We have established a screening platform predicated on melatonin receptor agonists, with the goal of harnessing synthetic biology principles to develop more efficacious treatments for global sleep disorders. Two essential oil, Clove Pod and Caraway, have been validated as MTNR1A agonist. To further probe the small molecule constituents within these essential oils that effectively interact with our platform, we conducted molecular docking experiments on the small molecules in conjunction with their respective receptor proteins. Molecular docking is a computational method used to simulate molecular interactions, particularly in the structure-based drug design. It predicts the optimal binding conformation between a ligand and a receptor, typically a protein, thereby identifying the state with the lowest binding free energy¹. By applying this approach to our study, we aimed to identify small molecules within Clove Pod and Caraway that effectively interact with MTNR1A receptors.
Melatonin receptors MTNR1A and MTNR1B are Gi protein-coupled receptors (GPCRs), which play critical roles in regulating circadian rhythms and sleep patterns. The three-dimensional structures of MTNR1A and MTNR1B have been elucidated by Vadim Cherezov and his group² and the Tao laboratory³ providing valuable insights into their functional properties. Notably, both receptors exhibit a high degree of sequence identity (68%), indicating significant overall structural similarity. Both MTNR1A and MTNR1B possess a characteristic seven-transmembrane helix bundle topology .(Figure 1), comprising three extracellular and intracellular loops, as well as a short amphipathic helix oriented parallel to the membrane.

Figure.1: Overall architecture of MTNR1A/MTNR1B binds with the central Ramelteon. (A) MTNR1A. (B) MTNR1B. Protein structures are shown as rainbow cartoon, colored by helix bundle, Ramelteon molecules are shown as red sticks. ECL2 closes off the binding site to the extracellular space is shown as blue-purple. Inter-helix links are illustrated as gray.

In terms of their functional properties, both MTNR1A and MTNR1B have been shown to bind ramelteon, a FDA approved medication for the treatment of insomnia. The binding site of ramelteon within MTNR1A、MTNR1B have been resolved by X-ray crystallography at resolutions of 2.8 Å, revealing a highly conserved orthosteric pocket that is formed by MTNR1A，the convergence of transmembrane helices TM3, TM5, TM6, TM7, as well as extracellular loop 2 (ECL2). This compact binding site exhibits distinctive ligand-receptor interactions, primarily mediated through strong π-π stacking with Phe179, in addition to auxiliary hydrogen bonds with Asn162 and Gln181 (Figure 2). MTNR1B, on the other hand, posseses a binding pocket that is approximately 50 ų larger than that of MTNR1A, primarily due to an expansion in the area surrounding the alkylamide tail and the hydrophobic subpocket. Although the composition of the binding site residues is conserved between MTNR1A and MTNR1B, notable conformational differences are observed, including variations in the side chains of Y200 ^5.38 and Y294 ^7.39 , and the backbone region around P174 ^4.59 .

Figure 2. Ramelteon interaction with MTNR1A/MTNR1B. (A) MTNR1A protein (PDB ID:6ME2) is shown as cyan cartoon. Residues around ramelteon are shown as cyan sticks. Ramelteon molecule is shown as yellow sticks. Hydrogen bonds between ligand and protein are shown as red dashes. (B) MTNR1B protein (PDB ID:7VH0) is shown as blue cartoon. Residues around ramelteon are shown as blue sticks. Ramelteon molecule is shown as yellow sticks.

Both MTNR1A and MTNR1B possess a membrane-buried lateral ligand entry channel, which facilitates access of ligands to their respective orthosteric binding sites. However, structural analyses of MTNR1B （Figure 2B）reveal an additional secondary access route that originates from the solvent-exposed extracellular region. This additional pathway may contribute to the distinct pharmacological properties of MTNR1B compared to MTNR1A².
Interestingly, MTNR1B exhibits some conformational differences relative to MTNR1A, which may be associated with variations in ligand selectivity. The ligand access channel in MTNR1B, formed by helix IV and V, is is characterized by a narrower diameter of approximately 2.6 Å. This constriction is likely due to the presence of a hydrogen bond between Y200 and N175, which restricts the channel's opening³. To investigate the binding affinity and specificity of constituents in the essential oils for MTNR1A and MTNR1B, we performed molecular docking studies of all compounds present in the oil against both receptors, aiming to identify potential MTNR1A agonists and understand their mechanism of action.

