2
Transcreener® ADP Assay: Simplifying
and Streamlining Kinase Profiling
from Experimental Design through Data Analysis
Meera Kumar, Thomas Zielinski, Karen Kleman, and Robert G. Lowery
BellBrook Labs, Madison, WI, USA
200
Figure 1. Transcreener assays are the only direct ADP detection
method available. ADP displaces tracer from a highly specific monoclonal
antibody resulting in a change in fluorescence, with fluorescence
polarization (FP), time resolved FRET (TR-FRET) and fluorescence
intensity (FI) formats available. All three are homogenous, mix and read
assays and use a red-shifted tracer to minimize compound interference.
ADP detection methods other than Transcreener (e.g., ADP Glo™, ADP
Quest™ ) rely on coupling enzymes, which convert the ADP to a detectable
product in a series of enzymatic steps.
0
40
80
200
150
50
50
0
0.1
0
120
0
Time, min
40
80
120
RFU
40000
10
1
100
1000
Figure 3. Transcreener assays can be run in real time to
measure initial progress curves. The equilibration reaction of ADP
Figure 4. Determining optimal enzyme concentration for inhibitor
profiling Shows the PKA titration curve at Km of ATP, 20µM, and 20µM Kemptide
binding to ADP2 antibody is less than ten minutes making kinetic
measurements possible with the Transcreener Assay. In the kinetic mode
one can save reagents, time and money making initial assay development
simple and easy.
after 1 hour incubation. The area within the two solid red lines represents 3%-20%
ATP conversion and is the recommended concentration of enzyme to generate
acceptable Z` (>0.7) for primary screens of inhibitors. The highlighted area within the
curve shows the concentration of enzyme one would choose to perform a dose
dependency inhibitor titration. This region is between EC60-EC80 concentration
(7-15mU PKA/Rxn) of the enzyme.
Determining IC50 Value directly from Raw
Fluorescence Data
0.86
0.71
0.92
ND
ND
0.85
0.72
0.88
0.3
ND
Linear Correlation between Raw Data and ADP
Formation
8000
5 (1)
16(10)
1.1(1)
11(1)
31(50)
3.1(1)
2.3(1)
1.7(1)
Substrate
0.01
1
100
H89
PKI
G06983
AG1478
1.5
1.0
0.5
0.0
0.0001
10000 1000000
0.01
Inh, nM
IC50- Raw Data (nM)
IC50- Converted Data (nM)
1
EC80
0.1 nM
Kemptide-KRRASKG
1.2 nM
Abltide-EAIYAAPFAKKK
2 nM
Dapktide-KKRPQRRYNVF
5 nM
MBP
2 nM
Poly(G-T)
3 nM
S6 Peptide-AKRRLSSLRA
RS Peptide-GRSRSRSRSRSRSR 4 nM
2.8 nM
poly [Glu, Tyr] 4:1
100
R Value 0.92
Slope 2.5 ± 0.12
19.6
1.2
1405
>10000
5.2
0.36
350
>10000
10
00
10
0
10
Concordance of Inhibitor Potencies
Between FP, FI and TR-FRET.
60
2
6000
4000
Figure 5. Determining IC50 values A) Shows the dose response
curves of representative inhibitors of PKA generated using the EC80
concentration of PKA (0.1 nM) in the presence of 20 µM ATP (Km) and 20
µM Kemptide . B) Shows the same dose response curves when the raw
data is converted to ADP produced. The IC50 of the raw data is
approximately 2.5 fold greater than the IC50 from the converted data.
R
40
TRF(nM)
FI(nM)
1.0 ± 0.05
0.82 ± 0.038
0.92
0.9
40
20
20
TRF(nM )
FI(nM )
2000
0
10000 1000000
Go6983 Tyrophostin AGI 1478
PKI
120
120
120
120
120
120
120
120
often screened using the Km concentration for ATP, but use of lower ATP
concentrations is sometimes desirable to bias screens more toward competitive
inhibitors and decrease enzyme usage. Less than 5 nM enzyme (EC80) can provide a
ΔmP of 100 or more using the Transcreener ADP2 Assay.
Slope
0
0
Inh, nM
H89
Reaction
Time
(Min) 30°C
Table 2. Detection of kinases at low ATP concentrations. Kinases are
2
2.0
100
0.0001
1
Robust Kinase Detection with Low ATP
IC50 FI (nM)
150
2.5
Raw IC50 (nM)
mP
200
0.
1
0.89
0.72
0.92
0.62
0.52
IC50 TRF (nM)
H89
PKI
G06983
AG1478
250
ADP produced, µM
300
0.
01
ADP, µM
60
B
0.
00
1
0.
00
01
10
00
0.
1
10
0
0
10
0.0
1
10000
0.
01
0.3
PKA, mU/Rxn
Time (Min)
A
1µM
0.1uM
30000
20000
0.
00
1
10
00
10
0
10
1
0.
1
0.6
50000
Figure 2. Standard curves for conversion of ATP to ADP demonstrate the outstanding
response of the Transcreener ADP2 Assays at different initial ATP concentrations.
Standard curves are used to mimic enzyme reactions. Starting at the indicated
concentrations of ATP, ADP is titrated and ATP is decreased proportionately, n = 24. A. FP
reactions were read in Tecan Safire2TM. B. TR-FRET reactions were read in BMG
Labtech`s PHERAstar Plus reader. C. FI assay was read in Perkin Elmer`s EnVision.
