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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.

Background:We demonstrated that oxidative stress plays a crucial role in cognitive impairment in klotho mutant mice, a genetic model of aging. Since down-regulation of melatonin due to aging is well documented, we used this genetic model to determine whether the antioxidant property of melatonin affects memory impairment.Methods:First, we examined the effects of melatonin on hippocampal oxidative parameters and the glutathione/oxidized glutathione (GSH/GSSG) ratio and memory dysfunction of klotho mutant mice. Second, we investigated whether a specific melatonin receptor is involved in the melatonin-mediated pharmacological response by application with melatonin receptor antagonists. Third, we examined phospho-extracellular-signal-regulated kinase (ERK) expression, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, Nrf2 DNA binding activity, and glutamate-cysteine ligase (GCL) mRNA expression. Finally, we examined effects of the ERK inhibitor SL327 in response to antioxidant efficacy and memory enhancement mediated by melatonin.Results:Treatment with melatonin resulted in significant attenuations of oxidative damage, a decrease in the GSH/GSSG ratio, and a significant amelioration of memory impairment in this aging model. These effects of melatonin were significantly counteracted by the selective MT2 receptor antagonist 4-P-PDOT. Importantly, 4-P-PDOT or SL327 also counteracted melatonin-mediated attenuation in response to the decreases in phospho-ERK expression, Nrf2 nuclear translocation, Nrf2 DNA-binding activity, and GCL mRNA expression in the hippocampi of klotho mutant mice. SL327 also counteracted the up-regulation of the GSH/GSSG ratio and the memory enhancement mediated by melatonin in klotho mutant mice.Conclusions:Melatonin attenuates oxidative stress and the associated memory impairment induced by klotho deficiency via signaling interaction between the MT2 receptor and ERK- and Nrf2-related antioxidant potential.

Aims/hypothesis A type 2 diabetes-risk-increasing variant, MTNR1B (melatonin receptor 1B) rs10830963, regulates the circadian function and may influence the variability in metabolic responses to dietary carbohydrates. We investigated whether the effects of carbohydrate quantity and dietary glycaemic index (GI) on glycaemic response during OGTTs varied by the risk G allele of MTNR1B- rs10830963. Methods This study included participants ( n =150) of a randomised crossover-controlled feeding trial of four diets with high/low GI levels and high/low carbohydrate content for 5 weeks. The MTNR1B- rs10830963 (C/G) variant was genotyped. Glucose response during 2 h OGTT was measured at baseline and the end of each diet intervention. Results Among the four study diets, carrying the risk G allele (CG/GG vs CC genotype) of MTNR1B -rs10830963 was associated with the largest AUC of glucose during 2 h OGTT after consuming a high-carbohydrate/high-GI diet (β 134.32 [SE 45.69] mmol/l × min; p =0.004). The risk G-allele carriers showed greater increment of glucose during 0–60 min (β 1.26 [0.47] mmol/l; p =0.008) or 0–90 min (β 1.10 [0.50] mmol/l; p =0.028) after the high-carbohydrate/high-GI diet intervention, but not after consuming the other three diets. At high carbohydrate content, reducing GI levels decreased 60 min post-OGTT glucose (mean –0.67 [95% CI: –1.18, –0.17] mmol/l) and the increment of glucose during 0–60 min (mean –1.00 [95% CI: –1.67, –0.33] mmol/l) and 0–90 min, particularly in the risk G-allele carriers ( p interaction <0.05 for all). Conclusions/interpretation Our study shows that carrying the risk G allele of MTNR1B -rs10830963 is associated with greater glycaemic responses after consuming a diet with high carbohydrates and high GI levels. Reducing GI in a high-carbohydrate diet may decrease post-OGTT glucose concentrations among the risk G-allele carriers. Graphical Abstract

