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The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT 1 and MT 2 . Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light–dark cycle 1 – 4 . The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep 5 , 6 and depression 1 – 4 , 7 – 9 . Despite their importance, few in vivo active MT 1 -selective ligands have been reported 2 , 8 , 10 – 12 , hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT 1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT 1 inverse agonists—which were topologically unrelated to previously explored chemotypes—that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT 1 -selective inverse agonists advanced the phase of the mouse circadian clock by 1.3–1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT 1 - but not in MT 2 -knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT 1 -selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries. A computational screen of an ultra-large virtual library against the structure of the melatonin receptor found nanomolar ligands, and ultimately two selective MT 1 inverse agonists that induced phase advancement of the mouse circadian clock when given at subjective dusk.

Melatonin receptors (MT 1 and MT 2 in humans) are family A G protein–coupled receptors that respond to the neurohormone melatonin to regulate circadian rhythm and sleep. Numerous efforts have been made to develop drugs targeting melatonin receptors for the treatment of insomnia, circadian rhythm disorder, and cancer. However, designing subtype-selective melatonergic drugs remains challenging. Here, we report the cryo-EM structures of the MT 1 –G i signaling complex with 2-iodomelatonin and ramelteon and the MT 2 –G i signaling complex with ramelteon. These structures, together with the reported functional data, reveal that although MT 1 and MT 2 possess highly similar orthosteric ligand-binding pockets, they also display distinctive features that could be targeted to design subtype-selective drugs. The unique structural motifs in MT 1 and MT 2 mediate structural rearrangements with a particularly wide opening on the cytoplasmic side. G i is engaged in the receptor core shared by MT 1 and MT 2 and presents a conformation deviating from those in other G i complexes. Together, our results provide new clues for designing melatonergic drugs and further insights into understanding the G protein coupling mechanism. Melatonin receptors (MT1 and MT2) are the targets for melatonin, the major neurohormone involved in circadian rhythm and sleep regulation. Here the authors describe the structures of 2-iodomelatonin and ramelteon bound MT1–Gi and MT2-Gi, revealing that MT1 and MT2 possess distinctive features within the ligand-binding pocket.

Melatonin is a neurohormone produced in both animals and plants. It binds at least three G-protein-coupled receptors: MT1 and MT2, and Mel1cGPR. Mammalian GPR50 evolved from the reptilian/avian Mel1c and lost its capacity to bind melatonin in all the therian mammal species that have been tested. In order to determine if binding is lost in the oldest surviving mammalian lineage of monotremes we investigated whether the melatonin receptor has the ability to bind melatonin in the platypus (Ornithorhynchus anatinus), and evaluated its pharmacological profile. Sequence and phylogenetic analysis showed that platypus has in fact retained the ancestral Mel1c and has the capacity to bind melatonin similar to other mammalian melatonin receptors (MT1 and MT2), with an affinity in the 1 nM range. We also investigated the binding of a set of melatoninergic ligands used previously to characterize the molecular pharmacology of the melatonin receptors from sheep, rats, mice, and humans and found that the general profiles of these compounds make Mel1c resemble human MT1 more than MT2. This work shows that the loss of GPR50 binding evolved after the divergence of monotremes less than 190MYA in therian mammals.

Melatonin is a neurohormone that has been claimed to be involved in a wide range of physiological functions. Nevertheless, for most of its effects, the mechanism of action is not really known. In mammals, two melatonin receptors, MT1 and MT2, have been cloned. They belong to the G‐protein‐coupled receptor (GPCR) superfamily. They share some specific short amino‐acid sequences, which suggest that they represent a specific subfamily. Another receptor from the same subfamily, the melatonin‐related receptor has been cloned in different species including humans. This orphan receptor also named GPR50 does not bind melatonin and its endogenous ligand is still unknown. Nevertheless, this receptor has been shown to behave as an antagonist of the MT1 receptor, which opens new pharmacological perspectives for GPR50 despite the lack of endogenous or synthetic ligands. Moreover, MT1 and MT2 interact together through the formation of heterodimers at least in cells transfected with the cDNA of these two receptors. Lastly, signalling complexes associated with MT1 and MT2 receptors are starting to be deciphered. A third melatonin‐binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). Inhibition of QR2 by melatonin may explain melatonin's protective effect that has been reported in different animal models and that is generally associated with its well‐documented antioxidant properties. British Journal of Pharmacology (2008) 154, 1182–1195; doi:10.1038/bjp.2008.184; published online 19 May 2008

