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Intro
du
ction
Daniel Müller, Frank Totzke, Thomas Weber, Marcel Pathe, Christoph Schächtele, and Michael H.G. Kubbutat
Reaction Biology Europe GmbH, Freiburg, Germany.
Introduction
Protein kinases belong to one of the largest families of evolutionarily related proteins. The human genome encodes more than 500 kinases. Due to the
structural similarity, especially within the ATP-binding site, many kinase inhibitors show limited selectivity. Still, sufficient selectivity within the human kinases
is critical e.g. to reduce the risk of adverse side effects during treatment.
Therefore, measuring and improving the selectivity of a compound within the kinome is of pivotal importance during drug discovery and optimization
phase in the development of therapeutically relevant kinase inhibitors.
Broad profiling of kinase inhibitors in biochemical activity assays of several hundred kinases is nowadays well established. Usually, kinase profiling is done
using one or two concentrations of a test compound and measurement of the relative inhibition of the kinase activity compared to a high and low control.
However, due to the limited dynamic range of this approach, and the challenge of selecting the most appropriate compound concentration, this profiling
approach oftengives limited information with respect to the potency of compounds against on- and off-target kinases.
We set up an IC50 kinase profiling approach that consists of measuring the impact of a compound on the activity of 320 human protein kinases at six
different concentrations with the33
PanQinaseTM
assay format. Here, we present data showing the effect of compound concentration on the selectivity
score in traditional profiling settings. IC50 kinase profiles of different approved and clinical stage kinase inhibitors demonstrate that an IC50 based
profiling allows a more accurate determination of selectivity of a compound based on the comparison of the IC50 values against the on-target kinases in
relation to the IC50 values of the off-target kinases providing significantly improved guidance in the further optimization of the test compound.
33PanQinase
TM: Principle of the assay
The assay is based on radiolabelled33
P-γ-
ATP. Kinase and substrate are incubated in
presence of ATP containing33
P-γ-ATP as
tracer in 96 well FlashPlates (Perkin Elmer).
After stopping the kinase reaction, the
proteins are immobilized to the plate surface
and bound radioactivity is quantified by
scintillation counting.
All assay steps are performed using a robotic
pipetting system 96 well FlashPlate per well:
• 10 µl ATP/33
P-γ-ATP mix
• 25 µl 2.5x assay buffer
• 5 µl compound in 10% DMSO
• 10 µl kinase-substrate mix
• - mix on a plate shaker
• - incubate for 60 min at 30°C
• - stop reaction with 50 µl 2% (v/v) H3PO
4
• mix on a plate shaker
• wash 2 times with 200 µl 0.9% (w/v) NaCl
• count dry plate with a scintillation counter
Final assay conditions were modified for those kinases
requiring specific cofactors for kinase activity.
• 70 mM HEPES pH 7.5
• 3 mM MgCl2
• 3 mM MnCl2
• 3 µM Na-orthovanadate
• 1.2 mM DTT
• 1 % (v/v) DMSO
• ATP*, substrate and kinase at variable concentration
*: [ATP]: apparent KM[ATP] of the respective kinase
Kinase and substrate combinations are selected for optimal results under the assay conditions described above. Kinase concentration is typically 1-40 nM (assuming
100% pure and active protein preparations), substrate concentration is 10-30 times higher than the kinase concentration.
