Single tracer-based protocol for broad-spectrum kinase profiling in live cells with NanoBRET

Summary This protocol is used to profile the engagement of kinase inhibitors across nearly 200 kinases in a live-cell context. This protocol utilizes one single kinase tracer (NanoBRET(TM) Tracer K10) that operates quantitatively at four different concentrations. Minimizing the number of tracers offers a significant workflow improvement over the previous protocol that utilized a combination of 6 tracers. Each NanoBRET(TM) kinase assay is built using commercially available plasmids and has been optimized for NanoLuc tagging orientation, diluent DNA, and tracer concentration. For complete details on the use and execution of this protocol, please refer to Vasta et al. (2018).


SUMMARY
This protocol is used to profile the engagement of kinase inhibitors across nearly 200 kinases in a live-cell context. This protocol utilizes one single kinase tracer (NanoBRET(TM) Tracer K10) that operates quantitatively at four different concentrations. Minimizing the number of tracers offers a significant workflow improvement over the previous protocol that utilized a combination of 6 tracers. Each NanoBRET(TM) kinase assay is built using commercially available plasmids and has been optimized for NanoLuc tagging orientation, diluent DNA, and tracer concentration. For complete details on the use and execution of this protocol, please refer to Vasta et al. (2018).

BEFORE YOU BEGIN
Prepare concentrated DNA stock solutions (0.2 mg/mL) and 23 working DNA solutions (20 mg/mL) Timing: 1-10 h 1. Table 1 describes the characteristics of each kinase and its performance in the NanoBRET(TM) assay with Tracer K10. Obtain this library of plasmids encoding NanoLuc/kinase fusions (from Promega individually or en masse as a collection).
Note: Each plasmid has been purified with low endotoxin levels. If this library is being generated de novo, be sure to utilize purification methods that yield low endotoxin. each plate in an air-tight plastic bag. When thawing the DNA plate, spin the DNA plates prior to removing the foil cover.
3. From the concentrated stock solutions, prepare 23 working DNA solutions for day-to-day experimentation. Dilute the stock 0.2 mg/mL plasmid DNA solutions 1:10 into nuclease-free TE buffer to achieve a total final concentration of 20 mg/mL DNA in TE buffer. 4. As described above, seal each plate with foil tape covers to avoid evaporation. To further minimize evaporation during storage, enclose each plate in an air-tight plastic bag. Ideally, plates stored long-term will be heat-sealed. 5. Store these 20 mg/mL solutions for up to 4 weeks at À80 C and avoid greater than 53 freeze/thaw cycles. When thawing the DNA plate for transfection, spin the DNA plates prior to removing the foil cover. This will minimize the cross-contamination. Reseal with fresh seals after each use. i. Technical replicates (recommended) can be used for each control and test compound sample ii. Transfection Control (recommended): Use this control to determine if the transfection was successful prior to executing the complete NanoBRET assay. Process this sample prior to the assay setup on day 2. Prepare this transfection on a separate plate. Transfect at least 4 wells for this purpose. The MET-NanoLuc plasmid is an ideal DNA for this purpose. Dilute the MET-NanoLuc control plasmid into Carrier DNA to match the conditions used for the experimental sample. b. Assemble 23 Kinase DNA solutions and transfer to a sterile 96-well polypropylene plate:

KEY RESOURCES TABLE
Note: For each well of analysis, you will require 3 mL of each 23 Kinase DNA solution at 20mg/mL.
Note: If frozen, we recommend thawing the DNA quickly at 37 C and centrifuging the plate or strip briefly to ensure the solution is at the bottom of the vessel. c. Prepare 23 Fugene HD solution: i. Dilute Fugene HD to a final concentration of 60mL/mL in room temperature (22 C-26 C) OptiMEM in a sterile conical tube, directly into the liquid.
Note: For each well of analysis, prepare 3 mL of 23 Fugene HD solution. d. Add equal parts of the 23 Fugene HD solution to the 23 Kinase DNA solutions. e. Mix on an orbital shaker for 15 s at 400 rpm. f. Allow complexes to form for 30 min at room temperature.
Note: For experienced users, the cells can be prepared during this step.
7. Transfer 5 mL per well of the Transfection Complex into 100 mL/well of the 96-well plate with the HEK293 cells. 8. Incubate 16-24 h at 37 C and 5% CO 2 .
Note: Allow a minimum of 16 h for transfection to occur, ideally between 20-24 h.

