TITLE: Quantitative, Real-Time Measurements of Intracellular Target Engagement Using Energy Transfer
AUTHORS: Matthew B. Robers, James D. Vasta, Cesear R. Corona, Rachel Friedman Ohana, Robin Hurst, Manisha A. Jhala, Kenneth M. Comess, Keith V. Wood

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Intracellular target affinity and residence time are fundamental aspects of pharmacological mechanism (Lu and Tonge, Curr Opin Chem Biol 14:467–474, 2010). Although various robust biochemical approaches exist to measure these binding characteristics, analysis of compound binding with isolated targets may not accurately reflect engagement in the milieu of living cells. To realize the influence of cellular context, methods are needed that are capable of quantifying affinity and residence time in the presence of the intracellular factors that may impact target engagement. Bioluminescence resonance energy transfer (BRET) offers a solution for intracellular target engagement when quantitative metrics or kinetic analyses are required.</div></div>

[STEPS]
?. [3.2.1 [Day 1]: Transient Transfection of HEK293 Cells with NanoLuc(R) Fusions]
Cultivate desired cells (e.g., HEK293) appropriately prior to assay and resuspend cells into a single-cell suspension using complete cell culture medium.
?. [3.2.1 [Day 1]: Transient Transfection of HEK293 Cells with NanoLuc(R) Fusions]
Adjust the cell density to 2 × 105/mL in cell culture medium in a sterile, conical tube.
?. [3.2.1 [Day 1]: Transient Transfection of HEK293 Cells with NanoLuc(R) Fusions]
Prepare plasmid DNAs for transfection. For first-time experiments, use a 1:10 dilution (mass:mass) of expression construct into a promoterless DNA (Transfection Carrier DNA). At this stage it may be useful to perform a titration of expression, by diluting the expression construct into Transfection Carrier DNA. Examples of extending this titration are shown in Fig. 6.
?. [3.2.1 [Day 1]: Transient Transfection of HEK293 Cells with NanoLuc(R) Fusions]
Prepare transfection complexes using the manufacturer’s protocol.
?. [3.2.1 [Day 1]: Transient Transfection of HEK293 Cells with NanoLuc(R) Fusions]
Mix one part (e.g., 1 mL) of transfection complex with 20 parts (e.g., 20 mL) of HEK293 cells in suspension at 2 × 105/mL. Mix gently by inversion five times in a sterile, conical tube.
?. [3.2.1 [Day 1]: Transient Transfection of HEK293 Cells with NanoLuc(R) Fusions]
Dispense cells/lipid–DNA complex into a sterile tissue culture flask, and incubate at least  to allow expression to occur. We recommend a cell density of approximately 55,000–80,000 cells/cm2 during the transfection (for example use approximately 4−6 million cells for a T75 flask). Larger or smaller bulk transfection should be scaled accordingly, using this ratio.
?. [3.2.2 [Day 2]: Preparation of Cells with 1x Tracer for BRET Assay]
Harvest cells via trypsinization, and resuspend in growth medium including serum. Centrifuge the cells and resuspend the pellet in Opti-MEM without serum or phenol red.
?. [3.2.2 [Day 2]: Preparation of Cells with 1x Tracer for BRET Assay]
Adjust the cell density to 2 × 105/mL in Opti-MEM without serum or phenol red.
?. [3.2.2 [Day 2]: Preparation of Cells with 1x Tracer for BRET Assay]
Prepare cells for target engagement analysis in live or permeabilized cells. For live cell target engagement experiments, dispense  per well into white, 96-well NBS plate. Periodically mix cells appropriately to avoid settling of the cell suspension. For live cell analysis, proceed to step 13. For permeabilized cell experiments dispense  per well into a white, 96-well plate. Add  (either 5× Passive Lysis Buffer or 250 μg/mL digitonin).
[cell suspension]
[cell suspension]
[lysis reagent ]
?. [3.2.2 [Day 2]: Preparation of Cells with 1x Tracer for BRET Assay]
Prepare the complete 20× tracer. For characterization of tracer affinity, perform a serial dilution of the tracer in DMSO at a 100× concentration (see Note 4). For characterization of unlabeled drug affinity (if the optimal concentration of tracer has already been determined) prepare a 100× concentration of the tracer in pure DMSO.
?. [3.2.2 [Day 2]: Preparation of Cells with 1x Tracer for BRET Assay]
Dilute the 100× tracers to 20× with Tracer Dilution Buffer according to the manufacturer’s instructions. Add 4 parts Tracer Dilution Buffer to 1 part of 100× tracer (from DMSO) to prepare the Complete 20× tracer reagent.
?. [3.2.2 [Day 2]: Preparation of Cells with 1x Tracer for BRET Assay]
Mix gently several times to ensure that the DMSO solution is mixed with the tracer dilution buffer.
?. [3.2.2 [Day 2]: Preparation of Cells with 1x Tracer for BRET Assay]
Add  per well to cells in suspension.
