Quantum mechanics insights into melatonin and analogs binding to melatonin MT1 and MT2 receptors.

Melatonin receptors MT1 and MT2 are G protein-coupled receptors that mediate the effects of melatonin, a hormone involved in circadian rhythms and other physiological functions. Understanding the molecular interactions between these receptors and their ligands is crucial for developing novel therapeutic agents. In this study, we used molecular docking, molecular dynamics simulations, and quantum mechanics calculation to investigate the binding modes and affinities of three ligands: melatonin (MLT), ramelteon (RMT), and 2-phenylmelatonin (2-PMT) with both receptors. Based on the results, we identified key amino acids that contributed to the receptor-ligand interactions, such as Gln181/194, Phe179/192, and Asn162/175, which are conserved in both receptors. Additionally, we described new meaningful interactions with Gly108/Gly121, Val111/Val124, and Val191/Val204. Our results provide insights into receptor-ligand recognition's structural and energetic determinants and suggest potential strategies for designing more optimized molecules. This study enhances our understanding of receptor-ligand interactions and offers implications for future drug development.

2-Arylmelatonin analogues: Probing the 2-phenyl binding pocket of melatonin MT1 and MT2 receptors.

In crystal structures of melatonin MT1 and MT2 receptors, a lipophilic subpocket has been characterized which accommodates the phenyl ring of the potent agonist 2-phenylmelatonin. This subpocket appears a key structural element to achieve high binding affinity and selectivity for the MT2 receptor. A series of 2-arylindole ligands was synthesized to probe the requirements for the optimal occupation and interaction with the 2-phenyl binding pocket. Thermodynamic integration simulations applied to MT1 and MT2 receptors in complex with the α-naphthyl derivative provided a rationale for the MT2-selectivity and investigation on the binding mode of a couple of atropisomers allowed to define the available space and arrangement of substituents inside the subpocket. Interestingly, more hydrophilic 2-aza-substituted compounds displayed high binding affinity and molecular dynamics simulations highlighted polar interaction with residues from the subpocket that could be responsible for their potency.

Design and Validation of the First Family of Photo-Activatable Ligands for Melatonin Receptors.

Melatonin is a neurohormone released in a circadian manner with peak levels at night. Melatonin mediates its effects mainly through G protein-coupled MT1 and MT2 receptors. Drugs acting on melatonin receptors are indicated for circadian rhythm- and sleep-related disorders. Tools to study the activation of these receptors with high temporal resolution are lacking. Here, we synthesized a family of light-activatable caged compounds by attaching o-nitrobenzyl (o-NB) or coumarin photocleavable groups to melatonin indolic nitrogen. All caged compounds showed the expected decrease in binding affinity for MT1 and MT2. The o-NB derivative MCS-0382 showed the best uncaging and biological properties, with 250-fold increase in affinity and potency upon illumination. Generation of melatonin from MCS-0382 was further demonstrated by its ability to modulate the excitation of SCN neurons in rat brain slices. MCS-0382 is available to study melatonin effects in a temporally controlled manner in cellular and physiological settings.

Structural basis of the ligand binding and signaling mechanism of melatonin receptors.

Melatonin receptors (MT1 and MT2 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 MT1-Gi signaling complex with 2-iodomelatonin and ramelteon and the MT2-Gi signaling complex with ramelteon. These structures, together with the reported functional data, reveal that although MT1 and MT2 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 MT1 and MT2 mediate structural rearrangements with a particularly wide opening on the cytoplasmic side. Gi is engaged in the receptor core shared by MT1 and MT2 and presents a conformation deviating from those in other Gi complexes. Together, our results provide new clues for designing melatonergic drugs and further insights into understanding the G protein coupling mechanism.

N-(Anilinoethyl)amide Melatonergic Ligands with Improved Water Solubility and Metabolic Stability.

