The increasing social pressures and workloads are disrupting the natural sleep cycles of an growing proportion of the population, with more and more young and old people suffering from insomnia caused by circadian rhythm sleep disorder. Melatonin, a potent remedy for sleep disorders stemming from these rhythmic imbalances, targets two specific receptors: MTNR1A and MT2. Considering the distinct role of the MTNR1A receptor in melatonin signaling, we have opted to focus on MTNR1A in the development of our targeted screening platform for the identification of melatonin receptor agonists.

In the 2024 iGEM competition, we conducted comprehensive human practices studies to gain insight into the clinical status of circadian rhythm disorders, the advantages and disadvantages of aromatherapy, patient expectations of treatment, potential ethical and safety issues that may arise in the developing programs, and how we can serve the synthetic biology community in the development of new potential drugs. In order to gain insight into these matters, we engaged with a range of stakeholders, including the general public, sleep specialists, synthetic biologists, and pharmaceutical researchers. The findings of our social research informed our decision-making process regarding the therapeutic target and the screened subject that we ultimately selected for use in our project. Furthermore, they influenced the experimental design and the discussion of our future implementation in a number of ways. Figure 1 provides a visual representation of the manner in which our human practice studies have been integrated into the process of our work throughout the iGEM competition.

Figure 1. Schematic outline of the HP Framework

The World Health Organization (WHO) has identified sleep disorders as the second most prevalent mental health condition globally, affecting 27% of the population that estimated. According to the White Paper on the Sleep Health of Chinese Residents in 2024,the highest two proportions of sleep disturbances were both related to Circadian rhythm sleep disorders, which are a distinct class of sleep disorders characterized by a mismatch between the desired timing of sleep and the ability to fall asleep and remain asleep. Since many patients are clinically misdiagnosed for insomnia, it is estimated that there are 10% of adult and 16% of adolescent sleep disorders patients may have a circadian rhythm sleep disorder (CRSD), which is much higher than the 3% who are diagnosed CRSD.

In our interview with Dr. Ouyang Xuan, a clinician of Xiangya Medical College, we learned that more and more old people are suffering from sleep disorders caused by CRSD against the backdrop of an increasingly aging population, and the number of young people with CRSD is higher and higher due to work and other reason in recent years.

Figure 2. Interview with Dr. Ouyang Xuan, a clinician at Xiangya Medical College

According to the internet survey, the frequency of searches for terms like insomnia, jet lag and sleep disorders has also been rising steadily each year. At the same time, the literature on circadian rhythm sleep disorders on the PubMed database website has also been increasing annually. This trend collectively indicate that CRSD is becoming an increasingly significant concern.

Figure 3.Background data. (A) the data of the White Paper on the Sleep Health of Chinese Residents. (B) The proportion of causes of insomnia. (C) An increase in the number of studies on circadian rhythm disorders.

In our research, the latest edition of the China Insomnia Diagnostic and Treatment Guidelines provides ideas for addressing CRSD. This guideline shows that melatonin is used for the short-term treatment of delayed circadian rhythm syndrome, insomnia caused by jet lag, and insomnia in elder adults aged 55 years and above, which is the main drugs used to treat sleep disorders caused by CRSD.

Melatonin supplements may be accompanied by a range of side effects such as headaches, nausea and dizziness. A review of the literature reveals that melatonin acts through two types of receptors: MTNR1A and MTNR1B². MTNR1A receptors are mainly expressed in the brain, whereas MTNR1B receptors are not only expressed in the brain, but are also widely present in organs such as the retina and the gastrointestinal tract^3,4.. This phenomenon aroused our great interest, so we specially interviewed Dr. Elaine Lei, whose expertise lies in the fields of medical statistics and traditional Chinese medicine. Dr. Lei pointed out that although both MTNR1A and MTNR1B can affect sleep, it is MTNR1A that is directly related to circadian rhythms, and MTNR1B, due to its wide distribution in the body, is involved in a variety of physiological activities including neurotransmission, cardiovascular system, and regulation of the immune system, which may be one of the reasons why melatonin causes side effects. Furthermore, Dr. Lei mentioned that a recent study has shown that while most current melatonin receptor agonists are broad-spectrum, molecules with the ability to selectively bind MTNR1A have greater potential for treating insomnia.

Through our discussions, we determined the general direction of the project is to screen molecules that can selectively bind to MTNR1A.

Figure 4. Interview with Dr. Yilin Lei, whose expertise lies in the fields of medical statistics and traditional Chinese medicine

The process of engineering a new cell design for screening purposes is inherently challenging. In our initial attempt to establish the MTNR1A-Ca²⁺-NFAT-Nluc pathway, we encountered difficulties with the functionality of our system. Although we tried changing the copy number of NFAT to 1×NFAT, 6×NFAT and 7×NFAT to see if this would affect the response, the result was not as expected. In our discussion with Dr. Yiming Dong, a synthetic biologist (Ph.D.) from Peking University whose research interests include the development of gene editing tools and their applications in metabolic engineering and DNA information storage. He suggested that we attempt to detect the concentration of Ca²⁺ upstream of NFAT, as NFAT is not particularly sensitive to Ca²⁺. He also proposed that we can use GCaMP to report the concentration of Ca²⁺. This discussion proved invaluable in enabling us to redesign our project, as we discovered that the up-regulation of Ca²⁺ concentration is remarkably brief (Please see our Results page, for more details ).