[complete 20× tracer reagent]
?. [3.2.2 [Day 2]: Preparation of Cells with 1x Tracer for BRET Assay]
Mix on orbital shaker for  at .  Mixing may vary between orbital shakers and should be optimized for each individual unit accordingly. Due to the viscosity of the Tracer Dilution Buffer, it is necessary to dispense this solution slowly.
33
?. [3.2.3 [Day 2]: Addition of Test Compounds for Competition Analysis and Determination of Specific BRET]
Prepare a suitable unlabeled test compound at 1000× final concentration in 100% DMSO. Typically, the 1000× concentration of compound is in the – range. Then dilute to a 10× final concentration in Opti-MEM without serum or phenol red.
?. [3.2.3 [Day 2]: Addition of Test Compounds for Competition Analysis and Determination of Specific BRET]
The characterization of BRET tracers can be performed as a single concentration of unlabeled compound for tracer dose–response curves. It is generally recommended that a 20−100× molar excess of parental drug is coincubated with the tracer to determine the nonspecific BRET signal.
?. [3.2.3 [Day 2]: Addition of Test Compounds for Competition Analysis and Determination of Specific BRET]
For characterization of unlabeled drug IC50, create a serial dilution of test compound for drug IC50 analysis.
?. [3.2.3 [Day 2]: Addition of Test Compounds for Competition Analysis and Determination of Specific BRET]
Add  per well to the 96-well plates containing cells with 1× tracer. Mix on orbital shaker for  at .
[10× inhibitor/test compound]
33
?. [3.2.3 [Day 2]: Addition of Test Compounds for Competition Analysis and Determination of Specific BRET]
Incubate the plate at  and 5% CO2 for . Allow plate to cool to  for approximately , then proceed to the next section below. (step 24)
37 °C
0 Room temperature
?. [3.2.3 [Day 2]: Addition of Test Compounds for Competition Analysis and Determination of Specific BRET]
For permeabilized cell assays, incubate the plate at , protected from light for −(depending on the characteristics of the test compound). Then proceed with the next step (see Section 3.2.4 "Detection in Microplate Luminometer Equipped with Appropriate BRET Filters").
0 Room temperature
?. [3.2.4 Detection in Microplate Luminometer Equipped with Appropriate BRET Filters]
Immediately prior to BRET measurements, prepare 3× Complete NanoBRET(TM) Nano-Glo(R) Substrate.
?. [3.2.4 Detection in Microplate Luminometer Equipped with Appropriate BRET Filters]
For live cell analysis, add Extracellular NanoLuc(R) Inhibitor in Opti-MEM and add to cells according to the manufacturer’s instructions (Promega) (see Note 5).
?. [3.2.4 Detection in Microplate Luminometer Equipped with Appropriate BRET Filters]
For Permeabilized cell analysis, prepare 3× NanoBRET(TM) Nano-Glo(R) Substrate in Opti-MEM without serum or phenol red, and omit the Extracellular NanoLuc(R) Inhibitor. This solution consists of only a 1:166 dilution of NanoBRET(TM) Nano-Glo(R) Substrate in Opti-MEM.
?. [3.2.4 Detection in Microplate Luminometer Equipped with Appropriate BRET Filters]
Following addition of , measure donor emission (e.g., 450 nm) and acceptor emission (e.g., 610 nm or 630 nm) using a NanoBRET(TM)-compatible luminometer.
[NanoBRET(TM) Nano-Glo(R) Substrate]
?. [3.2.4 Detection in Microplate Luminometer Equipped with Appropriate BRET Filters]
To generate raw BRET ratio values, divide the acceptor emission value (e.g., 610 nm) by the donor emission value (e.g., 450 nm) for each sample. Convert raw BRET units to milliBRET units (mBU) by multiplying each raw BRET value by 1000.
?. [3.2.4 Detection in Microplate Luminometer Equipped with Appropriate BRET Filters]
(Optional) If a background correction is desired, use the NanoBRET Equation (Eq. 1).(\frac{Acceptor_{sample}}{Donor_{sample}}-\frac{Acceptor_{no\ tracer\ control}}{Donor_{no\ tracer\ control}}) \times 1000 (1)
?. [3.4.1 [Day 1]: Pre-dispense of Test Compounds to Assay Plate]
Aliquot test and control compound stocks in 100% DMSO at high concentration (typically either  or  depending on desired final assay concentration of  or , respectively) to acoustic dispense qualified mother plates. Depending on equipment, one or more additional dilutions likely are required.
?. [3.4.1 [Day 1]: Pre-dispense of Test Compounds to Assay Plate]
Adjust instrument to dispense 80 nL of highest concentration, then 1:3 dilutions using 100% DMSO as backfill in order to create a 12-point dilution series of 80 nL total volume in each well. Since the lowest dispense amount may be 5 nL, several intermediate dilutions of the initial 5 or 10 mM stock likely are required to achieve the required dispenses.
?. [3.4.1 [Day 1]: Pre-dispense of Test Compounds to Assay Plate]
Seal plates tightly and store at  until day 2 of assay.
