R b t Detection
Robust
D t ti off ATP
ATP-Utilizing
Utili i Enzymes
Utilizing
E
with
ith an
n IImproved
d
Fluorescence Polarization ADP Detection Assay
y
Karen M. Kleman‐Leyer, Tony A. Klink, Andrew L. Kopp, Tracy J. Wor
K
M Kl
L y , T y A Kli k, A d
L K pp, T y J W rzella, ll ,
C i A.
Cori
Cori A. Pinchard, and Robert G. Lowery
A Pinchard,
Pi h d and
d Robert
R b G.
G Lowery
L
BellBrook Labs Madison Wisconsin USA 53711
BellBrook Labs, Madison, Wisconsin, USA, 53711 Introduction
d
The Transcreener ADP2 FP Assay allows the facile detection and screening of established drug targets including protein and
lipid kinases as wells as emerging targets such as heat shock proteins and other ATPases by directly measuring ADP
formation. This homogenous, single-step competitive fluorescence polarization immunoassay employs a selective ADP
antibody and a red-shifted ADP-Alexa Fluor®633 tracer. Here we describe the recent development of an improved
monoclonal antibody with >100-fold selectivity for ADP vs. ATP, which enables robust detection of initial velocity rates (Z’ >
0.7 at ≤ 20 % substrate consumption) at ATP concentrations ranging from 0.1 μM to 1,000 μM. This generic ADP detection
system enables robust detection of enzymes with low activity or low ATP requirements, saving on reagents and improving
data quality, especially relative to ATP depletion methods.
Figure 1.
Fi
1 Th
The Transcreener
T
ADP2 FP A
Assay enables
bl direct
di t detection
d t ti off ADP using
i a
competitive fluorescence polarization immunoassay. ADP produced by kinases or other ATP
ATPutilizing enzymes displaces a far red fluorescent tracer from a highly selective mAb against ADP, reducing its polarization.
The method is a single addition, homogenous format that has been extensively validated in pharmaceutical HTS laboratories
with more than 40 million data points.
Nucleotide
Old ADP Ab
N ADP Ab
New
NDP Affinity
Affi it
Improvement
ADP
180
79
7.9
23x
ATP
17,300
,
915
ATP/ADP
GDP
GTP
GTP/GDP
UDP
UDP-Glc
96
50
1 500
1,500
30
ND
ND
92
10.7
1 400
1,400
131
214
744,000
,
UDP-Glc/UDP
ND
3,476
14x
ND
Figure 2
2. Standard curves for conversion of ATP to ADP show the increased
sensitivity
iti it off the
th ADP2 Ab Standard curves mimicking enzyme reactions were constructed for conversion of ATP
Table 1
1. The new ADP Ab increases sensitivity of ADP detection
more than
th 20-fold
20 f ld and
d enables
bl detection
d t ti off other
th NDPs.
NDP Nucleotides
to ADP using initial ATP concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM, and 1,000 μM ATP; as ADP was added, ATP was
decreased proportionately. Antibody was present at its EC85 concentration for each initial ATP concentration; Alexa-Fluor® 633ADP tracer was present at 2 nM (n=24).
were titrated in the presence of ADP antibodies and an Alexa-Fluor® 633-ADP tracer in 384 well
plates, fluorescence polarization measurements were made on a Tecan Safire™, and the resulting
competition curves were used to generate IC50 values. Solution conditions were optimized
separately for each antibody.
A))
B)
A
ABL1T315I, 4
ABL1T315I
4.0
0 μ M ATP
5.7%
% ATP Conversion
C
i
Z' = 0.77
C)
mP
m
P
B
ZAP 70
70, 0
0.1
1 μ M ATP
8 0% ATP C
8.0%
Conversion
i
Z' = 0.74
D)
300
300
250
250
200
200
mP
m
P
IC50 ((nM))
150
150
100
100
5 ng/ml ABL1T315I
no enzyme control
50
12 ng/ml ZAP70
no enzyme control
50
0
0
0
5
10
15
20
0
25
5
10
15
20
25
Replicate Number
Replicate Number
Figure 3
3. Enhanced ADP detection translates to robust detection of enzymes with low ATP re
equirements or low specific activity
activity.
Dose response curves for kinases in the presence of A) ATP at its corresponding
Km concentration
t ti , or B) 0
0.1
1 μM.
M Assay
A
windows
i d
for
f initial
i iti l velocity
l it detection
d t ti
off C) ABL1T3151 using
i 4 μM
M ATP and
d D) ZAP70 usin
ing 0
0.1
1 μM
M ATP.
ATP All experiments
i
t were performed
f
d iin C
Corning
i ® 384 Well
W ll Black
Bl k Round
R
dB
Bottom
tt
Low
L Volume
V l
Polystyrene
P l t
Non-Binding
N Bi di SSurface
f
Mi
Microplates
l t (Part
(P t # 3676).
