The design of a melatonin-responsive gene circuit and the construction of a designer-cell based drug screening assay are the key milestones of achieving engineering success in our project. Following the design-build-test-learn (DBTL) cycle (Figure 1) we have successfully constructed a drug screening plat from integrating in silico virtual screening and a mammalian cell-based assay. We have demonstrated that our screening pipeline helped identifying eugenol, a constituent of Clove Pod essential oil, from 37 candidate essential oils we purchased from the market.. In this section, we describe in detail how this was achieved through multiple iterations of DBTL cycle. Figure 1. Design-Build-Test-Learn Engineering Workflow (you can scroll horizontally to see more information)

Design

As a G protein-coupled receptor, human melatonin receptor 1a (MTNR1A) has been identified to activate PKC/Ca²⁺ signaling¹, which results in the elevation of intracellular Ca²⁺ levels. Hence, we designed a mammalian genetic circuit that utilizes the Nuclear Factors of Activated T cells (NFAT) to rewire the elevation of calcium signaling to transcriptional activation of the reporter gene. The elevated Ca²⁺ triggers the dephosphorylation of NFAT, which would then bind to the NFAT binding sites on the chimeric promoter and activate the downstream gene expression ² (Fig. 2). To simplify the quantification of reporter gene transcription activation, we used secreted Nanoluc luciferase (Nluc) as our reporter gene. By fusing Nanoluc coding gene with the DNA encoding secretion signaling peptide from IgK protein, we would be able to quantify the activation of target gene by measuring the Nanoluc activity in the culture medium.

Build & Test

To evaluate the functionality and sensitivity of our genetically engineered pathway, we co-transfected HEK293T cells with pNC099 (P_CMV-MTNR1A), alongside either pNC102 (P_{NFAT_1}-IgK-Nluc), pNC100 (P_{NFAT_5}-IgK-Nluc), pNC104 (P_{NFAT_6}-IgK-Nluc), or pNC103 (P_{NFAT_7}-IgK-Nluc), which encode a P_NFAT-driven Nluc-expressing cassette harboring different copy numbers of NFAT binding sites. Cells were then treated with 1 nM melatonin, a concentration that approximates physiological levels, at 6 hours post transfection. The nanoluc activity in the culture medium was measured after 48-hour to assess the melatonin-induced activation of reporter gene expression. Unfortunately, we observed no significant Nluc expression was observed in cells treated with melatonin compared to untreated controls (Fig. 3A). In contrast, robust Nluc expression could be detected in cells treated with Thapsigargin (Fig. 3B), a known inducer of intracellular calcium release via ER calcium depletion³. In addition, cells transfected with pNC107 (lacking NFAT binding sites) showed no significant response to Thapsigargin, confirming the specificity of the response. These findings suggest that MTNR1A may not effectively induce sufficient calcium signaling in HEK-293T cells to activate the NFAT-dependent calcium-responsive promoters.

Figure 3. (A) The P_NFAT promoters respond poorly to melatonin receptor activation. HEK293T cells were co-transfected with melatonin receptor plasmid pNC099(P_CMV-MTNR1A) and either pNC102(P_{NFAT_1}-IgK-Nluc), pNC100(P_{NFAT_5}-IgK-Nluc), pNC104(P_{NFAT_6}-IgK-Nluc) or pNC103(P_{NFAT_7}-IgK-Nluc). Cells were treated with either DMSO or melatonin 6 hours post transcription. Data represents mean±SD of nanoluc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments). (B) The P_NFAT promoters respond robustly to the Thapsigargin-induced elevation of intracellular calcium level. HEK293T cells were transfected with either pNC102(P_{NFAT_1}-IgK-Nluc), pNC100(P_{NFAT_5}-IgK-Nluc), pNC104(P_{NFAT_6}-IgK-Nluc) or pNC103(P_{NFAT_7}-IgK-Nluc). Cells were treated with either DMSO or Thapsigargin 6 hours post transcription. Data are mean±SD of nanoluc expression levels measured 48 h after thapsigargin stimulation (n = 3 independent experiments).

To investigate why the calcium-based circuit did not perform as expected, we consulted Dr. Yiming Dong, a synthetic biologist, and Chief Scientific Officer of Atantares. Drawing from her experience with calcium-sensing promoters in mammalian cells, Dr. Dong suggested that we examine the calcium dynamics triggered by melatonin receptors. She noted that, in her experience, most calcium-responsive promoters required a sustained elevation of intracellular calcium levels for proper activation. By utilizing GCaMP, a fluorescent protein that generate fluorescent signal in the existence of calcium ions⁴, we were able measured the intracellular calcium dynamics upon melatonin stimulation. We observed no significant change in GCaMP fluorescence in cells treated with melatonin for 48 hours compared to the untreated control cells (Fig. 4A and B), which is in consistent with data of the P_NFAT-Nluc circuit. Interestingly, real-time live-cell calcium imaging revealed a significant spike in intracellular calcium levels immediately after melatonin administration. This calcium surge was short-lived, dissipating within 100 seconds of melatonin stimulation. In contrast, Thapsigargin induced a prolonged elevation of intracellular calcium levels (Fig. 4C).