To establish a reliable benchmark for our docking methodology, we incorporated melatonin and its well-characterized analogs in the docking experiments. These ligands include ramelteon, Iodomelatonin, 5HEAT and CTL 01-05-B-A05, which are known ligands of MTNR1A/MTNR1B receptors and have been extensively studied for their pharmacological properties. The molecules used in the docking experiments are listed in Table 1. The redocking results for ramelteon against MTNR1A/MTNR1B receptors yielded RMSD values of 1.126 and 1.151, compared to the solved structure, indicating good reliability of the docking protocol (Table 2 and Figure 3A and 3B).

*RMSD values were calculated using DockRMSD4 for ligand atoms only.

Results also show that CTL 01-05-B-A05 outperforms other ligands in binding affinity to both MTNR1A and MTNR1B receptors. Notably, CTL 01-05-B-A05 demonstrate a clear preference for MTNR1A over MTNR1B. In contrast, the results suggest that melatonin is a relatively weak binder towards MTNR1A/MTNR1B receptors, consistent with its role in promoting sleep and being naturally degraded during the sleep cycle. These findings validate the feasibility of the docking parameters employed. Then, we performed in silico screening of all constituents of Clove Pod⁵ and Caraway⁶essential oils by molecular docking. Interestingly, the binding affinity data suggest that major molecules present in essential oils can bind to both MTNR1A/MTNR1B receptors. Only Eugenol exhibited a preference towards MTNR1A. Caryophyllene and α-humulene exhibit comparable affinities to melatonin (Figure 3C). One thing needs to mention is that in Clove Pod, Eugenol is up to 55.6%, while checking its docking result, it exhibits weak binding to MTNR1A but not MTNR1B. To further investigate why Clove Pod can activate MTNR1A, we proceeded with further cell experiments using both Caryophyllene, eugenyl-acetate and eugenol.

Figure 3. Overview of molecular docking results. Redocking result of Rameltenon with the MTNR1A (A) and MTNR1B (B) receptor protein. Protein structures are shown as green (MTNR1A) and cyan (MTNR1B) cartoons. Ramelteon molecules in complex with melatonin receptors are shown as magenta sticks and redocked molecules are shown as yellow sticks. (C) Heatmap of binding affinities of various melatonin receptor agonists and natural small molecules from Clove Pod and Caraway towards MTNR1A and MTNR1B. The main constituents’ ratios were presented within parentheses. Redocking result of Ramelteon with the MTNR1A.

AutoDock Vina⁷ and AutoDock Tools⁸ were employed to dock small molecules into the MTN1RA/ MTN1RB proteins. Macromolecules were processed using AutoDock Tools to remove water molecules and add polar hydrogens. Subsequently, Kollman Charges were added and the macromolecule receptor was saved as PDBQT format. Small molecule, being the main constituents of essential oils, were retrieved from the PubChem database according to the literature. Next, ligand files were prepared using AutoDock Tools, where charges were added and rotatable bonds were determined.
To perform docking, a grid box was selected based on its original ligand ramelteon. Figure 3 illustrates the details of the grid box. A size sufficient for covering all residues potentially involved in binding was determined. To ensure precise docking, we set exhaustiveness to 72 and output 20 modes for each docking run. All docking runs were carried out three times to eliminated random effects. The results were visualized and analyzed using open-source Pymol (Figure 3A and 3B).

Figure 4.（A） Docking box for small molecules with the MTNR1A receptor protein. (B) Docking box for small molecules with the MTNR1B receptor protein.