This year, we aimed to construct cell-based high-throughput drug screening platform that identifies potential melatonin receptor agonists to address the widespread issue of sleep disorders. The activation of melatonin receptors MTNR1A contributes to their downstream physiological and therapeutic effects by initiating several classic intracellular signaling pathways, including the cAMP/PKA/CREB pathway and calcium (Ca2+) signaling pathway. To sense and assess the activation of these signaling pathways in mammalian cell chassis, we developed synthetic melatonin-responsive circuit, which couples the activation of MTNR1A to downstream detectable reporter gene expression.

For such, we submitted 10 composite parts. By integrating these parts in various combinations, we can build different synthetic melatonin-responsive circuit and characterized the downstream signaling pathways of melatonin receptors upon stimulation of melatonin.

Figure 1. Concrete schematic diagram of BBa_K5267040 composite part

The composite part P_4xCRE->IgK->Nluc->bGH_polyA (Part: BBa_K5267040) is essential for evaluating the responsiveness of the melatonin signaling pathway. Standard approaches for studying signaling pathways typically involve cloning the response element of the relevant transcription factor into a luciferase reporter gene vector, such as pCRE-luc2. Due to the limited transcriptional impact of a single response element, we incorporated multiple tandem copies of the element near the reporter gene's genomic location. This amplification enhanced the initiation efficacy of the signaling cascade3. Therefore, we constructed the Pmini_4xCRE sequence, which includes a 5' minimal promoter incorporating 4 copies of CREs4.

To validate and characterize the composite part P_4xCRE->IgK->Nluc->bGH_polyA (Part: BBa_K5267040), we co-transfected it with MTNR1a (Basic Part: BBa_K5267001) into HEK-293T cell lines. This setup aims to mimic the melatonin receptor-mediated cAMP/PKA/CREB signaling transduction observed in human SCN cells. Theoretically, melatonin-activated MTNR1A receptors should activate CREB (cAMP response element-binding protein), thereby binding to 4xCREs and initiating NanoLuc expression from the composite part.

HEK-293T cell lines was co-transfected with PCMV->MTNR1a->bGH_polyA (part: BBa_K5267047) and the composite part P_4xCRE->lgK->Nluc->bGH polyA(Part:BBa_K5267040) or P_5xCRE->lgK-> Nluc -> bGH polyA(Part:BBa_K5267041) or P_6xCRE->lgK-> Nluc -> bGH polyA(Part:BBa_K5267042). After transfection, both groups of cells were stimulated with 1 nM melatonin, and medium samples were collected at 24 and 48 hours to detect NanoLuc expression.

The results showed a significant increase in Nluc expression in the melatonin-stimulated group compared to those without stimulation, indicating that the composite part can correctly respond to melatonin stimulation as expected. Notably, the NanoLuc expression level detected in the group transfected with P_4xCRE->IgK->Nluc->bGH polyA (Part: BBa_K5267040) showed a higher expression level than other experimental groups, with the highest fold change compared to the control. Therefore, we selected this composite part as the optimal component and constructed a cell-based screening platform based on P_4xCRE->IgK->Nluc->bGH polyA (Part: BBa_K5267040). Based on its performance and importance of this functional part, we chose this composite part as our favorite composite part.

Meanwhile, we also validate the influence of different transfection ratios(Figure 3B), stimulation concentrations and stimulation time(Figure 3C).