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

Consider Eurofins DiscoverX’s custom development capabilities for custom cell lines, assays, & enzyme development.

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Assay Principles

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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).

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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.