A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state.

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.

Xylazine is an agonist at kappa opioid receptors and exhibits sex-specific responses to opioid antagonism.

Xylazine is in the unregulated drug supply at increasing rates, usually combined with fentanyl, necessitating understanding of its pharmacology. Despite commentary from politicians, and public health officials, it is unknown how xylazine impacts naloxone efficacy, and. few studies have examined it alone. Here, we examine the impact of xylazine alone and in combination with fentanyl on several behaviors in mice. Surprisingly, naloxone precipitates withdrawal from xylazine and fentanyl/xylazine coadministration, with enhanced sensitivity in females. Further, xylazine is a full agonist at kappa opioid receptors, a potential mechanism for its naloxone sensitivity. Finally, we demonstrate surprising effects of xylazine to kappa opioid antagonism, which are relevant for public health considerations. These data address an ongoing health crisis and will help inform critical policy and healthcare decisions.

A proximity proteomics pipeline with improved reproducibility and throughput.

Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

Molecular glues as potential GPCR therapeutics.

"Molecular Glues" are defined as small molecules that can either be endogenous or synthetic which promote interactions between proteins at their interface. Allosteric modulators, specifically GPCR allosteric modulators, can promote both the association and the dissociation of a given receptor's transducer but accomplishes this "at a distance" from the interface. However, recent structures of GPCR G protein complexes in the presence of allosteric modulators indicate that some GPCR allosteric modulators can act as "molecular glues" interacting with both the receptor and the transducer at the interface biasing transducer signaling in both a positive and negative manner depending on the transducer. Given these phenomena we discuss the implications for this class of allosteric modulators to be used as molecular tools and for future drug development.

AlphaFold2 structures guide prospective ligand discovery.

AlphaFold2 (AF2) models have had wide impact but mixed success in retrospective ligand recognition. We prospectively docked large libraries against unrefined AF2 models of the σ2 and serotonin 2A (5-HT2A) receptors, testing hundreds of new molecules and comparing results with those obtained from docking against the experimental structures. Hit rates were high and similar for the experimental and AF2 structures, as were affinities. Success in docking against the AF2 models was achieved despite differences between orthosteric residue conformations in the AF2 models and the experimental structures. Determination of the cryo-electron microscopy structure for one of the more potent 5-HT2A ligands from the AF2 docking revealed residue accommodations that resembled the AF2 prediction. AF2 models may sample conformations that differ from experimental structures but remain low energy and relevant for ligand discovery, extending the domain of structure-based drug design.

MRGPRX4 mediates phospho-drug-associated pruritus in a humanized mouse model.

The phosphate modification of drugs is a common chemical strategy to increase solubility and allow for parenteral administration. Unfortunately, phosphate modifications often elicit treatment- or dose-limiting pruritus through an unknown mechanism. Using unbiased high-throughput drug screens, we identified the Mas-related G protein-coupled receptor X4 (MRGPRX4), a primate-specific, sensory neuron receptor previously implicated in itch, as a potential target for phosphate-modified compounds. Using both Gq-mediated calcium mobilization and G protein-independent GPCR assays, we found that phosphate-modified compounds potently activate MRGPRX4. Furthermore, a humanized mouse model expressing MRGPRX4 in sensory neurons exhibited robust phosphomonoester prodrug-evoked itch. To characterize and confirm this interaction, we further determined the structure of MRGPRX4 in complex with a phosphate-modified drug through single-particle cryo-electron microscopy (cryo-EM) and identified critical amino acid residues responsible for the binding of the phosphate group. Together, these findings explain how phosphorylated drugs can elicit treatment-limiting itch and identify MRGPRX4 as a potential therapeutic target to suppress itch and to guide future drug design.

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.

Virtual Screening of a Chemically Diverse "Superscaffold" Library Enables Ligand Discovery for a Key GPCR Target.

The advent of ultra-large libraries of drug-like compounds has significantly broadened the possibilities in structure-based virtual screening, accelerating the discovery and optimization of high-quality lead chemotypes for diverse clinical targets. Compared to traditional high-throughput screening, which is constrained to libraries of approximately one million compounds, the ultra-large virtual screening approach offers substantial advantages in both cost and time efficiency. By expanding the chemical space with compounds synthesized from easily accessible and reproducible reactions and utilizing a large, diverse set of building blocks, we can enhance both the diversity and quality of the discovered lead chemotypes. In this study, we explore new chemical spaces using reactions of sulfur(VI) fluorides to create a combinatorial library consisting of several hundred million compounds. We screened this virtual library for cannabinoid type II receptor (CB2) antagonists using the high-resolution structure in conjunction with a rationally designed antagonist, AM10257. The top-predicted compounds were then synthesized and tested in vitro for CB2 binding and functional antagonism, achieving an experimentally validated hit rate of 55%. Our findings demonstrate the effectiveness of reliable reactions, such as sulfur fluoride exchange, in diversifying ultra-large chemical spaces and facilitate the discovery of new lead compounds for important biological targets.

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.

Making Sense of Psychedelics in the CNS.

For centuries, ancient lineages have consumed psychedelic compounds from natural sources. In the modern era, scientists have since harnessed the power of computational tools, cellular assays, and behavioral metrics to study how these compounds instigate changes on molecular, cellular, circuit-wide, and system levels. Here, we provide a brief history of psychedelics and their use in science, medicine, and culture. We then outline current techniques for studying psychedelics from a pharmacological perspective. Finally, we address known gaps in the field and potential avenues of further research to broaden our collective understanding of physiological changes induced by psychedelics, the limits of their therapeutic capabilities, and how researchers can improve and inform treatments that are rapidly becoming accessible worldwide.