4 °C
?. [3.4.2 [Day 2]: Preparation of Cells with 1× Tracer for BRET Assay]
Harvest cells as described in Section 3.2.2 "[Day 2]: Preparation of Cells with 1× Tracer for BRET Assay" and adjust the cell density to 2 × 105/mL in Opti-MEM without serum or phenol red.
?. [3.4.2 [Day 2]: Preparation of Cells with 1× Tracer for BRET Assay]
Dispense  per well into white, 384-well plate containing previously dispensed, serially diluted test and control compounds.
[cell suspension/extracellular NanoLuc Inhibitor]
?. [3.4.2 [Day 2]: Preparation of Cells with 1× Tracer for BRET Assay]
Prepare the BRET tracer at the lowest concentration determined to yield a useable signal:background and Z’ greater than 0.5, optimally 50−80% of full target occupancy.
?. [3.4.2 [Day 2]: Preparation of Cells with 1× Tracer for BRET Assay]
Add  per well to cells in suspension. Mix thoroughly using appropriate instrumentation.
[Complete 20× tracer]
?. [3.4.2 [Day 2]: Preparation of Cells with 1× Tracer for BRET Assay]
Incubate plates in a humidified, /5% CO2 incubator for .
37 °C
?. [3.4.2 [Day 2]: Preparation of Cells with 1× Tracer for BRET Assay]
Add  per well for a 384-well plate. Incubate – at  prior to first read; readings are stable at least .
[3× Complete NanoBRET(TM) Nano-Glo(R) Substrate]
0 Room temperature
?. [3.4.2 [Day 2]: Preparation of Cells with 1× Tracer for BRET Assay]
Measure BRET as described in Section 3.2.4  "Detection in Microplate Luminometer Equipped with Appropriate BRET Filters."
?. [3.5.1 Adding Test Compound to Cells]
After transfecting cells (as described in Section 3.2.1 "[Day 1]: Transient Transfection of HEK293 Cells with NanoLuc(R) Fusions"), resuspend cells using prewarmed assay medium (e.g., Opti-MEM without serum or phenol red).
?. [3.5.1 Adding Test Compound to Cells]
Adjust the density to 2 × 105 cells/mL in assay medium in a 15 mL sterile polypropylene conical tube. We recommend a minimum volume of 1 mL of cells, ideally 5 mL of cells.
?. [3.5.1 Adding Test Compound to Cells]
Prepare test compound at 10× final concentration in appropriate solvent (e.g., 1% DMSO).
?. [3.5.1 Adding Test Compound to Cells]
To determine the optimal concentration of test compound for residence time analysis, it is recommended to first determine the compound IC50 using the equilibrium-based Target Engagement Protocol (see Section 3.2.2 "[Day 2]: Preparation of Cells with 1× Tracer for BRET Assay").  Introduce the test compound at a final concentration of 10–20× Ki,app determined under equilibrium conditions. This insures that adequate washout conditions can be achieved prior to off-rate measurements. Add  to each 1 mL of cells in assay medium and mix gently. (Optional) Include a positive control treatment of a compound at a high and saturating dose as a full-occupancy control, as well as a negative control treatment with compound vehicle.
[10× test compound]
?. [3.5.1 Adding Test Compound to Cells]
Place the conical tube in a rack, and incubate the tube with the cap loosened at , 5% CO2 for a minimum of  or until sample is expected to reach equilibrium.
37 °C
?. [3.5.2 Preparing BRET Reagents]
To prepare Complete 20× tracer reagent, first prepare a 100× solution of BRET tracer in 100% DMSO. Add 4 parts Tracer Dilution Buffer to 1 part of 100× tracer to generate Complete 20× tracer reagent.
?. [3.5.2 Preparing BRET Reagents]
For analysis of residence time in live cells, prepare a 2× solution of NanoBRET(TM) Nano-Glo(R) Substrate plus Extracellular NanoLuc Inhibitor by diluting the NanoBRET(TM) Nano-Glo(R) Substrate 1:250 and the Extracellular NanoLuc(R) Inhibitor 1:750 in a conical tube with Opti-MEM without serum or phenol red. For example:For a 96-well plate, mix , , and  to produce 10 mL of 2× NanoBRET™ Nano-Glo(R) Substrate plus Extracellular NanoLuc(R) Inhibitor Solution. Mix gently by inversion 5–10 times. For analysis of residence time in permeabilized cells, include 100 μg/mL digitonin and omit the Extracellular NanoLuc(R) inhibitor in the 2× solution.
[NanoBRET(TM) Nano-Glo(R) Substrate]
[ Extracellular NanoLuc(R) Inhibitor]
[assay medium]
?. [3.5.3 Preparing Cells and Dispensing Reagents for the Residence Time Assay]
After incubation with test compound is complete, centrifuge cells at .
Centrifuge: 200 34, 5 min
?. [3.5.3 Preparing Cells and Dispensing Reagents for the Residence Time Assay]
Optional Wash: To remove unbound test compound, resuspend cells in prewarmed assay medium, centrifuge cells and remove medium. Maintain the concentration of the positive control compound for the full-occupancy control.
