NanoBRET™ Technology for Target Engagement

The NanoBRET™ Target Engagement (TE) Assay measures compound binding at select target proteins in intact cells, in real time. The assay uses bioluminescence resonance energy transfer (BRET), achieved by transferring the luminescent energy from NanoLuc® luciferase to the fluorescent tracer that is bound to the target protein-NanoLuc® fusion. This energy transfer makes it possible to directly measure compound binding affinity as well as compound-target residence time.

Assay Overview

In the first step of the NanoBRET™ TE Assay, a fixed concentration of tracer is added to cells expressing the desired NanoLuc® fusion protein. Introduction of competing compounds results in a dose-dependent decrease in NanoBRET™ energy transfer allowing estimation of intracellular affinity at the target protein.

The NanoBRET™ TE Assay analyzes the apparent binding affinity and permeability of test compounds by competitive displacement of a NanoBRET™ Tracer reversibly bound to a NanoLuc® fusion protein in cells.

The NanoBRET™ TE Assay directly measures test compound affinity using a cell-permeable NanoBRET™ Tracer.

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Panel A. Compound engagement is measured in a competitive format using a cell-permeable NanoBRET™ tracer. Binding of the test compound results in a loss of NanoBRET™ signal between the target protein and the tracer in intact cells. Panel B. The optimal NanoBRET™ Tracer affinity is determined for each target protein. For analysis of target engagement by a test compound, cells are treated with a fixed concentration of NanoBRET™ Tracer that is near the EC50 value of the NanoBRET™ Tracer dose response curve. Panel C. To determine test compound affinity, cells are titrated with varying concentrations of the test compound in the presence of a fixed concentration (EC50–EC80) of tracer.

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Panel A. Compound engagement is measured in a competitive format using a cell-permeable NanoBRET™ tracer. Binding of the test compound results in a loss of NanoBRET™ signal between the target protein and the tracer in intact cells. Panel B. The optimal NanoBRET™ Tracer affinity is determined for each target protein. For analysis of target engagement by a test compound, cells are treated with a fixed concentration of NanoBRET™ Tracer that is near the EC50 value of the NanoBRET™ Tracer dose response curve. Panel C. To determine test compound affinity, cells are titrated with varying concentrations of the test compound in the presence of a fixed concentration (EC50–EC80) of tracer.

The optimal NanoBRET™ Tracer affinity is determined for the bromodomain (BRD)-NanoLuc® luciferase fusion protein.

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Apparent intracellular NanoBRET™ tracer affinity in HEK293 cells transiently expressing NanoLuc®-BRD4 full-length fusion protein. HEK293 cells expressing NanoLuc®-BRD4 FL fusion protein were resuspended in Assay Medium, seeded into 96-well plates and mixed with increasing concentrations of tracer. Cells were treated with a molar excess of unlabeled iBET-151 (20µM) as a competitive inhibitor for 2 hours before adding 3X Complete Substrate plus Inhibitor Solution. BRET measurements were made on a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted vs. NanoBRET™ BRD Tracer concentration to determine apparent intracellular affinity of the tracer.

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Apparent intracellular NanoBRET™ tracer affinity in HEK293 cells transiently expressing NanoLuc®-BRD4 full-length fusion protein. HEK293 cells expressing NanoLuc®-BRD4 FL fusion protein were resuspended in Assay Medium, seeded into 96-well plates and mixed with increasing concentrations of tracer. Cells were treated with a molar excess of unlabeled iBET-151 (20µM) as a competitive inhibitor for 2 hours before adding 3X Complete Substrate plus Inhibitor Solution. BRET measurements were made on a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted vs. NanoBRET™ BRD Tracer concentration to determine apparent intracellular affinity of the tracer.

The NanoBRET™ TE Assay directly measures the durability of compound-target engagement after binding in real time and provides information on full-length protein targets.

Determining compound residence time with the NanoBRET™ TE Assay.

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Compound residence time is determined from the rate of NanoBRET™ signal between the tracer and the NanoLuc® fusion protein.

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Compound residence time is determined from the rate of NanoBRET™ signal between the tracer and the NanoLuc® fusion protein.

The NanoBRET™ TE Intracellular HDAC Assay analyzes compound residence time at a histone deacetylase (HDAC)-NanoLuc® fusion protein.