Type 2 diabetes mellitus (T2DM) poses a significant global health challenge. Genome-wide Association Studies have linked T2DM to genetic variants in the melatonin receptor 1a ( MTNR1A) and 1b ( MTNR1B ) genes, which encode the MT1 and MT2 receptors, respectively. Our results found that the rs2119882 MT1 mutation was associated with higher blood glucose levels and increased body mass index (BMI) in humans. Metabolomic analysis showed elevated levels of palmitic acid (a saturated fatty acid) and reduced levels of oleic acid (an unsaturated fatty acid) in individuals with this mutation. In contrast, the rs10830963 MT2 mutation did not show the significant differences in blood glucose level or BMI compared to normal control individuals. Inhibition of MTNR1A and MTNR1B expression led to lower GLUT-4 mRNA and insulin receptor protein levels in human liver cells, resulting in decreased glycogen synthesis and metabolic disruptions. We used CRISPR/Cas9 to create MTNR1A and MTNR1B knockout (KO) mice, which also exhibited reduced GLUT-4 and INSR mRNA levels, decreased glucose tolerance, and increased insulin resistance. These mice also developed obesity, liver lipid deposition, increased abdominal white adipose tissue, and lower androgen levels. Metabolomic and proteomic analyses of the KO mice revealed increased triglycerides and phospholipids, and decreased unsaturated fatty acids. Proteomic studies showed reduced levels of insulin receptor tyrosine kinase, lipid droplet-associated hydrolase, and glucose-6-phosphate dehydrogenase, disrupting fatty acid metabolism and increasing liver lipid deposition. Additionally, a high-fat diet challenge in MTNR1A and MTNR1B KO male mice accelerate the INSR protein expression suppression, hepatic triglyceride accumulation, blood glucose elevation and weight gain. Finally, we generated AANAT over-expressing sheep, which showed improved glucose tolerance and higher insulin levels after glucose injection compared to WT sheep. These findings underscore the importance of melatonin and its receptors in glucose and lipid metabolism, suggesting their deficiencies may contribute to T2DM.

This study evaluated the receptor- and/or antioxidant stress-mediated mechanisms by which melatonin prevents the ovarian toxicity of cisplatin treatment. The expression of the MT1 receptor in mouse ovaries was investigated by immunohistochemistry. Pretreatment with melatonin (5, 10, or 20 mg/kg body weight, i.p.) before cisplatin (5 mg/kg body weight, i.p.) was administered to mice once daily for 3 days (phase I). The pharmacological modulation via melatonin type 1 and/or 2 receptors was analyzed by administration of receptor antagonists (luzindole: nonselective MT1/MT2 antagonist; 5 mg/kg body weight or 4-phenyl-2-propionamidotetralin: selective MT2 antagonist; 4mg/kg body weight) once daily for 3 days, 15 min before the treatment with melatonin and cisplatin (phase II). Thereafter, the ovaries were harvested and used for histological (morphology and activation), immunohistochemical (PCNA, activated caspase-3 and bcl-2 expression), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and fluorescence (reactive oxygen species [ROS], glutathione [GSH], and active mitochondria levels) analyses. The expression of the MT1 protein in mouse ovaries was documented. Pretreatment with 20 mg/kg melatonin before cisplatin administration preserved the normal follicular morphology and cell proliferation rate, reduced apoptosis, ROS production, mitochondrial damage and increased GSH expression, as compared to the cisplatin treatment alone. Additionally, administration of the nonselective MT1/MT2 receptor antagonist inhibited the melatonin ovarian protection from the cytotoxic effects of cisplatin. However, administration of a selective MT2 antagonist did not modify the protective effects observed at 20 mg/kg melatonin. In conclusion, pretreatment with 20 mg/kg melatonin effectively protected the ovaries against cisplatin-induced damage. Moreover, the MT1 receptor and melatonin antioxidant effects mediated this cytoprotective activity. Summary Sentence Melatonin attenuated cisplatin-induced ovarian damage in mice, and the MT1 receptor could be used as a promising therapeutic target to the development of novel agents for preserving ovarian function during chemotherapy.