Figure 4. Melatonin-triggered alteration of intracellular Calcium dynamics in HEK-293T cells. (A and B) Long-term calcium response to melatonin stimulation in HEK-293T cells. HEK293T cells were co-transfected with the MTNR1A-expressing plasmid pNC099 (P_CMV-MTNR1A) and the plasmid expressing the calcium indicator GCaMP6 (P_CMV-GCaMP). Cells were treated with either DMSO or Melatonin 6 hours post transfection. For (A), Fluorescent images were taken 48 h after stimulation, scale bar: 100 µm. Data are representative images of 3 independent experiments. For (B), cells were subjected to fluorescence detection with a plate reader 48 h after stimulation. Data are mean±SD of relative fluorescence intensity (in RFU) measured 48 h after stimulation (n = 8 independent experiments). (C) Short-term calcium response to melatonin stimulation in HEK-293T cells. HEK293T cells were co-transfected with the MTNR1A-expressing plasmid pNC099 (P_CMV-MTNR1A) and the plasmid expressing the calcium indicator GCaMP6 (P_CMV-GCaMP) and treated with the indicated reagent at the 48 h post transfection. Cells were imaged through a fluorescent microscope every 5 seconds, and 20 cells were selected for fluorescent analysis in each group with ImageJ software. The cellular fluorescent levels of each group were normalized to the steady state level before chemical induction, Data shows Mean±SEM.

Learn:

Our experimental results suggest that the design based on NFAT responding to the calcium signaling downstream of MTNR1A may not have met expectations because the calcium spike generate by melatonin cannot activate P_NFAT efficiently. Since developing new calcium-inducible switches specifically for such calcium spike could be time-consuming, we decided to explore alternative signaling pathways.

Design

The MTNR1A receptor has the unique ability to couple to both Gs and Gi proteins, thereby enabling the bidirectional regulation of adenylyl cyclase and modulating cAMP signaling in a differential mannerr^4,5. The intricate balance of cAMP levels, in turn, influences the activation state of CREB (cAMP-response element-binding protein), which acts as a pivotal transcription factor in various cellular processes, including gene expression related to metabolism, cell survival, and neuronal plasticity.
We have engineered a novel genetic circuit utilizing the cAMP-responsive element (CRE) as the signaling responsive element to drive the expression of the downstream reporter gene NanoLuc. This genetic circuit is capable of reporting the upregulation of cAMP signals within cells, based on our literature-derived confirmation that upon melatonin stimulation, cAMP levels in HEK293 cells are upregulated rather than downregulated following the activation of MTNR1A⁵.(Figure 5)

Figure 5. Schematic diagram of the gene circuit design that responds to the MTNR1A-induced alteration of cAMP level. The activation of MATNR1A triggers the elevation of intracellular cAMP level, which triggers the activation of CREB, which would then bind to the CRE sites on the chimeric promoter and activate the downstream gene expression.

Build & Test

To assess the functionality and sensitivity of our genetic circuit, we engineered a series of genetic constructs incorporating varying copy numbers of the cAMP-responsive element (CRE) as the reporter component and assessed the expression efficiency of NanoLuc (Nluc) under the stimulation of 1 nM melatonin Our results demonstrated that the CRE element-based design responded effectively to the increased intracellular cAMP levels, with the tetrameric CRE element showing the most robust response (Figure 6). It is gratifying that the group without melatonin stimulation exhibited a relatively low basal level of NanoLuc expression. In contrast, upon stimulation with 1 nM melatonin, the cells stably and robustly expressed the nanoluciferase protein. This confirms that the genetic circuit we designed is responsive to the activating signal of melatonin on its receptor and can stably report it for our detection

Figure 6. Co-expression of MTNR1A and P_CRE-promoter variants enables robust transcriptional activation upon melatonin stimulation. HEK293T cells were co-transfected with pNC099(P_CMV-MTNR1A) and either pNC101(P_{CRE_4}-IgK-Nluc), pNC105(P_{CRE_5}-IgK-Nluc), or pNC106(P_{CRE_6}-IgK-Nluc). Cells were treated with DMSO or melatonin at 6 hours post transfection; data are mean±SD of nanoluc expression levels measured 24 h after melatonin stimulation (n = 3 independent experiments).