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HeLa cells expressing HDAC1-NanoLuc® fusion protein were resuspended in Assay Medium, combined with 100nM FK228, 10μM SAHA or 10µM mocetinostat and incubated for 2 hours. Cells were washed, seeded in 96-well plates and Complete 20X NanoBRET™ Tracer and 2X NanoBRET™ Nano-Glo® Substrate plus Extracellular NanoLuc® Inhibitor Solution dispensed into each well. BRET was measured repeatedly using a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP).

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HeLa cells expressing HDAC1-NanoLuc® fusion protein were resuspended in Assay Medium, combined with 100nM FK228, 10μM SAHA or 10µM mocetinostat and incubated for 2 hours. Cells were washed, seeded in 96-well plates and Complete 20X NanoBRET™ Tracer and 2X NanoBRET™ Nano-Glo® Substrate plus Extracellular NanoLuc® Inhibitor Solution dispensed into each well. BRET was measured repeatedly using a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP).

The NanoBRET™ TE Intracellular Assay analyzes compound residence time at a bromodomain (BRD)-NanoLuc® luciferase fusion protein.

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HeLa cells expressing NanoLuc®-BRD4 FL fusion protein were resuspended in Assay Medium, combined with 1,000nM or 20,000nM JQ1 and incubated for 2 hours. For samples treated with 1,000nM JQ-1, cells were washed, seeded in 96-well plates and Complete 20X NanoBRET™ Tracer and 2X NanoBRET™ Nano-Glo® Substrate plus Extracellular NanoLuc® Inhibitor Solution dispensed into each well. Full-occupancy control samples were treated with 20,000nM JQ-1 without washing. BRET was measured repeatedly using a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP).

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HeLa cells expressing NanoLuc®-BRD4 FL fusion protein were resuspended in Assay Medium, combined with 1,000nM or 20,000nM JQ1 and incubated for 2 hours. For samples treated with 1,000nM JQ-1, cells were washed, seeded in 96-well plates and Complete 20X NanoBRET™ Tracer and 2X NanoBRET™ Nano-Glo® Substrate plus Extracellular NanoLuc® Inhibitor Solution dispensed into each well. Full-occupancy control samples were treated with 20,000nM JQ-1 without washing. BRET was measured repeatedly using a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP).

The NanoBRET™ Target Engagement Assay can be used to evaluate binding of compounds to key drug target classes, such as HDACs, BRDs and kinases.

The NanoBRET™ TE Assay analyzes compound binding to a histone deacetylase (HDAC)-NanoLuc® fusion protein.

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HEK293 cells expressing individual NanoLuc® fusions with Class I or Class IIb HDAC isozymes were seeded into 96-well plates and mixed with various concentrations of tracer. Cells were treated with increasing concentrations of unlabeled SAHA inhibitor and incubated for 2 hours before adding 3X Complete Substrate plus Inhibitor Solution. BRET was measured using a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP).

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HEK293 cells expressing individual NanoLuc® fusions with Class I or Class IIb HDAC isozymes were seeded into 96-well plates and mixed with various concentrations of tracer. Cells were treated with increasing concentrations of unlabeled SAHA inhibitor and incubated for 2 hours before adding 3X Complete Substrate plus Inhibitor Solution. BRET was measured using a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP).

NanoBRET™ Tracer competition in transiently transfected HEK293 cells expressing NanoLuc®-BRD4 full-length fusion proteins.

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HEK293 cells expressing NanoLuc®-BRD4 fusion protein were mixed with various concentrations of tracer. Cells were treated with increasing concentrations of iBET-151 as a competitive inhibitor before adding 3X Complete Substrate plus Inhibitor Solution. BRET was measured using a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted versus iBET-151 inhibitor concentration to determine apparent intracellular affinity of the compound. IC50 value generated using the recommended concentration of 0.5µM NanoBRET™ BET BRD Tracer is shown in bold.

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HEK293 cells expressing NanoLuc®-BRD4 fusion protein were mixed with various concentrations of tracer. Cells were treated with increasing concentrations of iBET-151 as a competitive inhibitor before adding 3X Complete Substrate plus Inhibitor Solution. BRET was measured using a GloMax® Discover System equipped with NanoBRET 618 filters (donor 450nm/8nm BP and acceptor 600nm LP). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted versus iBET-151 inhibitor concentration to determine apparent intracellular affinity of the compound. IC50 value generated using the recommended concentration of 0.5µM NanoBRET™ BET BRD Tracer is shown in bold.