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

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JAK type I cytokine receptor YYYYYY YYYYYY Y Y JAK kinase extracellular intracellular JAK-STAT signalling Slide 2 YYYYYY YYYYYY JAK Y Y cytokine P P JAK-STAT signalling Slide 3 YYYYYY YYYYYY JAK Y Y P P P P P P P P JAK-STAT signalling Slide 4 YYYYYY YYYYYY JAK Y Y P P P P P P P P Y Y STAT JAK-STAT signalling Slide 5 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 Slide 6 JAK2 FFFFFF FFFFFF Y Y cytokine receptor leptin receptor (Y>F) MAPPIT Slide 7 ligand JAK2 FFFFFF Y FFFFFF Y P P MAPPIT Slide 8 ligand JAK2 FFFFFF Y FFFFFF Y P P MAPPIT Slide 9 ligand JAK2 FFFFFF Y FFFFFF Y P P bait MAPPIT Slide 10 ligand JAK2 FFFFFF Y FFFFFF Y P P YYYYYYYY prey gp130 MAPPIT Slide 11 ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P P P P P P MAPPIT Slide 12 ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P PP P P P STAT3 Y Y MAPPIT Slide 13 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 Slide 14 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 Slide 15 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 Slide 16 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 Slide 17 MAPPIT toolbox can be applied in a drug discovery pipeline protein target interaction lead compound MAPPITReverse MAPPITMASPIT optimised lead compound Slide 18 EpoR LR-F3 DHFR Epo JAK2 FFFFFF Y FFFFFF Y P P MASPIT Slide 19 Epo JAK2 FFFFFF Y FFFFFF Y P P methotrexate compound MASPIT PEG linker Slide 20 Epo JAK2 FFFFFF Y FFFFFF Y P P YYYYYYYY YYYYYYYY MASPIT Slide 21 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 Slide 22 Parent molecule Target protein Luciferase reporter (fold induction) AP1867FKBP F36V 305 PD 173955Abl359 RGB-285961CDK225 RGB-286147CDK2213 RGB-285978GSK3b218 E7070CAII49 Slide 23 MASPIT Fold induction MFC concentration (M) Parent molecule Target protein Luciferase reporter (fold induction) AP1867FKBP F36V 305 PD 173955Abl359 RGB-285961CDK225 RGB-286147CDK2213 RGB-285978GSK3b218 E7070CAII49 Slide 24 MASPIT Fold induction gp-130-CDK2 pMG-CDK2 plasmid (mg) Fold induction MFC concentration (M) Time (hours) Parent molecule Target protein Luciferase reporter (fold induction) AP1867FKBP F36V 305 PD 173955Abl359 RGB-285961CDK225 RGB-286147CDK2213 RGB-285978GSK3b218 E7070CAII49 Slide 25 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] M % Control Target : DHFR MFC: RGB-286649 Epo JAK2 FFFFFF Y FFFFFF Y P P methotrexate PD173955 YYYYYYYY Abl-prey [PD-173955] M % Control MFC: RGB-286649 Target : Abl MFC: RGB-286649 Target : Abl % Control [Imatinib] M EC 50 0.03M EC 50 0.07M Slide 26 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 FFFFFF Y FFFFFF Y P P methotrexate PD173955 MASPIT Target gene In vitro kinase activity (IC 50 M) Lyn 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 Slide 32 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 Slide 33 hIL5R Y anti-hIL5R Y Y anti-PE magnetobead baitLR-F3 CMV hIL5Rcyt rPAP1 hIL5R Y anti-mIgG-PE FACS sort mEcoR Y Y MACS enrichment preygp130 5LTR CMV CD90 retroviral prey cDNA library MAPPIT cDNA library screening Slide 34 towards an efficient screening format: reverse transfection Slide 35 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