The MT2 -selective melatonin receptor ligand UCM765 (N-(2-((3-methoxyphenyl)(phenyl)amino)ethyl)acetamide), showed interesting sleep inducing, analgesic and anxiolytic properties in rodents, but suffers from low water solubility and modest metabolic stability. To overcome these limitations, different strategies were investigated, including modification of metabolically liable sites, introduction of hydrophilic substituents and design of more basic derivatives. Thermodynamic solubility, microsomal stability and lipophilicity of new compounds were experimentally evaluated, together with their MT1 and MT2 binding affinities. Introduction of a m-hydroxymethyl substituent on the phenyl ring of UCM765 and replacement of the replacement of the N,N-diphenyl-amino scaffold with a N-methyl-N-phenyl-amino one led to highly soluble compounds with good microsomal stability and receptor binding affinity. Docking studies into the receptor crystal structure provided a rationale for their binding affinity. Pharmacokinetic characterization in rats highlighted higher plasma concentrations for the N-methyl-N-phenyl-amino derivative, consistent with its improved microsomal stability and makes this compound worthy of consideration for further pharmacological investigation.

Chiral Recognition of Flexible Melatonin Receptor Ligands Induced by Conformational Equilibria.

N-anilinoethylamides are a class of melatoninergic agents with the aniline portion mimicking the indole ring of the natural ligand and the ethylamide chain reproducing that of melatonin. The simplest compound in this class, N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide (UCM793), has nanomolar binding affinity for MT1 and MT2 membrane receptors. To explore the effect of chain conformation on receptor binding, a methyl group was inserted on the methylene alpha or beta to the amide nitrogen and conformational equilibria were investigated by NMR spectroscopy and molecular dynamics simulations. Receptor affinity was conserved only for the beta-methyl derivative, which also showed significant stereoselectivity, with the (S) enantiomer being the eutomer. Molecular dynamics simulations, validated by NMR spectroscopy, showed that the beta-methyl group affects the conformational preferences of the ethylamide chain. Docking into the receptor crystal structure provides a rationale for the observed chiral recognition, suggesting that the (S)-beta-methyl group favors the conformation that better fits the receptor binding site.

Melatonin receptor ligands: A pharmaco-chemical perspective.

Melatonin MT1 and MT2 receptor ligands have been vigorously explored for the last 4 decades. Inspection of approximately 80 publications in the field revealed that most melatonergic ligands were structural analogues of melatonin combining three essential features of the parent compound: an aromatic ring bearing a methoxy group and an amide side chain in a relative arrangement similar to that present in melatonin. While several series of MT2 -selective agents-agonists, antagonists, or partial agonists-were reported, the field was lacking MT1 -selective agents. Herein, we describe various approaches toward the development of melatonergic ligands, keeping in mind that most of the molecules/pharmacophores obtained were essentially melatonin copies, even though diverse tri- or tetra-cyclic compounds were explored. In addition to lack of structural diversity, only few studies examined the activity of the reported melatonergic ligands in vivo. Moreover, an extensive pharmacological characterization including biopharmaceutical stability, pharmacokinetic properties, specificity toward other major receptors to name a few remained scarce. For example, many of the antagonists described were not stable in vivo, were not selective for the melatonin receptor subtype of interest, and were not fully characterized from a pharmacological standpoint. Indeed, virtual screening of large compound libraries has led to the recent discovery of potent and selective melatonin receptor agonists and partial agonists of new chemotypes. Having said this, the melatonergic field is still lacking subtype-selective melatonin receptor antagonists "active" in vivo, which are critical to our understanding of melatonin and melatonin receptors' role in basic physiology and disease.

Anticancer Melatplatin Prodrugs: High Effect and Low Toxicity, MT1-ER-Target and Immune Response In Vivo.

Multitargeted therapy could rectify various oncogenic pathways to block tumorigenesis and progression. The combination of endocrine-, immune-, and chemotherapy might exert a highly synergistic effect against certain tumors. Herein, a series of smart Pt(IV) prodrugs 3-6, named Melatplatin, were rationally designed not only to multitarget DNA, MT1, and estrogen receptor (ER) but also to activate immune response. Melatplatin, conjugating first-line chemotherapeutic Pt drugs with human endogenous melatonin (MT), significantly enhanced drug efficacy especially in ER high-expression (ER+) cells, among which 3 presented the most potent cytotoxicity toward ER+ MCF-7 with nanomolar IC50 values 100-fold lower than cisplatin. Melatplatin could bind well to melatonin receptor (MT1) according to molecular docking. Besides, 3 evidently increased intracellular accumulation and DNA damage, upregulated γH2AX and P53, and silenced NF-κB to induce massive apoptosis. Most strikingly, 3 effectively inhibited tumor growth and attenuated systemic toxicity compared to cisplatin in vivo, promoting lymphocyte proliferation in spleen to achieve immune modulation.

