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JAK JAK
type I cytokine receptor
Y
Y
Y
Y
Y
Y
Y
YJAK kinase
extracellular
intracellular
JAK-STAT signalling
Y
Y
Y
Y
Y
Y
JAKY
JAKY
cytokine
P
P
JAK-STAT signalling
Y
Y
Y
Y
Y
Y
JAKY
JAKY
P
P
P
P
P
P
P
P
JAK-STAT signalling
Y
Y
Y
Y
Y
Y
JAKY
JAKY
P
P
P
P
P
P
P
P
Y
Y
STAT
JAK-STAT signalling
Y
Y
Y
Y
Y
Y
JAKY
JAKY
P
P
P
P
P
P
P
P YPYP
YPYP
STAT responsive promoter
cytoplasm
nucleus
JAK-STAT signalling
JAK2 JAK2
F
F
F
F
F
F
Y
Y
cytokine receptor
leptin receptor (Y>F)
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
bait
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
YYYY
prey
gp130
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
STAT3
Y
Y
MAPPIT
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
Y
Y
reporter generPAP1 promoter
P
P
MAPPIT
Y PYP
ligand
JAK2
F
F
F
YJAK2
F
F
F
Y
YY
YY
P
P
PP
PP
Y PYP
reporter generPAP1 promoter Y PYP
MAPPIT
MAPPIT assets
• operates in intact human cells, a close to normal physiological context
• works in different cell types (epithelial, haematopoietic, neuronal)
• tight background control
– interaction and effector zone are separated
– ligand-inducible system
• easy to perform, simple readout, can be automated
MAPPIT constitutes a toolbox of related interaction mapping technologies
• MAPPIT (2H): protein-protein interaction
-> protein interaction mapping
• ReverseMAPPIT (R2H): disruption of protein-protein interaction
-> compound screening
• MASPIT (3H): compound-protein interaction
-> compound target profiling
MAPPIT toolbox can be applied in a drug discovery pipeline
proteintarget
interactionlead
compoundMAPPIT Reverse MAPPIT MASPIT optimised lead compound
EpoR
LR-F3
DHFR
Epo
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
MASPIT
Epo
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
methotrexate
compound
MASPIT
PEG linker
Epo
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
YY
YY
YY
YY
MASPIT
Epo
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
YY
YY
YY
YY
luciferaserPAP1 promoter Y PYP
PP
PP
YPP
PP
P
Y P
Y PYP
MASPIT
MASPIT
Parent molecule
Target protein
Luciferase reporter
(fold induction)
AP1867 FKBP F36V 305
PD 173955 Abl 359
RGB-285961 CDK2 25
RGB-286147 CDK2 213
RGB-285978 GSK3b 218
E7070 CAII 49
MASPIT
0.001 0.01 0.1 10
100
200
300
400
Fold
in
du
ctio
n
MFC concentration (µM)
Parent molecule
Target protein
Luciferase reporter
(fold induction)
AP1867 FKBP F36V 305
PD 173955 Abl 359
RGB-285961 CDK2 25
RGB-286147 CDK2 213
RGB-285978 GSK3b 218
E7070 CAII 49
MASPIT
Fold
in
du
ctio
n
0 5 10 15 20 25 300
5
10
15
20
25
0.001 0.010 0.100 1.0000
5
10
15
20
25
30
Fold
in
du
ctio
n
0.000 0.001 0.010 0.100 1.0000
2
4
6
8
10
12
14
16
18
gp-130-CDK2
pMG-CDK2 plasmid (mg)
Fold
in
du
ctio
n
MFC concentration (µM)
Time (hours)
Parent molecule
Target protein
Luciferase reporter
(fold induction)
AP1867 FKBP F36V 305
PD 173955 Abl 359
RGB-285961 CDK2 25
RGB-286147 CDK2 213
RGB-285978 GSK3b 218
E7070 CAII 49
MASPIT competition assays
affinity determination in intact cells
>> MASPIT EC50 better reflects reality than IC50 obtained with in vitro assays (e.g.
compound uptake, interference of other proteins with binding)
0.0001 0.001 0.01 0.1 1
0
20
40
60
80
100
[Methotrexate] µM
% C
ontr
ol Target : DHFR
MFC: RGB-286649
Epo
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
methotrexatePD173955
YY
YY
Abl-prey
0.0001 0.001 0.01 0.1 1 10
0
20
40
60
80
100
[PD-173955] µM0.01 0.1 1 10
0
20
40
60
80
% C
ontr
ol
MFC: RGB-286649Target : Abl
MFC: RGB-286649Target : Abl
% C
ontr
ol
[Imatinib] µM
EC50 0.03µM EC50 0.07µM
MASPIT screening with PD173955
• all identified preys are kinases• known (Abl, Lyn, Fyn, FGFR1) and novel (Eph, GAK)• validated with in vitro kinase activity assays
Epo
JAK2
F
F
F
YJAK2
F
F
F
Y
P
P
methotrexate
PD173955
MASPIT Target gene
In vitro kinase activity (IC50 mM)
Lyn <0.001
Fyn 0.001
FGFR1 0.016
EphA2 0.02
EphB1 nd
EphB2 0.018
EphB4 0.034
GAK nd
Abl <0.003
MAPPIT validation of Y2H protein network maps
empirical confidence score
from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009
empirical confidence score
from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009
mapping the human interactome
• 3 year NIH grant
• Y2H: 16.000 x 16.000 full lenght human ORFs (~ 50% of total matrix of 22.000 x 22.000)
• interaction toolkit re-test: ~25-30.000 interactions (~10.000/year; ~20% of the map)
benchmarking binary interaction mapping methods
>> MAPPIT performance is similar to that of the other tested methods
from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009
benchmarking binary interaction mapping methods
from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009
>> the interaction mapping methods are highly complementary
hIL5Rα
Y
anti-hIL5Rα
Y
Y
anti-PEmagnetobead
baitLR-F3CMV hIL5RαΔcytrPAP1
hIL5Rα
Yanti-mIgG-PE FACS sort
mEcoR
YY
MACS enrichment
preygp1305’LTR CMVCD90
retroviral prey cDNA
library
MAPPIT cDNA library screening
towards an efficient screening format: reverse transfection
nucleic acid
transfectionreagent
spotaddcells incubate
ArrayMAPPIT screening
MAPPIT prey collection
prey (+reporter)plasmid transfection
reagent
reverse transfection mix
MAPPIT prey array(stable for months !)
luciferase read-out MAPPIT baitcell line
-/+ ligand
human ORFeome collection