3676) Fl
Fluorescence polarization
l i ti was read
d iin a Tecan
T
S fi 2 or P
Safire
PerkinElmer
ki El
E Vi i multiwell
EnVision
lti ll reader.
d The
Th ffree tracer
t
re
eference
f
was sett tto 20 mP,
P and
d buffer
b ff containing
t i i antibody
tib d was used
d as a bl
blankk ffor sample
l and
d reference
f
wells.
ll Kinase
Ki
enzyme reactions
ti
were
ere rrun
n in CK b
buffer
ffer (50 mM HEPES (pH 7.5),
7 5) 0.01%
0 01% Brij
Brij-35,
35 2 mM EGTA,
EGTA 4 mM MgCl2) in 10 l final volume;
ol me final read volume
ol me after addition of Stop/Dete
Stop/Detectt Mix
Mi was
as 20 μl.l Stop and Dete
Detectt Mi
Mix contained
ontained (final assa
assay concentrations)
on entrations) 10 mM EDTA,
EDTA 2 nM Alexa
Ale a 633-ADP
633 ADP and ADP Ab at
concentrations appropriate for the starting [ATP].
[ATP] Standard curves similar to those shown in Figure 2 were used to determine ADP formattion from enzyme titrations and enzyme amounts were chosen for initial velocity conditions used for Z’ determination (n=24).
(n 24)
0.8
40
0.6
30
0.4
4
20
0.2
2
10
Z Factor
Z'-Factor
% ATP Conversion
0
10 12 14 16
0
2
4
6
8
mU/Rxn PKA
1.0
150
0.8
Z Fac
Z'-F
ctorr
50
100
0.6
0.4
50
0.2
Z Factor
Z'-Factor
% ATP Conversion
0.0
0
50
100
150
200
%A
ATP Con
C nve
ers
sion
n
1.0
0.0
Luciferase Assay
B)
% ATP
A P Con
C nve
ers
sion
n
Z Fac
Z'-F
ctorr
A))
Transcreener Assay
0
250
mU/Rxn PKA
Fi
Figure
5.
5 Transcreener
T
ADP2 FP Assay
A
iis more sensitive
iti th
than Luciferase-based
L if
b d ATP d
depletion
l ti methods.
th d
Figure 4. Dose Response for Gleevec ® with ABL1 and the ABL1
T315I shows the expected potency difference.
difference Enzyme
E
reactions
i
and
d
detection with Transcreener ADP2 FP Assay were as described for Figure 3 in the presence of
inhibitor titrations. IC50 profiles for the full panel of inhibitors used is shown below.
ABL1
PKA enzyme reactions
r
were performed in 384-well plates in a 10 μL reaction volume with 10 μM ATP and 50 μM Kemptide substrate for one hour at
room temperaature. The enzyme buffer recommended by the Luciferase Assay vendor was used for both assays and fluorescence polarization and
luminescence
e were measured on a BMG LABTECH PHERAstar Plus plate reader. A) Transcreener: an equal volume of ADP Detection Mixture was added to
stop the reacttion, followed by a 10 minute equilibration before reading. The equilibration period was shortened from the usual one hour period to be
consistent witth the Luciferase Assay protocol. B) Luciferase: an equal volume of Luciferase Assay reagent was added to the reaction, followed by a 10
minute equilib
bration before reading.
ABL1 T315I
Gleevec
GO-6983
H-89
Iressa
PP2
Ro-31-8220
Roscovatine
SB 202190
S
Staurosporine
T
Tyrphostin
h ti AG1478
Y27632
S mmarry:
Summar
r :
< 1 µM
1 - 10 µM
10 -100 µM
No Inhibition
•A
A higher affinity monoclonal antibody that enhances sensitivity for ADP more than 20-fold
20 fold (IC50 = 7
7.9
9 nM)
h b
has
been developed
d l
d and
d incorporated
i
t d iinto
t th
the recently
tl iintroduced
t d d Transcreener
T
ADP2 FP A
Assay.
•The Transcreener ADP2 FP Assay enables robust detection (Z
(Z’ > 0.7)
0 7) of kinase velocity using ATP
concentrattions
i
as llow as 100 nM and
d as hi
high
gh as 1 mM.
•The enhanced sensitivity and flexibility for ATP allows facile screening of enzymes with low ATP
requireme
q
ents ((<5 μM)
μ ) or low specific
p
activityy ((ABL 1 T315I).)
Acknowledgements:
c o edge e ts:
This work is supported by NIH SBIR grants R44CA110535 and R44GM073290.
©2008
2008 BellBrook Labs. Transcreener is a registered trademark of BellBrook Labs.
AlexaFluor
e a uo iss a registered
eg ste ed trademark
t ade a of
o Molecular
o ecu a Probes/Invitrogen.
obes/
t oge .
BellBrook Labs 5500 Nobel Drive,, Suite 250,, Madison,, WI 53711
866.313.7881 or 608.443.2400 www.BellBrookLabs.com
www bellbrooklabs com
www.bellbrooklabs.com
•In
I di
directt comparisions
c
ii
with
ith luciferase
l if
b d ATP depletion
based
d l ti methods,
th d th
the Transcreener
T
ADP2 FP A
Assay
enabled ro
obust PKA detection (Z’>0.7)
(Z >0.7) using 30-fold
30 fold less enzyme (2 mU vs. 60 mU) and much lower ATP
conversion
n (10% vs
vs. 100%)
•The
The impro
oved ADP antibody also enables highly selective detection of GDP and UDP,
UDP allowing generic
d t ti ffor GTPases
detection
GTP
and
d UDP
UDP-sugar dependent
d
d t glycosyltransferases.
l
lt
f