Evaluation of the anticonvulsant effect of novel melatonin derivatives in the intravenous pentylenetetrazol seizure test in mice.

The design of new pharmacologically active compounds with affinity to melatonin receptors has become an area of great interest during the last decade. Recently, we reported that newly synthesized melatonin derivatives, containing aroylhydrazone moiety in the indole scaffold, with the highest affinity to the elaborated pharmacophore model, possess an anticonvulsant activity in the maximal electroshock (MES) and 6Hz test in mice. We aimed further to explore the effect of these melatonin derivatives and the role of melatonin receptors on seizure threshold measured by the timed intravenous pentylenetetrazole (iv PTZ) infusion test in mice. Carbamazepine (CBZ) and melatonin were used as positive controls. Three out of six compounds, 3c, 3f, and 3e, respectively, dose-dependently increased the PTZ-induced seizure thresholds for myoclonic twitch, clonic, and tonic seizures comparable to the effect of CBZ and melatonin. The anticonvulsant effect of 3c, 3f, and 3e was blocked by the non-selective melatonin receptor antagonist luzindol suggesting the involvement of melatonin receptors in the activity of these compounds. Also docking study of 3c, 3f and 3e in the melatonin-binding site of melatonin receptor confirm the possible mechanism of action of these compounds involving melatonin receptors. Our previous and present results suggest that 3c, 3f, and 3e can be considered promising agents with anticonvulsant activity on melatonin receptors in the brain.

Melatonin receptor structures shed new light on melatonin research.

The tryptophan derivative melatonin is an evolutionary old molecule that is involved in a pleiotropy of physiological functions. In humans, age-related decline of circulating melatonin levels and/or dysregulation of its circadian synthesis pattern have been associated with several disorders and disease states. Several molecular targets have been proposed for melatonin since its discovery, in 1959. Among them, melatonin MT1 and MT2 receptors are the best characterized melatonin targets, mediating melatonin effects in a variety of tissues. They belong to the superfamily of G protein-coupled receptors. Two back-to-back articles published in the "Nature" Journal earlier this year present the first crystal structures of the human MT1 and MT2 in its inactive states. Here, we will briefly outline the discovery path of melatonin receptors until their structural elucidation and discuss how these new findings will guide future research toward a better understanding of their function and rational drug design.

XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity.

The human MT1 and MT2 melatonin receptors1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns3. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer3, and MT2 has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure8 of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [3H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.

Structural basis of ligand recognition at the human MT1 melatonin receptor.

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 manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Characterization of the Mel1c melatoninergic receptor in platypus (Ornithorhynchus anatinus).

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.

Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50.

BACKGROUND AND PURPOSE: Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT1 receptor and in the inactivation of the closely related orphan receptor GPR50. EXPERIMENTAL APPROACH: Chimeric MT1 -GPR50 receptors were generated and functionally analysed in terms of 2-[125 I]iodomelatonin binding, Gi /cAMP signalling and ß-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations. KEY RESULTS: MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of the MT1 receptor (ii) the most suitable features for melatonin binding in MT1 receptors and (iii) major predicted rearrangements upon MT1 receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 receptors and GPR50 respectively. CONCLUSIONS AND IMPLICATIONS: Our work demonstrated the crucial role of the E2 loop for MT1 receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

New coumarin-based fluorescent melatonin ligands. Design, synthesis and pharmacological characterization.

The design and synthesis of a series of new fluorescent coumarin-containing melatonin analogues is presented. The combination of high-binding affinities for human melatonergic receptors (h-MT1R and h-MT2R) and fluorescent properties, derived from the inclusion of melatonin pharmacophoric elements in the coumarin scaffold, yielded suitable candidates for the development of MT1R and MT2R fluorescent probes for imaging in biological media.

Synthesis and functional characterization of substituted isoquinolinones as MT2-selective melatoninergic ligands.

