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The orexin system regulates sleep and arousal in humans, with orexin receptor antagonists becoming promising therapeutics for insomnia; now, the X-ray crystal structure of the human OX 2 receptor in the presence of the insomnia drug suvorexant is solved. Antagonst-bound orexin receptor structures The orexin(hypocretin) system regulates sleep and arousal in humans, and orexin receptor antagonists are seen as promising therapeutics for insomnia. Here the authors solve the X-ray crystal structure of the human OX 2 orexin receptor in the presence of the suvorexant, a dual-orexin receptor antagonist anti-insomnia drug. The structure provides a molecular framework for understanding dual-orexin receptor antagonist binding. The authors use two different docking programs to predict how three other antagonists with different core structures may bind to this G-protein-coupled receptor. The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) respond to orexin neuropeptides in the central nervous system to regulate sleep and other behavioural functions in humans 1 . Defects in orexin signalling are responsible for the human diseases of narcolepsy and cataplexy; inhibition of orexin receptors is an effective therapy for insomnia 2 . The human OX 2 receptor (OX 2 R) belongs to the β branch of the rhodopsin family of GPCRs 3 , and can bind to diverse compounds including the native agonist peptides orexin-A and orexin-B and the potent therapeutic inhibitor suvorexant 4 . Here, using lipid-mediated crystallization and protein engineering with a novel fusion chimaera, we solved the structure of the human OX 2 R bound to suvorexant at 2.5 Å resolution. The structure reveals how suvorexant adopts a π-stacked horseshoe-like conformation and binds to the receptor deep in the orthosteric pocket, stabilizing a network of extracellular salt bridges and blocking transmembrane helix motions necessary for activation. Computational docking suggests how other classes of synthetic antagonists may interact with the receptor at a similar position in an analogous π-stacked fashion. Elucidation of the molecular architecture of the human OX 2 R expands our understanding of peptidergic GPCR ligand recognition and will aid further efforts to modulate orexin signalling for therapeutic ends.

G-protein-coupled receptors do not only feature the orthosteric pockets, where most endogenous agonists bind, but also a multitude of other allosteric pockets that have come into the focus as potential binding sites for synthetic modulators. Here, to better characterise such pockets, we investigate 557 GPCR structures by exhaustively docking small molecular probes in silico and converting the ensemble of binding locations to pocket-defining volumes. Our analysis confirms all previously identified pockets and reveals nine previously untargeted sites. In order to test for the feasibility of functional modulation of receptors through binding of a ligand to such sites, we mutate residues in two sites, in two model receptors, the muscarinic acetylcholine receptor M 3 and β 2 -adrenergic receptor. Moreover, we analyse the correlation of inter-residue contacts with the activation states of receptors and show that contact patterns closely correlating with activation indeed coincide with these sites. G-protein-coupled receptors bind endogenous ligands at sites that are frequently highly conserved. Here, authors computationally describe alternative allosteric pockets, several of which have not been targeted by synthetic ligands before.

Temperature-related words such as cold-blooded and hot-headed can be used to describe criminal behavior. Words associated with coldness describe premeditated behavior and words associated with heat describe impulsive behavior. Building on recent research about the close interplay between physical and interpersonal coldness and warmth, we examined in a lab experiment how ambient temperature within a comfort zone influences judgments of criminals. Participants in rooms with low temperature regarded criminals to be more cold-blooded than participants in rooms with high temperature. Specifically, they were more likely to attribute premeditated crimes, ascribed crimes resulting in higher degrees of penalty, and attributed more murders to criminals. Likewise, participants in rooms with high temperature regarded criminals to be more hot-headed than participants in rooms with low temperature: They were more likely to attribute impulsive crimes. Results imply that cognitive representations of temperature are closely related to representations of criminal behavior and attributions of intent.

Orexins are neuropeptides that activate the rhodopsin-like G proteincoupled receptors OX1R and OX2R. The orexin system plays an important role in the regulation of the sleep-wake cycle and the regulation of feeding and emotions. The nonselective orexin receptor antagonist suvorexant has been the first drug on the market targeting the orexin system and is prescribed for the treatment of insomnia. Subtype-selective OX1R antagonists are valuable tools to further investigate the functions and physiological role of the OX1R in vivo and promising lead compounds for the treatment of drug addiction, anxiety, pain or obesity. Starting from the OX1R and OX2R crystal structures bound to suvorexant, we exploited a single amino acid difference in the orthosteric binding site by using molecular docking and structure-based drug design to optimize ligand interactions with the OX1R while introducing repulsive interactions with the OX2R. A newly established enantiospecific synthesis provided ligands showing up to 75-fold selectivity for the OX1R over the OX2R subtype. The structure of a new OX1R antagonist with subnanomolar affinity (JH112) was determined by crystallography in complex with the OX1R and corresponded closely to the dockingpredicted geometry. JH112 exhibits high selectivity over a panel of different GPCRs, is able to cross the blood–brain barrier and acts as slowly diffusing and insurmountable antagonist for Gq protein activation and in particular β-arrestin-2 recruitment at OX1R. This study demonstrates the potential of structure-based drug design to develop more subtype-selective GPCR ligands with potentially reduced side effects and provides an attractive probe molecule and lead compound.

