Our team performs small-, mid-, and large-scale screens of compounds on over 560 validated kinase assays including protein and lipid kinases.

Biochemical assay formats to measure kinase activity of any class of inhibitors:

• TR-FRET, ADP-Glo
• Assay setups include single-dose, multiple-dose, or IC50 dose-response formats
• Turnaround time for small projects is 5 business days

Kinase panel options for selectivity screening:

• Full Panel (563 wild-type & mutant kinases)
• Wild-Type Panel (389 wild-type kinases)
• Mini Kinase Panel (73 kinases)
• Customized Panels (>15 kinases)

Assay setups include single-dose/duplicate screening including a positive control compound.

Turnaround time for selectivity screening is 10 to 15 business days, depending on panel size.

Cellular Phosphorylation Assays help determine kinase activity in the physiological environment of cells via quantification of substrate phosphorylation.

• Options for cell lines include:
  • Naturally kinase target-expressing cell lines
  • Knock-in kinase-expressing cell lines
  • Kinase-overexpressing cell lines
• Substrate phosphorylation can be monitored by a variety of readout options, such as:
  • ELISA
  • Real-time imaging
  • High-content imaging for localization of phosphorylated targets
  • Signaling pathway elucidation
  • Fully automated high-throughput screening

Ba/F3 Oncogenic Driver Assays utilize cell lines that are genetically engineered to express a specific kinase it depends on for survival. Ba/F3 assays enable the quantification of kinase downstream signaling, therefore, providing a functional readout.

• 294 Ba/F3 cell lines established
  
• Readout options include:
  • Cell viability testing
  • Real-time imaging
  • in vivo testing in mice

NanoBRET Target Engagement (TE) Assays quantify test article binding to the target kinase by displacing a fluorescent tracer bound to the ATP-binding pocket.

• 90+ NanoBRET TE cell lines
• New NanoBRET cell lines can be engineered by our team for customized assays
• Binary or ternary setups
• Readout options include:
  • Binding affinity
  • Binding kinetics
  • Residence time
  • Target occupancy

Cellular Thermal Shift Assays quantify the binding affinity for any class of kinase inhibitor by investigating how binding to the kinase stabilizes the newly formed complex.

• Readout options include:
  • Western blotting: in-cell Western, Jess automated Western, conventional Western
  • MSD MesoScale

Pharmaron provides comprehensive signal pathway research to assess how kinase inhibitors modulate signaling pathways and to identify biomarkers for successful target inhibition.

Methods for identification and screening of proximal markers:

• RPPA assay (reverse phase protein array) for phospho-proteomics, enabling the testing of hundreds of signaling proteins, including phospho-targets, on a microarray
• Western blotting (manual, Jess, in-cell)
• MSD MesoScale
• AlphaLisa, HTRF, ELISA
• Mass spectrometry
• dPCR, qPCR
• Nanostring, RNAseq, WGS, WES

Signaling network analysis from genomic and proteomic data is supported by our in-house bioinformatics teams.