A series of substituted isoquinolinones were synthesized and their binding affinities and functional activities towards human melatonin MT1 and MT2 receptors were evaluated. Structure-activity relationship analysis revealed that substituted isoquinolinones bearing a 3-methoxybenzyloxyl group at C5, C6 or C7 position respectively (C5>C6>C7 in terms of their potency) conferred effective binding and selectivity toward the MT2 receptor, with 15b as the most potent compound. Most of the tested compounds were MT2-selective agonists as revealed in receptor-mediated cAMP inhibition, intracellular Ca2+ mobilization and phosphorylation of extracellular signal-regulated protein kinases. Intriguingly, compounds 7e and 7f bearing a 4-methoxybenzyloxyl group or 4-methylbenzyloxyl at C6 behaved as weak MT2-selective antagonists. These results suggest that substituted isoquinolinones represent a novel family of MT2-selective melatonin ligands. The position of the substituted benzyloxyl group, and the substituents on the benzyl ring appeared to dictate the functional characteristics of these compounds.

Efficient synthesis of hexahydroindenopyridines and their potential as melatoninergic ligands.

Hexahydroindenopyridine (HHIP) is an interesting tricyclic piperidine nucleus that is structurally related to melatonin, a serotonin-derived neurohormone. Melatonin receptor ligands have applications in several cellular, neuroendocrine and neurophysiological disorders, including depression and/or insomnia. We report herein an efficient two-step method to prepare new HHIP via enamine C-alkylation-cyclization. The influence of substituents on the benzene ring and the nitrogen atom on melatoninergic receptors has been studied. Among the 25 synthesized HHIPs, some of them containing methylenedioxy (series 2) and 8-chloro-7-methoxy substituents (series 4) on the benzene ring revealed affinity for the MT1 and/or the MT2 receptors within the nanomolar range or low micromolar. Similar activities were also encountered for those presenting urea (4g), N-aryl (2e) and N-alkyl (2f) acetamide functions. Therefore, new synthesized compounds with a HHIP nucleus have emerged as new promising leads towards the discovery of melatoninergic ligands which could provide new therapeutic agents.

Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes.

The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs)-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.

MT1 and MT2 melatonin receptors: ligands, models, oligomers, and therapeutic potential.

Numerous physiological functions of the pineal gland hormone melatonin are mediated via activation of two G-protein-coupled receptors, MT1 and MT2. The melatonergic drugs on the market, ramelteon and agomelatine, as well as the most advanced drug candidates under clinical evaluation, tasimelteon and TIK-301, are high-affinity nonselective MT1/MT2 agonists. A great number of MT2-selective ligands and, more recently, several MT1-selective agents have been reported to date. Herein, we review recent advances in the field focusing on high-affinity agonists and antagonists and those displaying selectivity toward MT1 and MT2 receptors. Moreover, the existing models of MT1 and MT2 receptors as well as the current status in the emerging field of melatonin receptor oligomerization are critically discussed. In addition to the already existing indications, such as insomnia, circadian sleep disorders, and depression, new potential therapeutic applications of melatonergic ligands including cardiovascular regulation, appetite control, tumor growth inhibition, and neurodegenerative diseases are presented.

A molecular and chemical perspective in defining melatonin receptor subtype selectivity.

Melatonin is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle. In addition to its intrinsic antioxidant property, the neurohormone has renowned regulatory roles in the control of circadian rhythm and exerts its physiological actions primarily by interacting with the G protein-coupled MT1 and MT2 transmembrane receptors. The two melatonin receptor subtypes display identical ligand binding characteristics and mediate a myriad of signaling pathways, including adenylyl cyclase inhibition, phospholipase C stimulation and the regulation of other effector molecules. Both MT1 and MT2 receptors are widely expressed in the central nervous system as well as many peripheral tissues, but each receptor subtype can be linked to specific functional responses at the target tissue. Given the broad therapeutic implications of melatonin receptors in chronobiology, immunomodulation, endocrine regulation, reproductive functions and cancer development, drug discovery and development programs have been directed at identifying chemical molecules that bind to the two melatonin receptor subtypes. However, all of the melatoninergics in the market act on both subtypes of melatonin receptors without significant selectivity. To facilitate the design and development of novel therapeutic agents, it is necessary to understand the intrinsic differences between MT1 and MT2 that determine ligand binding, functional efficacy, and signaling specificity. This review summarizes our current knowledge in differentiating MT1 and MT2 receptors and their signaling capacities. The use of homology modeling in the mapping of the ligand-binding pocket will be described. Identification of conserved and distinct residues will be tremendously useful in the design of highly selective ligands.