Compound-Target Engagement Assays

Universal Cell-Based Compound-Target Binding Assays
 

Monitoring cellular drug penetration and drug-target interaction enables assessment of compound efficacy and confirmation of mode of action.  InCELL Hunter™ and InCELL Pulse™ cellular compound-target engagement assays provide the ability to confirm compound cell entry and target binding. In addition, these assay are validated and designed for screening inhibitors, validating hits identified in biochemical assays, measuring cellular EC50 values, and ranking compounds – all in the native cellular environment. These simple binding assays require no custom chemical tracer or antibody reagents and provide a convenient solution when functional assays are difficult or unavailable.

 
Target Engagement Images

InCELL Pulse and InCELL Hunter Platforms

DiscoverX offers two InCELL target engagement assay platforms, InCELL Hunter and InCELL Pulse, that are both based on the proprietary Enzyme Fragment Complementation (EFC) technology and allow for the determination of compound engagement to an intracellular protein target in a native cellular environment. Only the InCELL Pulse platform expands on the EFC technology incorporating compound binding detection based on protein thermal stability.

InCELL Hunter Stabilized Compound-Protein Complex Assay Format



The intracellular target protein in these assays is fused with a small enzyme donor fragment of β-galactosidase (β-gal) called enhanced ProLabel® (ePL). 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. The detection reagents include a chemiluminescent substrate added with a large enzyme acceptor (EA) fragment that 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 these assays, 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.
 

InCELL Pulse Assay Principle

InCELL Pulse Assay Principle

The 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 fused to the ePL tag are treated with test compound and then subjected to elevated temperatures during a pulse denaturation step. Compound binding protects the target protein from thermal denaturation, which enhances complementation between EA and ePL 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.


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.

Products

View the available stable cell lines, assay-ready kits, starter kit, and detection reagents.
Cell Lines
Assay Ready Kits
Starter Kit for creating your own cell-based target engagement assays
Detection Reagents Expression vectors for 25 under-studied and well-studied kinases available upon request for creating your own InCELL Pulse cell-based target engagement assays.  These include off-target kinases.

Services

Kinases
Epigenetics
Custom Assay Development

InCELL Compound-Target Engagement Assay Highlights

  • Easily measure compound binding to your protein target with a chemiluminescent readout
  • Rapidly make your own target engagement cell-based assays in <2 weeks
  • Generate biologically relevant data for more confident drug discovery decisions
  • Simple cell-based assays to automate and accurately screen compounds in a high throughput format

Download application note to learn about automating InCELL Hunter assays for epigenetic targets.
 

Key Resources

  • Webinars: Learn how to make your own compound-target engagement, cell-based assays
  • Publication: Review how customers created a high-throughput, InCELL Pulse assay
  • Blog: Read about benefits and challenges of do-it-yourself cell-based assays
  • Reference Set: See validation data for over 30 kinase InCELL Pulse assays
  • Application Note: Learn about automating InCELL Hunter assays for epigenetic targets.
 

Validation Data for Diverse Kinases

  • Cellular target engagement potency data for multiple kinase inhibitors
  • Dose response curves for under-studied and well-studied kinases
  • Quantitative EC50s for kinases represented across the human kinome including off-target kinases identified in biochemical inhibitor selectivity screens

Learn more about InCELL Pulse kinase data

Validated Kinase Assays Across Human Kinome

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 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 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
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
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.]