Protein kinase analysisKinome
G protein-coupled receptor
Tyrosine kinase receptor
Ser/Thr kinase receptor
Cytokinereceptor
Intracellular Signal Transduction
GAC
cAMP
PKA
CREB
PLCG
Ca2+
CaM
CaMK
DAG
Sos
Grb2
Ras
Raf
MEK
ERK
PKC
Pi
Pi
Pi
Pi
C-MycPi
ELKPi
PI3K
PKBFAK
Pi
Pi
SmadPi
JAKPiSTAT
Pi
Cyt-cApaf-1Casp-9
Casp-3Bcl-2
Bad
Pi
GSK3
PiApoptosis
Glycogensynthesis
AdhesionExtension
Transcription
Protein kinases
1)ca. 500 kinds of putative kinases are coded in human genome 2) 1/3 of whole proteins are phosphorylated.3)Certain protein kinase is hyper-activated in particular diseases, such as cancers, cardiovascular diseases
Protein kinase signaling will be crucial target in diagnostics and drug discovery
P
+ ATP + ADP
Protein kinases
Disease cells and hyper-activated protein kinases
Cancer ： Protein kinase Cα, Src, Akt, ALC, c-Met
Inflammation ： I-κ-kinase
Cardiovascular diseases ： Rho kinase, Src
Diabetes ： Protein kinase Cβ
Protein kinase PP Aqnti-phospho antibodyPP
Increase of the fluorescence polarization
Protein kinasePP
PP
PPPP
+
Fluorescence-labeled peptide
Protein substrate Increase of the fluorescence polarization
a)
b)
Assay of protein kinase activity by using fluorescence polarization
GFP GFP
PP Anti-phospho antibody
GFP
PP
TbTb TbTb
Time-resolved fluorescence assay using GFP fluorescence
Protein kinase
FRETa)
fluoreophore
quencher FRET
Quenching of the fluorescence
Protein kinase PP elastase PP
Non-fluorescent
elastase
Recovery of the fluorescence
b)
Assay of protein kinase activity by using FRET
GST MoesineLRRK2
GST
PP
MoesineDonor bead
Acceptor bead
GST
PP
Moesine
hn1O2
O
O
O
N
CO2- CO2
-
ON
CO2-
CO2-
n
Ga3+
Protein kinase PP
Ga Ga
Quencher
Quenching of the bead due to FRET with the quencher
Other protein kinase assay
a-Screen Assay
QTL assay
Fluorescent probe for protein kinase using D-RECS strategy
OHN
NH
O
HN
O
O
O-Na+
HN
O
NH2
HN
O
NH
O
OO+Na-O
+Na-O
O
l m n
LPEI-KS10.3(l = 79.6, m = 10.3, n = 10.1)
OHN
NH
O
HN
O
O
O-Na+
HN
O
NH2
HN
O
NH
NH
S
O
O
O-Na+
O-Na+
Ol m n
pAsp-F3.7(l = 91.8, m = 4.5, n = 3.7)
pAsp-F1.1(l = 97.2, m = 1.7, n = 1.1)
HN
NN
N NN O
NH
ALRRASLGW
l m n
LPEI-KS10.3(l = 79.6, m = 10.3, n = 10.1)
OHN
NH
O
HN
O
O
O-Na+
HN
O
NH2
HN
O
NH
NH
S
O
O
O-Na+
O-Na+
Ol m n
pAsp-F3.7(l = 91.8, m = 4.5, n = 3.7)
pAsp-F1.1(l = 97.2, m = 1.7, n = 1.1)
LPEI-Kemp-S10.3(l=79.6, m=10.3, n=10.1)
Polycation with substrate
Polyanion labeled with fluoresceine
Polyion complex(quenching)
Recovery of fluorescence
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 2 4 6 8
Rel
ativ
e F
luo
resc
ence
In
ten
sity
C/A