?. [3.5.3 Preparing Cells and Dispensing Reagents for the Residence Time Assay]
Remove medium, resuspend cells in prewarmed assay medium, and adjust cell density to 2 × 105 cells/mL.
?. [3.5.3 Preparing Cells and Dispensing Reagents for the Residence Time Assay]
Dispense  per well into white, 96-well plates. Periodically mix cells to avoid cell settling in the tube.(Optional) For background correction, dispense  in triplicate as no-tracer control samples.
[cell suspension with test compound or controls]
[cell suspension]
?. [3.5.3 Preparing Cells and Dispensing Reagents for the Residence Time Assay]
Add  prepared above (see Section 3.5.2 "Preparing BRET Reagents").
[2× NanoBRET(TM) Nano-Glo(R) Substrate]
?. [3.5.3 Preparing Cells and Dispensing Reagents for the Residence Time Assay]
Dispense  per well to cells. Mix the 96-well plate on an orbital shaker for .(Optional) Prepare a separate set of samples without tracer for optional background correction steps.
[Complete 20× NanoBRET™ Tracer Reagent]
33
?. [3.5.3 Preparing Cells and Dispensing Reagents for the Residence Time Assay]
Immediately proceed to the next step (see Section 3.5.4 "BRET Detection in Kinetic Mode").
?. [3.5.4 BRET Detection in Kinetic Mode]
Set a NanoBRET™ Assay-compatible instrument to perform repeat measurements for the desired time interval. As a starting point, use  intervals between each measurement, and be sure to include a sufficient number of measurements to determine compound dissociation. Set the instrument at  to minimize sample evaporation. However, as temperature may impact residence time profiles, it may be valuable to evaluate physiological temperatures.
25 °C
?. [3.5.4 BRET Detection in Kinetic Mode]
Measure donor emission wavelength (e.g., 450 nm) and acceptor emission wavelength (e.g., 600 nm), and determine the BRET ratio as described earlier (see Section 3.2.4 "Detection in Microplate Luminometer Equipped with Appropriate BRET Filters").
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Mix desired cells with lipid–DNA complexes as described in Section 3.2.1 "[Day 1]: Transient Transfection of HEK293 Cells with NanoLuc(R) Fusions".
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Dispense  into a sterile, tissue-culture treated 96-well plate, and incubate at least  to allow expression. We recommend a cell density of approximately 55,000–80,000 cells/cm2 during the transfection (for example, use approximately 20,000 cells/well for a 96-well Corning #3917 assay plate).
[cells and the lipid–DNA complex]
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
On day 2, gently remove medium from the assay plate with transfected HEK293 cells via aspiration.
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Add back  per well. Include suitable controls as indicated in Sections 3.5.1 "Adding Test Compound to Cells" and 3.5.3 "Preparing Cells and Dispensing Reagents for the Residence Time Assay."
[prewarmed assay medium]
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Add  to each assay well. For full-occupancy control, include a sample with a saturating dose of a positive control compound. For zero-occupancy control, add only DMSO. If the optional “No tracer control samples” were added to the experiment, do not add test compound.
[10× test compound]
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Mix on orbital shaker for .
33
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Incubate the assay plate at , 5% CO2 for a minimum of  or until sample is expected to reach equilibrium. Shorter or longer incubation times may be required depending on the characteristic of the test compound.
37 °C
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
After incubation with test compound is complete, gently aspirate to remove the medium and test compound from the assay plate. For the full-occupancy control, do not perform this step. Test compound should remain in the sample.
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
To remove residual unbound test compound, gently add  to each well and incubate at  for . For the full-occupancy control, do not perform this step. Test compound should remain in the sample.
[assay medium]
0 Room temperature
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Gently aspirate the medium and dispense  per well.
[assay medium]
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Add  to each well of the 96-well plate (prepared as described in Section 3.5.2 "Preparing BRET Reagents" in the protocol for residence time in NBS format).
[2× NanoBRET(TM) Nano-Glo(R) Substrate plus Extracellular NanoLuc(R) Inhibitor solution]
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Dispense  per well. Mix the 96-well plate on an orbital shaker for .
[Complete 20× NanoBRET(TM) Tracer Reagent]
33
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Immediately measure BRET in kinetic mode as described in Section 3.5.4 "BRET Detection in Kinetic Mode" in the protocol for residence time in NBS format.
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
Process data as described above in the protocol for residence time in NBS format (see Section 3.5.4 "BRET Detection in Kinetic Mode").