Aims/hypothesis The aim of this study was to assess the interaction between melatonin receptor 1B gene (MTNR1B) rs10830963 polymorphism and lifestyle intervention during pregnancy on occurrence of gestational diabetes mellitus (GDM) in high-risk women. Methods This is a secondary analysis of the randomised controlled gestational diabetes prevention trial ‘RADIEL’, conducted between 2008 and 2014 in four maternity hospitals in southern Finland. A total of 226 women with a history of GDM and/or a pre-pregnancy BMI ≥ 30 kg/m 2 were enrolled at <20 weeks of gestation (mean 13 weeks) and randomised into an intervention group receiving counselling on diet, physical activity and weight control and a control group receiving standard antenatal care. The main outcome was incidence of GDM, defined as one or more pathological glucose values in a standard 75 g 2-h OGTT. The MTNR1B rs10830963 was genotyped for further analyses. Results No significant differences were found in the genotype distribution between the intervention and the control group. A significant interaction was observed between the rs10830963 genotypes and the lifestyle intervention on age-adjusted occurrence of gestational diabetes ( p  = 0.038). Among women homozygous for the C allele of rs10830963, the OR for GDM was significantly lower in the intervention group than in the control group (OR 0.16 [95% CI 0.03, 0.85], p  = 0.014). This difference was not seen in women heterozygous (OR 0.88 [95% CI 0.32, 2.41], p  = 0.798) or homozygous (OR 2.25 [95% CI 0.34, 14.69], p  = 0.384) for the risk allele G. Conclusions/interpretation In women at high risk of GDM, only those not carrying the risk allele G benefited from the lifestyle intervention. Our results indicate that certain genetic risk variants may modify the effectiveness of lifestyle interventions. This may provide important information when planning GDM prevention studies in the future.

Abnormalities in the melatonin signaling pathway and the involvement of melatonin receptor MT2 have been reported in patients with adolescent idiopathic scoliosis (AIS). Whether these abnormalities were involved in the systemic abnormal skeletal growth in AIS during the peripubertal period remain unknown. In this cross-sectional case-control study, growth plate chondrocytes (GPCs) were cultured from twenty AIS and ten normal control subjects. Although the MT2 receptor was identified in GPCs from both AIS and controls, its mRNA expression was significantly lower in AIS patients than the controls. GPCs were cultured in the presence of either the vehicle or various concentrations of melatonin, with or without the selective MT2 melatonin receptor antagonist 4-P-PDOT (10 µM). Then the cell viability and the mRNA expression of collagen type X (COLX) and alkaline phosphatase (ALP) were assessed by MTT and qPCR, respectively. In the control GPCs, melatonin at the concentrations of 1, 100 nM and 10 µM significantly reduced the population of viable cells, and the mRNA level of COLX and ALP compared to the vehicle. Similar changes were not observed in the presence of 4-P-PDOT. Further, neither proliferation nor differentiation of GPCs from AIS patients was affected by the melatonin treatment. These findings support the presence of a functional abnormality of the melatonin signaling pathway in AIS GPCs, which might be associated with the abnormal endochondral ossification in AIS patients.

Background Primary headache disorders such as migraine and cluster headache exhibit circadian and circannual variations in attack onset. Melatonin plays a central role in regulating biological rhythms and likely influences the timing of headache attacks. Recent evidence suggests melatonin may be a promising treatment for migraine and cluster headache; however, underlying mechanisms need to be elucidated. Because of the importance of the trigeminovascular system (TVS) and trigeminal-parasympathetic ganglia in these disorders, we proposed that melatonin receptors (MT1 and MT2) are expressed in these pathways. Methods The trigeminal ganglion (TG), sphenopalatine ganglion (SPG), dorsal root ganglion (DRG), basilar artery, dura mater and hypothalamus were dissected from adult male and female rats. RT-qPCR and immunohistochemistry were used to assess the expression of mRNA and protein, respectively, for melatonin MT1 and MT2 receptors. Results Immunohistochemical analysis showed MT1 and MT2 were differentially expressed in the TG, SPG and DRG. MT1 was widely distributed in the cytoplasm and nuclei of neurons and satellite glial cells (SGCs) in the TG, while MT2 localized mainly in the cytoplasm of neurons and Aδ-fibers. Both MT1 and MT2 co-localized with CGRP and the CGRP receptor component RAMP1. MT2 immunoreactivity was also found in Aδ-fibers throughout the dura mater and was co-expressed with Contactin-associated protein 1(CASPR) at the paranodal regions of Aδ-fibers. No significant sex differences were found in receptor expression in the TG, although mRNA levels of MT1 were approximately twice as high as those for MT2. In SPG, both receptors co-localized with the neuropeptides VIP and PACAP. In cerebral arteries, only MT1 was detected, and it was localized mainly in endothelial and smooth muscle cells. Conclusions This study demonstrates that MT1 and MT2 receptors are expressed in the TVS and SPG, key components involved in migraine and cluster headache. Melatonin receptor co-localization with neuropeptides involved in autonomic and sensory neuronal regulation supports a potential mechanism by which melatonin influences headache onset and progression. Together, these findings support a role for melatonin influences in primary headache pathophysiology and point to its potential for novel headache treatment.

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.

The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer’s disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo . We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices or primary neurons treated with an MT2 receptor selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3 β /CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3 β /CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons.

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.