The establishment of a molecular screening platform for melatonin receptor agonists has given rise to a new challenge, namely the question of which substances should be screened with the platform.

In response to our inquiry, Dr. Gengyao Hu, a clinician at The First Affiliated Hospital of AFMU, highlighted that the prevailing clinical approach in China is pharmacological therapy. However, she underscored the necessity of considering the significant prevalence of insomnia, which is estimated to affect approximately 300 million individuals, while the number of individuals seeking medical attention remains relatively low at 19 million. Individuals presenting with mild to moderate symptoms are more likely to purchase over-the-counter healthcare products, such as aromatherapy essential oils, to self-manage their condition.

A review of the literature reveals that aromatherapy, based on plant essential oils, has a long history worldwide. Its safety, natural, convenient and environmentally friendly characteristics have earned it widespread acceptance⁵. Nevertheless, the uncertainty surrounding the underlying mechanisms and the existence of both efficacious and inefficacious products on the market present obstacles to its sustained expansion⁶

Figure 5. Interview with Dr. Gengyao Hu, a clinician of The First Affiliated Hospital of AFMU

Inspired by the aforementioned findings, we designed a social survey based on a questionnaire to investigate the acceptability of existing drugs and natural substances among patients with sleep disorders. Among the 2,580 individuals who filled out our questionnaire, there are 1919 samples facing with sleep disorders. (Figure 5A)The data showed that 73.7% of patients were amenable to the use of natural substances, a figure that is nearly double that of those willing to use drugs (41.7%) (Figure 5B). The results demonstrated that, in comparison to pharmaceuticals, a greater proportion of patients expressed a willingness to accept natural substances, including Chinese herbs and aromatherapy plant essential oils, as a means of improving their sleep disorders.

Figure 6. Questionnaire-based social study. (A) People suffering from sleep disorders accounted for 74.4% of the total sample. (B) Questionnaire-based social study on insomnia. Aromatherapy is the treatment that people faced with insomnia were most willing to try,which accounts for 73.7%.

So we decided to use our platform Knight: a designer-cell based drug screening platform for MTNR1A agonist discovery to screen essential oils that work by activating melatonin receptors.

Using MTNR1A receptor agonists screening platform, we identified two essential oils with therapeutic properties among the 34 plant essential oils. During the subsequent visit, the subject reiterated the issue of structural similarity between MTNR1A and MTNR1B receptors, which could lead to the same side effects as melatonin. She emphasised the necessity for further investigation to ascertain the specificity of the current results, particularly in regard to whether they are solely responsive to MTNR1A and not MTNR1B. In light of Dr. Lei's proposal and the findings in the literature, we have initiated the development of a molecular screening platform based on MTNR1B. The intention is to conduct a reverse verification of the screening results with MTNR1A in order to ensure the identification of specific substances that activate MTNR1A but not MTNR1B. This will facilitate more targeted treatment of insomnia associated with circadian rhythm disorders.

Building upon the implementation of our platform to successfully identify two of the 34 plant essential oils with established efficacy, our objective is to expeditiously determine small molecules that possess genuinely efficacy in activating melatonin receptors. In the course of the interview with Yangze River Pharmaceutical (Group) Co., Ltd., the concept of computer-aided drug design (CADD) was introduced by Ms Guangju Jiang, the company's marketing manager. This provided us with a valuable insight into the potential for CADD in the screening of small molecules. Computational chemistry-based CADD facilitates the discovery and rational design of novel drug molecules by simulating, calculating, and predicting the interaction between ligand and receptor biomolecules, as well as analysing the interaction between drug activity and structure. Given the complex nature of essential oils, conducting comprehensive synthetic or biological tests is a formidable undertaking. The utilisation of virtual screening can effectively streamline the process by narrowing down the potential small molecule candidates and expediting the identification of effective ones. This approach not only reduces the time and cost investment but also enhances the overall efficiency of the screening process.

Following our attendance at the Building the Future with Unity - 2024 iGEMer Central China Regional Exchange Conference, we were advised that essential oils are mixtures of various small molecules. Furthermore, a judge suggested that virtual molecular interconnection could be employed initially to simulate and identify the small molecules with the highest structural affinity for melatonin receptors. This approach may prove time- and cost-effective, while also offering an environmentally friendly and economical solution (Please refer to our result and model page,for further details).

Figure 7. Questionnaire-based social study. (A) Our interviews with Ms. Guangju Jiang. (B) The judge gave a critical suggestion on our project

In order to consider the future implementation of our project, we made a return visit to Dr. Hu. In a subsequent interview, we updated Dr. Hu on the progress of our project, and we further discussed with her the future implementation of our project. In response to our questions, she noted that each strategy may have its own advantages or advantages for different ingredients, and the key aspect is the quantification of the active pharmaceutical ingredient (API), which will help evaluate results more accurately. It is important for us to determine the future dosage form. In terms of concentration determination of apis, we have reviewed the literature and intend to use high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) to overcome the limitations of traditional UV-VIS spectroscopy. (Please see the implementation page for more details,for further details.)