Learn

To this end, we have demonstrated the success engineering of a melatonin-responsive genetic circuit in mammalian cells. However, it came to our knowledge that the circuit itself is not sufficient for us to build a cell-based drug screening platform. During our interview with Dr. Yiming Dong, she mentioned that the endogenous signaling pathways we chose (eg. Ca²⁺ and cAMP/PKA pathways) are also tightly coupled with a set of endogenous signaling pathways, which might also be affected by the compound we add into the cells. Therefore, we should also design a control assay to identify these potential false-positive hits.

Design

Building on the melatonin-responsive circuits, we then went on to design a cell-based drug screening platform to discover MTNR1A agonist. Learning from the interview with Dr. Dong, we design a platform consists of two stable cell lines, HEK_MT and HEK_CTR The HEK_MT cells express both MTNR1A and P_CRE-Nluc cassette, while the HEK_CTR cells were incorporated with P_CRE-Nluc cassette only. During the screening process, we would treat both cells simultaneously with identical compound library. The ones that activate Nluc expression in HEK_MT instead of HEK_CTR cells will be considered as potential hits.

Build & Test

To generate the 1.0 version of MTNR1A agonist screening platform, we first generated the HEK_MT and HEK_CTR cell lines with the sleeping beauty transposon system. Then, we decided to validate the feasibility of our platform by running a minibatch of screening. While we were building the HEK_CTR cells, we interviewed the front-line psychiatrists and engaged the potential user of the drug we might discover through our screening platform to gain some hints about what we can start testing our screening platform on. Interestingly, the term “aromatherapy” pops up as its been widely used around the globe for thousands of years to help falling asleep, and the general public feels more comfortable using these natural product as a daily supplement (See Human Practices page for more information). Hence, we purchased 37 plant essential oils online and hope to see if we can get lucky finding some new agonists for MTNR1A.

Figure 7. The characterization of a cAMP/CREB-based melatonin-responsive gene circuit in HEK-293T cells. (A) Finetuning of P_CRE4-Nluc allows the tuning of system behavior. HEK293T cells were co-transfected with pNC099(P_CMV-MTNR1A) and pNC101(P_{CRE_4}-IgK-Nluc) under the indicated plasmid ratio. Cells were treated with DMSO or melatonin at 6 hours post transfection. Data are mean±SD of nanoluc expression levels measured at 24 h after melatonin stimulation (n = 3 independent experiments). (B) Step-response dynamics of HEK_MT cells under melatonin treatment. HEK_MT cell line carrying both P_CRE4-Nluc and P_CMV-MTNR1A expressing cassette was stimulated with the indicated amount of melatonin 16 hours post seeding. Nanoluc expression levels were measured at different time points after melatonin stimulation (Data are mean±SD, n = 3 independent experiments). (C) Dose-dependence curve of HEK_MT cells under melatonin treatment. HEK_MT cell line was stimulated with the indicated amount of melatonin 16 hours post seeding. Nanoluc expression levels were measured at 24 h after melatonin stimulation (Data are mean±SD, n = 3 independent experiments, EC₅₀=0.4228 nM). (D) Ramelteon response of the HEK_MT cells. HEK_MT cells were treated with ramelteon 16 h post seeding. Nanoluc expression levels were measured at 24 h after ramelteon stimulation (Data are mean±SD, n = 3 independent experiments).

Intriguingly, while most of the plant essential oils showed no significant alteration in the Nluc production of either the HEK_MT cells or the HEK_CTR cells, we surprisingly found that the HEK_MT cells treated with Clove Pod oil showed a robust elevation in Nluc production compared to the DMSO-treated control (~5.25-fold). In comparison, Nluc production in HEK_CTR cells showed no significant alteration, suggesting that Clove Pod oil activates Nluc production in a MTNR1A-dependent manner (Fig. 8A). Similar results were also observed on Caraway Oil, with a milder induction rate of Nluc production in HEK_MT cells (~2.32-fold, Fig. 8A). Further analysis revealed that both Clove Pod oil and Caraway Oil could robustly induce MTNR1A-dependent Nluc production, but no clear dose-dependence could be observed on Caraway Oil (Fig. 8B and 8C). These findings not only showed the feasibility of our designer-cell-based platform for rapid, robust, and resource-efficient high-throughput drug screening but also identified two essential oil hits for agonist discovery. (Figure 8)

Figure 8. Designer cell-based high throughput screen of plant essential oils to identify MTNR1A agonism. (A) High-throughput analysis. HEK_CTR refers to stable HEK293T cells stabling expressing P_{CRE_4}-Nluc, HEK_MT refers to the HEK293T cells shown in Fig. 3D-3F. Cells were treated with 1×10⁴ v/v of either DMSO or indicated essential oil 16 h post seeding. Cells treated with 1 nM melatonin were used as positive control. Nanoluc expression levels were measured at 24 h after exposure to essential oils (Data are mean±SD, n = 3 independent experiments). (B, C) Dose-dependent validation of the most active essential oil hits. (B) HEK_CTR and (C) HEK_MT were treated with different essential oil dilutions (v/v) 16 h post seeding. Nanoluc expression levels were measured at 24 h after exposure to essential oils (Data are mean±SD, n = 3 independent experiments).

