TARGET ENGAGEMENT ASSAYS

Validation Data for Diverse Kinases

• Cellular target engagement potency data for multiple kinase inhibitors
• Dose-response curves for established as well as under-studied kinases
• Quantitative EC₅₀ values for kinases represented across the human kinome, including off-target kinases identified in biochemical inhibitor selectivity screens

InCELL Hunter and InCELL Pulse Platforms

Eurofins DiscoverX offers two InCELL target engagement assay platforms—InCELL Hunter and InCELL Pulse—both based on the industry validated Enzyme Fragment Complementation (EFC) technology and used to determine compound cell entry and measure compound engagement to an intracellular protein target in a native cellular environment. Both assay types have similar underlying principles, but crucial differences that suit them for different targets. In general, the InCELL Pulse platform is built on the principle of protein thermal stability, while InCELL Hunter is built on the principle of protein stabilization related to the protein turnover.

To learn more about the differences of each assay platform, read our blog Comparison of InCELL Hunter vs InCELL Pulse for Analyzing Compound-Target Engagement.

InCELL Pulse Assay Principle

InCELL Pulse compound-target engagement assays are based on a novel cellular application of the EFC technology incorporating compound binding detection based on protein thermal stability. Cells expressing a protein of interest is fused to the small EFC enzyme donor of β-galactosidase (β-gal) called ePL (enhanced ProLabel®). These cells are treated with test compound and then subjected to elevated temperatures during a pulse denaturation step. The larger EFC enzyme donor (EA) of β-gal is then added with a chemiluminescnet substrate and EFC complementation. Compound binding protects the target protein from thermal denaturation, which enhances complementation between added EA and ePL components and increases the chemiluminescent signal measured using the EFC-based detection system. In the absence of compound binding, the target protein forms denatured aggregates that poorly complement with EA, which results in a low chemiluminescent signal.

InCELL Hunter Stabilized Compound-Protein Complex Assay Format

In the InCELL Hunter stabilization assay, the intracellular target protein is fused to the small EFC enzyme donor (ePL) similar to InCELL Pulse. Upon addition of a compound that binds the target, protein levels are stabilized or altered in the cell, and this change can be monitored by measuring target protein abundance using chemiluminescent detection. Upon addition of the large EFC enzyme acceptor (EA) fragment and chemiluminescent substrate, EA naturally complements with the ePL tag on the target protein to create an active β-gal enzyme. The resulting active enzyme hydrolyzes the substrate to generate a chemiluminescent signal. A larger signal corresponds to greater presence of compound-target engagement in the cell.

InCELL Hunter Destabilized Partner Protein Assay Format

In the InCELL Hunter destabilization assay, cell lines are engineered to co-express an untagged target protein and an interacting partner protein fused with ePL. In the absence of an inhibitor compound, the untagged target and ePL-tagged partner protein interact, resulting in stable steady-state levels of ePL-tagged partner protein. Addition of inhibitors that bind the target protein and disrupt the target-partner protein interaction results in ePL-tagged partner protein degradation. The abundance of ePL-tagged partner protein is measured by chemiluminescent detection similar to the format above. The greater the signal corresponds to less compound-target engagement in the cell.

Measure and Rank Inhibitory Potency Against Your Intracellular Protein Target

Analyze a variety of intracellular protein targets such as kinases, hydrolases, methyltransferases, and bromodomains.

ABL1 Tyrosine Kinase InCELL Pulse Assay

ABL1 tyrosine kinase cellular target-engagement dose-response curves for type I, type II, and allosteric inhibitors using the InCELL Pulse assay with one pulse denaturation cycle at 48°C for 4 minutes. A. The type I and II inhibitors dasatinib and imatinib, respectively, show the correct rank-order potencies. The lipid kinase inhibitor PI103 was included as a negative control. B. The allosteric inhibitor GNF2, which targets the myristate binding site in the C-terminal kinase lobe, is detected using the ABL1 InCELL Pulse assay.

MTH1 Hydrolase InCELL Pulse Assay

MTH1 hydrolase cellular target-engagement dose-response curves for the diverse inhibitors TH588, S-crizotinib, and SCH 51344 show the correct rank order potencies using the InCELL Pulse assay with one pulse denaturation cycle at 48°C for 2.5 minutes. The lipid kinase inhibitor PI103 was included as a negative control.

BRD4(1) Bromodomain InCELL Hunter Assay

Bromodomains, such as BRD4(1), are epigenetic proteins that have no enzymatic activity and less number of assays available. BRD4(1) cellular target-engagement dose-response curves for the inhibitors JQ1(+), I-BET, and E12348 show the correct rank order potencies using the InCELL Hunter assay.

Identify Cell Permeable Compounds with the Best Cellular Potency

Confirm compound cell entry and target binding in the native cellular environment.

G9a Methyltransferase InCELL Hunter Assay

InCELL Hunter G9a methyltransferase target engagement assay identifies compounds with the best cellular potency. Published biochemical IC₅₀’s indicate compounds bind with defined affinities outside the cell. The InCELL Hunter assay shows compounds with similar biochemical IC₅₀ do not have the same binding affinity to the target inside a cell. Results suggests the compound UNC0321 is not as cell permeable as UNC0638 or not as good at engaging the target in an intracellular environment. [Cellular IC₅₀ reference: Nature Chemical Biology. 2011; 7(8): 566-574.]

Kinase Target Engagement Assay Targets

Epigenetic Proteins Target Engagement Assay Targets