Results
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
E PH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
ST K17A
ST K25
ST K39
TAOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-ATRK-C
T SK2TTBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
E PH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
ST K17A
ST K25
ST K39
TAOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-ATRK-C
T SK2TTBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
EPH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST 1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
STK17A
ST K25
ST K39
T AOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-AT RK-C
T SK2T TBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2WNK1WNK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
EPH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST 1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
STK17A
ST K25
ST K39
T AOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-AT RK-C
T SK2T TBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2WNK1WNK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
EPH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST 1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
STK17A
ST K25
ST K39
T AOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-AT RK-C
T SK2T TBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2WNK1WNK3 ZAK
ABL1
ACK1 ACV-R1BACV-R2BAKT1 AKT3AM PK-alpha1
ASK1Aurora -B
AXLBMPR1A
B-RAFBRSK1
BTKCAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
EPH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST 1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
STK17A
ST K25
ST K39
T AOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-AT RK-C
T SK2T TBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2WNK1WNK3 ZAK
0.1 nM 1 nM 10 nM
100 nM 1000 nM 10000 nM
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
E PH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
ST K17A
ST K25
ST K39
TAOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-ATRK-C
T SK2TTBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
E PH A2
E PH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
ST K17A
ST K25
ST K39
TAOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-ATRK-C
T SK2TTBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
E PH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
ST K17A
ST K25
ST K39
TAOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-ATRK-C
T SK2TTBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
E PH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
ST K17A
ST K25
ST K39
TAOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-ATRK-C
T SK2TTBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
E PH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
ST K17A
ST K25
ST K39
TAOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-ATRK-C
T SK2TTBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK
ABL1ACK1 ACV-R1BACV-R2BAKT1 AKT3
AM PK-alpha1ASK1
Aurora -BAXL
BMPR1AB-RAF
BRSK1BTK
CAMK1D
CAMK2B
CAMK2G
CAMKK1
CDC42BPA
CDC7/DBF4
CDK1/Cy cB1
CDK16/Cyc Y
CDK2/Cy cA2
CDK3/Cy cC
CDK4/Cy cD1
CDK5/p25NCK
CDK6/Cy cD1
CDK7/Cy cH/M AT1
CDK9/Cy cK
CHK1
CK1-al pha 1
CK1-epsi lon
CK1-ga mma 2
CK2-al pha 1
CLK1
CLK3
COT
CSK
DAPK2
DCAM KL 2
DM PK
DY RK1A
DY RK2
DY RK4
EGF-R
E IF2AK3
EPH A2
E PH A4
EPH A6
EPH A8
EPH B2
EPH B4
ERBB4
ERK2
ERK7
FER
FG F-R1
FG F-R3
FG R
FRK
GRK2
GRK4
GRK6
GS G2
GS K3-beta
HI PK1
HI PK3HRI
IKK-a lphaIKK-epsilon
IN SR-RIRAK4
JAK1JAK3
JNK2KITLI MK1LRRK2LY NM AP3K10
M AP3K7/M AP3K7IP1M AP4K2M AP4K5M APKAPK3M ARK1M ARK3
M ATKM EK2
M EKK2M ELK
M ETM KK4
M KK7M KN K2
M ST1
M ST 3
mT OR
M YLK
M YLK3
NEK11
NEK3
NEK6
NEK9
NL K
p38-beta
p38-ga mma
PAK2
PAK4
PAK7
PBK
PDGF R-beta
PHKG1
PIM 1
PIM 3
PKC-al pha
PKC-beta 2
PKC-epsi lon
PKC-ga mma
PKC-mu
PKC-theta
PKM YT 1
PLK3
PRK2
PRKG 1
PRKX
RAF1 Y 340D/Y341D
RIPK2
ROCK1
RON
RPS6KA1
RPS6KA3
RPS6KA5
S6K
SAK
SG K2
SI K1
SI K3
SN ARK
SRC
SRPK1
ST K17A
ST K25
ST K39
TAOK2
T BK1
T GFB-R1
T IE2T LK2
T RK-ATRK-C
T SK2TTBK1
T TKT YK2
VEGF-R1VEGF-R3 VRK2W NK1W NK3 ZAK
0.1 nM 1 nM 10 nM
100 nM 1000 nM 10000 nM
320 kinases were tested with Crizotinib (left) and Dinaciblib (right) in 6 concentrations. For each compound concentration, the percent inhibition was
determined for the respective kinase relative to the non-inhibited positive control. The circles represent from the inside 25, 50, 75, and 100 % inhibition.
