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Accurate snow cover data is crucial for understanding climate change, managing water resources, and calibrating models. The MODIS (Moderate-resolution Imaging Spectroradiometer) and its cloud-free snow cover datasets are widely used, but they have not been systematically evaluated due to different benchmark data and evaluation parameters. Conventional methods using station observations as a ground truth suffer from underrepresentation and mismatches in temporal and spatial scales. This study established a scale-matched spatial benchmark dataset, compiling from 18,433 Landsat series and 11,172 Sentinel-2 images over two decades, totaling â¼1.86 billion samples and â¼320 million snow samples. We evaluated seven MODIS cloud-free snow cover datasets for seasons, elevation zones, land covers and subregions using this benchmark data. For the clear-sky part, NIEER_MODIS_SCE (MODIS snow cover extent product over China) performs best due to its use of optimal NDSI thresholds suitable for each land use type. This highlights the importance of regional customization in snow mapping algorithms, and it can be further improved in spring, forests and zone 1 by combining it with M*D10A1GL06. For the cloud removed part, one-step integrated spatiotemporal cloud removal datasets perform better than any other approach does. The second-best dataset is obtained from a simple but effective single temporal cloud removal method using nearby time information. For the whole dataset, the best NIEER_MODIS_SCE has an overall accuracy of 0.82 and snow retrieval accuracy of 84.56 %. It performs excellently in most settings but weakest in forests thus requiring more efficient strategies. This research provides new perspectives and methods for objectively assessing MODIS snow cover products and other relevant datasets. These methods can be readily extended to other regions and adapted to future satellite missions. And such findings may guide the selection of more valid snow cover data and the developing of even better snow detecting strategies.
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The persistence of chronic pain and continuing overdose deaths from pain-relieving opioids targeting µ opioid receptor (µOR) have fueled the need for reliable long-term analgesics which use different targets and mechanisms. The δ opioid receptor (δOR) is a potential alternative target for non-addictive analgesics to alleviate chronic pain, made more attractive by its lack of respiratory depression associated with µOR agonists. However, early δOR full agonists were found to induce seizures, precluding clinical use. Partial δOR agonists may offer more controlled activation of the receptor compared to full agonists, but the development of such ligands has been hindered by uncertainty over the molecular mechanism mediating partial agonism. Using a structure-based approach, we explored the engagement of the sodium binding pocket in δOR and developed a bitopic ligand, C6-Quino, predicted to be a selective δOR partial agonist. Functional studies of C6-Quino revealed that it displayed δOR partial agonist activity at both G-protein and arrestin pathways. Its interaction with the sodium pocket was confirmed and analyzed using a single particle cryo-EM. Additionally, C6-Quino demonstrated favorable chemical and physiological properties like oral activity, and analgesic activity in multiple chronic pain models. Notably, µOR-related hyperlocomotion and respiratory depression, and δOR-related convulsions, were not observed at analgesic doses of C6-Quino. This fundamentally new approach to designing δOR ligands provides a blueprint for the development of partial agonists as safe analgesics and acts as a generic method to optimize signaling profiles of other Class A GPCRs.
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The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.
Assuntos
Analgésicos Opioides , Avaliação Pré-Clínica de Medicamentos , Naloxona , Receptores Opioides mu , Bibliotecas de Moléculas Pequenas , Animais , Humanos , Masculino , Camundongos , Regulação Alostérica/efeitos dos fármacos , Analgésicos Opioides/antagonistas & inibidores , Analgésicos Opioides/farmacologia , Sítios de Ligação/efeitos dos fármacos , Microscopia Crioeletrônica , Fentanila/antagonistas & inibidores , Fentanila/farmacologia , Cinética , Ligantes , Modelos Moleculares , Morfina/antagonistas & inibidores , Morfina/farmacologia , Naloxona/administração & dosagem , Naloxona/química , Naloxona/metabolismo , Naloxona/farmacologia , Antagonistas de Entorpecentes/administração & dosagem , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/metabolismo , Antagonistas de Entorpecentes/farmacologia , Overdose de Opiáceos/tratamento farmacológico , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Células Sf9 , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Camundongos Endogâmicos C57BLRESUMO
The concept of ligand bias is based on the premise that different agonists can elicit distinct responses by selectively activating the same receptor. These responses often determine whether an agonist has therapeutic or undesirable effects. Therefore, it would be highly advantageous to have agonists that specifically trigger the therapeutic response. The last two decades have seen a growing trend towards the consideration of ligand bias in the development of ligands to target the κ-opioid receptor (κOR). Most of these ligands selectively favor G-protein signaling over ß-arrestin signaling to potentially provide effective pain and itch relief without adverse side effects associated with κOR activation. Importantly, the specific role of ß-arrestin 2 in mediating κOR agonist-induced side effects remains unknown, and similarly the therapeutic and side-effect profiles of G-protein-biased κOR agonists have not been established. Furthermore, some drugs previously labeled as G-protein-biased may not exhibit true bias but may instead be either low-intrinsic-efficacy or partial agonists. In this review, we discuss the established methods to test ligand bias, their limitations in measuring bias factors for κOR agonists, as well as recommend the consideration of other systematic factors to correlate the degree of bias signaling and pharmacological effects. This article is part of the Special Issue on "Ligand Bias".
