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Crystals, ligand electron density maps, and packing of MT1 a, b, Bright field (a) and cross-polarized (b) images of representative MT1–2-PMT crystals, optimized for synchrotron data collection (representing three independent crystallization setups). c, Cross-polarized image of representative MT1–ramelteon crystals used for XFEL data collection (representing two independent crystallization setups). d, 2mFo − DFc ligand electron density maps of MT1 co-crystallized with 2-PMT (orange), 2-iodomelatonin (yellow), and agomelatine (cyan), contoured at 1.0σ (grey mesh). e, 2mFo − DFc (blue, contoured at 1.0σ) and mFo − DFc (green/red, ±3.5σ) electron density maps of MT1–ramelteon (ligand purple, protein yellow) illustrating the small, unassigned electron density close to N2556.52 that is tentatively attributed to the essential additive 2-propan-ol. The distance from this electron density to the closest ligand atom is approximately 4.8 Å. f, Packing of MT1–PGS crystallized in the P4 21 2 space group. The receptor is shown in green and the PGS fusion protein is shown in purple. g, Simulated annealing mFo − DFc omit maps (green mesh) of 2-PMT (orange sticks), 2-iodomelatonin (yellow), and agomelatine (cyan), contoured at 3.0σ.
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Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms¹ by synchronization to environmental cues and is involved in diverse physiological processes² such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function³. Melatonin is formed in the pineal gland in a light-regulated...
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Objective: The antidepressant agomelatine agent is a melatonin receptor (MT1 and MT2) agonist and a serotonin receptor (5-HT2C) antagonist. Increasing evidence shows that agomelatine has neuroprotective and neuromodulatory effects. In this study, the potential effects of agomelatine in rats with scopolamine-induced cognitive impairment were investi...
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... The performance of interaction fingerprints was compared with the automated Site-Finder function to guide docking site selection in the MOE software [35]. Receptor and ligand structure sources for each docking calculation, as well as the fingerprints tested, are shown in Table S2 [ [37][38][39][40][41][42][43][46][47][48][49][50]52,59,81,85,88,91,100,102,[121][122][123][124][125]148,149,161,162,175,176,191,192]. The percentage of docking calculations that produced ligand poses with RMSD values in the successful (<2 Å), acceptable (2-3 Å), successful + acceptable (<3 Å), and unsuccessful (>3 Å) categories were used to compare fingerprint performance to the automated SiteFinder performance. ...
... As such, the internal dataset used to develop the random forest classifier in this work contained 1820 class A GPCR-ligand complexes of mixed origin that were grouped into six categories that reflect the different types of structures that can be encountered during a GPCR ligand discovery workflow ( Figure 2). The first structural category within the internal dataset contained 342 experimentally determined GPCR structures possessing orthosterically bound nonpeptide ligands that were publicly available from the Protein Data Bank [23] as of 31 December 2021 (Table S4 [ 103,104,106,111,113,115,116,[121][122][123][124][125][126][127][128][129][131][132][133][134][135][136]140,142,[145][146][147][148][149][150][151][152][153]158,159,[161][162][163][164]166,167,[169][170][171][172][173][174][175][176][177][178][179][180][181][182][183][184][185][186][191][192][193][194][196][197][198]200,). Although experimentally determined ligand-receptor complexes are unlikely to be available for GPCR targets with few known ligands, the inclusion of these complexes in the dataset allowed for the classifier to be trained on ligand interactions observed in vitro. ...
G protein-coupled receptor (GPCR) transmembrane protein family members play essential roles in physiology. Numerous pharmaceuticals target GPCRs, and many drug discovery programs utilize virtual screening (VS) against GPCR targets. Improvements in the accuracy of predicting new molecules that bind to and either activate or inhibit GPCR function would accelerate such drug discovery programs. This work addresses two significant research questions. First, do ligand interaction fingerprints provide a substantial advantage over automated methods of binding site selection for classical docking? Second, can the functional status of prospective screening candidates be predicted from ligand interaction fingerprints using a random forest classifier? Ligand interaction fingerprints were found to offer modest advantages in sampling accurate poses, but no substantial advantage in the final set of top-ranked poses after scoring, and, thus, were not used in the generation of the ligand–receptor complexes used to train and test the random forest classifier. A binary classifier which treated agonists, antagonists, and inverse agonists as active and all other ligands as inactive proved highly effective in ligand function prediction in an external test set of GPR31 and TAAR2 candidate ligands with a hit rate of 82.6% actual actives within the set of predicted actives.