ACK1
ALK
CAMK2G
CAMKK2
CLK2
FAKFER
FES IGF1-RINS-R
INSR-R
LRRK2
LTK
MKNK2
ROS TNK1
TSSK1
5,0
5,5
6,0
6,5
7,0
7,5
8,0
8,5
9,0
pIC
50
Kinase
Ceritinib
CDK19/CycC
CDK8/CycCDDR2
ERK7
FGF-R2
FLT3
HIPK4
MUSKPDGFR-alpha PDGFR-beta
RAF1
RET
TAOK2
TRK-ATRK-BVEGF-R1
VEGF-R2
ZAK
5,0
5,5
6,0
6,5
7,0
7,5
8,0
8,5
9,0
pIC
50
Kinase
pIC50 Threshold Value
Sorafenib
ALK
AXLFAK
LCK
LTK
MAP4K5MERTK
MET
MUSK RON
ROS
SLK
TRK-A
TRK-BTRK-C
5,0
5,5
6,0
6,5
7,0
7,5
8,0
8,5
9,0
pIC
50
Kinase
Crizotinib
IC50
values were determined for compound Crizotinib, Ceritinib, and
Sorafenib by determining the residual activities in the presence of 6 increasing,
logarithmically diluted concentrations of the respective compounds. From these six
values IC50
values were calculated using Graphpad Prism 5.04. The mathematical
model used was "Sigmoidal response (variable slope)" with parameters "top" fixed at
100 % and "bottom" at 0 %. pIC50
(negative decimal logarithm of IC50
values) were
depicted in the graphs for better visualization of the range of the compound
potency. An arbitrary threshold value 20 fold below the pIC50
of the most sensitive
kinase was set to select kinases showing significant inhibition by the respective
compound. Red circles indicate the primary target of the respective compound.
Summary & Conclusion
1. Apparent kinase inhibitor selectivity depends on the compound concentration used to measure
percental inhibition of kinases
2. Accuracy and reproducibility of profiling are limited by the dynamic range and assay variability
of single compound concentration measurement.
3. IC50
selectivity profiling based on six compound concentrations represents a significant
improvement to assess differences in the potency of a given compound and allows a more
precise judgment of the selectivity of protein kinase inhibitors
0,0
0,1
0,2
0,3
0,4
0,5
0,1 1 10 100 1000 10000
Sele
ctiv
ity
Sco
re S
50
Compound concentration [nM]
Sorafenib Crizotinb
Selectivity scores were
determined for Sorafenib, Crizotinib
and Ponatinib for a panel of 320
protein kinases at different compound
concentrations based on residual
activities. Selectivity scores were
calculated using the formula:
(count of data points < 50 %) / (total
number of data points).
0,0360,048
0,0770,081
0,130
0,1880,188
0,217
0,270
0,0
0,1
0,2
0,3
10x 20x 50x
IC5
0 S
elec
tivi
ty S
core
Threshold x IC50 (target)
Sorafenib (VEGFR2) Crizotinib (ALK) Ponatinib (ABL1)
Selectivity scores were determined
for Sorafenib, Crizotinib and Ponatinib using a
panel of 320 protein kinases based on IC50
values calculated from six different compound
concentrations in the assay. Selectivity scores
were compared with different threshold values
of 10/20/50 fold of the target IC50
value
based on the calculation:
count of IC50
values < threshold) / total
number of IC50
values determined
R² = 0,9614
5,0
5,5
6,0
6,5
7,0
7,5
8,0
8,5
9,0
5,00 5,50 6,00 6,50 7,00 7,50 8,00 8,50 9,00
The pIC50
profile of compound
Ponatinib was determined
twice in independent
experiments and the
resulting pIC50 values for
each kinase were
compared
pIC50 values of experiment 2
pIC
50
val
ues
or
exp
erim
ent
1