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Prurido , Receptores Opioides kappa , Receptores Opioides kappa/agonistas , Humanos , Animais , Prurido/tratamento farmacológico , Dor/tratamento farmacológico , Analgésicos Opioides/uso terapêutico , Analgésicos Opioides/farmacologia , Ligantes , Manejo da Dor/métodosRESUMO
While in the process of designing more effective synthetic opioid rescue agents, we serendipitously identified a new chemotype of potent synthetic opioid. Here, we report that conformational constraint of a piperazine ring converts a mu opioid receptor (MOR) antagonist into a potent MOR agonist. The prototype of the series, which we have termed atoxifent (2), possesses potent in vitro agonist activity. In mice, atoxifent displayed long-lasting antinociception that was reversible with naltrexone. Repeated dosing of atoxifent produced antinociceptive tolerance and a level of withdrawal like that of fentanyl. In rats, while atoxifent produced complete loss of locomotor activity like fentanyl, it failed to produce deep respiratory depression associated with fentanyl-induced lethality. Assessment of brain biodistribution demonstrated ample distribution of atoxifent into the brain with a Tmax of approximately 0.25 h. These results indicate enhanced safety for atoxifent-like molecules compared to fentanyl.
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Analgésicos Opioides , Fentanila , Receptores Opioides mu , Insuficiência Respiratória , Animais , Camundongos , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Insuficiência Respiratória/induzido quimicamente , Insuficiência Respiratória/tratamento farmacológico , Analgésicos Opioides/farmacologia , Analgésicos Opioides/síntese química , Analgésicos Opioides/química , Ratos , Masculino , Fentanila/farmacologia , Fentanila/síntese química , Fentanila/química , Relação Estrutura-Atividade , Piperazinas/farmacologia , Piperazinas/química , Piperazinas/síntese química , Piperazinas/uso terapêutico , Piperazinas/farmacocinética , Humanos , Ratos Sprague-Dawley , Distribuição Tecidual , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Naltrexona/farmacologia , Naltrexona/análogos & derivados , Naltrexona/síntese química , Naltrexona/química , Naltrexona/uso terapêuticoRESUMO
The liver gene expression of the peroxisomal ß-oxidation enzyme acyl-coenzyme A oxidase 1 (ACOX1), which catabolizes very long chain fatty acids (VLCFA), increases in the context of obesity, but how this pathway impacts systemic energy metabolism remains unknown. Here, we show that hepatic ACOX1-mediated ß-oxidation regulates inter-organ communication involved in metabolic homeostasis. Liver-specific knockout of Acox1 (Acox1-LKO) protects mice from diet-induced obesity, adipose tissue inflammation, and systemic insulin resistance. Serum from Acox1-LKO mice promotes browning in cultured white adipocytes. Global serum lipidomics show increased circulating levels of several species of ω-3 VLCFAs (C24-C28) with previously uncharacterized physiological role that promote browning, mitochondrial biogenesis and Glut4 translocation through activation of the lipid sensor GPR120 in adipocytes. This work identifies hepatic peroxisomal ß-oxidation as an important regulator of metabolic homeostasis and suggests that manipulation of ACOX1 or its substrates may treat obesity-associated metabolic disorders.
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Acil-CoA Oxidase , Fígado , Camundongos Knockout , Obesidade , Animais , Fígado/metabolismo , Camundongos , Acil-CoA Oxidase/metabolismo , Acil-CoA Oxidase/genética , Obesidade/metabolismo , Obesidade/genética , Masculino , Resistência à Insulina , Camundongos Endogâmicos C57BL , Oxirredução , Metabolismo dos Lipídeos , Tecido Adiposo/metabolismo , Dieta Hiperlipídica , Metabolismo Energético , Ácidos Graxos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
Understanding elevation changes on the Tibetan Plateau is crucial to comprehend the changes in topography, landscape, climate, environmental conditions, and water resources. However, some of the current products that track elevation changes only cover specific surface types or limited areas, and others have low spatial resolution. We propose an algorithm to extract ICESat-2 crossover points dataset for the Tibetan Plateau, and form a dataset. The crossover points dataset has a density of 2.015 groups/km², and each group of crossover points indicates the amount of change in elevation before and after a period of time over an area of approximately 17 meters in diameter. Comparing ICESat-2 crossover points data with existing studies on glaciers and lakes, we demonstrated the reliability of the derived elevation changes. The ICESat-2 crossover points provide a refined data source for understanding high-spatial-resolution elevation changes on the Tibetan Plateau. This dataset can provide validation data for various studies that require high-precision or high-resolution elevation change data on the Tibetan Plateau.
