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Signaling Reporter Assays
A Simple, Orthogonal Screening Tool for Understanding Therapeutic MOAs
Cell-based reporter assays are well established and used to evaluate the cellular impact of therapeutics targeting a variety of proteins in signaling pathways. Researchers utilize these standard assays to study gene expression at the transcription level, and they offer an orthogonal screening method for understanding a therapeutic's MOA (mechanism of action).
The Eurofins DiscoverX® PathHunter® Signaling Reporter assay platform provides a simple, functional cell-based assay platform for screening small molecules or biologic therapeutics, and quantifying the activation and inhibition of various signaling pathways. These assays provide a downstream (transcriptional/translational) read-out complementary to receptor proximal-based assay read-outs to ultimately gain a comprehensive understanding of your drug molecule’s MOA targeting NFAT, NF-κB, PD-1, STAT3, CD27, GM-CSF, ILs, BCMA, TSLP, and more.
Product Highlights
- Greater Understanding – Assess both receptor-upstream and -downstream responses for a comprehensive understanding of the drug molecule’s MOA
- Versatile – Suitable for screening agonists or antagonists, and capable of building assays to interrogate other pathways
- Simple Protocol, Fast Results — Easy-to-run, rapid homogeneous protocol amenable to implementation in multiple labs and high throughput format for increased efficiency
- Biologically Relevant — MOA-reflective, functional assays for monitoring diverse signaling and testing of targeted therapeutics
Assay Principles
Targets
Applications
Resources
Related Pages
Assay Principles
PathHunter signaling reporter assay principle. These assays detect target pathway signaling through the activation of endogenous receptors or receptors introduced into cells with a reporter gene construct. Ligand-mediated stimulation of these receptors initiates pathway signaling and subsequent activation of transcription factors, which bind to a regulatory transcriptional element controlling reporter gene expression. In this assay, the activated signaling pathway drives the expression of the reporter protein tagged with the small enhanced ProLabel® (ePL) β-galactosidase (β-gal) enzyme donor fragment. Reporter activity is measured by lysing reporter pathway cells with a detection reagent containing the complementary β-gal enzyme acceptor (EA) fragment and luminescent enzyme substrate. The enzyme activity is then detected as a result of enzyme fragment complementation (EFC).
The PathHunter PD-1 reporter assay principle. This assay monitors effects of PD-1 inhibitors by measuring T-cell activation resulting in increased NFAT-dependent expression of a reporter gene tagged with the ePL enzyme donor fragment. PD-1 is heterologously expressed in the PathHunter Jurkat NFAT reporter cell line, and co-cultured with U2OS PD-L1 ligand cells co-expressing a TCR activator, resulting in attenuated TCR activation. Pre-incubation of Jurkat PD-1 cells with an antagonist PD-1 antibody promotes PD-1 inhibition of TCR signaling, and NFAT-controlled reporter expression induced by the TCR activator is detected.