In this year's project, we aim to utilize synthetic biology principles to engineer a mammalian cell-based high-throughput drug screening platform for melatonin receptor agonists. The melatonin receptors (MTs), specifically MTNR1A (MT1), belong to family A of G protein-coupled receptors (GPCRs), with melatonin serving as their endogenous ligand. These receptors play a pivotal role in regulating the human circadian rhythm and are significant therapeutic targets for treating sleep disorder. Notably, MTNR1A is believed to primarily modulate non-rapid eye movement (NREM) sleep. We have chosen to focus on MTNR1A to assess its activity upon interaction with effective ingredients in essential oils and other aromatic drugs, screening for compounds capable of effectively treating sleep disorders¹.

Our favored basic part, BBa_K5267001, is one of the most crucial part in our parts collection, as it encodes the melatonin receptor MTNR1A and act as a sensor of compounds capable of effectively activating melatonin receptor. It lays the foundational basis for cell-based screening platform and subsequent experiments in our project for screening melatonin receptor agonist.

According to previous studies, upon melatonin-mediated activation of the melatonin receptor MTNR1A, several pathways, such as cAMP/PKA/CREB pathways and Ca²⁺ signaling pathways are activated². (Figure 2). The cAMP/PKA/CREB pathway is activated when melatonin binds to the MTNR1A. This binding activates the associated G proteins, which in turn stimulate adenylate cyclase to convert ATP into cyclic adenosine monophosphate (cAMP). The increase in cAMP levels activates protein kinase A (PKA), which phosphorylates various target proteins, leading to a cascade of downstream effects. For instance, PKA can modulate the activity of transcription factors such as cAMP response element-binding protein (CREB), thereby influencing gene expression related to cellular processes such as metabolism, growth, and apoptosis³. Besides, previous studies demonstrate an increase in intracellular Ca²⁺ levels upon melatonin receptor activation, which can occur through several mechanisms, including the release of Ca²⁺ from the endoplasmic reticulum into the cytoso⁴.

Based on these well-characterized signaling pathways, we therefore utilized two distinct methodologies to validate the activation of Melatonin receptor, specifically focusing on the downstream signaling transduction of cAMP/PKA/CREB pathway and the Ca²⁺< signaling pathway.

We first focused on the cAMP/PKA/CREB pathway, employing the synthetic CRE promoter to sense pathway activation. Plasmids expressing MTNR1A part (Basic part: BBa_K5267047) and a chimeric cAMP-sensing promoter (Composite part: BBa_K5267040) containing multiple repeats of CREB binding sites followed by a miniCMV promoter were co-transfected into HEK-293T cells, and the melatonin was added post-transfection. 48 hours post-transfection, the Nanoluc activity was assessed to characterize the activation of the cAMP/PKA/CREB pathway. Theoretically, Activation of MTNR1A by melatonin would lead to an increase in cAMP and the phosphorylation of the endogenous CREB protein. The phosphorylated CREB protein would then bind to the CREB site of the PCre promoter, activating the expression of the downstream reporter genes.

The results showed that, upon activation of melatonin receptors by adding 1nM melatonin, the activity of NanoLuc was significantly elevated compared to the control group without melatonin stimulation. This demonstrates that the basic part MTNR1A is capable of sensing melatonin stimulation and subsequently initiating transcription of chimeric PCre promoter as expected.

The functionality of the our basic part was also characterized based on a cell-based platform the sense the downstream Ca²⁺ signaling pathway upon activation of Melatonin receptors. For such matter, we used GCaMP (Composite part: BBa_K3755007), an ultra-sensitive protein-based sensor that allows visualization of Ca²⁺ dynamics within living cells. Thapsigargin was used as a positive control to stimulate the cells, as it is known to induce endoplasmic reticulum stress, leading to an up-regulation of Ca²⁺ in the cytoplasm⁵.

The results showed that the addition of both melatonin and Thapsigargin induced a significant surge in the Ca²⁺ fluorescent signal. The Ca²⁺ surge was short-lived, dissipating within 100 seconds of melatonin stimulation. In contrast, Thapsigargin induced a prolonged elevation of intracellular calcium levels. This demonstrates that our basic part is capable of activating the Ca²⁺ signaling pathway upon melatonin stimulation as expected.