Target Engagement Assays
Universal Cell-Based Compound-Target Binding Assays
Target engagement assays measure compound binding to a specific protein target. In a cellular environment, these assays monitoring compound cellular penetration and compound-target interaction to enable the assessment of compound efficacy and confirmation of mode-of-action.
InCELL cell-based compound-target engagement assays provide the ability to confirm compound cell entry and protein target binding, screen and rank inhibitors, validate hits identified in biochemical assays, and measure cellular EC50 values all in the native cellular environment. These simple binding assays require no custom chemical tracers nor antibody reagents and provide a convenient solution when functional assays are difficult or unavailable.
Access two leading Eurofins DiscoverX® assay platforms, InCELL Pulse™ and InCELL Hunter™ — both based on the industry validated EFC technology with similar underlying principles, but crucial differences that suit them for different protein targets. 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 protein turnover or interaction disruption.
Discover the power of the InCELL assays and take advantage of stable cell lines, ready-to-use kits, and a do-it-yourself, starter kit perfect for creating your own compound-target engagement assay.
- Biologically Relevant - Obtain quantitative cellular compound entry and target binding data for more confident drug discovery decisions
- Easy-to-Use - Simple, homogeneous protocol to easily measure compound binding to your protein target with a chemiluminescent readout
- High-Throughput Amenable - Easily automate assays to screen multiple compounds accurately
- Quick & Thorough Protocol - Rapidly make your own target engagement cell-based assays in less than 2 weeks following the detailed, step-by-step protocol
Eurofins DiscoverX offers two target engagement assay platforms, InCELL Pulse and InCELL Hunter. The InCELL Hunter platform offers researchers cell lines, ready-to-use kits and profiling services for specific targets. The InCELL Pulse platform offers researchers ready-to-use kits for specific targets and a do-it-yourself, starter kit perfect for creating your own compound-target engagement assay.
- InCELL Hunter Cell Lines – Stable cell lines confirmed for past 10 passages and rigorously and continuously validated for accurate pharmacology through Eurofins Discovery services with billions of data points screened.
- InCELL Hunter eXpress Kits – Complete assay-ready kits with specific cell line, optimized reagents, and plates for quick Enzyme Fragment Complementation (EFC) based chemiluminescent detection.
- InCELL Pulse Starter Kit – Create your own cell-based, target engagement assays with reagents and protocol for the design and development of InCELL Pulse assays for the measurement of inhibitor cellular potency against user-defined protein targets of interest.
- InCELL Detection Kit – Measure compound binding to a specific protein target in InCELL Hunter and InCELL Pulse cell lines
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. The greater the 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
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The webinar focuses on utilizing an InCELL Pulse to characterize small-molecule cellular target engagement. As an example, a case study…Watch Now
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