Transcript
Page 1: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

JAK JAK

type I cytokine receptor

Y

Y

Y

Y

Y

Y

Y

YJAK kinase

extracellular

intracellular

JAK-STAT signalling

Page 2: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

Y

Y

Y

Y

Y

Y

JAKY

JAKY

cytokine

P

P

JAK-STAT signalling

Page 3: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

Y

Y

Y

Y

Y

Y

JAKY

JAKY

P

P

P

P

P

P

P

P

JAK-STAT signalling

Page 4: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular 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

Page 5: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular 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

Page 6: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

JAK2 JAK2

F

F

F

F

F

F

Y

Y

cytokine receptor

leptin receptor (Y>F)

MAPPIT

Page 7: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

ligand

JAK2

F

F

F

YJAK2

F

F

F

Y

P

P

MAPPIT

Page 8: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

ligand

JAK2

F

F

F

YJAK2

F

F

F

Y

P

P

MAPPIT

Page 9: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

ligand

JAK2

F

F

F

YJAK2

F

F

F

Y

P

P

bait

MAPPIT

Page 10: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

ligand

JAK2

F

F

F

YJAK2

F

F

F

Y

P

P

YYYY

prey

gp130

MAPPIT

Page 11: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

ligand

JAK2

F

F

F

YJAK2

F

F

F

Y

YY

YY

P

P

PP

PP

MAPPIT

Page 12: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

ligand

JAK2

F

F

F

YJAK2

F

F

F

Y

YY

YY

P

P

PP

PP

STAT3

Y

Y

MAPPIT

Page 13: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 14: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

ligand

JAK2

F

F

F

YJAK2

F

F

F

Y

YY

YY

P

P

PP

PP

Y PYP

reporter generPAP1 promoter Y PYP

MAPPIT

Page 15: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 16: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 17: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

MAPPIT toolbox can be applied in a drug discovery pipeline

proteintarget

interactionlead

compoundMAPPIT Reverse MAPPIT MASPIT optimised lead compound

Page 18: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

EpoR

LR-F3

DHFR

Epo

JAK2

F

F

F

YJAK2

F

F

F

Y

P

P

MASPIT

Page 19: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

Epo

JAK2

F

F

F

YJAK2

F

F

F

Y

P

P

methotrexate

compound

MASPIT

PEG linker

Page 20: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

Epo

JAK2

F

F

F

YJAK2

F

F

F

Y

P

P

YY

YY

YY

YY

MASPIT

Page 21: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 22: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 23: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 24: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 25: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 26: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 27: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

MAPPIT validation of Y2H protein network maps

Page 28: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

empirical confidence score

from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009

Page 29: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

empirical confidence score

from Braun et al. An experimentally derived confidence score for binary protein-protein interactions. Nature Methods 2009

Page 30: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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)

Page 31: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 32: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 33: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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

Page 34: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

towards an efficient screening format: reverse transfection

nucleic acid

transfectionreagent

spotaddcells incubate

Page 35: JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling

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


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