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
To achieve quantitation in living cells, the BRET technique exploits permeable fluorescent reporter probes (or tracers), derived from chemical matter with appropriate binding characteristics for the select target protein or protein family. Tracers may be derived from compounds with high selectivity for the target protein, but in many cases, broad-spectrum inhibitors or published tool compounds can be utilized to achieve greater coverage if desired [4].The chemical synthesis of functional tracers involves the covalent conjugation of a biologically active small molecule to a fluorescent organic dye. Certain organic dyes have proven optimal in maintaining cell permeability with spectral properties required for NanoBRET(TM) (Fig. 2a). These dyes are readily coupled to base compounds containing pendant amine groups via a one-step reaction of a highly reactive, activated ester group on the dye to produce a stable amide linkage.Fig. 2The structures of NanoBRET(TM) activated ester dyes (a) and example linker building blocks (b). NanoBRET(TM) dyes balance permeability and energy transfer efficiency, resulting in higher signal, stability and reactivity. Dyes directly coupled to the inhibitor yield the tracers, though it may be necessary to install a linker group to provide separation between the core and the fluorescent tag. In general, C3 to C6 alkyl, as well as O1 to O4 PEGs are suitable as starting pointsTracers must permeate live cells and function in a complex intracellular environment. Thus, there are several physical requirements that must be considered during the design phase. First, high affinity compounds may yield the most useful intracellular tracers. Within the milieu of cells, strong affinity may be required to overcome competition from endogenous metabolites or enzymatic substrates that may compete directly with the tracer. Second, the intrinsic binding kinetics of tracer for the target may also be important for consideration. In general, compounds with rapid equilibration kinetics also offer better capabilities for real-time analysis of target engagement (i.e., residence time analysis). Finally, the base scaffold must have a solvent exposed reactive group for conjugation. This site can be determined through cocrystal structure, SAR analysis, or homology models. Although a variety of synthetic approaches can be employed, NanoBRET(TM) dyes prepared as activated esters can be readily conjugated to the scaffold via a primary or secondary aliphatic amine. These amine groups have been shown to react quickly and quantitatively with activated ester dyes and amino acid linkers.A variety of dye classes have been evaluated in BRET assay formats, and dyes representing two fluorophore classes (Fig. 2a) have been identified that demonstrate consistently robust cell permeability and optimal spectral overlap for energy transfer. Derivatives containing varied linkers and dyes may perform differently in the assay (examples provided in Fig. 2). Therefore, as a starting point for assay development, it is initially recommended that at least four derivatives be prepared and evaluated in the BRET configuration: Minimal/no linker derivatives conjugated to NanoBRET™ 618 and NanoBRET™ 590 dyes and extended linkers (e.g., multi-PEG or alkyl derivatives) conjugated to NanoBRET™ 618 and NanoBRET™ 590 dyes. This panel of tracer candidates can be created from common intermediates and will provide structural diversity required to ensure the highest probability of success, and if required, guide further optimization. To serve as examples, these principles have been employed to create a panel of candidate cell-permeable tracers based on crizotinib, a multikinase inhibitor scaffold (Fig. 3).Fig. 3Chemical structures of four candidate tracer derivatives based upon the crizotinib scaffold. Similar dye/linker variants can be created for a wide variety of base molecules by conjugation to solvent exposed primary or secondary amines
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
3.1.1 Example Synthesis of Amine-Linked Tracers:
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Charge the small molecule precursor, functionalized with an amine, () into a 5 mL amber vial.
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Add  and stir.
[anhydrous dimethylformamide (DMF) ]
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Add , allowing the mixture to stir for .
[N,N-Diisopropylethylamine]
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Add . Cap and allow to react in the dark for .
[NanoBRET™ 590 SE]
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Monitor the reaction for consumption of the starting amine by analytical HPLC. The reaction is judged complete when this material has been consumed. The reaction progresses quickly and is often near completion after (see Note 1).
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Dilute the crude mixture with the reaction quenching solution.
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Purify the compound using reverse-phase preparative HPLC. Most tracers can be purified using standard HPLC methods. Use a gradient of trifluoroacetic acid (TFA) HPLC Buffer (or 0.1% formic acid in water) in acetonitrile as an eluent.
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Pool product containing fractions and concentrate to dryness under reduced pressure.
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Dissolve the resulting film in approximately  and concentrate to dryness a total of three times (see Note 2).
[acetonitrile]
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Dry the resulting film for a minimum of  to remove trace volatile solvents.
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Dissolve the resulting solid in an appropriate volume of DMSO to give a  solution.
?. [3.1 Development and Characterization of NanoBRET™ Tracers with Optimal Cell Permeability]
Calculate the concentration of the solution using the following molar extinction coefficients: εNanoBRET™ 590 = 83,000 M−1 cm−1, εNanoBRET™ 618 = 96,000 M−1 cm−1.
