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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.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT₁), its associated G protein, and β-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT₁ signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT₁ overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, “automitocrine,” analogous to “autocrine” when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand–receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by dopaminergic neuron loss and Lewy bodies in the substantia nigra. Abnormal mitochondrial function and accumulated α-synuclein (α-syn) are key etiological factors of PD. Melatonin type 1 receptor (MT1) regulates sleep upon activation by melatonin and may be reduced in PD patients. However, the role of MT1 in PD pathogenesis remains elusive. In this study, we found knockdown of MT1 caused mitochondrial dysfunction, mitochondrial fission and mitophagy in SH-SY5Y cells. Expression of mitochondrial fission protein dynamin-related protein 1 (DRP1) was increased and expression of fusion proteins optic atrophy 1 (OPA1), mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were decreased. This was probably attributed to decreased phosphorylation of DRP1 at S637 by protein kinase A (PKA) and increased phosphorylation at S616 by extracellular-regulated kinase 1/2 (ERK1/2). Loss of MT1 exacerbated mitochondrial fission without influencing mitophagy, TH expression and movement in an MPTP-induced mouse model. Neuronal MT1 deficiency aggravated preformed fibrils induced autophagy inhibition and α-syn aggregation. Overexpression of MT1 reduced mitochondrial fission, increased LC3II expression and decreased P62 accumulation to promote autophagy in HEK293T cells, thus mitigating aggregation of α-syn. This study demonstrates the function of MT1 in mitochondria and autophagy, which sheds further light on PD prevention targeting MT1.

Ovarian cancer (OC) is the most lethal gynecologic malignancy, and melatonin has shown various antitumor properties. Herein, we investigated the influence of melatonin therapy on energy metabolism and mitochondrial integrity in SKOV-3 cells and tested whether its effects depended on MT1 receptor activation. SKOV-3 cells were exposed to different melatonin concentrations, and experimental groups were divided as to the presence of MT1 receptors (melatonin groups) or receptor absence by RNAi silencing (siRNA MT1+melatonin). Intracellular melatonin levels increased after treatment with melatonin independent of the MT1. The mitochondrial membrane potential of SKOV-3 cells decreased in the group treated with the highest melatonin concentration. Melatonin reduced cellular glucose consumption, while MT1 knockdown increased its consumption. Interconversion of lactate to pyruvate increased after treatment with melatonin and was remarkable in siRNA MT1 groups. Moreover, lactate dehydrogenase activity decreased with melatonin and increased after MT1 silencing at all concentrations. The UCSC XenaBrowser tool showed a positive correlation between the human ASMTL gene and the ATP synthase genes, succinate dehydrogenase gene (SDHD), and pyruvate dehydrogenase genes (PDHA and PDHB). We conclude that melatonin changes the glycolytic phenotype and mitochondrial integrity of SKOV-3 cells independent of the MT1 receptor, thus decreasing the survival advantage of OC cells.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets.Key messages• MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines.• Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex.• Tumors with a high MT1/MT2 expression ratio have significantly better survival rates.• Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.

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 (Mel), an important mediator of photoperiodic annual rhythm regulation and seasonal reproduction in animals, directly modulates the expression of specific genes in the epididymis and protects sperm from oxidative damage. Bactrian camel is a dominant species in desert and semi-desert areas, exhibiting the unique reproductive regulation patterns. However, the underlying regulation mechanism of Mel on Bactrian camel is still unclear. This study isolated the epididymal caput epithelial cells of Bactrian camels and investigated the expression of specific genes involving sperm protection after Mel treatment and overexpression/knockdown of Mel receptor MT1/MT2 using real-time quantitative PCR assay (qPCR), ELISA, and western blotting assay. The results showed that MT1, MT2, clock genes cryptochrome 1/2 (Cry1/Cry2) were all positively expressed in the epididymal lumen epithelial cells, peritubular myoid cells, and luminal spermatozoa. Intriguingly, Mel treatment activated receptor MT1 in epididymal caput epithelial cells, indicating that Mel treatment regulated genes expression mainly via MT1-dependent manner. Mel treatment or overexpression of MT1 both increased secretion of glutathione peroxidase 5 (GPX5) and prostaglandin D2 synthase (PTGDS), and MT1 silencing induced downregulation of GPX5 and PTGDS expression, indicating that the expression of GPX5 and PTGDS were regulated by Mel-MT1. Overexpression of MT1 or MT2 promoted Cry2 expression, and overexpression of Cry2 also activated the MT1/MT2 expression by feedback regulation. Finally, the double luciferase reports assay showed that the activation of MT1 by Cry2 occurred during transcription. These results help to understand the regulatory effect of Mel on the epididymis in Bactrian camels. Graphical Abstract

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.

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.