Sleep disorders have been linked to a range of adverse outcomes, including emotional lability, periorbital hyperpigmentation. According to the World Health Organization, the global rate of sleep disorders is 27%, which has become the second most common mental disorder in the world. Through our human practice study, we found that existing drug screening methods have certain limitations. Therefore, we designed a screening platform for melatonin receptor (MTR) agonist based on MTR and the downstream signaling pathways. In this part , we are main going to discuss how to decompose out goal into modules and parts.
Melatonin (MLT) is ubiquitous molecule with wide distribution in nature and is produced by many living organisms. In human beings, pineal gland is the major site for melatonin production. Melatonin mostly exerts its effect through different pathways with melatonin receptor 1 (MTNR1A) and melatonin receptor 2 (MTNR1B) being the predominant type of receptor that are mainly expressed by many mammalian organs.
The two types guide in different downstream signaling pathways. For example, the MTNR1A can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP , protein kinase A signaling, and CREB phosphorylation levels in cell. The MTNR1A receptor can also activate the phospholipase–calcium (PLC/ Ca2+) signaling pathway. MTNR1B melatonin receptor activation inhibits both cAMP production and cGMP formation and activates protein kinase C (PKC) in the SCN.
Through our investigations, we found that MTNR1A plays a more extensive and important role in mediating sleep-related physiological processes, compared to MTNR1B. Therefore, we preferred to consider MTNR1A and its downstream signaling pathways when designing.
The reporting module includes two parts: response elements and detectable reporting elements. As for the response elements, we chose two major signaling pathways for the response to melatonin receptor agonists: cAMP response element (CRE) and nuclear factor of activated T-cells (NFAT).
CRE is a specific DNA sequence that binds transcription factors, primarily CRE-binding protein (CREB), to regulate gene transcription. CRE plays a key role in signal transduction pathways that rely on the cAMP (cyclic adenosine monophosphate) signaling cascade. When the potential melatonin receptor agonists bind to the melatonin receptors, a series of chain signaling reaction between AC-cAMP-PKA will activate the CREB (CRE binding protein) into activation state, then the activated CREB will bind to the CER, initiating the transcription and ultimately causing downstream gene expression.
NFAT is a member of transcription factors that play a critical role in regulating gene expression, particularly in immune cells, such as T cells, as well as in other cell types. The NFAT pathway is primarily activated by calcium (Ca2+) signaling. When MTNR1A is activated, the 4,5-bisphosphate (PIP2) is decomposed into two second messengers: Diacylglycerol (DAG), which activates Protein Kinase C (PKC) and Inositol 1,4,5-trisphosphate (IP3). IP3 stimulates the release of Ca²⁺ from intracellular stores (like the endoplasmic reticulum). Then the release of calcium from intracellular stores increases cytosolic calcium levels, which leads to the activation of NFAT. The activated NFAT then triggers the expression of our reporting genes1.
The reporting module majorly includes two reporting genes: Nluc and GCaMP. They help to generate enzymes capable of reacting biochemically with the substrate, thus enabling visual detection of the product, and ultimately helping our project to translate the activation of the melatonin receptor into signals that can be easily detected, thus helping to enable the screening and detection of melatonin receptor agonists2.
Figure 2.The pathways of the reporting module.
1
2
Ahmad, S. B. et al. Melatonin and Health: Insights of Melatonin Action, Biological Functions, and Associated Disorders. Cell Mol Neurobiol 43, 2437-2458 (2023). https://doi.org/10.1007/s10571-023-01324-w
Stauch, B., Johansson, L. C. & Cherezov, V. Structural insights into melatonin receptors. FEBS J 287, 1496-1510 (2020). https://doi.org/10.1111/febs.15128
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