Day 2: Verify transfection efficiency with the transfection control samples
Timing: 30 min 9. Prepare 33 Complete NanoBRET TM Nano-Gloâ Substrate (included in the NanoBRET(TM) K10 Complete Kit) in OptiMEM without serum or phenol red. This solution consists of a 1:166 dilution of NanoBRET TM Nano-Gloâ Substrate plus a 1:500 dilution of Extracellular NanoLuc Inhibitor in OptiMEM without serum or phenol red. Mix gently by inverting 5-10 times in a conical tube. (The final concentration of Extracellular NanoLuc inhibitor in the 33 solution is 60 mM, for a working concentration of 20 mM.) Note: 33 solutions should be used within 1.5 h of preparation.
10. To wells with transfected cells: Add 50mL per well of 33 Complete NanoBRET TM Nano-Gloâ Substrate with NanoBRET TM Extracellular Inhibitor for a 96-well plate. Incubate 2-3 min at room temperature (RT). 11. To the empty wells that are on the opposite side of the plate: Add 50mL per well of 33 Complete NanoBRET TM Nano-Gloâ Substrate with NanoBRET TM Extracellular Inhibitor for a 96-well plate.

OPEN ACCESS
Incubate 2-3 min at RT. Note that measuring background wells adjacent to sample wells may result in signal bleed through. Therefore, to accurately quantify background luminescence, use wells of the plate that are not adjacent to wells that contain NanoLuc expression.. 12. Following addition of NanoBRET TM Nano-Gloâ Substrate, measure donor emission (e.g., 450 nm) and acceptor emission (e.g., 610 nm or 630 nm) using a NanoBRETä-compatible luminometer. 13. Determine the signal-to-background (S/B) in the 450 nM channel.

Timing: 2-3 h
This step is required for the cellular system to achieve equilibrium. This involves introduction of NanoBRET(TM) Tracer K10 at concentrations appropriate to quantify occupancy of each kinase. NanoBRET(TM) tracers generally equilibrate after short incubation times, however 2 h is recommended as a general incubation time for most kinase inhibitors. Longer incubations may be required for slow-binding inhibitors.

Addition of NanoBRET tracers
14. Based on the plate layout, calculate the number of data points needed for each concentration bin. a. Refer to Table 1 for a list of the four tracer conditions recommended (25 nM, 100 nM, 250 nM, 1000 nM). These concentrations are recommended based on kinase affinity measurements.
CRITICAL: For quantitative target engagement analysis, use NanoBRET(TM) tracers at or below their apparent affinity for each kinase. Therefore, tracer concentration should not exceed the values provided in Table 1. Although increasing tracer concentration will often produce a stronger BRET signal, the occupancy results may deviate due to over-saturation of the kinase target with tracer.  Figure 1) and pan-kinase inhibitor CC1 (at 300 nM; Figure 2). For first time users, it may be valuable to compare assay results to this table to ensure accurate experimental setup and BRET detection.

LIMITATIONS
The NanoBRET(TM) method may not be capable of detecting all modes of target engagement. For example, allosteric inhibitors that bind via a mechanism that is non-competitive with the ATP-site tracer may result in undetectable occupancy, thus representing false negatives. It is critical to recognize therefore, that complementary methods may be required to deconvolute some assay results that fail to correlate with those observed using alternate phenotypic or pathway analysis methods.

Potential solution
Weak expression observed in step 14 may be a result of: Inconsistent dispensing of tracer. Ensure that liquid handlers are accurately delivering the tracer to each well.

Potential solution
Negative fractional occupancy of test compound may be a result of: Inaccurate dispensing of tracer for DMSO samples (100% BRET, or 0 % fractional occupancy controls). Ensure liquid handling is accurately dispensing the NanoBRET(TM) tracer Figure 1. Results of live cell kinase profiling using control compound crizotinib at 1000 nM. Each dot represents a kinase occupied > 50% by the control compound Results are the mean of three independent experiments (n = 3) and summarized in Table 1. Illustrations were reproduced courtesy of Cell Signaling Technologies, Inc.
Auto-fluorescent or light scattering properties of the test compound. Optical effects may increase the BRET value. This is often determined by using an irrelevant BRET control assay. If the compound has the same effect on an irrelevant BRET assay, this is likely a spurious optical effect. Although rare, global / nonspecific impacts on kinase activation state may be observed. Nonspecific kinase inhibitors may indirectly impact the target of interest, thus increasing the activation state of the kinase. In some cases, increasing the kinase activation state may increase the apparent affinity of the NanoBRET(TM) tracer leading to a non-specific increase in BRET. It may be possible to run specific NanoBRET(TM) kinase assays in digitonin-treated cells to determine if this increase in BRET is due to such non-specific pathway influences as described in earlier studies Vasta et al., 2018).