LPEI-Kemp10.3-S/pAsp-F3.7
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0 20 40 60 80Rel
ativ
e F
luo
resc
ence
In
ten
sity
Time (min)
LPEI-Kemp-S10.3/pAsp-F3.7=3.0
PKA (+)
PKA (-)
Inactivated PKA
Protein kinase assay using Au nano-particle
Phosphorylated substrate
Substrate peptide
B
0
0.05
0.1
0.15
0.2
0.25
0.3
0 0.5 5
OD
Inhibitor concentration (μM)
Phosphorylation ratio
(%)
OD
Phosphorylation ratio OD
0
20
40
60
80
100
0
0.05
0.1
0.15
0.2
0.25
0.3
Tumor Normalp-PKCαactin
ANormal (skin) Tumor （ B16 ）
4 % 70 % 33 % 0 %
p-PKCa
actin
tumor normal
inhibitor （ mM ） - - 0.5 5.0
Inhibitor (Ro-31-7549)
Detection of PKCα activity in tumor tissue
Normal tissue tumor tissue
Tissue lysate
Detected with Au-particle system
Tumor Normal
Tumor Normal
OD
0
0.05
0.1
0.15
0.2
0.25
0.3
0
0.05
0.1
0.15
0.2
0.25
0.3
0 5 10 15 20
Normal
Normal
Application to breast cancer prognosis
Breast cancer(18 samples)
Lysis of tissues
PKCa was detected with the assay
P < 0.05
OD
@ 6
70nm
tumor normal0
0.1
0.2
0.3
Biosensors Bioelectronics, 2010, 25, 1869.
Normal tissue(12 samples)
Low (6)
Middle (6)
High (6)
Low
Middle
High
Evaluation of kinase inhibitors
Protein kinse A inhibitors
H-89 [M]
0 30
IC50: 125 nM*
H-89
Rottlerin
IC50: 2.18 M
101 102 103 1040.0
0.1
0.2
0.3
0.4
active p38 + inactive MAPKAPK-2
active MAPKAPK-2
[SB-202190] (nM)
OD
65
0
active p38 +inactive MAPKAPK-2
active MAPKAPK-2
3000 1000 300 100 30 10
SB 202190 (nM)
active p38 +inactive MAPKAPK-2
active MAPKAPK-2
3000 1000 300 100 30 10
SB 202190 (nM)
p38 inhibitor (SB202190)
101 102 103 1040.05
0.15
0.25
0.35 SU6656PP2Y27632
inhibitor (nM)
OD
650
Src inhibitors
J. Oishi et al, Anal. Biochem. 373, 161-163 (2008)
Screening of PKA inhibitors1 2 3 4
5 6 7 8
9 10 11 12
13 14 15 16
Candidates found by the assay － IC50 －
8
11
H89
Staurosporine
IC50 (nM)calculated reported
95
4.8
144
7.0
Each compound was added in 0, 10, 30, 100, 300, 1000, 3000, 10000 nM to MCF-7 lysate, respectively and after the phosphorylation reaction, Au-particle dispersion was added to the reaction mixture.
No. Name
Ｋｉｎｏｍｅ ＆ Ｋｉｎｏｍｉｃｓ
Cancer chemotherapy and molecular target drug
2/10
３ . problem with acquisition of resistanceSome tumors acquire a resistances against molecular targeted drug with repeated administration. However, its mechanism has not been clarified.
２ . survival benefit of molecular target drugTwo drugs combination （ Avastin®+ Erbitax® ） for colon cancer brought about 1.3 months shorter life expectancy comparing with the conventional anticancer drug （ 5-FU+OxPt) administration. → Is molecular target drug also effective?
