NanoBRET™ Target Engagement
Establishing that a 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 the NanoBRET™ Target Engagement assay platform that uses bioluminescence energy transfer to quantitatively measure target occupancy, compound affinity, residence time and permeability in live cells.
What is NanoBRET™ Target Engagement?
Target engagement describes the interaction or binding of a chemical compound with its target protein in a living system. Target engagement assays are needed at multiple stages of drug development, including target validation, establishing structure activity relationships, and confirming mechanism of action (MOA).
Promega has developed a novel live-cell binding assay, called the NanoBRET™ Target Engagement Assay. It uses an energy transfer technique known as bioluminescence resonance energy transfer (BRET) to measure binding between the target protein and small molecules in live cells. 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 binds reversibly to the target protein.
An overview of NanoBRET™ technology and its application for target engagement assays.
Advantages of NanoBRET™ TE Assays:
- Get started quickly with over 360 ready-to-use assays for multiple target classes, including kinases, E3 ligases, HDACs, bromodomains, RAS, PARP and many other proteins.
- Develop a NanoBRET™ TE assay to your target protein of interest using NanoBRET™ TE Do-It-Yourself Tools.
- Target specific assays due to tight distance constraints of BRET.
- Compatible with multiple cell types
NanoBRET™ Target Engagement (TE) Assays 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.
- Calculate cellular compound permeability.
- Generate high‐quality data with low error rate and high reproducibility.
How Does the NanoBRET™ TE Assay Work?
Quantitate Affinity and Occupancy
Unlike other cellular target engagement assays, NanoBRET™ TE assays are quantitative for intracellular compound affinity, not just potency. Quantitative affinity measurements are achieved by use of an appropriate NanoBRET™ Tracer concentration, which is less than or equal to Tracer Kd. For each target specific NanoBRET™ TE assay developed by Promega, the NanoBRET™ Tracer Kd and recommended Tracer concentration is provided.
NanoBRET™ TE is also quantitative for fractional occupancy. By using the appropriate experimental controls, the BRET ratio can be converted to occupancy. Use of fractional occupancy allows quantifying compound selectivity across many similar targets, such as kinases.
The poster Cellular Kinase Assays that Deliver Quantitative Compound Affinity, Occupancy, and Selectivity in Live Cells using NanoBRET provides details on how quantitation is achieved, as well as supporting data using NanoBRET™ TE Kinase assays.
Determine Residence Time
NanoBRET™ Target Engagement Assays have the unique ability to assess compound residence time in live cells. This measurement involves using cells expressing the target-NanoLuc® fusion protein and equilibrating them with a near-saturating concentration of compound. Next, unbound compound is removed and cells are treated with a near-saturating concentration of tracer. The binding is followed kinetically such that compounds with slow dissociation kinetics from the target impede tracer binding, which slows production of the BRET signal. The real-time monitoring of binding kinetics and drug-target residence time in live cells is enabled by fast-binding NanoBRET™ tracers and the long signal half-Life for NanoLuc® Luciferase.
A published study using the NanoBRET™ TE HDAC1 Assay examines isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. The study concludes that:
- Mocetinostat has greater durable binding (residence time) compared to SAHA, despite its weaker equilibrium binding affinity.
- The clinically approved HDAC prodrug FK228 and the related natural product thailandepsin A (TDP-A) had very slow dissociation rates from HDAC1. This long residence time suggests that these strong inhibitor-HDAC complexes are the cause of the prolonged phenotypic effect previously reported.
An example of one method for measuring compound residence time using NanoBRET™ technology.
Correlation of phenotypic potency with target engagement to intracellular HDAC isozymes. Treatment with HDAC inhibitors for 48 hours results in antiproliferative effects in HeLa cells, as measured by intracellular ATP levels.
Measuring the intracellular residence time of HDAC inhibitors at HDAC1. Measuring compound affinity under equilibrium conditions is one aspect of a live-cell NanoBRET™ TE assays. By incorporating residence time analysis, new insights are obtained.
Design 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.
Additional Resources
Quantifying Residence Time and Target Binding
Learn how a novel BRET assay is able to assess cellular target engagement and residence time in this webinar.
Measuring Kinase Target Engagement in Live Cells
This webinar describes how to examine target engagement for kinases.
The Surprising Landscape of CDK Inhibitor Selectivity in Live Cells
This blog post describes a study to repurpose some kinase inhibitors as selective chemical probes for lesser-studied CDK family members.