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The µ-opioid receptor (µOR) is an important target for pain management1 and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.
Assuntos
Ligantes , Conformação Proteica , Receptores Opioides mu , Humanos , beta-Arrestina 1/química , beta-Arrestina 1/metabolismo , Sítios de Ligação , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Guanosina Difosfato/metabolismo , Guanosina Difosfato/química , Modelos Moleculares , Ligação Proteica , Receptores Opioides mu/metabolismo , Receptores Opioides mu/química , Imagem Individual de MoléculaRESUMO
Alpine grassland is the main vegetation on the Qinghai-Tibetan Plateau (QTP) and exhibits high sensitivity to extreme weather events. With global warming, extreme weather events are projected to become more frequent on the QTP. However, the impact of these extreme weather events on the carbon cycle of alpine grassland remains unclear. The long-term in-situ carbon fluxes data was collected from 2013 to 2022 at an alpine grassland site to examine the impact of extreme low air temperature (ELT) and reduced moisture (including air and soil) on carbon fluxes during the growing season. Our findings indicated that a significant increase in net ecosystem production (NEP) after 2019, with the average NEP increasing from 278.91 ± 43.27 g C m-2 year-1 during 2013-2018 to 415.45 ± 45.29 g C m-2 year-1 during 2019-2022. The ecosystem carbon use efficiency (CUE) increased from 0.38 ± 0.06 during 2013-2018 to 0.62 ± 0.11 during 2019-2022. By combining concurrently measured environmental factors and remote sensing data, we identified the factors responsible for the abrupt change in the NEP after 2019. This phenomenon was caused by an abrupt decrease in ecosystem respiration (Reco) after 2019, which resulted from the inhibition imposed by ELT and reduced moisture. In contrast, gross primary production (GPP) remained stable from 2013 to 2022, which was confirmed by the remotely sensed vegetation index. This study highlights that combined extreme weather events associated with climate change can significantly impact the NEP of alpine grassland, potentially affecting different carbon fluxes at different rates. These findings provide new insights into the mechanisms governing the carbon cycle of alpine grassland.
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Ciclo do Carbono , Monitoramento Ambiental , Pradaria , Tibet , Mudança Climática , Temperatura Baixa , EcossistemaRESUMO
Receptor-G protein promiscuity is frequently observed in class A G protein-coupled receptors (GPCRs). In particular, GPCRs can couple with G proteins from different families (Gαs, Gαq/11, Gαi/o, and Gα12/13) or the same family subtypes. The molecular basis underlying the selectivity/promiscuity is not fully revealed. We recently reported the structures of kappa opioid receptor (KOR) in complex with the Gi/o family subtypes [Gαi1, GαoA, Gαz, and Gustducin (Gαg)] determined by cryo-electron microscopy (cryo-EM). The structural analysis, in combination with pharmacological studies, provides insights into Gi/o subtype selectivity. Given the conserved sequence identity and activation mechanism between different G protein families, the findings within Gi/o subtypes could be likely extended to other families. Understanding the KOR-Gi/o or GPCR-G protein selectivity will facilitate the development of more precise therapeutics targeting a specific G protein subtype.
Assuntos
Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G , Receptores Opioides kappa , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Opioides kappa/metabolismo , Receptores Opioides kappa/química , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Ligação Proteica , Animais , Conformação Proteica , Modelos MolecularesRESUMO
Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-ß-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-ß-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-ß-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.
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Analgésicos Opioides , Receptores Opioides kappa , Masculino , Camundongos , Animais , Receptores Opioides kappa/metabolismo , Ligantes , Analgésicos Opioides/química , Receptores Opioides mu/metabolismo , Peptídeos Cíclicos/químicaRESUMO
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.
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Dinoprostona , Receptores de Prostaglandina , Humanos , Camundongos , Animais , Fagocitose , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Dor/tratamento farmacológico , Anti-Inflamatórios não Esteroides/farmacologiaRESUMO
The leaf area index (LAI) played a crucial role in ecological, hydrological, and climate models. The normalized difference vegetation index (NDVI) has been a widely used tool for LAI estimation. However, the NDVI quickly saturates in dense vegetation and is susceptible to soil background interference in sparse vegetation. We proposed a multi-angular NDVI (MAVI) to enhance LAI estimation using tower-based multi-angular observations, aiming to minimize the interference of soil background and saturation effects. Our methodology involved collecting continuous tower-based multi-angular reflectance and the LAI over a three-year period in maize cropland. Then we proposed the MAVI based on an analysis of how canopy reflectance varies with solar zenith angle (SZA). Finally, we quantitatively evaluated the MAVI's performance in LAI retrieval by comparing it to eight other vegetation indices (VIs). Statistical tests revealed that the MAVI exhibited an improved curvilinear relationship with the LAI when the NDVI is corrected using multi-angular observations (R2 = 0.945, RMSE = 0.345, rRMSE = 0.147). Furthermore, the MAVI-based model effectively mitigated soil background effects in sparse vegetation (R2 = 0.934, RMSE = 0.155, rRMSE = 0.157). Our findings demonstrated the utility of tower-based multi-angular spectral observations in LAI retrieval, having the potential to provide continuous data for validating space-borne LAI products. This research significantly expanded the potential applications of multi-angular observations.