?. [3.2 Verification of Tracer Permeability and Optimization of BRET]
BRET is highly sensitive to molecular distance and orientation between the donor (luciferase) and acceptor (fluorescent tracer) partners. This feature enables a bona fide measurement of direct molecular interactions inside intact cells. However, optimization may be warranted to identify the conditions producing optimal BRET between the donor/acceptor partners. This can generally be achieved without a laborious effort, via transient transfection of plasmids encoding target/NanoLuc(R) fusion proteins (see Note 3) [4]. The following basic protocol can be used to determine the optimal orientation of NanoLuc(R) luciferase for target engagement analysis via BRET. It is generally recommended that novel NanoBRET(TM) tracers are evaluated in both live cells, as well as permeabilized cells (via addition of digitonin or Passive Lysis buffer), to assess tracer affinity in the absence of interfering cellular factors. Figure 4 provides an example where the protocol is used to determine the optimal tracer for a kinase target, and Figs. 5 and 6 provide examples wherein the protocol is used to determine optimal NanoLuc(R) fusion orientation or expression level, respectively.Fig. 4Verification of tracer permeability and affinity. HEK293 cells transfected with NanoLuc(R) fusion plasmids were titrated with tracers. Cells treated with 50 μg/mL digitonin were included as controls for engagement of poorly permeable tracers. Each tracer demonstrated specific BRET with MET kinase in live cells and permeabilized cells. Both of the NanoBRET(TM) 590 tracers yielded the strongest cellular engagement and proved to be optimal for MET kinase. In certain cases, alternate dye/linker combinations may prove optimalFig. 5Optimization of NanoLuc(R) orientation for BRET. (a) For HDAC10, BRET was optimal with NanoLuc(R) on the C-terminus of the target protein. For (b) TNK2 both N- and C-terminal NanoLuc(R) fusions performed optimally in BRET experiments. (c) For ABL2, both N- and C-terminal NanoLuc(R) fusions performed in BRET experiments, but theN-terminal placement of NanoLuc(R) performed optimally. N- and C-terminal orientations should be tested when possibleFig. 6Optimization of expression levels for BRET studies. Expression levels can be easily optimized by dilution of the expression plasmids into a promoterless carrier DNA (Transfection Carrier DNA). This maintains a constant amount of DNA for transfection, but allows for lower expression levels across the population of transfected cells. (a) For the GPCR example (Histamine H1 receptor), BRET signal and tracer engagement was sensitive to expression levels, supporting use of lower expression levels for the receptor of interest. (b) For DDR1 kinase, BRET and tracer engagement was unaffected by expression levels. In general, it is recommended that low expression levels be used to ensure physiological conditions and avoid ligand depletion
?. [3.3 Considerations for Quantitation of Compound Affinity by Competitive Displacement of Tracers]
As described above (see Section 3.2 "Verification of Tracer Permeability and Optimization of BRET"), the basic protocol details the measurement of the intracellular engagement potency (IC50 of BRET attenuation) of a test compound at a single concentration of tracer. In general, compound affinity is measured under steady-state conditions between the test compound, the tracer, and the intracellular target protein. For the simple determination of compound rank-order affinity, it is recommended to use the tracer at a fixed concentration approximating 50−80% occupancy at the target. However, tracers can be exploited to provide a more quantitative apparent affinity value, as engagement potency is often dependent on the concentration and affinity of the tracer used in the assay. As the tracer and test compound bind to the target in a competitive and mutually exclusive manner, the relationship between the apparent IC50of the test compound and the concentration of tracer used in the assay is governed by the Cheng–Prusoff Equation [8] (Eq. 2).
  (2)K_{i,app} = \frac{IC_{50}}{1 + \frac{[tracer]}{K_{d,app}}}Equation 2 provides the quantitative capability to extract an apparent dissociation constant (Ki,app) for the test compound. This analysis yields a value that is insensitive to tracer concentration. Considering the limits of Eq. 2 with respect to tracer concentration, in situations where the concentration of tracer is much less than its affinity (Kd,app), the measured IC50 for the test compound will generally be within twofold of the Ki,app (i.e., if [tracer] Kd,app, then IC50 ≅ Ki,app). Conversely, when the concentration of tracer increases above the Kd,app, the apparent IC50 value of the test compound shifts to higher concentrations.The Cheng–Prusoff relationship can be used to determine the Ki,app for a test compound in multiple ways. For example, if the apparent affinity of the tracer (Kd,app) has already been measured, Eq. 2 can be used to convert an IC50 value measured at a single, known concentration of tracer to the Ki,app for the test compound. Alternatively, Eq. 2 can be algebraically linearized (Eq. 3) and used experimentally to extract the Ki,app, a method known as linearized Cheng–Prusoff analysis [9]. In practice, the apparent IC50 value for the test compound is measured at multiple tracer concentrations, after which the IC50 values are replotted as a function of tracer concentration. Regression analysis yields a linear plot with a y-intercept equal to the Ki,app, and slope equal to Ki,app/Kd,app. Figure 7 provides examples in which linearized Cheng–Prusoff analysis was used to quantify the Ki,app for the binding of the broad spectrum bromodomain inhibitor bromosporine to NanoLuc-bromodomain fusions. 