Table 1. The high sensitivity of the Transcreener ADP2 Assays allows detection of
10% conversion of ATP even at low levels of ATP. Z’ values were calculated and
compared with ADP Glo and Kinase Glo (Promega) reactions run under identical conditions.
PKA
ABL1
DAPK1
CLK1
Src
ROCK1
CLK4
Zap70
250
100
0
0.9
ATP Km
Enzyme
(Bin) (µM)
3%-------20% ATP Conversion
EC85
EC40
300
mP
∆ mP
∆ mP
50
1.2
1000µM
100µM
10µM
ADP µM
Transcreener FP
Transcreener TR-FRET
Transcreener FI
Luc-ADP Detection Assay
Luc-ATP Depletion Assay
150
3 nM Rock 1
1 nM Rock 1
1000µM
100µM
10µM
1µM
0.1µM
Z` at 10% Conversion
1 µM ATP/ADP 10 µM ATP/ADP 100 µM ATP/ADP
200
100
0.
01
ADP µM
0.75 nM ABL1
100
0.
00
1
0
1 nM ABL1
150
∆ Ratio 670/620
∆mP
100
EC60 -EC80
B
60000
1.5
1000µM
100µM
10µM
1µM
0.1µM
200
Optimal Enzyme Concentration for Initial
Velocity Reactions: EC60-EC80
Transcreener Allows Assays in Kinetic Mode
A
300
0.
00
01
ADP detection is an attractive approach for screening kinases and other ATP-utilizing
enzymes because it provides a universal platform that can be used for any member of the
kinase superfamily as well as many other ATP-dependent enzymes, regardless of the
acceptor substrate. The Transcreener ADP2 Assays use direct immunodetection of ADP
with three different fluorescent detection modes (FP, TR-FRET and FI) to provide a highly
sensitive and versatile HTS method for kinases and other ATP-utilizing enzymes. It is the
only ADP detection method that does not rely on the use of coupling enzymes to convert
ADP to a secondary analyte, which makes it less prone to interference from screening
compounds. In this poster, we compare the available ADP detection methods with
respect to assay principle, protocol, and performance and highlight the approach that we
have developed at BellBrook Labs to streamline kinase assay development with the
Transcreener ADP Assay, with an emphasis on minimizing experimental time and reagent
consumption and simplifying data analysis. Using a panel of six kinases, we outline a
streamlined approach for assay development including optimization of enzyme
concentration and reaction time to yield an outstanding signal window and enable
accurate determination of inhibitor potencies. Some of the key points include: a) the
ability to use Transcreener assays in kinetic or end point mode reduces experimental
time and reagent consumption during optimization; b) enzyme concentrations of less than
5nM can be achieved even with low-activity enzymes, insuring accurate IC50
measurements for high potency inhibitors; c) by using an empirically determined
conversion factor, IC50 values can be obtained directly from raw fluorescence data,
eliminating the need for standard curves; d) there is a good concordance between IC50
values determined using the three different detection formats. All of the methods outlined
are consistent with HTS assay guidelines set forth by the NIH Chemical Genomics
Center, and adhere to Michaelis-Menten kinetics assumptions.
Sensitive, Robust Initial Velocity Detection at Low
ATP Conversion in Any Plate Reader
0.
00
01
Direct Versus Coupled Enzyme Assay ADP
Detection Methods
Overview
20
40
60
IC50 FP (nM)
0
0
1000
2000
3000
Converted IC50 (nM)
Figure 6. Linear correlation of IC50 values determined
from raw polarization and ADP formation. The IC50 of raw data
and IC50 of converted data was generated using EC60, EC70 or EC80 concentrations
of the target enzymes and reference inhibitors. Enzymes used in the study were
PKA, AKT2, Rock1, Clk4, Zap 70 and Abl1 with ATP Km being 20µM , 300µM,
2µM,4µM, 3µM and 4µM respectively. The inhibitors used were Staurosporine,
K252a, H89, Tyrophostin AGI 1478, PKI and Gleevac. The slope of the line shows
that the IC50 of the raw data is approximately 2.5- fold greater than the IC50 of the
converted data.
Km of Kemptide for PKA ( µM)
Km of ATP for PKA(µM)
EC50 of PKA (mU/Rxn)
FP
FI
TRF
2
23.65
6
1.8
16.7
10
2.03
20
5
Figure 7. Equivalency of the three detection modes There is a
linear correlation for the IC50 values obtained with the three detection formats.
The slope of the line is close to 1 in both cases. The enzyme used in the study
was PKA and the inhibitors were Staurosporine, K252a, H89, Tyrophostin AGI
1478, PKI and Gleevac.
Table 3. Shows the three fluorescent detection modes generating similar Km
values and EC50 by using PKA and its inhibitors as an example.
Conclusions
•The Transcreener ADP2 Assay is the simplest ADP detection method available, relying on direct immunodetection instead of enzyme-coupled assays.
•The greater sensitivity of the Transcreener ADP2 Assay allows the practical use of ATP concentrations as low as 100 nM.
• For Inhibitor dose dependency curves we recommend using EC60-EC80 concentrations of target enzymes.
•There is a linear correlation between IC50 values determined from raw polarization data and ADP formation with a slope of 2.5.
•Similar inhibitor potencies, Km values and IC50 values were observed for the three detection modes of Transcreener assay.
Acknowledgments: This work is supported
by NIH SBIR grant :1 R43 GM073290-01A1
© 2012 BellBrook Labs. All Rights Reserved.
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