In delving into how melatonin triggers adverse reactions when regulating biological rhythms, we learned from Dr. Lei that MTNR1A play a critical role in sleep disorders instead of MTNR1B. (see our Human Practices page for more details). MTNR1A receptors are more directly related to regulating rapid eye movement (REM) sleep, while MTNR1B receptors promote non-rapid eye movement (NREM) sleep ¹⁰. These two receptors belong to the G protein-coupled receptor family, and MTNR1A receptors are particularly abundantly expressed in the brain's suprachiasmatic nucleus (SCN) region, which is the center of circadian regulation and essential for rhythmic regulation. Meanwhile, MTNR1B receptors are more widely expressed in peripheral organs such as the retina, cardiovascular system, liver, kidney, spleen, and intestine ¹¹. In addition to playing a pivotal role in controlling the cyclic release of melatonin, MTNR1B receptors are also involved in regulating blood glucose, cardiovascular function, the nervous system, and the reproductive system, among other physiological activities. This wide range of functions of MTNR1B receptors may be a critical factor in the multiple side effects of melatonin in regulating biological rhythms ¹⁰. This means that the development of an agonist specifically targeting the MTNR1A receptor might be able to modulate circadian rhythms while avoiding the side effects caused by existing melatonin receptor agonist drugs.

MLT receptors, MTNR1A, is G-protein-coupled receptor that mediate the effects of the hormone melatonin. The MTNR1A can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to modulate cAMP differentially, protein kinase A signaling, and CREB phosphorylation levels in cells ¹². The MTNR1A receptor also increases phosphorylation of mitogen-activated protein kinase 1/2 and extracellular signal-regulated kinase 1/2, as well as activation of the phospholipase–calcium (PLC/ Ca ²⁺) signaling pathway. Melatonin receptor activation decreases neuronal firing through activation of the MTNR1A receptor in the SCN and areas of the limbic system, which may mediate the sleep-promoting properties of melatonin ^13,14.(Figure 2)

Figure 2: Signal pathway of MTNR1A.

To build up a platform that can be used for screening of potential melatonin receptor agonists, we utilized HEK293T cells as chassis cells and target the melatonin receptor subtype MTNR1A. Following extensive screening and optimization efforts (see our Engineering page for more details), we designed and constructed a series of gene circuits based on key components in the cAMP-PKA signaling pathway downstream of the MTNR1A receptor, with the goal of developing a designer cell line that can rapidly detect MTNR1A receptor activation. Upon the activation of MTNR1A receptor on the surface of these designer cells, the production of an readily measurable reporter protein, Nanoluc luciferase, is trigered, allowing for accurate quantification and analysis of its expression level through an enzyme marker. The experimental results showed that this screening platform, constructed based on designer cells (HEK _MT,HEK _CTL ), effectively responds to MTNR1A activation by melatonin. This approach provides an effective strategy for developing agonist drug molecules explicitly targeting the MTNR1A receptor and screening functionally closely related drugs.

To assess the effectiveness of our screening platform, we leveraged insights gained from our previous research on sleep disorder interventions (see our Human Practices page for more details). Utilizing this knowledge, we applied the constructed screening platform to identify molecules that activating melatonin receptor MTNR1A among aromatic essential oils with better public acceptance（73.7%）. Fortunately, we successfully identified two essential oils (Kudzu and Clove Pod), from a pool of 34 plant essential oils, that demonstrate significant activating effects on the melatonin MTNR1A receptor.

As we further explored the key active ingredients in these two screened essential oils, we drew upon the insights gained during our participation in the Building the Future with Unity - 2024 iGEMer Central China Regional Exchange Conference and our interactions with Ms. Guangju Jiang(see our Human Practices page for more details), both of whom emphasized the potential of molecular docking technology in improving the efficiency of small molecule interaction screening (see our Human Practices page for more details). Under the guidance of Ms. Zhang from our team, we employed molecular docking technology to analyze in detail the interactions between two small molecules from these essential oils and melatonin MTNR1A receptors. This analysis successfully revealed two naturally occurring active small molecules with significant binding ability. Meanwhile, following the expert recommendations (see our Human Practices page for more details), we also evaluated the binding ability of these two small molecules with the melatonin MTNR1B receptor, thus providing a reliable theoretical basis for these small molecules as potential target molecules for the melatonin MTNR1A receptor.

We hope that that future developments will enable us to incorporate high-throughput screening for selective receptor agonists, allowing us to integrate signaling pathways of different receptors using synthetic biology's logic operations within the same cell. This potential breakthrough could lead to the discovery of more selective melatonin receptor agonists, ultimately providing individuals, who struggle with busy lifestyles and limited self-care time with a tranquil, gentle, and comfortable night’s rest.