... Recent advances include the determination of MT 1 and MT 2 receptor structures bound to various ligands (Stauch et al., 2019) or in complex with Gα i/o proteins . These structures are crucial for initiating virtual screens to identify new specific modulating ligands for these receptors (Stein et al., 2020). ...
G protein‐coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. This overview emphasises the GPCRs of the nervous system, which are the research focus of the members of ERNEST COST action (CA18133) working group ‘Biological roles of signal transduction’. First, the (patho)physiological role of the nervous system GPCRs in the modulation of synapse function is discussed. We then debate the (patho)physiology and pharmacology of opioid, acetylcholine, chemokine, melatonin and adhesion GPCRs in the nervous system. Finally, we address the orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed to deorphanize them.
... C1403.52L, and L108ECL1F , were introduced to MT2, which are not contagious to the coupling interface of G-protein and ligand binding pocket. The ligand interaction with receptor and G-proteins coupling interferes minimally with mutations (Johansson et al., 2019). An EM density map was obtained at 3.5 nominal resolution by using the triple mutant complex of ramelteouun-MT2-Gi-scFv16, enabling to model ramelteon, scFv16, significant portions of the receptor, MT1 and MT2 Gi protein receptors are assembled similarly to Gi protein, GPCRS, and G-protein complexes. ...
... Homologous pair N1754.60 and Y2005.38 also underwent structural changes in MT2 during the transition. This hydrogen bond's absence demonstrates that MT2 does not require an entrance-restricting hydrogen bond similar to the one found in MT1 for activation, which is consistent with the earlier finding that the N1754.60 protein does not need such a hydrogen bond (Stauch et al., 2019). No functional consequences resulted from a mutation (Johansson et al., 2019). ...
... This hydrogen bond's absence demonstrates that MT2 does not require an entrance-restricting hydrogen bond similar to the one found in MT1 for activation, which is consistent with the earlier finding that the N1754.60 protein does not need such a hydrogen bond (Stauch et al., 2019). No functional consequences resulted from a mutation (Johansson et al., 2019). Because N4.60-Y5.38 can be altered in conformation thanks to a conserved proline (P4.59) located close by in MT1 and MT2, changing P4.59 impairs MT2's ability to bind ligands (Mazna et al., 2008). ...
Melatonin (5-methoxy-acetyl tryptamine) sleep-inducing hormone, and the pineal gland produces it in response to the circadian clock of darkness. In the body, MT1 and MT2 receptors are mostly found, having an orthosteric pocket and ligand binding determinants. Melatonin acts by binding on melatonin receptors, intracellular proteins, and orphan nuclear receptors. It inhibits adenyl cyclase and activates phospholipase C, resulting in gene expression and an intracellular alteration environment. Melatonin signaling pathways are also associated with other intracellular signaling pathways, i.e., cAMP/PKA and MAPK/ERK pathways. Relative expression of different proteins depends on the coupling profile of G protein, accounting pharmacology of the melatonin receptor bias system, and mediates action in a Gi-dependent manner. It shows antioxidant, antitumor, antiproliferative and neuroprotective activity. Different types of melatonin agonists have been synthesized for the treatment of sleeping disorders. Researchers have developed therapeutics that target melatonin signaling, which could benefit a wide range of medical conditions. This review focuses on melatonin receptors, pharmacology, and signaling cascades; it aims to provide basic mechanical aspects of the receptor's pharmacology, melatonin functions in cancer and neurodegenerative diseases, and any treatments and drugs designed for these diseases. This will allow a basic comparison between the receptors in question, highlighting any parallels and differences that may exist and providing fundamental knowledge about these receptors to future researchers.
... NMR experiments were performed using a Bruker AVIII HD 600 MHz spectrometer equipped with a triple resonance Prodigy N2 cryoprobe with z-axis pulse field gradient. The 3D structural model of the MT 1 inactive state was obtained using as template the crystal structure of MT 1 bound to ramelteon (PDB ID: 6ME2) [21]. For the active MT 1 receptor the three-dimensional model was obtained using as template (i) the cryo-EM structure of the human mu-opioid receptor for the residues 21-290 of the transmembrane bundle (TM1-TM7) (PDB ID: 6DDE) [22] and (ii) the crystal structure of the human adenosine A2A receptor (PDB ID: 2YDO) [23] for the region from 291 to 308 comprising ICL4 and helix 8. ...
