Signaling Reporter Assays -

Applications & Products

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. These standard assays are used to study gene expression at the transcription level, and they offer an orthogonal screening method for understanding the therapeutic's MOA (mechanism-of-action).

The 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, and more.

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Assay 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

Key Resources

PathHunter Signaling Reporter assays are offered as stable cell lines and used with PathHunter detection reagents for the assessment of diverse signaling pathways.

Target	Description	Cat. No.
NFAT	PathHunter® Jurkat NFAT Reporter Cell Line	93-1141C19
NFkB	PathHunter® U2OS NF-KB Reporter Cell Line	93-1157C3
RANK NF-kB	PathHunter® U2OS RANK-NF-KB Reporter Cell Line	93-1156C3
STAT3	PathHunter® HepG2 STAT3 Reporter Cell Line	93-1144C44
PD-1 NFAT	PathHunter® Jurkat PD1-NFAT Reporter Cell Line	inquire
NF-κB	PathHunter® HEK293 NF-κB Reporter Cell Line	inquire
CD27 NF-κB	PathHunter® HEK293 CD27-NF-κB Reporter Cell Line	inquire
TREM1 NFAT	PathHunter® Jurkat TREM1-NFAT Reporter Cell Line	inquire
IL-11R STAT3	PathHunter® HepG2 IL11R-STAT3 Reporter Cell Line	inquire

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.

PathHunter Signaling Reporter assays have diverse applications to help you obtain a comprehensive understanding of your therapeutics’ MOA.

Quantify the Activation and Inhibition of Signaling Pathways

Pathway signaling reporter assays for endogenous or heterologously-expressed target receptors. A. The PathHunter U2OS NF-κB Reporter Assay detects CD40L-mediated activation of endogenous CD40 receptors that signal through the NF-κB pathway. B. The PathHunter HEK293 CD27-NF-κB Reporter Assay was created by heterologous expression of the co-stimulatory receptor, CD27, in the PathHunter HEK293 NF-κB Pathway Reporter cells, resulting in a sensitive assay to characterize CD27 agonists. C. The TREM1 ligand PGLYRP1 in combination with PGN-EK was used to activate TREM1, but PGLYRP1 TREM1 activation was inhibited with an Anti-PGLYRP1 antibody. Anti-PGLYRP1 Antibody + 6.7 μg/mL PGLYRP1 + 13.4 μg/mL PGN-EK exhibited a decrease in signal with an signal-to-background (S:B) ratio of 4.4, while PGLYRP1+PGN-EK in a 1:2 ratio resulted in an increase in signal with an S:B ratio of 4.9.

Measure Sensitive Responses, from either Distal or Proximal Events

Comparison of antagonist testing using an anti-PD-1 antibody results from two different PathHunter Jurkat PD-1 Assays. A. The PathHunter PD-1 Reporter Assay was used to measure downstream effects of PD-1 receptor signaling resulting in NFAT-regulated reporter protein expression. Conversely, B. The PathHunter PD-1 SHP2 Signaling Assay was used to measure upstream PD-1 signaling events independent of T cell receptor activation. Both assays are robust and measure inhibition with sensitive responses, from either downstream or upstream events.

Develop Potent Agonist and Antagonist Therapeutics

Develop therapeutics targeting receptors with the various signaling pathway reporter assays. The PathHunter HepG2 STAT3 Signaling Reporter Assay was used to detect the potent antagonist therapeutic, Tocilizumab (Actemra®, registered trademark of Genentech), which has been approved for clinical trials to treat patients with severe Covid-19 pneumonia.