Recent structural and molecular studies have both transformed and expanded classical concepts of receptor pharmacology and have begun to illuminate the distinct mechanisms by which structurally, chemically, and functionally diverse ligands modulate GPCR function. These molecular insights into ligand engagement and action have enabled new computational methods and accelerated the discovery of novel ligands and tool compounds, especially for understudied and orphan GPCRs. These advances promise to streamline the development of GPCR-targeted medications.

Crystal Structure of an LSD-Bound Human Serotonin Receptor.

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 slow 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 ß-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. PAPERCLIP.

Built-in functional selectivity in neurons is mediated by the neuronal protein, GINIP.

In this issue of Molecular Cell, Park et al.1 comprehensively profile how neurons utilize the Gα inhibitory interacting protein, GINIP, to modulate neurotransmission at a systems level through bias of downstream G protein-coupled receptor (GPCR) signaling.

Alternative splicing of latrophilin-3 controls synapse formation.

The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Structures of the human dopamine D3 receptor-Gi complexes.

The dopamine system, including five dopamine receptors (D1R-D5R), plays essential roles in the central nervous system (CNS), and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders. Here, we report two cryo-EM structures of human D3R in complex with an inhibitory G protein and bound to the D3R-selective agonists PD128907 and pramipexole, the latter of which is used to treat patients with Parkinson's disease. The structures reveal agonist binding modes distinct from the antagonist-bound D3R structure and conformational signatures for ligand-induced receptor activation. Mutagenesis and homology modeling illuminate determinants of ligand specificity across dopamine receptors and the mechanisms for Gi protein coupling. Collectively our work reveals the basis of agonist binding and ligand-induced receptor activation and provides structural templates for designing specific ligands to treat CNS diseases targeting the dopaminergic system.

Conformational ensembles in GPCR activation.

Recent advances in G-protein-coupled receptor structural biology have provided only limited insight into the active conformations of these key signaling molecules. A paper from Nygaard et al. reveals the dynamic nature of GPCRs along the activation pathway by complementing NMR experiments with ultralong-timescale molecular dynamics simulations.