... We therefore built a 3D model for the inactive and active state of MT 1 receptors ( Fig. 4C-E; Supplemental Fig. 8A-C; Supplemental Fig. 1A-B) by homology modeling. Consistent with previous studies [21,29], the 3D structural model of inactive MT 1 indicates that the receptor presents the typical GPCR architecture ( Fig. 4C; Supplemental Fig. 8A-B) with the presence of helical kinks in TM5 (Pro 198 ), TM6 (Pro 253 ), and TM7 (Ala 292 ) (Supplemental Fig. 8C), which are essential for coupling ligand binding to conformational changes needed to reach the active state [30,31]. In contrast, TM3 is straight, and forms the structural core of the receptor interacting with many neighboring TMs (Supplemental Fig. 6C). ...
... Human pose evaluation techniques have long been an important research component in the field of computer vision and pattern recognition [1][2][3]. With the proliferation of camera surveillance devices and motion capture devices, advances in computer technology, and the rise of artificial intelligence techniques, pose evaluation has become an increasingly popular topic [4][5]. If a computer can accurately recognize the movements that occur in humans, it can plan and interact with humans [6]. ...
In this paper, we first analyze full domain convolution and LSTM to evaluate human pose by convolutional neural network and LSTM network. Secondly, graph structure skeleton image and skeleton point image classifier based on CNN and LSTM is constructed. The two-dimensional pose assessment method and three-dimensional pose assessment method were used to empirically analyze the human pose assessment. The results show that the average accuracy mAP values of the traditional evaluation methods are 69.7, 72.3, 71.4, and 74.4, respectively, while the average accuracy mAP value of the method used for 2D pose evaluation is 74.6. Where the average error of LReLU is the smallest. This shows that full-domain convolution and LSTM can be effective for human pose evaluation.
... The melatonin MT1 receptor also binds a hydrophobic ligand, which is proposed to enter through a gap between TM4 and TM5. In particular, the conformational change of Y187 5.38 in TM5 of MT1 during its transition to the activated state is suggested to reduce the size of the ligand entrance and ligand dissociation rate 47,48 (Supplementary Fig. 16b). In the HCA2 structures, W188 5.38 in TM5 is located in the groove between TM4 and TM5, completely closing off ligand access (Fig. 6a, b and Supplementary Fig. 16c). ...
Hydroxycarboxylic acid receptors (HCA) are expressed in various tissues and immune cells. HCA2 and its agonist are thus important targets for treating inflammatory and metabolic disorders. Only limited information is available, however, on the active-state binding of HCAs with agonists. Here, we present cryo-EM structures of human HCA2-Gi and HCA3-Gi signaling complexes binding with multiple compounds bound. Agonists were revealed to form a salt bridge with arginine, which is conserved in the HCA family, to activate these receptors. Extracellular regions of the receptors form a lid-like structure that covers the ligand-binding pocket. Although transmembrane (TM) 6 in HCAs undergoes dynamic conformational changes, ligands do not directly interact with amino acids in TM6, suggesting that indirect signaling induces a slight shift in TM6 to activate Gi proteins. Structural analyses of agonist-bound HCA2 and HCA3 together with mutagenesis and molecular dynamics simulation provide molecular insights into HCA ligand recognition and activation mechanisms.
... Molecular dynamics simulations revealed substantial flexibility of the extracellular part of TM5 controlling a gate between TM4 and TM5, sufficient for a lipid ligand to enter the pocket directly from the membrane (28). In most other lipid GPCRs as well as melatonin receptors, a direct access to the orthosteric ligandbinding pocket from the lipid membrane was proposed (65)(66)(67). The structures revealed that the acidic headgroups of CysLTR ligands interact with mostly hydrophilic and positively charged top part of the ligand pocket formed by residues from TMs1-3,5,7 and ECL2 (28,29) (Fig. 4, D-F). ...
