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Kinase Assay Overview

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Kinase Slide Set - Overview of assays for drug discovery and receptor biology.

Kinase Slide Set - Overview of assays for drug discovery and receptor biology.

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  • Receptor Activation: Ligand binding causes receptor activation. Cytokine receptors lack kinase domain. They signal through the JAK/STAT pathwayJAK kinase phosphorylates the receptor. Phosphorylated cytokine receptors activate the SH2 containing domains to bind to the phosphorylated receptor..Cytokine receptors, growth factor..T and B-cell receptors, Integrins, Interferons, Interleukins GP130 (LIF, CSF etc.) GH, EPO, TPO, Leptin, PR
  • Prolactin is a class I cytokine receptor
  • Transcript

    • 1. Target-Specific Cell Based Kinase Assays DiscoveRx Corporation
    • 2. DiscoveRx EFC Technology &Target-Specific Cell-Based Assays Active Enzyme Inactive Fragments Active Enzyme PK PK + GPCRs Kinases NHRs Proteases
    • 3. Studying kinases at the receptor:PathHunterTM Tyrosine kinase Assay Principle 1 2 3 4 Grb2-EA, Shc1-EA,PLCG-EA 1 Receptor Activation 2 Receptor Dimerization 3 Receptor Phosphorylation 4 Interaction with SH2 domain protein .
    • 4. RTK Assay Approach Thaw & Plate Cells U2OS TRKC Pool Screen 2000 SHC1 PLCG1 PLCG2 Mean RLU 12-48h 1000 GRB2 SYK-C PTPN11 0 1 hr 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 1 hr NT3 (g/ml) TrkC Clone Performance 2500 SHC1 2000 Mean RLU 1500 1000 500 0 10 -12.5 10 -11.5 10 -10.5 10 -9.5 10 -8.5 10 -7.5 10 -6.5 NT-3 (g/ml)
    • 5. Benefits of PathHunterTM RTK assays Assay Measures.. Assay Benefits Full length proteins  Non-ATP pocket binders  Antibody Therapeutics Ligand Binding  Ligand binding inhibitors Phosphorylation P P  Dimerization inhibitors Signal Transduction: Natural activation process Adapter protein binding  Unactive and active kinase  Cellular processes Cell Permeability of compounds
    • 6. Application: Identify Small Molecule inhibitorsMost small-molecule inhibitors of tyrosine kinases are ATP pocket-binders. Some of thecommon small molecule inhibitors are Tarceva, Glivec, Iressa, Sutent..This is proof data toshow PathHunter capabilities in small molecule inhibitor discovery.
    • 7. Uncover Compound Specificity & Selectivity Inhibitors Kinases • Correlates with existing Biochemical Data: • Staurosporine – Least selective inhibitor • Clustering of “hits” by family • Promiscuity is target dependent
    • 8. Comparison of Biochemical and PathHunterAssays PathHunter Data Purified Kinase Data PathHunter TrkA 80000 70000 60000 50000 RLU 40000 30000 20000 10000 0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 Inhibitor [M]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.
    • 9. Novel Platform for therapeutic Antibody testing Anti-ligand Ab (Trk A) EGFR 10000 111.6 528 8000 225 Mean RLU 6000 Use of a full length receptor 4000 protein allows identification of 2000 novel antibodies in PathHunter 10 -11 10 -10 10 -9 10 -8 10 -7 Blocking Ab (g/ml) 10 -6 10 -5 assays. Anti-receptor Ab (EGFR)
    • 10. Summary: Target-specific Tyrosine Kinase Assays Activating Ab Assay Menu Assay  TrkA +/- P75 -Whole cell Homogenous Assay  TrkB +/- P75 -High throughput  TrkC +/- P75 --Chemiluminescent Readout  EGFR -Non-Specialized Reader  Blocking Ab ErbB2/3  ErbB4  INSR  IGF1R Full Length Receptors  FGFR4 -Ideal for antibody Screening  EPHB4 -Kinase activity measured  DDR1  Small molecule inhibitors  Cell permeability intact  DDR2  Natural activation/deactivation Profiling capabilities  FLT3 processes  PDGFRb  c-MET  Jak1  Jak2  CSF3R  PRLR
    • 11. Cellular platforms @ the receptor: 2. PathHunterTM Cytokine receptors or Cytosolic Tyrosine Kinases Ligand 1 1 Receptor activation 2 3 2 Role of cytosolic tyrosine kinases• 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..
    • 12. Assay Approach: PathHunter Assay for the Jak-coupled ProLactin Receptor. PRLR Red = + Jak2 Blue = - Jak2 PLCG2 PLCG1 SHC1 •PRLR activation monitored using PathHunter technology •High specificity for SH2 adapter
    • 13. PROLACTIN Receptor with JAK 1 & JAK2 U2OS PLCG2 PRLR Jak1 U2OS PLCG2 PRLR Jak2 15000 9000 8000 7000 10000 6000 RLU RLU 5000 4000 5000 3000 2000 1000 0 0 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 Prolactin (g/mL) Prolactin (g/mL) EC50 1.0723e-008 EC 50 9.0311e-009 S/B 17.2 S/B 6.6PathHunter Prolactin Functional Assay can be used to identify prolactin receptoragonists or inhibitors to either Prolactin receptor or inhibitors to JAK 1 and JAK2
    • 14. Assay Validation Clonal Selection 5000 4000 Mycoplasma testing RLU 3000 2000 Passage stability testing 1000 10 -4 10 -3 10 -2 10 -1 TNF-a [ng/mL] 10 0 10 1 10 2 10 3 upto 10 passages Functional Validationwith reference agonist 100000 U2OS PK2-FOXO3 EA-Nuc clone Wortmannin LY294002 Akt inhibitor XFunctional validation 75000 RLU 50000with 25000 knows inhibitors 0 -12 -11 -10 -9 Wortmannin -8 Inhibitor (M) -7 LY 294002 -6 -5 -4 Akt inhibitor XDocumentation that EC50 5.1758e-008 1.1559e-005 1.2035e-005 involves a cell guide.
    • 15. DiscoveRx Assay Capabilities Biological Events Targets Functional Read-outs 2nd Messenger Detection Arrestin Activation GPCRs Internalization Protein Levels OrthologsCore EFC Kinase Binding assaysTechnolo Kinases ADP Accumulation Protein Translocation Phosphoprotein binding gy NHRs Nuclear Translocation Protein Interactions SRC-Cofactor Recruitment DNA Pathway Damage Assays Cell Cycle Signaling PathHunter Platform Technology: Similar detection methodologies Library of over 600 Cell-Based Assays
    • 16. Thank You!