  (3)IC_{50} = {K_{i,app}}({1 + \frac{[tracer]}{K_{d,app}}})Fig. 7Linearized Cheng–Prusoff analysis to determine bromosporine affinity to CECR2 Bromodomain (BD) and TAF1 BD1-BD2. HEK293 cells transiently expressing NanoLuc(R) fusions to bromodomains were seeded into 96-well plates and then treated with a dilution series of bromosporine at increasing concentrations of tracer. Panel A depicts dose–response curves for bromosporine at each individual tracer concentration. Individual curves were fitted with the sigmoidal dose–response curve to determine the apparent IC50. Replots of the IC50values as a function of tracer concentration (panel B) show linearity and allowed calculation of the Ki,app for bromosporine and Kd,app of the tracer using Eq. 3The linearized Cheng–Prusoff analysis is generally recommended for determinations of Ki,app for test compounds, particularly if binding of the tracer is saturable. For targets where tracer binding is weak and not saturable, but signal window (SW) is still adequate for IC50 determination (in practice, SW ≥ 1.6), the measured IC50 will often be a good approximation for the Ki,app (generally within twofold, as discussed above) (see Note 6).
?. [3.4 Liquid Handling Requirements and Considerations for High-Throughput Screening (HTS) and Lead Optimization]
Cellular target engagement assays can provide critical information for hit prioritization at both the HTS and lead optimization stages, particularly when the biochemical and cell-based assays fail to correlate. Accordingly, compounds that are similarly active in biochemical, cellular target engagement, and cellular activity assays can be assigned highest priority, while those that are very weak or inactive in a cellular target engagement assay, yet active in the other two formats, should be binned as suspicious. Such compounds are likely to trigger cellular activity assay inhibition through indirect effects. A sensitive cellular target engagement assay may also show correlative activity with the biochemical assay for certain compounds, yet fail to demonstrate cellular activity. In this case, the compound or compound series may be valid, yet require chemical optimization to allow cellular activity. It would therefore be valuable to apply the NanoBRET™ target engagement assay for HTS and as a key aid for dissection of mechanism in lead optimization.It is possible to configure the steps and equipment to higher throughput, in order to enable efficient intracellular target engagement-based HTS and lead optimization. In order to afford the throughput required for HTS and lead optimization, several steps to streamline the NanoBRET™ process are suggested. Strategies to maximize efficiency include switching from 96-well to 384-well format to reduce the number of plates required and the plate “real estate” occupied by controls, reducing the number of steps by using assay-ready plates, and implementation of robotic liquid handling protocols. Preparation of assay-ready plates requires acoustic droplet liquid dispensing technology. While relatively expensive, it is becoming increasingly available in core facilities within both academic and industrial research centers.It is recommended that each 384-well plate contain a set of controls occupying one entire row of 24 wells and comprising a dose response with a 12-point concentration range. Be sure to include appropriate controls including a zero BRET control (i.e., no tracer) as well as a “BRET specificity control” (i.e., a high concentration of a control inhibitor in the presence of tracer) (see Notes 7 and 8).Table 1 summarizes the results of target engagement analysis for a panel of full-length intracellular kinases in a scaled format (i.e., automated, 384-well format). As a measure of statistical robustness, each assay yielded a Z’ above 0.4, supporting suitability in HTS work flows. As a future direction, scaling the BRET technique to higher density (e.g., 1536-well plates) may be possible.TargetMAP4K1BTKLTKITKMAP4K2TBK1DDR1CDK2TECTBK1Plus tracer signal (mBRET)92.69174.6104.778.4655.41124.354.8236.22134.655.7Plus tracer signal stdev3.853.123.871.572.385.413.082.6013.14.01No tracer signal (mBRET)20.0821.8620.3620.8019.8520.4119.8017.4024.5620.90No tracer signal stdev0.543.150.610.910.552.132.170.803.512.71Z′0.820.880.840.870.750.780.550.460.550.42S:B4.627.995.143.772.796.092.772.085.482.67Tracer Kd (M)1.50E-066.00E-071.70E-069.70E-085.40E-074.90E-079.80E-072.70E-063.20E-071.70E-06
?. [3.5 Optimization of Energy Transfer for Residence Time Studies in Live Cells]
Although equilibrium-based analysis is predictive of engagement in a closed system (i.e., conditions of static drug concentrations), nonequilibrium analysis may better predict target occupancy in open systems, where drug concentrations are dynamic. The compound residence time (i.e., koff−1) may better dictate target occupancy in the dynamic regime of a living organism [10, 11]. The energy transfer technique can be readily configured to measure real-time binding kinetics in live cells, thus providing an approach to assess drug residence in an artificial open system. The open system can be simulated by applying an acute treatment of test compound to the cells, followed by removal of unbound compound and a real-time measurement of residence time using energy transfer (Fig. 8).Fig. 8Illustration of BRET for intracellular residence time analysisPrior to performing a residence time analysis, it is important to determine a few key parameters. First, it is important to select an optimal tracer and determine a working concentration of the tracer for BRET measurement after compound washout. In general, it is recommended to use a saturating concentration of tracer that has fast association and dissociation kinetics with the target of interest. While this may be achievable in practice, the binding of the tracer might not be saturable for some targets. Thus, it is important to optimize the association kinetics at the highest tracer concentration possible. For new tracers and/or new target combinations, it is recommended to initially evaluate the kinetic parameters in a dose-responsive manner to ensure that an optimal tracer and working concentration are selected for the downstream residence time analysis. This is accomplished via observed rate constant (kobs) analysis, wherein the establishment of BRET at the target is measured kinetically over time at increasing tracer concentrations. Subsequently, the kobs is determined by nonlinear regression analysis of the kinetic curves for each tracer concentration using the one-phase association equation (Eq. 4), assuming pseudo-first order association [12]. The kobs values are replotted with respect to tracer concentration, which yields a linear relationship. Fitting Eq. 5 to the data allows dissection of the kon (from the slope) and koff (from the y-intercept) of the tracer. Figure 9 provides an example in which a kobs analysis is performed to choose between two different tracers for kinetic analysis of the BRD9 Bromodomain.