Targeting the Frizzled family (FZD 1-10 ) of WNT receptors pharmacologically has, despite substantial therapeutic potential, proven difficult. Given an almost complete lack of validated, effective small molecules targeting FZDs, no putative ligand binding site has so far been identified. In order to target FZD 7 , a potential target for the treatment of intestinal tumors, we combine an approach of adapted docking setups and large molecular library docking screens, identifying compound C407. Applying pharmacological assays, genetically-encoded biosensors, site-directed mutagenesis, cryo-electron microscopy and molecular dynamics simulations, the compound binding site in the core of the seven transmembrane bundle is validated and C407 is confirmed as a negative allosteric modulator of WNT-induced and FZD-mediated WNT/ β -catenin signaling. In summary, we provide here the proof-of-principle that targeting FZDs with small molecule compounds is possible and effective. Future hit optimization and functional validation in disease-relevant in vitro and in vivo models will pave the way towards clinical exploration. Although FZDs are promising drug targets, so far no small molecules targeting them were described. Here, the authors report the a FZD7 core-targeting small molecule negative allosteric modulators of WNT-induced signaling, confirmed by pharmacology, structure determination and MD simulations.

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H 2 R) in an active conformation with bound histamine and in complex with G s heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E . coli lysates. Structural comparison with the inactive conformation of H 2 R and the inactive and G q -coupled active state of H 1 R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H 2 R, its agonist-dependent internalization and its interaction with endogenously synthesized H 1 R and H 2 R in HEK293 cells by applying a recently developed nanotransfer technique. The study describes the molecular structure of the human histamine 2 receptor in active conformation and in complex with G s heterotrimer, synthesized in a cell-free system and co-translationally inserted into preformed nanodiscs.

G protein-coupled receptors (GPCRs) are sophisticated signaling machines able to simultaneously elicit multiple intracellular signaling pathways upon activation. Complete (in)activation of all pathways can be counterproductive for specific therapeutic applications. This is the case for the serotonin 2 A receptor (5-HT 2A R), a prominent target for the treatment of schizophrenia. In this study, we elucidate the complex 5-HT 2A R coupling signature in response to different signaling probes, and its physiological consequences by combining computational modeling, in vitro and in vivo experiments with human postmortem brain studies. We show how chemical modification of the endogenous agonist serotonin dramatically impacts the G protein coupling profile of the 5-HT 2A R and the associated behavioral responses. Importantly, among these responses, we demonstrate that memory deficits are regulated by G αq protein activation, whereas psychosis-related behavior is modulated through G αi1 stimulation. These findings emphasize the complexity of GPCR pharmacology and physiology and open the path to designing improved therapeutics for the treatment of stchizophrenia. Here authors aim to understand the 5-HT 2A R coupling signature in response to different signaling probes and their physiological impacts using computational modeling, in vitro and in vivo experiments, and analysis of human brain tissue.

Extra virgin olive oil (EVOO) possesses a high-value rank in the food industry, thus making it a common target for adulteration. Hence, several methods have been essentially made available over the years. However, the issue of authentication remains unresolved with national and food safety organizations globally struggling to regulate and control its market. Over the course of this study, the aim was to determine the origin of EVOOs suggesting a high-throughput, state-of-the-art method that could be easily adopted. A rapid, NMR-based untargeted metabolite profiling method was applied and complemented by multivariate analysis (MVA) and statistical total correlation spectroscopy (STOCSY). STOCSY is a valuable statistical tool contributing to the biomarker identification process and was employed for the first time in EVOO analysis. Market samples from three Mediterranean countries of Spain, Italy, and Greece, blended samples from these countries, as well as monocultivar samples from Greece were analyzed. The NMR spectra were collected, with the help of chemometrics acting as “fingerprints” leading to the discovery of certain chemical classes and single biomarkers that were related to the classification of the samples into groups based on their origin.

The Apelin receptor (APLNR), a class A G-protein coupled receptor, plays a crucial role during cardiovascular development and tumor angiogenesis. To understand its spatiotemporal activity in health and disease is fundamental for the development of drugs to manipulate its activation state. To obtain this understanding, here we develop a tool box of various APLNR conformation biosensors, based on FRET, BRET and the conformation-sensitive fluorophore circularly permuted GFP (cpGFP), with further focus on its in vivo application. We demonstrate the functionality of our biosensors by pharmacological characterization and signal transduction analysis in vitro. Two APLNR-cpGFP biosensors show superior signal-to-noise ratio and are further analyzed for their in vivo applicability. In zebrafish embryos, APLNR-cpGFP biosensors are able to bind both endogenous ligands, Apelin and Apela, and visualize endogenous Aplnr activity in growing blood vessels. Moreover, we are able to measure an Apelin ligand gradient across cellular distances in vivo. Hence, these APLNR conformation biosensors are powerful tools to resolve the spatiotemporal Apelin signaling activity in health and disease. The Apelin receptor (APLNR) plays a key role during cardiovascular development. Here, authors develop genetically encoded APLNR conformation biosensors, which enable the measurement of temporally and spatially resolved APLNR activity in model cell lines and living organisms.

Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β 1 -adrenoceptors, arrestin-biased signalling via β 2 -adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol’s cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through β 2 ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the β-adrenoceptor system. How carvedilol, a β1-blocker, activates β2-adrenoceptors, is unclear. Here, the authors resolve this enigma and show that carvedilol drives all of its detectable cellular β2-adrenoceptor signals by slow and low efficacy G protein activation.