Poster LANCE Ultra Tyrosine kinases

Flexibility in the Development of LANCE® Ultra Assays for Tyrosine Kinases using General and Specific Substrates
Francesco Lipari, Mireille Caron, Anja Rodenbrock, Anne Labonté, Mireille Legault, Christian Fafard, Véronique Brechler and Philippe Roby
PerkinElmer Life and Analytical Sciences, Montreal (QC), Canada H3J 1R4
General substrates
Cat.#
TRF0100
TRF0101
TRF0120
TRF0121
TRF0122
AD0066
AD0068
various
CR97-100
S4400
A2383
15575-020
6007290
6005250
2103-0010
2100-4160
2100-5060
2100-5090
2100-5110
EC50 = 55 pM
50000
0
-∞ -13
-12
-11
-10
-9
10
Enzyme time course
ULight-IRS-1
ULight-JAK-1
300000
250000
150000
EC50 = 388 pM
100000
50000
0
-8
INSR kinase
100000
1 00 00 0
350000
200000
Interference by Tyr kinase in detection
-∞ -13 -12 -11 -10
-9
-8
-7
50000
40nM
20nM
10nM
5nM
2.5nM
30000
20000
10000
250000
-6
50
100
150
200
250
0 nM
0.25 nM
150000
0.50 nM
100000
1.0 nM
2.0 nM
50000
0
0
[JAK3]
200000
0
log [c-Src Kinase] (M)
log [c-Src Kinase] (M)
[INSR]
40000
300
0
25
50
8
6 00 00
4 00 00
75
100
125
+ ATP - no inhibition
- ATP - no inhibition
2 00 00
-∞ -13
Time (min)
Time (min)
ATP titration and staurosporine curve
8 00 00
0
-12
11
-8
-7
-6
-5
-4
-3
100000
50000
0
- ∞ -8
-7
LANCE Signal (665 nm)
100000
80000
60000
IC50 = 19 nM
40000
20000
0
- ∞ -10
-9
-8
-7
-6
6
-4
-3
-5
-4
200000
150000
40000
50000
0
+ 10 μM Staurosporine
0
5
10
15
Well number
20
25
-9
-8
-7
-6
-5
-3
Z' = 0.87
50000
+ 10 μ M Staurosporine
5
10
15
20
Receptor
Km app = 4.6 μ M
20000
10000
-∞
IC50 = 61 nM
30000
20000
10000
-10
-9
-8
-7
25
Well number
Summary:
Src kinase assays using ULight-polyGT and ULight-polyGAT as substrates
have been developed. The enzyme is more efficient in phosphorylation of
ULight-polyGT, but the apparent Km for ATP in the presence of the different
substrates is similar. Excellent precision is obtained in a Z` experiment for
both substrates.
-7
-6
-5
-4
-6
-5
-4
IC50 = 34 pM
30000
-∞
-13
-12
-11
-10
-9
50000
40000
Z'=0.74
30000
20000
+ 10 μM Staurosporine
10000
0
0
5
10
15
Well number
20
25
-7
no straurosporine
Z' = 0.82
+ 10 µM straurosporine
0
-6
+ ATP : EC50 = 13 nM
- ATP : EC50 > 30 nM
-∞ -13 -12 -11 -10
-9
-8
-7
-6
log [JAK3 Kinase] (M)
ALK
AXL
EGFR
EPHB4
FGFR1
FLT1
HER2
IGF1R
INSR
KIT
MET
PDGFRA
RET
TIE2
BRK
BTK
FAK
ITK
JAK1
JAK2
JAK3
LCK
LYNa
SRC
TYK2
ZAP70
Kinase assay signal/background (+ATP / -ATP)
[Kinase] (nM) ULight -poly GT ULight -poly GAT ULight -CDK1 ULight -IRS-1 ULight -JAK-1
10
7
7
2
3
8
10
5
13
1
2
2
0,5
4
13
2
2
17
0,5
7
11
5
6
24
1
2
13
1
7
25
1
15
18
1
3
15
20
7
8
1
1
1
0,5
8
7
1
4
2
20
6
12
2
6
3
5
10
14
1
1
4
1
9
15
1
2
5
1
12
15
1
4
19
0,5
3
10
1
6
20
1
6
15
2
3
19
5
14
10
1
1
3
0,5
11
15
3
1
2
20
8
18
1
1
2
0,5
8
10
2
3
7
20
11
15
1
4
24
5
5
9
2
9
20
10
3
9
1
1
23
0,5
16
19
5
1
15
1
9
11
8
2
18
20
8
12
3
1
9
20
9
5
1
7
1
20
4
9
1
1
13
S/B > 4
Summary: ULight-polyGAT gives acceptable S/B values for all kinases tested (26/26) and ULightpolyGT works for 21/26 kinases. The other substrates provide acceptable S/B values with certain
kinases only.