  (4)Y=Y_o+(Plateau−Y_o)×(1−e^{−k_{obs}∗t})
  (5)k_{obs}=k_{on}×[tracer]+k_{off}Fig. 9Kinetic optimization of bromodomain tracers. HEK293 cells transiently expressing NanoLuc(R)-BRD9 bromodomain were seeded into 96-well plates, after which a dilution series of tracer was added and the BRET measured kinetically. Panels A and B depict tracer association curves for two different bromodomain tracers. Individual points represent the mean of three technical replicates. Nonlinear regression analysis using the one-phase association equation (Eq. 4) was performed to extract an observed rate constant (kobs) at each tracer concentration. Observed rate constants were replotted with respect to tracer concentration (panel C), yielding linear relationships. Kinetic parameters extracted by linear regression using Eq. 5 are tabulated in panel D. Tracer 2 gave faster association and dissociation kinetics compared to tracer 1 and was chosen for downstream residence time analyses at a working concentration of 1 μMGenerally, higher values of both kon and koff are desired for residence time analysis to ensure that the tracer binding is at equilibrium with the target at any given time once the test compound dissociates. However, in many cases, a simple comparison of the relative kobs value itself may be sufficient to select an optimal tracer among a few lead candidates. Though equilibration for tracers may vary greatly (nearly instantaneous to hours), equilibration of the tracer at the target within 20−30 min is sufficient for a qualitative assessment (i.e., rank-order assessment) of test compound residence time.After the tracer and working concentrations are established, it is recommended to determine an appropriate acute dose of the test compound, as well as the length of time required to achieve equilibration with the target in the cellular context (see Note 9). Figure 10 provides an example for the determination of the acute dose and equilibration time for the binding of two test compounds to the histone deacetylase, HDAC1. Figures 11 and 12 provide example residence time analyses for the binding of the same two test compounds at HDAC1, with cell permeabilization controls to evaluate the effect of intracellular trapping, respectively.Fig. 10Determination of optimal inhibitor concentrations for compound treatment prior to residence time analysis. BRET analysis was performed in real-time to determine an appropriate equilibration time and inhibitor concentration for cellular washout experiments. In general, an inhibitor concentration approximating 90% occupancy (e.g., 10−20× Ki,app) is optimal prior to washout studies. (a) For HDAC1, equilibration with SAHA was proven to be rapid, indicating that a concentration of 1 μM SAHA is suitable following a short incubation time. (b) For FK228, equilibration kinetics were slower, indicating that a dose of 100 nM is appropriate, and 3 h of equilibration is required to achieve HDAC1 occupancyFig. 11Residence time analysis of HDAC1 with SAHA and FK228 in live cells. SAHA shows rapid dissociation, as expected. The occupancy of HDAC1 with FK228 following washout was equivalent to the full occupancy control (SAHA, no washout), thus indicating durable binding. Thus, FK228 shows protracted residence time at HDAC1, consistent with previous reportsFig. 12Control residence time experiments in permeabilized cells to ensure that kinetic profiles are not influenced by intracellular trapping of the test compound. FK228 maintains a long residence time after permeabilization of the cells, thus supporting previous results in live cells of durable binding
?. [3.5.5 Adapting the Residence Time Protocol to Adherent (ADH) Format]
In addition to the residence time protocol described above, residence time analysis can be performed in adherent (ADH) format in which the cells are adhered to a tissue culture-treated assay plate and the compound incubations are performed directly in that assay plate. Adherent format has both benefits and drawbacks compared to the conventional format in the nonbinding surface assay plate (NBS format) described above that include the ability to test significantly more experimental conditions simultaneously (test compound concentrations and/or targets), and improved workflow for washout that reduces time between the initial washout and the tracer addition step and preserves kinetic information at early time points. However, in some cases the kinetics of the BRET signal may not achieve a constant value under steady state conditions, or may appear biphasic compared to those assessed in NBS-treated plates.Users should consider their specific goals for the residence time analysis to help select between the assay formats. Figure 13 depicts an example residence time determination for dasatinib and ibrutinib at BTK kinase in both NBS and ADH formats for comparison.