Melatonin, a molecule with diverse biological functions, is ubiquitously present in living organisms. There is significant interest in understanding melatonin signal transduction pathways in humans, particularly due to its critical role in regulating the sleep-wake cycle. However, a knowledge gap remains in fully elucidating the mechanisms by which melatonin influences circadian regulation. To bridge this gap, there is a growing need for a model system to study the role of melatonin in circadian clocks, with Neurospora crassa being a promising candidate. As a first step in this investigation, we focused on identifying melatonin receptors in N . crassa . Given the lack of sequence similarity between potential receptors in this fungus and known human melatonin receptors, we utilized structural similarity analysis through AlphaFold2. This approach led to the identification of a strong candidate gene, gpr-3 , which shares structural similarities with human melatonin receptors. Experimental validation confirmed that the removal of GPR-3 from cells results in the absence of melatonin signaling. This proof-of-concept study underscores the potential of N . crassa as a model organism for circadian research and demonstrates the broader applicability of using AlphaFold2, especially when sequence similarity does not lead to candidate genes, for identifying novel receptors across different species.

Melatonin receptors (MTNRs) belonging to the G protein-coupled receptor family are considered to consist of three subtypes in vertebrates: MTNR1a, MTNR1b and MTNR1c. Additionally, MTNR1a-like genes have been identified in teleostean species as a fish-specific subtype of MTNR1a. However, similar molecules to this MTNR1a-like gene can be found in some reptiles upon searching the DNA database. We hypothesized that a vertebrate can essentially have four functional subtypes of MTNR as ohnologs. Thus, in the present study we examined the molecular phylogeny, expression patterns and pharmacological profile(s) using the teleost medaka ( Oryzias latipes ). The four conserved subtypes of MTNR (MTNR1a, MTNR1b, MTNR1c and MTNR1a-like) in vertebrates were classified based on synteny and phylogenetic analysis. The fourth MTNR, termed MTNR1a-like, could be classified as MTNR1d. It was observed by using RT-qPCR that expression patterns differed amongst these subtypes. Moreover, mtnr1a , mtnr1c and mtnr1a-like / mtnr1d expression was elevated during short days compared to long days in diencephalons. All the subtypes were activated by melatonin and transduced signals into the Gi pathway, to perform a cAMP-responsive reporter gene assay. It was shown that MTNR originally consisted of four subtypes: MTNR1a, MTNR1b, MTNR1c and MTNR1d. These subtypes were functional, at least in fish, although some organisms, including mammals, have lost one or two subtypes.

Melatonin is a known modulator of follicle development; it acts through several molecular cascades via binding to its two specific receptors MT1 and MT2. Even though it is believed that melatonin can modulate granulosa cell (GC) functions, there is still limited knowledge of how it can act in human GC through MT1 and MT2 and which one is more implicated in the effects of melatonin on the metabolic processes in the dominant follicle. To better characterize the roles of these receptors on the effects of melatonin on follicular development, human granulosa-like tumor cells (KGN) were treated with specific melatonin receptor agonists and antagonists, and gene expression was analyzed with RNA-seq technology. Following appropriate normalization and the application of a fold change cut-off of 1.5 (FC 1.5, p  ≤ 0.05) for each treatment, lists of the principal differentially expressed genes (DEGs) are generated. Analysis of major upstream regulators suggested that the MT1 receptor may be involved in the melatonin antiproliferative effect by reprogramming the metabolism of human GC by activating the PKB signaling pathway. Our data suggest that melatonin may act complementary through both MT1 and MT2 receptors to modulate human GC steroidogenesis, proliferation, and differentiation. However, MT2 receptors may be the ones implicated in transducing the effects of melatonin on the prevention of GC luteinization and follicle atresia at the antral follicular stage through stimulating the PKA pathway.

Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT 1 and MT 2 receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT 1 or MT 2 receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT 1 and MT 2 ) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.