12
ULight-JAK-1
60000
-8
Log [Staurosporine] (M)
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
-7
0
General protocol: same as other kinase assays. For kinase reaction the concentration of kinase
was chosen to avoid interference. Used 200 μM ATP, 100 nM ULight-substrate, incubation 4 hours.
10000
Z’-factor determination
no Staurosporine
-8
20000
20000
Log [Staurosporine] (M)
70000
-3
40000
0
-∞
-8
Log [ATP] (M)
ULight-IRS-1
150000
100000
30000
0
-2
40000
80000
no Staurosporine
0
-4
50000
-4
9
0
-5
60000
0
- ∞ -10
ULight-polyGAT
Z' = 0.87
20000
0
Staurosporine
Inhibition
IC50 = 68 nM
100000
200000
no Staurosporine
-6
log [Staurosporine] (M)
ULight-polyGT
60000
-∝
40000
log [ATP] (M)
Z’-factor determination
80000
20000
0
-2
LANCE signal (665nm)
In LANCE Ultra kinase assays, the
phosphorylation of a ULight-labeled
peptide substrate is detected with a
specific anti-phospho-peptide antibody
(Ab) labeled with europium chelate
molecules (Eu). ULight is an innovative
low-molecular weight red-shifted dye.
The binding of the Eu-antibody (donor)
to the phosphorylated ULight peptide
(acceptor) substrate brings both the
donor and acceptor dye molecules into
close proximity. Upon irradiation at 320
or 340 nm, the excited europium
chelate transfers its energy to the
nearby ULight dye molecule that will in
turn emit light at 665 nm. The intensity
of light emission is proportional to the
level
of
the
ULight
peptide
phosphorylation.
-5
40000
70000
log [Staurosporine] (M)
Assay Principle
-6
Km app = 134 μM
60000
log [ATP] (M)
log [ATP] (M)
Staurosporine
Inhibition
ATP
Titration
150000
Cytoplasmic
-9
200000 Km app = 1.5 µM
LANCE signal (665 nM)
-∞
250000
LANCE signal (665nm)
0
ULight-JAK-1
50000
80000
LANCE signal (665 nM)
50000
300000
LANCE signal (665 nM)
Km app = 1.6 µM
100000
ULight-IRS-1
350000
LANCE signal (665nm)
LANCE Signal (665nm)
LANCE Signal (665 nm)
ATP
Titration
150000
-9
40000
Kinase assays with ULight substrates
Type Kinase
200000
-10
60000
Summary:
JAK3 Tyr kinase interferes in phospho-Tyr detection by binding to the anti-phospho-Tyr
antibody, whereas INSR kinase does not interfere.
ATP titration and staurosporine curve
ULight-polyGAT
ULight-polyGT
-11
log [INSR Kinase] (M)
JAK3 kinase
80000
General protocol:
10 µL 50 nM biotin-phospho-Tyr-peptide plus 0-30 nM kinase ± 100 μM ATP
5 µL of 4X Eu-Antibody diluted in detection buffer (final 2 nM)
5 µL of 4X ULight -SA diluted in detection buffer (final 50 nM)
Incubate 60 min at RT and read on EnVision instrument
Peptide sequences: IRS-1 (CKKSRGDYMTMQIG), JAK-1 (CAGAGAIETDKEYYTVKD)
General Protocol:
5 µL of 2X INSR or JAK3 kinase in kinase buffer
5 µL of 2X ULight -Substrate/ATP mix (final 50 nM ULight-JAK-1 or 200 nM ULight-IRS-1)
Incubate up to 240 min at RT
5 µL of 4X EDTA in detection buffer (final 10 mM EDTA)
Incubate 5 min at RT
5 µL of 4X Eu-Antibody (AD0068) in detection buffer (final 2 nM)
Incubate 60 min at RT and read on EnVision instrument
General Protocol:
5 µL of 2X Src kinase in kinase buffer (50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2,
2 mM DTT and 0.01% Tween-20)
5 µL of 2X ULight -Substrate/ATP mix in kinase buffer (final 50 nM substrate)
Incubate 60 min at RT
5 µL of 4X EDTA in detection buffer (final 10 mM EDTA)
Incubate 5 min at RT
5 µL of 4X Eu-Antibody (AD0066) in detection buffer (final 2 nM)
Incubate 60 min at RT and read on EnVision instrument
LANCE signal (665nm)
100000
Kinase profiling
LANCE signal (665nm)
150000
LANCE signal (665nm)
LANCE Signal (665 nm)
LANCE Signal (665 nm)
200000
LANCE Signal (665nm)
3
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ULight-polyGAT
ULight-polyGT
5
Materials
7
Enzyme titration
Lance Signal (665 nm)
2
Item
ULight™-poly GT (4:1)
ULight-poly GAT (1:1:1)
ULight-IRS-1(Tyr983)
ULight-JAK-1(Tyr1023)
ULight-CDK1(Tyr15)
Eu-anti-phospho-Tyr (PY20) antibody
Eu-anti-phospho-Tyr (PT66) antibody
Kinases
LANCE Detection Buffer 10X
Staurosporine
ATP
0.5 M EDTA pH 8
White OptiPlate™-384
TopSeal™-A
EnVision® Multilabel Reader
Mirror: LANCE/DELFIA Dual
Excitation Filter: UV2(TRF) 320 nm
Emission Filter: Eu 615 nm
Emission Filter: LANCE 665 nm
4
Specific substrates
LANCE signal (665 nm)
Introduction
LANCE Signal (665 nm)
1
Of the 518 known human kinases, 90 are tyrosine (Tyr) kinases. Protein Tyr
kinases play a key role in signal transduction and normal cell growth. They are
also involved in numerous proliferative diseases like cancer and atherosclerosis, in
addition to a number of autoimmune diseases. For these reasons, Tyr kinases are
often targets for new drug discovery in many laboratories involved in basic R&D,
disease and therapeutics research, and HTS.
PerkinElmer now offers LANCE Ultra, a new ultra-HTS compatible TR-FRET
technology highly suited for kinase inhibitor screening. Five new Tyr kinase
substrates will soon be available in the Ultra format. ULight-poly GT (4:1) and
ULight-poly GAT (1:1:1) are general substrates that can be phosphorylated nonspecifically by Tyr kinases and can be used to quickly set up a kinase assay for an
enzyme with unknown physiological substrate. Alternatively, specific ULightpeptide substrates corresponding to the phosphorylation site sequence from the
physiological protein are available: ULight-IRS-1(Tyr983), ULight-JAK-1(Tyr1023),
and ULight-CDK1(Tyr15). The phosphorylated substrates are detected using Eulabeled anti-phospho-Tyr antibodies.
HTS-compatible assays have been developed for each substrate. Tyr kinase
profiling of the five substrates illustrates the applicability of the substrates for a
wide variety of Tyr kinases.
10
20
30
40
50
Well number
Summary:
Kinase assays using ULight-IRS-1 and ULight-JAK-1 as substrates have
been developed using INSR and JAK3 kinases, respectively. Excellent
precision is obtained in a Z` experiment for both substrates.
Summary and Conclusions
• Two types of ULight-labeled substrates for Tyr kinases were developed: general
and specific.
• The applicability of the substrates to the development of HTS assays is illustrated
by demonstrating enzyme kinetic data, inhibition curves, and Z` experiments. The
assays developed provide ideal parameters for HTS – only 3-4 additions per well,
low quantities of substrate and antibody, and Z` values > 0.7.
• Tyr kinases were assayed in detection assays to determine the interference of
the enzyme on antibody binding. The kinase interferes in antibody-based kinase
assays due to the presence of phospho-Tyr residues on the kinases. The
concentration of the kinase in the reaction must be adjusted accordingly.
• The substrates were tested with a variety of receptor and cytoplasmic Tyr
kinases. All the kinases were active with ULight-polyGAT as substrate and 21/26
kinases were active with ULight-polyGT. The applicability of the general substrates
to a wide variety of kinases illustrates their advantage in setting up Tyr kinase
assays in a short period of time. The specific substrates showed activity only
towards certain substrates. ULight-CDK1 prefers kinases form the Src family: LCK,
LYNa, and SRC. ULight-IRS-1 contains the YMxM motif and select kinases of both
receptor and non-receptor type were active with this peptide. Interestingly, ULightJAK-1 is active with a variety of kinases.
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