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Solo , Zea mays , Folhas de PlantaRESUMO
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.
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Analgésicos Opioides , Receptores Opioides , Transdução de Sinais , Humanos , Analgésicos Opioides/farmacologia , AnimaisRESUMO
The Soil Moisture Ocean Salinity (SMOS) was the first mission providing L-band multi-angular brightness temperature (TB) at the global scale. However, radio frequency interferences (RFI) and aliasing effects degrade, when present SMOS TBs, and thus affect the retrieval of land parameters. To alleviate this, a refined SMOS multi-angular TB dataset was generated based on a two-step regression approach. This approach smooths the TBs and reconstructs data at the incidence angle with large TB uncertainties. Compared with Centre Aval de Traitement des Données SMOS (CATDS) TB product, this dataset shows a better relationship with the Soil Moisture Active Passive (SMAP) TB and enhanced correlation with in-situ measured soil moisture. This RFI-suppressed SMOS TB dataset, spanning more than a decade (since 2010), is expected to provide opportunities for better retrieval of land parameters and scientific applications.
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G protein-coupled receptors (GPCRs) are the largest superfamily of human membrane target proteins for approved drugs. GPCR ligands can have a complex array of pharmacological activities. Among these activities, biased agonists have potential to serve as both chemical probes to understand specific aspects of receptor signaling and therapeutic leads with more specific, desired activity. Challenges exist, however, in the development of new biased activators due, in part, to the low throughput of traditional screening approaches. DNA-encoded chemical libraries (DELs) dramatically improve the throughput of drug discovery by allowing a collective selection, rather than discrete screening, of large compound libraries. The use of DELs has been largely limited to affinity-based selections against purified protein targets, which identify binders only. Herein, we report a split protein complementation approach that allows direct identification of DNA-linked molecules that induce the dimerization of two proteins. We used this selection with a DEL against opioid receptor GPCRs on living cells for the identification of small molecules that possess the specific function of activation of either ß-arrestin or G protein signaling pathways. This approach was applied to δ-, µ-, and κ-opioid receptors and enabled the discovery of compound [66,66], a selective, G-protein-biased agonist of the κ-opioid receptor (EC50 = 100 nM, E max = 82%, Gi bias factor = 6.6). This approach should be generally applicable for the direct selection of chemical inducers of dimerization from DELs and expand the utility of DELs to enrich molecules with a specific and desired biochemical function.
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The κ-opioid receptor (KOR) represents a highly desirable therapeutic target for treating not only pain but also addiction and affective disorders1. However, the development of KOR analgesics has been hindered by the associated hallucinogenic side effects2. The initiation of KOR signalling requires the Gi/o-family proteins including the conventional (Gi1, Gi2, Gi3, GoA and GoB) and nonconventional (Gz and Gg) subtypes. How hallucinogens exert their actions through KOR and how KOR determines G-protein subtype selectivity are not well understood. Here we determined the active-state structures of KOR in a complex with multiple G-protein heterotrimers-Gi1, GoA, Gz and Gg-using cryo-electron microscopy. The KOR-G-protein complexes are bound to hallucinogenic salvinorins or highly selective KOR agonists. Comparisons of these structures reveal molecular determinants critical for KOR-G-protein interactions as well as key elements governing Gi/o-family subtype selectivity and KOR ligand selectivity. Furthermore, the four G-protein subtypes display an intrinsically different binding affinity and allosteric activity on agonist binding at KOR. These results provide insights into the actions of opioids and G-protein-coupling specificity at KOR and establish a foundation to examine the therapeutic potential of pathway-selective agonists of KOR.
Assuntos
Microscopia Crioeletrônica , Proteínas Heterotriméricas de Ligação ao GTP , Ligantes , Receptores Opioides kappa , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacologia , Receptores Opioides kappa/química , Receptores Opioides kappa/metabolismo , Receptores Opioides kappa/ultraestrutura , Transdução de Sinais , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/ultraestrutura , Especificidade por Substrato , Regulação Alostérica/efeitos dos fármacos , Alucinógenos/metabolismo , Alucinógenos/farmacologiaRESUMO
The µ-opioid receptor (µOR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi.
RESUMO
The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR.