Cellular Kinase Assays that Deliver Quantitative Compound
Affinity, Occupancy, and Selectivity in Live Cells using NanoBRETJames Vasta, Cesear Corona, Jennifer Wilkinson, Morgan Thomas, Chad Zimprich, Mei Cong, Frank Fan,
Kristin Huwiler, and Matthew Robers
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711

4. Homogenous, Scalable Method to Assay>340 Kinases in Live Cells

NanoBRET™ Target Engagement is a real-time biophysicaltechnique that enables quantitative assessment of compound-target
protein binding in live cells, without disruption of cellular membrane
integrity. Specifically, cellular compound affinity, occupancy, andresidence time can be measured in a multi-well plate format. The
method utilizes energy transfer from a NanoLuc® luciferase-tagged
target protein and a cell-permeable fluorescent tracer that reversibly
engages the target protein of interest. The quantitative capability isachieved for select target kinases by simply adjusting theconcentration of the NanoBRET tracer to an appropriate level basedon its predetermined target affinity.

A.

This technique has now been broadly applied to the kinome. Cellularassays for >300 full-length protein kinases are available, including
integral membrane receptors and over 30 specific CDK-cyclin
pairings. Assay conditions that enable quantitative binding
measurements for each kinase have been determined. Quantitative
cellular affinity for various types of kinase inhibitors will bedemonstrated, including type I, II, and allosteric compounds. We willshow how this cellular method can also be applied to quantitative
measurement of compound selectivity against numerous kinases.

B.

NanoBRET™ Target Engagement
Kinase Assay Work-flowA.
Selectivity Among Similar Kinase Targets
TrkB
AXL
TrkA
ABL
TIE2INSRLCK

Normalized BRET

120100806040METALK

20010 -5C.

Kinases assayedusing NanoBRET TE

B.
Correlation to Phenotypic Data
Target Engagement, Log IC50 (nM)

1. Introduction

7. Determine Cellular Compound Selectivity
Against Similar Kinases

10 -4Z’ Analysis

10 -3

10 -2

10 -1

10 0

10 1

10 2

4
ABL1 LCK

3INSR
TRKA
TRKB

TIE2AXL

2ALK

1MET

R2 = 0.95
001234

Phoshpo-ELISA, Log IC50 (nM)

[Crizotinib] (M)

LIMK1 NanoBRET TE Assay (384-well)• The multi-kinase inhibitor Crizotinib’s cellular selectivity wasdetermined using NanoBRET TE (A). The primary targets, MET andALK, showed the strongest affinity.

0.025

BRET Ratio

0.020
0.015

Without Compound (Vehicle)

Z' = 0.75

With Competing Compound

0.010• Cellular selectivity of Crizotinib could be determined against multiple
kinases because NanoBRET TE

0.005

o Assays are quantitative for compound affinity, not just rank order
potency (see panel 3)0.0000102030Sample NumberTime-dependent target-compound occupancy (or residence time)
can also be obtained with using the NanoBRET method. An
assessment of kinetic and equilibrium selectivity of various kinase
inhibitors revealed different residence times for compounds withsimilar equilibrium affinity. By monitoring both cellular affinity andresidence time for a compound, unique inhibitor development
opportunities can be revealed.

o Assays are available for >340 kinases• Addition only cellular assay method performed in multi-well plates isscalable from 96-well to 384-well (A).

• NanoBRET TE data correlates well with cellular functional assays
such as phospho-ELISA (B)• >340 live cell kinase assays spanning the kinome, including full-length
wild-type and mutant kinases. Each kinase has assay conditions and data
available (B).

o NanoBRET TE assays are more specific and scalable than somefunctional assays• The ratiometric BRET data and addition only format for these assays
leads to excellent data quality (C).

5. Evaluate Diverse Chemical Matter andEffect of Clinical Kinase Mutations
A.

Affinity / Potency Determinations in Live Cells

A.

B.

1210810

BRET Ratio (mBu)

BRET Ratio (mBu)

BRET[Compound]

1464

Short Residence

RIPK1

ABL1
ABL-001 (IV)
Dasatinib (I)Ponatinib (II)GNF2(IV)
Imatinib (II)Residence Time Determinations

Ponatinib(II)Necrostatin I (III)KW-2449(I)
APY69(I)987Long Residence

BRET

2. NanoBRET Target Engagement

8. Intracellular Residence Time & Affinity
May Not Always CorrelateTime65410 -4 10 -3 10 -2 10 -1 10 0

210 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2

10 1

10 2

[Compound], M[Compound], M

B.

Equilibrium Binding, ABL1 Kinase

C.

Residence Time, ABL1 Kinase
Live Cells• NanoBRET TE assays are specific for the target fused to NanoLuc, as
BRET assays are governed by tight distance constraints between
energy donor (NanoLuc) and energy acceptor (Tracer).

[Tracer] ≤ Kd-apparent[Target] << compoundKd-apparent

010 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 020100
10 -5 10 -4 10 -3 10 -2 10 -1 10 080

6. Intracellular CDK Selectivity and Cyclin
Bias for Clinical Drugs

BRET Ratio (mBu)

Tracer Only
Tracer + excess unlabeled

60Window ofQuantitation2010 -210 -1

B.

A. Dinaciclib (SCH727965)

25
20151050
10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2[Compound], uM

100

500Occupancy (%) =[1−(X−Z)/(Y−Z)]*100

10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2[Compound], uM• Quantitative compound affinity is achieved when (A):o [Tracer] ≤ Kd-apparent, which is provided for each kinaseo [Target] << compound Kd-apparent, achieved by low kinase expression.
• NanoBRET TE Kinase-specific application notes allows quick start-up byproviding assay conditions including Kd (B)o Compound IC50 approach Kiapparent by using tracer concentration < Kd
o Therefore, NanoBRET TE measures more than rank order potency• NanoBRET TE is quantitative for compound occupancy, by (C):o Using controls for background (Z) & max (Y) BRET
o Converting BRET Ratio of Sample (X) to Occupancyo Fractional occupancy allows quantifying compound selectivity across
many kinases

July 2020

10 0[Compound] ,M

10 2200-2020Imatinib
Vehicle406080Time (m)Dasatinib

100120Ponatinib• Intracellular residence time is evaluated using NanoBRET TE via real-time
kinetic analysis (A)• The chronic myelogenous leukemia (CML) drug Imatinib (1st generation)shows weaker binding (B) & shorter residence time (C) at ABL1, compared
to Dasatinib and Ponatinib (2nd & 3rd generation drugs).
• Ponatinib displays similar affinity (B) to Dasatanib, but much longerresidence time (C).

9. Conclusions

NanoBRET TE Kinase assays are quantitative for compound
affinity and occupancy inside live cells• Kinase inhibitor types I, II, III, and IV have been studied• Equilibrium cellular assay measures direct binding of compound tokinase, not a downstream event

Omipalisib
Taselisib
Gedatolisib
Copanlisib
PF-502
PI-103
Dactolisib
Alpelisib
Apitolisib
AZD-8835
Buparlisib

100

C D K 2 / cycEC D K 2 / cycA

50010-410-310-210-1100[ D in a c ic lib ],  M101102

N o r m a liz e d B R E T (% )3010 -2

40NanoBRET TE cellular assays have been developed for >340
full-length kinases• The assays are ready-to-use with application notes for each kinase• Multi-well plate addition only assay format is simple & scalable• BRET read-out provides excellent data quality

10 0

N o r m a liz e d B R E T (% )35

10 -460

Abemaciclib (LY2835219)

PIK3CA/PIK3R1
Live HEK293 Cells

Omipalisib
Taselisib
Gedatolisib
Copanlisib
PF-502
PI-103Dactolisib
Alpelisib
Apitolisib
AZD-8835
Buparlisib

410 -6

Ponatinib
Imatinib

80

40010 -3

Occupancy (%)

BRET Ratio (mBU)

408Dasatinib[Compound], M

Converting BRET Ratio to Fractional Occupancy
PIK3CA/PIK3R1
Live HEK293 Cells

1210 1[Tracer K-3], M

C.

16100

Tracer Titration & Kd
Live HEK293 Cells

Signal1030• The ability wild-type MET and the clinical MET mutant D1228V to bind various type I& II kinase inhibitors were evaluated using NanoBRET TE. The type I inhibitors
Capmatinib and Savolitinib bound potently to WT MET, as did the type II inhibitors(C). The MET (D1228V) mutant failed to bind these type I inhibitors, but retained
ability to bind the type II inhibitors Glesatinib, Foretinib, Merestinib, & Cabozantinib.

PIK3CA/PIK31[Tracer]20

Capmatinib (I)Savolitinib(I)
Glesatinib(II)
Foretinib(II)
Merestinib(II)
Cabozantinib (II)• NanoBRET TE Kinase Assays can be used to characterize type I, II, and allosteric(III, & IV) kinase inhibitors as shown for ABL1 and RIPK1 kinases (A & B).

B. Kinase Specific Application Notes
Provide Assay Conditions
Application Note3040[Compound], M

3. NanoBRET TE Kinase Assays Quantitativefor Affinity & Occupancy••40

BRET Ratio (mBu)

BRET Ratio (mBu)• BRET is achieved by luminescent energy transfer from NanoLuc®
luciferase to the cell-permeable fluorescent Tracer that is bound to thetarget-NanoLuc fusion protein.

A. Conditions for Quantitation

MET (D1228V)

MET, Wild-Type
MET

12020
Normalized ResponseC.

BRET Ratio (mBU)

Live Cells• NanoBRET Target Engagement (TE) assays quantitatively monitor
compound binding (affinity & occupancy) to a target protein in live
cells in a multi-well plate format using BRET.

Very LongResidence orCovalent

100
C D K 6 / cycD 1
C D K 6 / cycD 3
50

Compound selectivity inside live cells is measured using
NanoBRET TE Kinase• Quantitative compound affinity measurements against multiple
kinases enable compound selectivity determination• NanoBRET TE selectivity data is physiologically relevant, correlating
to cellular functional data

010-510-410-310-210-1

100101102[A b e m a c ic lib ],  M

Residence time in live cells is measured with NanoBRET TE• Using both equilibrium & residence time methods, selectivity may berevealed- offering unique inhibitor development opportunities.

• Cellular selectivity profile of Dinaciclib identifies primary targets of CDKs
1 - 6 & 9, in addition to binding several cyclin Y CDKs (CDKs 14-18).
Dinaciclib intracellular potency can depend on cyclin pairing (A).
• Cellular selectivity profile of the selective CDK inhibitor Abemaciclib
reveals the primary targets CDKs 4 & 6. Also, it binds CDKs 14-18.
Abemaciclib intracellular potency can depend on cyclin pairing (B).

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NanoBRET is a trademark of Promega Corporation
NanoLuc is a registered trademark of Promega Corporation
For Research Use Only. Not for use in diagnostic procedures.

Corresponding author: kristin.huwiler@promega.com