Learn

Most essential oils are a mixture of multiple natural product molecules contributing differently to the overall therapeutic function. However, screening over all constituents in the essential oil hits to identify the functioning molecule can sometimes be difficult due to the availability of the constituents and the time- or financial-constraints. Similar concerns were also raised by Dr. Li, the instructors of HZAU-CHINA, during our presentation in the Central China iGEMer’s Meetup in Wuhan, China. Dr.Li suggested us to screen over the essential oil constituents in silico before moving towards the cellular experiments (refer to the Human Practice Page for more information). Almost concurrently, through interviews with experts and scholars in various fields, we have noticed that we can pay more attention to MTNR1B, as selective melatonin receptor agonists have a broader and safer application prospect in clinical therapy. Therefore, we plan to introduce both MTNR1A and MTNR1B in the virtual screening.

Design

To achieve a more robust and efficient identification of the putative active constituents in the essential oils that account for the activation of melatonin receptors, we generated an AutoDock Vina⁶-based pipeline to predict the binding affinity of the essential oil constituents against the known structure of melatonin receptor 1A (PDB ID 6ME2). Based on the input from our human practices, we also included the docking process with melatonin receptor 1B (PDB ID 7VH0) to identify the possible selectivity between MTNR1A and MTNR1B (Fig. 9). Following the previous iteration, we then validated the functionality of the improved screening platform by identifying the functional constituents from the essential oil hits we generated in our last in vitro screening.

Figure 9. (A) Docking box for small molecules with the MTNR1A receptor protein. (B) Docking box for small molecules with the MTNR1B receptor protein.

Build & Test

To validate the molecule docking parameters, we first preformed docking utilizing some known binders of MTNR1A and MTNR1B. The result suggest that CTL 01-05-B-A05 is a highly potent binder to both MTNR1A and MTNR1B (Fig. 10A, upper panel, also see the Modeling page for more information), outperforming other ligands in binding affinity. Notably, It displays a clear preference for MTNR1A over MTNR1B. In contrast, melatonin was found to be 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 Pod and Caraway essential oils by molecular docking. Interestingly, the major constituents of Clove Pod oil were found to bind to both MTNR1A/ MTNR1B receptors. Only Eugenol exhibited a preference towards MTNR1A (Fig. 10A). Structural analysis revealed that the hydroxy group of Eugenol may form a hydrogen bond with the main chain oxgen of Ala104 in MTNR1A (Fig. 10B). More importantly, in vitro assay showed a significantly increased Nluc production in HEK_MT cells treated with eugenol, while no Nluc induction was observed in HEK_CTR cells (Fig. 10C). Together, these results demonstrated the feasibility of our drug-screening pipeline that incorporates both designer cell-based in vitro screening and molecular docking-based in silico screening, and also suggested that eugenol might serve as a promising selective agonist for MTNR1A.

Figure 10. In silico and in vitro screening of MTNR1A selective constituents from essential oils. (A) 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. (B) Structural visualization of Eugenol binds with MTNR1A (left panel). MTNR1A protein is shown as cartoon, displayed as rainbow colors by different helix bundle. Eugenol molecule is shown as magenta sticks. 2D-interaction of Eugenol with MTNR1A (right panel). Potential hydrogen bond is shown between Eugenol and Ala104. (C) Quantification of MTNR1A agonism by eugenol using designer cells. HEK_CTR (left) and HEK_MT (right) were treated with the indicated concentration of Eugenol 16 h post seeding. Nanoluc expression levels were measured at 24 h after Eugenol induction (Data are mean±SD, n = 3 independent experiments).

Learn

Although we have demonstrated the feasibility of our screening platform, we have also noticed that it is necessary to further incorporate the MTNR1B receptor-based cell assay so we can further validate the selectivity of drug candidates experimentally. Unfortunately, due to time constraints, we could not complete the construction of the new circuit before the WIKI FREEZE deadline. We have decided to keep going on designing the MTNR1B-responsive circuit after iGEM.