Dihydroxy acid leukotriene (LTB4) and cysteinyl leukotrienes (LTC4, LTD4, and LTE4) are inflammatory mediators derived from arachidonic acid via the 5-lipoxygenase pathway. While structurally similar, these two types of leukotrienes (LTs) exert their functions through interactions with two distinct G protein–coupled receptor (GPCR) families, BLT and CysLT receptors, which share low sequence similarity and belong to phylogenetically divergent GPCR groups. Selective antagonism of LT receptors has been proposed as a promising strategy for the treatment of many inflammation-related diseases including asthma and chronic obstructive pulmonary disease, rheumatoid arthritis, cystic fibrosis, diabetes, and several types of cancer. Selective CysLT1R antagonists are currently used as antiasthmatic drugs, however, there are no approved drugs targeting CysLT2 and BLT receptors. In this review, we highlight recently published structures of BLT1R and CysLTRs revealing unique structural features of the two receptor families. X-ray and cryo-EM data shed light on their overall conformations, differences in functional motifs involved in receptor activation, and details of the ligand-binding pockets. An unexpected binding mode of the selective antagonist BIIL260 in the BLT1R structure makes it the first example of a compound targeting the sodium-binding site of GPCRs and suggests a novel strategy for the receptor activity modulation. Taken together, these recent structural data reveal dramatic differences in the molecular architecture of the two LT receptor families and pave the way to new therapeutic strategies of selective targeting individual receptors with novel tool compounds obtained by the structure-based drug design approach.
... Room-temperature SX at XFELs (SFX) and synchrotron sources (SSX) is applicable to membrane protein samples crystallized in lipidic cubic phase (LCP) (Caffrey & Cherezov, 2009;Liu et al., 2014). High-resolution structural data generated for integral membrane proteins (IMPs) (Axford et al., 2022;Johansson et al., 2019;Hosaka et al., 2022;Liu et al., 2013;Nango & Iwata, 2023;Nogly et al., 2015;Stauch et al., 2019;Zhang et al., 2017, to mention a few) are critical for understanding structure-function relationships, intermolecular interactions and protein dynamics, and for rational drug design (Hauser et al., 2018;Reis & Moraes, 2019). Structure determination of IMPs by traditional X-ray diffraction methods is challenging, since the crystals are usually extremely fragile and exceptionally sensitive to radiation damage. ...
Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.
... Recent advances include the determination of MT1 and MT2 structures bound to various ligands (Stauch et al., 2019) or in complex with Gαi/o proteins . These structures are crucial for initiating virtual screens to identify new specific modulating ligands for these receptors (Stein et al., 2020). ...
G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. In this review, we will first discuss the role of the nervous system GPCRs in the modulation of tripartite synapse function and how GPCRs control energy metabolism in the brain. We will then discuss the (patho)physiology and pharmacology of opioid, cannabinoid, acetylcholine, chemokine, and melatonin GPCRs in the nervous system. Furthermore, we will briefly report on adhesion GPCR function in nervous tissues. Finally, we will address orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed in the future to deorphanize them.
... An increased cAMP level and activation of PKA influenced the Kir channels and decreases their activities in the cardiomyocytes [48]. Also, luzindole and 4P-PDOT competitively block MT1 melatonin receptors, and both are inverse agonists in systems with constitutively active MT1 receptors [10,[49][50][51][52]. These luzindole effects could lead to a reduction in the amplitude of the IK1 current shown in this study. ...
Melatonin has been reported to cause myocardial electrophysiological changes and prevent ventricular tachycardia or fibrillation (VT/VF) in ischemia and reperfusion. We sought to identify electrophysiological targets responsible for the melatonin antiarrhythmic action and to explore whether melatonin receptor-dependent pathways or its antioxidative properties are essential for these effects. Ischemia was induced in anesthetized rats given a placebo, melatonin, and/or luzindole (MT1/MT2 melatonin receptor blocker), and epicardial mapping with reperfusion VT/VFs assessment was performed. The oxidative stress assessment and Western blotting analysis were performed in the explanted hearts. Transmembrane potentials and ionic currents were recorded in cardiomyocytes with melatonin and/or luzindole application. Melatonin reduced reperfusion VT/VF incidence associated with local activation time in logistic regression analysis. Melatonin prevented ischemia-related conduction slowing and did not change the total connexin43 (Cx43) level or oxidative stress markers, but it increased the content of a phosphorylated Cx43 variant (P-Cx43368). Luzindole abolished the melatonin antiarrhythmic effect, slowed conduction, decreased total Cx43, protein kinase Cε and P-Cx43368 levels, and the IK1 current, and caused resting membrane potential (RMP) depolarization. Neither melatonin nor luzindole modified INa current. Thus, the antiarrhythmic effect of melatonin was mediated by the receptor-dependent enhancement of impulse conduction, which was associated with Cx43 phosphorylation and maintaining the RMP level.
