SIGNALING PATHWAY PRODUCT SOLUTIONS

These apoptosis signaling pathway assays are based on the InCELL Hunter™ target engagement, destabilization Destabilized Partner Protein assay. Cell lines are engineered to co-express an untagged target protein and an interacting partner protein fused with ePL (enhanced ProLabel®; the small EFC enzyme donor fragment of β-galactosidase (β-gal)). In the absence of an inhibitor compound and addition of the large EFC enzyme acceptor (EA) β-gal fragment, the untagged target and ePL-tagged partner protein interact, protecting the partner protein from degradation and resulting in stable steady-state levels of ePL-tagged partner protein . Upon addition of inhibitors that bind the target protein and disrupt the target-partner protein interaction, results in ePL-tagged partner protein is degraded. degradation. The abundance of ePL-tagged partner protein in the presence of a substrate that is hydrolyzed is measured by chemiluminescent detection indicated by complementation of the ePL and EA fragments to make an active β-gal enzyme. Levels of ePL-tagged partner protein are assayed by application of the large EFC enzyme acceptor (EA) and a chemiluminescent substrate. ePL and EA combine to hydrolyze the substrate and provide luminescent signal. The greater the signal, the more ePL-tagged partner protein was protected from degradation, corresponding to less compound-target engagement in the cell.

PathHunter TORC1 or TORC2 assays measure TORC1 or TORC2 translocation from the cytosol to the nucleus and subsequent binding to TAZ1 protein. The cell lines have been engineered to express the two complementing EFC fragments of the β-gal enzyme within different cellular compartments. Full length human CRTC1 (TORC1) gene or CRTC2 (TORC2) is N-terminally tagged with ProLink™ (PK; the small enzyme donor of β-gal). TAZ1 (transcriptional adaptor zinc-binding domain) is C-terminally tagged with EA (the large enzyme acceptor of β-gal) and is localized in the cell nucleus. Phosphorylated TORC does not translocate, but the non-phosphorylated TORC translocates into the nucleus and binds to TAZ1 protein. Activation of the pathway initiates TORC1 translocation to the nucleus that forces EFC complementation of the two β-gal enzyme fragments (PK and EA). This action results in the formation of fully active β-gal enzyme, which can hydrolyze a substrate to generate a chemiluminescent signal that can be easily measured on any standard luminometer.

The DNA damage PathHunter assay monitors the translocation of p53 from the cytosol to the nucleus. The cell line has been engineered to express the small EFC enzyme donor fragment PL (ProLabel) of β-gal attached to p53 and the larger EFC enzyme acceptor fragment (EA) localized in the nucleus. Activation of the DNA damage pathway initiates protein translocation of p53 to the nucleus that forces complementation of the two β-gal enzyme fragments (PL and EA). This action results in the formation of fully complemented β-gal enzyme, the activity of which is measured using a chemiluminescent substrate. The signal can be read on any standard luminometer.

NF-κB and IκB signaling pathways assay principles. A. NF-κB signaling reporter assay construct regulates PathHunter EFC reporter expression via upstream NF-κB response elements controlled by NF-κB transcription factor binding. Upstream signaling events result in NF-κB nuclear translocation and subsequent response-element binding. The signaling activity is measured by monitoring the abundance of a reporter protein, which is tagged with the small EFC enzyme donor PL (ProLabel) fragment. Upon addition of the larger EFC enzyme acceptor (EA) fragment, complementation of the two β-gal enzyme fragments (PL and EA) occurs creating an active β-gal enzyme, which is capable of substrate hydrolysis and production of a chemiluminescent signal. B. PathHunter IκB degradation and RelA translocation assays are direct functional assays. In the IκB degradation assay (left pathway), IκB is tagged with the small EFC enzyme donor PL fragment. The assay measures human IκB degradation in response to activation of the NF-kB pathway. In the presence of TNFα or other compounds, cells signal through downstream IκB proteins and the IκB protein is degraded in response to compound stimulation. Upon addition of EA, complementation of the two β-gal enzyme fragments occurs creating an active β-gal enzyme, which is capable of substrate hydrolysis and production of a chemiluminescent signal. In the RelA (NF-kB p65) nuclear translocation assay (right pathway), p65 (RelA) is tagged with the small EFC enzyme donor Pk (ProLink) fragment and a nuclear protein (TAZ1) is tagged with EA. The assay measures RelA translocation to the nucleus in response to activation of the NF-κB pathway. In the presence of TNFα or other compounds, cells signal through downstream IκB proteins and causes RelA to translocate to the nucleus, where complementation of the two β-gal enzyme fragments occurs. This creates creating an active β-gal enzyme, which is capable of substrate hydrolysis and production of produces a chemiluminescent signal proportional to the amount of RelA translocation.

The PathHunter KEAP1-NRF2 Translocation Assay incorporates the small EFC enzyme donor PK (Prolink) fragment fused to the NRF2 protein in a recombinant cell line, while the larger EFC enzyme acceptor (EA) fragment is anchored in the nucleus. Activation of the Keap1-NRF2 protein complex results in NRF2 stabilization and translocation to the nucleus, resulting in PK-EA complementation and formation of a functional β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate, which is hydrolyzed to produce a chemiluminescent signal.

The PathHunter FOXO3 Nuclear Translocation Assay consists of a recombinant cell line which incorporates the small EFC enzyme donor PK (Prolink) fragment fused to the FOXO-3 protein in a recombinant cell line, while the larger EFC enzyme acceptor (EA) fragment is anchored in the nucleus. Activation of the pathway initiates FOXO-3 translocation to the nucleus, forces the complementation of PK-EA fragments, and leads to the formation of a functional β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate that is hydrolyzed to produce a chemiluminescent signal.

The PathHunter SREBP2 Nuclear Translocation Assay is based on a cell line that incorporates the small EFC enzyme donor PK (Prolink) fragment fused to the SREBP2 protein in a recombinant cell line, while and the larger EFC enzyme acceptor (EA) fragment is anchored in the nucleus. Low cholesterol activates the PK-SREB2/Scap protein complex that, which blebs off from the endoplasmic reticulum and traverses to the Golgi, where it is cleaved by S1P. The SREBP2-PK protein then translocates to the nucleus, forcing complementation of the PK-EA fragments and formation of a functional β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate, which is hydrolyzed to produce a chemiluminescent signal.

The PathHunter dimerization assays incorporate the small EFC enzyme donor PK (Prolink) fragment fused to one receptor, TGFβR1, and the larger EFC enzyme acceptor (EA) fragment fused to the other receptor, TGFβR2. Agonist binding to TGFβR2 induces its heterodimerization with TGFβR1 forcing the complementation of the PK with EA fragment resulting in a fully complemented active β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate, which is hydrolyzed to produce a chemiluminescent signal.

The PathHunter Wnt-frizzled assay consists of a recombinant cell line which incorporates the small EFC enzyme donor PL (ProLabel) fragment fused to the β-catenin protein in a recombinant cell line, while the larger EFC enzyme acceptor (EA) fragment is anchored in the nucleus. Activation of the Wnt-frizzled signaling pathway initiates β-catenin translocation to the nucleus, forcing complementation of the PK-EA fragments and resulting in the formation of a functional β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate, which is hydrolyzed to produce a chemiluminescent signal.