(New England Journal of Medicine, 360, 563-572(2009) より )
１ . survival benefit of cancer chemotherapy
(Journal of the National Cancer Institute, 98, 1108-17(2006) より )
When chemotherapy is used with operation, the survival benefit is just 5% for 5 years and only 1% for 15 years comparing with operation only
→Is cancer chemotherapy really effective ？
Molecular target drugDrug which is effective only to target cell
19
Pathways in Human Cancer
From R.A. Weinberg Appendix: The Biology of Cancer (Garland Science 2007, Taylor & Francis)
20
22Human Kinome
TK ・・・ tyrosine kinaseTKL ・・・ tyrosine kinase likeSTE ・・・ homologs of yeast sterile 7,11,20CK1 ・・・ casein kinase-1AGC ・・・ members of protein kinase A, G, C familiesCAMK ・・・ Ca+ calmodulin-dependent protein kinases CMGC ・・・ containing CDK, MAPK, GSK-3 and CLK families
From R.A. Weinberg: The Biology of Cancer, Fig.16-12 (Garland Science 2007, Taylor & Francis)
Peptide array for kinome
23
Peptide array:30µL / assay1/2000 分
384 plate: → 80mL / assay
パターン A
パターン B
Cell lysate
Cell lysate
Phosphorylation pattern
Profile A
Profile B
Normal cell
Disease or drug administered cell
Diagnostics based on the profile
Drug discovery using the profile
Concept of kinome profiling
PepChip ArrayEach peptide is immobilized in micro-well （ 50 μL ）1152 kinds of peptides can be immobilizedDetect with 33P-ATP
PamChip ArraySubstrates are immobilized on a porous supportRepeat the putting in and out repeatedly to promote the reaction Detect with 33P-ATP144 kinds of substrate can be immobilized.
Barrett’s esophagus
green ： acive in BEred ： inactive in BE
・ MAPK signaling was suppressed and glycolytic metabolism was activated in BE
Commercial available kinome arrays
CelluSpot ArrayEach substrate is synthesized on a cellulose membrane (SPOT synthesis)Radius of each spot: 1.2 mmDetect with HRP-labeled anti-phosphotyrosine antibody (CL)384 kinds of peptides can be immobilized.
Our peptide array
Phos-tag
Cy3-streptavidin
O
H
HO
O
NN
NN
NH
NHS
NH
NH
N NO-
Zn2+ Zn2+
P OO
O-O-R
Phos-tag
細胞破砕液
P P P P
P P P PP P P P
Detect with Microarray scanner
Surface chmistry
cover
PhosphorylationN
O
O
O
O
OP
O
O
O-
N+
O
OHN
OO
ONH
OO
NH
O
N
O
O
l
m
n
PY
Y
Y
Application to Iressa sensitivity
EGFR
Akt ERK
iressa
HCC827
EGFR
Akt ERK
A549
・ Highly sensitive・ EGFR kinase with mutation
・ Resisitant・ Other pathway is activated.
Other receptors
iressa
Cell death Cell survive
Protocol
Iressa2 hour
EGF15min
phosphorylation3 hour phostag-SA
1000 peptides
lysis
HCC827IC50=16 nM
A549IC50=30 μM
[Iressa]3 nM 300 nM
Comparison between A549 and HCC827
・ Iressa inhibited the phosphorylation of many substrate in HCC827.
Iressa(-)
iress
a 30
0 nM
A549
HCC827
Tyr peptide
Ser/Thr peptide
Tyr peptide のうち良く反応
Iressa(-)
iress
a 30
0 nM
Alternative pathway is Src-pathway?
A549 HCC827
target peptide no.
Src 9
EGFR 183
EGFR 264
EGFR 345
Src 657
Src 661
Abl 669
Src 822
Src 1062
Src 1144
Src 1148
Src 1228
iress
a(+
)/ir
ess
a(-
)
iress
a(+
)/ir
ess
a(-
)
EGFR Src EGFR Src
- 4.000
- 2.000
0.000
2.000
4.000
1 51 101 151 2 01 251 301 35 1 401 451 501 5 51
N o
１ ← peptide No. → ５５７
activation
suppression
＜ Kinome Profile modification with metformin ＞ (Dr. Osamu Okitsu)
- 4.000
- 2.000
0.000
2.000
4.000
1 51 101 151 201 251 301 351 401 451 501 551
N o
１ ← ペプチド ID 番号 → ５５７
activation
suppression
＜ Drug A ＞
