DiscoveRx Corporation

Target-Specific Cell BasedKinase Assays

DiscoveRx EFC Technology & Target-Specific Cell-Based Assays

+

Inactive Fragments Active Enzyme

PKPK

Active Enzyme

Enzyme Complementation

GPCRs

Kinases

NHRs Proteases

Studying kinases at the receptor: PathHunterTM Tyrosine kinase Assay Principle

Receptor Activation

Receptor Dimerization

Receptor Phosphorylation

Interaction with SH2 domain

protein

.

1

2

3

4

12

3

4

Grb2-EA, Shc1-EA,PLCG-EA

RTK Assay ApproachU2OS TRKC Pool Screen

10-13 10 -12 10-11 10 -10 10-9 10 -8 10-7 10 -6 10-50

1000

2000

SHC1PLCG1PLCG2GRB2SYK-CPTPN11

NT3 (g/ml)

Mea

n R

LUTrkC Clone Performance

10-12.5 10-11.5 10-10.5 10-9.5 10-8.5 10-7.5 10-6.50

500

1000

1500

2000

2500SHC1

NT-3 (g/ml)

Me

an

RL

U

1 hr1 hr

12-48h

Thaw & Plate Cells

Benefits of PathHunterTM RTK assays

Ligand Binding Phosphorylation

Signal Transduction: Adapter protein binding

Assay Measures.. Assay Benefits

Full length proteinsNon-ATP pocket binders Antibody Therapeutics Ligand binding inhibitors Dimerization inhibitors

Natural activation process Unactive and active kinase Cellular processes

Cell Permeability of compounds

P P

Application: Identify Small Molecule inhibitors

Most small-molecule inhibitors of tyrosine kinases are ATP pocket-binders. Some of the common small molecule inhibitors are Tarceva, Glivec, Iressa, Sutent..This is proof data to show PathHunter capabilities in small molecule inhibitor discovery.

Uncover Compound Specificity & Selectivity

• Correlates with existing Biochemical Data:• Staurosporine – Least selective inhibitor• Clustering of “hits” by family• Promiscuity is target dependent

Inhibitors

Kin

ases

Comparison of Biochemical and PathHunter Assays

PathHunter TrkA

10-9 10-8 10-7 10-6 10-5 10-40

10000

20000

30000

40000

50000

60000

70000

80000

Inhibitor [M]

RL

U

Purified Kinase DataPathHunter Data

Novel compound pharmacology using PathHunter Kinase Assays:• Biochemical assay all 9 compounds showed nM potency, • PathHunter assay only 3 compounds showed nM potency and the rest of the

compounds showed uM potency.

• This variation in rank potency illustrates the difference between in vivo and in vitro assays and may afford additional information on compound permeability.

Novel Platform for therapeutic Antibody testing

Anti-receptor Ab (EGFR)

Anti-ligand Ab (Trk A)

Use of a full length receptor protein allows identification of novel antibodies in PathHunter assays.

EGFR

10-11 10-10 10-9 10-8 10-7 10-6 10-52000

4000

6000

8000

10000111.6528225

Blocking Ab (g/ml)

Me

an

RL

U

Summary: Target-specific Tyrosine Kinase Assays

Full Length Receptors -Ideal for antibody Screening -Kinase activity measured

Small molecule inhibitors Cell permeability intact Natural

activation/deactivation processes

Assay-Whole cell Homogenous Assay-High throughput --Chemiluminescent Readout-Non-Specialized Reader

Assay Menu TrkA +/-

P75 TrkB +/-

P75 TrkC +/-

P75 EGFR ErbB2/3 ErbB4 INSR IGF1R FGFR4 EPHB4 DDR1 DDR2 FLT3 PDGFRb c-MET Jak1 Jak2 CSF3R PRLR

Blocking Ab

Activating Ab

Profiling capabilities

Cellular platforms @ the receptor:

1

2

1Ligand

23

• Cytokine receptors lack kinase domain. They signal through the JAK/STAT pathway.

• JAK kinase phosphorylates the receptor.

• Phosphorylated cytokine receptors activate the SH2 containing domains to bind to the phosphorylated receptor..

2. PathHunterTM Cytokine receptors or Cytosolic Tyrosine Kinases

Receptor activationRole of cytosolic tyrosine kinases

Assay Approach: PathHunter Assay for the Jak-coupled ProLactin Receptor.

PLCG2 PLCG1 SHC1

Red = + Jak2Blue = - Jak2

PRLR

• PRLR activation monitored using PathHunter technology• High specificity for SH2 adapter

PROLACTIN Receptor with JAK 1 & JAK2

U2OS PLCG2 PRLR Jak1

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-50

5000

10000

15000

S/B 17.2

EC50 1.0723e-008

Prolactin (g/mL)

RL

U

U2OS PLCG2 PRLR Jak2

10-13 10 -12 10-11 10 -10 10-9 10-8 10-7 10-6 10-50

1000

2000

3000

4000

5000

6000

7000

8000

9000

S/B 6.6

EC50 9.0311e-009

Prolactin (g/mL)

RL

U

PathHunter Prolactin Functional Assay can be used to identify prolactin receptor agonists or inhibitors to either Prolactin receptor or inhibitors to JAK 1 and JAK2

Assay Validation

Clonal Selection Mycoplasma testing Passage stability testing upto 10 passages Functional Validation with reference agonistFunctional validation with knows inhibitorsDocumentation that involves a cell guide.

10-4 10 -3 10 -2 10 -1 100 101 102 1031000

2000

3000

4000

5000

TNF-a [ng/mL]

RL

U

-12 -11 -10 -9 -8 -7 -6 -5 -40

25000

50000

75000

100000WortmanninLY294002

U2OS PK2-FOXO3 EA-Nuc clone

Akt inhibitor X

Inhibitor (M)

RL

U

EC50

Wortmannin5.1758e-008

LY2940021.1559e-005

Akt inhibitor X1.2035e-005

DiscoveRx Assay Capabilities

Core EFC Technolo

gy

Protein Levels

Protein Translocation

Protein Interactions

GPCRs

Kinases

NHRs

Pathway Assays

TargetsBiological Events

2nd Messenger DetectionArrestin ActivationInternalizationOrthologs

Kinase Binding assaysADP AccumulationPhosphoprotein binding

Nuclear TranslocationSRC-Cofactor Recruitment

Functional Read-outs

DNA DamageCell CycleSignaling

PathHunter Platform Technology: Similar detection methodologiesLibrary of over 600 Cell-Based Assays

Thank You!