Development of a Multimodal Assay System for GDF15/GFRAL Drug Discovery

Transforming Receptor Biology into a Screening-Ready Discovery Platform

Successful drug discovery begins with a detailed understanding of target biology and the signaling pathways that underpin disease. Translating this understanding into robust cellular models and screening assays is critical for identifying and progressing new therapeutics. For emerging targets, this requires more than assay development alone. It demands cellular systems that accurately reproduce complex signaling biology while delivering the robustness required for screening, lead optimization and characterization.

This challenge was central to a program focused on the GDF15-GFRAL pathway, an emerging therapeutic target attracting significant interest in metabolic disease, obesity, cachexia and oncology. GDF15 signaling is mediated through binding to GFRAL, which subsequently recruits the RET co-receptor to activate downstream signaling pathways, including ERK (Figure 1). Successfully modelling this biology requires coordinated expression of multiple receptor components while maintaining functional coupling to intracellular signaling mechanisms, creating a significant challenge for cell line generation and assay development.

To address these challenges, Sygnature Discovery combined expertise in cell line generation, receptor pharmacology and assay development to establish a stable inducible GDF15-GFRAL-RET cellular platform and complementary assay formats that support both compound screening and pharmacological confirmation.

Project Highlights:

• Stable inducible GDF15-GFRAL-RET cellular platform generated
• Extensive functional clone selection and validation
• Biologically relevant pERK assay measuring proximal pathway activation
• High-throughput SRE reporter assay suitable for compound screening
• Consistent pharmacology demonstrated across orthogonal assay formats

Converting Cell Engineering into Functional Biology

Establishing receptor expression is only the first stage of development. For a cell line to support drug discovery, it must demonstrate robust, reproducible and pharmacologically meaningful responses to pathway activation.

Our team therefore implemented an extensive clone selection strategy focused on functional performance. HEK cells were engineered to co-express GFRAL and RET under an inducible system and expressing (induced) and non-expressing (uninduced) clones were screened using a pERK ELISA assay to identify cell populations capable of generating robust and reproducible responses to GDF15 stimulation (Figure 2).

Screening revealed significant differences in pathway responsiveness between candidate clones, highlighting the importance of rigorous functional validation during cell line development. Through systematic evaluation, scientists identified clones that combined strong signal generation, reproducible pharmacology and the stability required for downstream screening applications.

For drug discovery teams, this stage is critical. Selecting the correct clone can determine whether an assay delivers meaningful insight or becomes limited by variability and poor assay performance.

Demonstrating Reproducibility: The Foundation of Screening Success

A robust signal alone is insufficient for modern drug discovery. As part of our quality control process, assay reproducibility is evaluated across multiple test days.

To evaluate platform stability, top-performing clones were tested across multiple independent experiments using GDF15 stimulation (Figure 3). The resulting concentration-response curves showed a high degree of agreement across studies, demonstrating stable pathway activation and sustained performance over time. This level of consistency is essential for drug discovery, providing confidence that compound activity can be measured reliably and reflects authentic target biology, while supporting progression into larger screening campaigns.

Expanding Utility Through Reporter Gene Technology

While pERK assays provide a biologically relevant measure of pathway activation, their transient nature makes them highly time dependent. To expand platform utility, Sygnature Discovery developed an SRE-luciferase reporter assay in selected GFRAL/RET clones, providing a robust high-throughput readout (Figure 4). Individual reporter clones were subsequently screened for assay performance (Figure 5).

The development of this reporter system transformed the platform from a single functional assay into a flexible, high-throughput screening solution capable of supporting multiple stages of a discovery program. Coupled with a sensitive luciferase readout, the assay can be readily deployed in 96-, 384- or 1536-well formats, enabling efficient screening of large compound libraries.

Establishing Pharmacological Consistency Across Orthogonal Assay Formats

A key objective was to ensure that both assay formats accurately reflected the underlying GDF15-GFRAL-RET biology. GDF15-mediated activation and inhibitor X-mediated pathway inhibition were therefore evaluated in both the pERK and SRE reporter assays (Figure 6).

GDF15 produced concentration-dependent activation in both assay formats, with comparable EC₅₀ values in the pERK and SRE assays. Inhibitor X likewise produced concentration-dependent inhibition across both readouts, demonstrating consistent pharmacology between proximal and downstream endpoints.

Together, the data supports the use of the SRE assay for high-throughput screening and the pERK assay for orthogonal confirmation of compound activity.

Why Sygnature?

This project demonstrates Sygnature’s ability to combine cell line generation, receptor pharmacology and assay development to enable efficient drug discovery. Our scientists transformed a complex multi-component receptor system into a screening-ready platform that supports compound screening, hit confirmation and further pharmacological investigation.

If you are looking to develop cell lines and screening assays for challenging targets, contact Sygnature Discovery to discuss how we can support your discovery program.