AlphaFold2 structures template ligand discovery.

AlphaFold2 (AF2) and RosettaFold have greatly expanded the number of structures available for structure-based ligand discovery, even though retrospective studies have cast doubt on their direct usefulness for that goal. Here, we tested unrefined AF2 models prospectively, comparing experimental hit-rates and affinities from large library docking against AF2 models vs the same screens targeting experimental structures of the same receptors. In retrospective docking screens against the σ2 and the 5-HT2A receptors, the AF2 structures struggled to recapitulate ligands that we had previously found docking against the receptors' experimental structures, consistent with published results. Prospective large library docking against the AF2 models, however, yielded similar hit rates for both receptors versus docking against experimentally-derived structures; hundreds of molecules were prioritized and tested against each model and each structure of each receptor. The success of the AF2 models was achieved despite differences in orthosteric pocket residue conformations for both targets versus the experimental structures. Intriguingly, against the 5-HT2A receptor the most potent, subtype-selective agonists were discovered via docking against the AF2 model, not the experimental structure. To understand this from a molecular perspective, a cryoEM structure was determined for one of the more potent and selective ligands to emerge from docking against the AF2 model of the 5-HT2A receptor. Our findings suggest that AF2 models may sample conformations that are relevant for ligand discovery, much extending the domain of applicability of structure-based ligand discovery.

Illuminating the understudied GPCR-ome.

G-protein-coupled receptors (GPCRs) are the target of >30% of approved drugs. Despite their popularity, many of the >800 human GPCRs remain understudied. The Illuminating the Druggable Genome (IDG) project has generated many tools leading to important insights into the function and druggability of these so-called 'dark' receptors. These tools include assays, such as PRESTO-TANGO and TRUPATH, billions of small molecules made available via the ZINC virtual library, solved orphan GPCR structures, GPCR knock-in mice, and more. Together, these tools are illuminating the remaining 'dark' GPCRs.

Psychedelics: preclinical insights provide directions for future research.

Recently, psychedelics have emerged as promising therapeutics for numerous neuropsychiatric disorders. While their potential in the clinic has yet to be fully elucidated, understanding their molecular and biological mechanisms is imperative as these compounds are becoming widely used both in therapeutic and recreational contexts. This review examines the current understanding of basic biology, pharmacology, and structural biology in an attempt to reveal both the knowns and unknowns within the field.

Docking for EP4R antagonists active against inflammatory pain.

The lipid prostaglandin E2 (PGE2) mediates inflammatory pain by activating G protein-coupled receptors, including the prostaglandin E2 receptor 4 (EP4R). Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce nociception by inhibiting prostaglandin synthesis, however, the disruption of upstream prostanoid biosynthesis can lead to pleiotropic effects including gastrointestinal bleeding and cardiac complications. In contrast, by acting downstream, EP4R antagonists may act specifically as anti-inflammatory agents and, to date, no selective EP4R antagonists have been approved for human use. In this work, seeking to diversify EP4R antagonist scaffolds, we computationally dock over 400 million compounds against an EP4R crystal structure and experimentally validate 71 highly ranked, de novo synthesized molecules. Further, we show how structure-based optimization of initial docking hits identifies a potent and selective antagonist with 16 nanomolar potency. Finally, we demonstrate favorable pharmacokinetics for the discovered compound as well as anti-allodynic and anti-inflammatory activity in several preclinical pain models in mice.

Development of a Peripherally Restricted 5-HT2B Partial Agonist for Treatment of Pulmonary Arterial Hypertension.

Ligands for the serotonin 2B receptor (5-HT2B) have shown potential to treat pulmonary arterial hypertension in preclinical models but cannot be used in humans because of predicted off-target neurological effects. The aim of this study was to develop novel systemically restricted compounds targeting 5-HT2B. Here, we show that mice treated with VU6047534 had decreased RVSP compared with control treatment in both the prevention and intervention studies using Sugen-hypoxia. VU6047534 is a novel 5-HT2B partial agonist that is peripherally restricted and able to both prevent and treat Sugen-hypoxia-induced pulmonary arterial hypertension. We have synthesized and characterized a structurally novel series of 5-HT2B ligands with high potency and selectivity for the 5-HT2B receptor subtype. Next-generation 5-HT2B ligands with similar characteristics, and predicted to be systemically restricted in humans, are currently advancing to investigational new drug-enabling studies.

Nonspecific membrane bilayer perturbations by ivermectin underlie SARS-CoV-2 in vitro activity.

Since it was proposed as a potential host-directed antiviral agent for SARS-CoV-2, the antiparasitic drug ivermectin has been investigated thoroughly in clinical trials, which have provided insufficient support for its clinical efficacy. To examine the potential for ivermectin to be repurposed as an antiviral agent, we therefore undertook a series of preclinical studies. Consistent with early reports, ivermectin decreased SARS-CoV-2 viral burden in in vitro models at low micromolar concentrations, five- to ten-fold higher than the reported toxic clinical concentration. At similar concentrations, ivermectin also decreased cell viability and increased biomarkers of cytotoxicity and apoptosis. Further mechanistic and profiling studies revealed that ivermectin nonspecifically perturbs membrane bilayers at the same concentrations where it decreases the SARS-CoV-2 viral burden, resulting in nonspecific modulation of membrane-based targets such as G-protein coupled receptors and ion channels. These results suggest that a primary molecular mechanism for the in vitro antiviral activity of ivermectin may be nonspecific membrane perturbation, indicating that ivermectin is unlikely to be translatable into a safe and effective antiviral agent. These results and experimental workflow provide a useful paradigm for performing preclinical studies on (pandemic-related) drug repurposing candidates.