JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling.

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Presentation transcript:

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

YYYYYY YYYYYY JAK Y Y cytokine P P JAK-STAT signalling

YYYYYY YYYYYY JAK Y Y P P P P P P P P JAK-STAT signalling

YYYYYY YYYYYY JAK Y Y P P P P P P P P Y Y STAT JAK-STAT signalling

YYYYYY YYYYYY JAK Y Y P P P P P P P P Y P Y P Y P Y P STAT responsive promoter cytoplasm nucleus JAK-STAT signalling

JAK2 FFFFFF FFFFFF Y Y cytokine receptor leptin receptor (Y>F) MAPPIT

ligand JAK2 FFFFFF Y FFFFFF Y P P MAPPIT

ligand JAK2 FFFFFF Y FFFFFF Y P P MAPPIT

ligand JAK2 FFFFFF Y FFFFFF Y P P bait MAPPIT

ligand JAK2 FFFFFF Y FFFFFF Y P P YYYYYYYY prey gp130 MAPPIT

ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P P P P P P MAPPIT

ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P PP P P P STAT3 Y Y MAPPIT

ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P PP P P P Y Y reporter gene rPAP1 promoter P P MAPPIT Y P Y P

ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P PP P P P Y P Y P reporter gene rPAP1 promoter Y P Y P 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 protein target interaction lead compound MAPPITReverse MAPPITMASPIT optimised lead compound

EpoR LR-F3 DHFR Epo JAK2 FFFFFF Y FFFFFF Y P P MASPIT

Epo JAK2 FFFFFF Y FFFFFF Y P P methotrexate compound MASPIT PEG linker

Epo JAK2 FFFFFF Y FFFFFF Y P P YYYYYYYY YYYYYYYY MASPIT

Epo JAK2 FFFFFF Y FFFFFF Y P P YYYYYYYY YYYYYYYY luciferase rPAP1 promoter Y P Y P P P P P Y P P P P P Y P Y P Y P MASPIT

Parent molecule Target protein Luciferase reporter (fold induction) AP1867FKBP F36V 305 PD Abl359 RGB CDK225 RGB CDK2213 RGB GSK3b218 E7070CAII49

MASPIT Fold induction MFC concentration (µM) Parent molecule Target protein Luciferase reporter (fold induction) AP1867FKBP F36V 305 PD Abl359 RGB CDK225 RGB CDK2213 RGB GSK3b218 E7070CAII49

MASPIT Fold induction gp-130-CDK2 pMG-CDK2 plasmid (mg) Fold induction MFC concentration (µM) Time (hours) Parent molecule Target protein Luciferase reporter (fold induction) AP1867FKBP F36V 305 PD Abl359 RGB CDK225 RGB CDK2213 RGB GSK3b218 E7070CAII49

MASPIT competition assays affinity determination in intact cells >> MASPIT EC 50 better reflects reality than IC 50 obtained with in vitro assays (e.g. compound uptake, interference of other proteins with binding) [Methotrexate] µM % Control Target : DHFR MFC: RGB Epo JAK2 FFFFFF Y FFFFFF Y P P methotrexate PD YYYYYYYY Abl-prey [PD ] µM % Control MFC: RGB Target : Abl MFC: RGB Target : Abl % Control [Imatinib] µM EC µM EC µM

MASPIT screening with PD all identified preys are kinases known (Abl, Lyn, Fyn, FGFR1) and novel (Eph, GAK) validated with in vitro kinase activity assays Epo JAK2 FFFFFF Y FFFFFF Y P P methotrexate PD MASPIT Target gene In vitro kinase activity (IC 50  M) Lyn<0.001 Fyn0.001 FGFR EphA20.02 EphB1nd EphB EphB GAKnd 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: x full lenght human ORFs (~ 50% of total matrix of x ) interaction toolkit re-test: ~ 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

hIL5R  Y anti-hIL5R  Y Y anti-PE magnetobead baitLR-F3 CMV hIL5RαΔcyt rPAP1 hIL5R  Y anti-mIgG-PE FACS sort mEcoR Y Y MACS enrichment preygp130 5’LTR CMV CD90 retroviral prey cDNA library MAPPIT cDNA library screening

towards an efficient screening format: reverse transfection

ArrayMAPPIT screening MAPPIT prey collection prey (+reporter) plasmid transfection reagent reverse transfection mix MAPPIT prey array (stable for months !) luciferase read-out MAPPIT bait cell line -/+ ligand human ORFeome collection