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Cellular Target Engagement

Establishing that a small molecule engages with its intended target protein in cells is an important step in drug discovery and chemical probe development. NanoLuc® luciferase is the basis of a novel target engagement assay platform that uses bioluminescence energy transfer to quantitatively measure target occupancy and compound affinity in live cells.

What is Target Engagement?

Target engagement describes the interaction or binding of a chemical compound with its target protein in a living system. Researchers use chemical probes to selectively target a protein and understand its function while screeners investigate small molecule compounds that may inhibit the activity of a target protein.

One such live-cell binding assay is the NanoBRET™ Target Engagement Assay that use an energy transfer technique called bioluminescence resonance energy transfer (BRET) to measure molecular proximity. This energy transfer is based on two elements: Cellular expression of the target protein fused to NanoLuc® luciferase and a cell-permeable fluorescent NanoBRET™ tracer, which reversible binds to the target protein. When the fluorescent tracer binds to the target-NanoLuc® fusion protein in cells, the tracer is in close proximity to NanoLuc and results in a BRET signal.

The apparent affinity of test compounds is measured by competitive displacement of the NanoBRET™ tracer. Thus, when a competing compound is introduced, there is a dose-dependent decrease in the BRET signal. Watch one of our R&D scientists describe the BRET-based method in this video.

Principle of Live-Cell Target Engagement Assay

Binding of test compound results in loss of NanoBRET signal

The basis of the NanoBRET™ Target Engagement Assay is the competitive displacement of a fluorescent NanoBRET™ tracer reversibly bound to a NanoLuc® fusion protein in cells. When the test compounds bind, they displace the tracer, resulting in loss of the NanoBRET™ signal. From this assay, you can determine apparent binding affinity, cell permeability and residence time.

With the NanoBRET™ Target Engagement Assay, you aren't limited by protein class or cellular background. You can:

  • Quantitate compound affinity (how tightly it binds to a protein) and target protein occupancy (how much compound binds to a protein) in live cells.
  • Assess how long a compound binds to the target protein (its residence time) under physiological conditions.
  • Scale the simple multiwell assay for your research.
  • Generate high‐quality data with low error rate and high reproducibility.
  • Get started quickly with available ready-to-use assays.

Measuring Kinase Target Engagement

Protein kinases are a large family of more than 500 enzymes that phosphorylate proteins. Because kinase activity has been associated with cancer and inflammatory diseases, these enzymes are key drug targets.

The NanoBRET™ TE Intracellular Kinase Assays detect target engagement of test compounds with kinases in live cells. Our suite of ready-to-use target engagement assays for over 300 full-length kinases have been successfully used to quantitate cellular affinity and occupancy for multiple inhibitors including types I, II and allosteric. In fact, using several of the NanoBRET™ TE Kinase Assays for selectivity profiling revealed the live-cell BRET-based binding assay to be a better predictor of cellular compound potency compared to biochemical profiling. You can read more about these assays in this Cell Chemical Biology article.

Cellular Affinity Assessed for Six Compounds

BTK Kinase compound affinity comparison with six chemical probes

Here a panel of six compounds were assessed for cellular affinity with Bruton's tyrosine kinase (BTK) in the live-cell NanoBRET™ TE Intracellular Kinase Assay.

Understanding Epigenetic Target Engagement

Post-translational modifications (PTMs) to histones that form the protein core of chromatin are critical to regulating gene expression. The proteins responsible for adding, erasing, or reading these PTMs play vital roles in normal and diseased states. Histone deacetylases (HDACs) and bromodomains are histone erasers and writers, respectively, and have been targeted for drug discovery.

A panel of NanoBRET™ Target Engagement HDAC Assays are available for analysis of compound affinity for a particular HDAC, selectivity of a compound against a panel of HDACs, or residence time of a compound with an HDAC.

Proteins within the BET Bromodomain family contain two tandem bromodomains (BRDs) and an extra-terminal domain. We have developed the NanoBRET™ TE BET BRD Assays for the full-length BRD proteins as well as assays for several segregated domains for different BRD proteins. Similar to the NanoBRET™ HDAC TE Assays, compound affinity, selectivity and residence time for these BET BRD proteins can be determined in live cells.

Residence Time Determined for Four Compounds

Residence time for HDAC1 among four compounds.

Residence time was assessed for four compounds and HDAC1. Measuring compound affinity is only one aspect of a live-cell-based target engagement assay. Residence time differs for each compound, demonstrating that compound affinity is only a single aspect of target-chemical binding.

Designing Your Own Target Engagement Assay

NanoBRET™ Target Engagement technology is applicable to multiple classes of target proteins, including kinases, HDACs, bromodomains and GPCRs. If there is no NanoBRET™ TE Assay for your target of interest, you can create one! There are two critical pieces you need to make: 1) a fluorescent tracer that will bind your protein target; and 2) a vector that expresses a fusion of your target protein with NanoLuc® luciferase.

If your target of interest is an intracellular protein, you will need both the NanoBRET™ Nano-Glo® Substrate and the Extracellular NanoLuc Inhibitor for your assay. These components will ensure that the BRET signal you measure is the result of intracellular interactions. The method needed to develop these DIY assays is detailed in this Systems Chemical Biology chapter.

Overview of the Process for Designing Your Own NanoBRET™ TE Assay.

Creating a Custom NanoBRET Target Engagement Assay

Find materials to develop your DIY TE Assay. See options>>