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

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

3. YYYYYY YYYYYY JAK Y Y P P P P P P P P JAK-STAT signalling

4. YYYYYY YYYYYY JAK Y Y P P P P P P P P Y Y STAT JAK-STAT signalling

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

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

7. ligand JAK2 FFFFFF Y FFFFFF Y P P MAPPIT

8. ligand JAK2 FFFFFF Y FFFFFF Y P P MAPPIT

9. ligand JAK2 FFFFFF Y FFFFFF Y P P bait MAPPIT

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

11. ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P P P P P P MAPPIT

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

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

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

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

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

17. MAPPIT toolbox can be applied in a drug discovery pipeline protein target interaction lead compound MAPPITReverse MAPPITMASPIT optimised lead compound

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

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

20. Epo JAK2 FFFFFF Y FFFFFF Y P P YYYYYYYY YYYYYYYY MASPIT

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

22. Parent molecule Target protein Luciferase reporter (fold induction) AP1867FKBP F36V 305 PD 173955Abl359 RGB-285961CDK225 RGB-286147CDK2213 RGB-285978GSK3b218 E7070CAII49

23. MASPIT Fold induction MFC concentration (µM) Parent molecule Target protein Luciferase reporter (fold induction) AP1867FKBP F36V 305 PD 173955Abl359 RGB-285961CDK225 RGB-286147CDK2213 RGB-285978GSK3b218 E7070CAII49

24. 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 173955Abl359 RGB-285961CDK225 RGB-286147CDK2213 RGB-285978GSK3b218 E7070CAII49

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] µM % Control Target : DHFR MFC: RGB-286649 Epo JAK2 FFFFFF Y FFFFFF Y P P methotrexate PD173955 YYYYYYYY Abl-prey [PD-173955] µM % Control MFC: RGB-286649 Target : Abl MFC: RGB-286649 Target : Abl % Control [Imatinib] µM EC 50 0.03µM EC 50 0.07µM

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<0.001 Fyn0.001 FGFR10.016 EphA20.02 EphB1nd EphB20.018 EphB40.034 GAKnd Abl<0.003

27. MAPPIT validation of Y2H protein network maps

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

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

30. 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)

31. 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

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

33. 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

34. towards an efficient screening format: reverse transfection

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