Selectivity Kinase Panels
|Panels run at German facility||Panel size||Panel|
|IC50 Wild Type Kinase Panel||340||view panel|
|Lipid Kinase Panel (add on) - also available as standalone Lipid Kinase Profiler - see table below||14||view panel|
|Mutant Kinase Panel (add on)||96||view panel|
|Wild Type Kinase Panel||340||view panel|
Speciality Kinase Panels
|Panels run at US facility||Panel size||Data|
|ABL1 Mutant Profiler||13||Data sheet|
|ALK Mutant Profiler||22||Data sheet|
|BRAF Mutant Profiler||16||Data sheet|
|c-KIT Mutant Profiler||22||Data sheet|
|c-MET Mutant Profiler||28||Data sheet|
|CDK Profiler||31||Data sheet|
|EGFR Mutant Profiler||52||Data sheet|
|ERBB2 Mutant Profiler||9||Data sheet|
|FGFR2 Mutant Profiler||11||Data sheet|
|FGFR3 Mutant Profiler||6||Data sheet|
|FGFR4 Mutant Profiler||5||Data sheet|
|FLT3 Mutant Profiler||9||Data sheet|
|RET Mutant Profiler||28||Data sheet|
|TIE Mutant Profiler||7||Data sheet|
|TRKA Mutant Profiler||15||Data sheet|
|TRKC Mutant Profiler||6||Data sheet|
|Panels run at German facility||Panel size||Data|
|ABL1 Mutant Profiler||10||Data sheet|
|ALK Mutant Profiler||9||Data sheet|
|CDK Profiler||28||Data sheet|
|EGFR Mutant Profiler||10||Data sheet|
|KIT Mutant Profiler||9||Data sheet|
|Lipid Kinase Profiler||14||Data sheet|
|MET Mutant Profiler||9||Data sheet|
|RET Mutant Profiler||11||Data sheet|
Kinase Panel Screening Details
HotSpot™ and ³³PanQinase™: two radiometric assay formats
Reaction Biology offers two radiometric assay formats that are performed in our screening facilities located in Malvern, PA, USA, and Freiburg, Germany. Both assays are based on the transfer of ³³P-labelled phosphate from ATP to the kinase substrate. The methods, however, differ with respect to retention and detection of the phosphorylated substrates. Proteins and peptides in the HotSpot™ assay (performed in the US) are captured via spotting of the reaction mix on a filter membrane. The ³³PanQinase™ assay (performed in Germany) is performed with FlashPlate microtiter plates, which are coated with scintillant for detection.
Lipid kinases are screened with ADP-Glo (Promega) at both facilities.
The IC50 values of a BCR-ABL kinase inhibitor were determined for 320 wild type kinases in two independent experiments and compared. The high R2 value shows a high correlation of data. IC50 value determination was performed with 6 compound concentrations in singlicate with the ³³PanQinase™ assay.
The control compound Staurosporine was tested over the course of 6 months in 12 independent experiments. IC50 determination was performed with 10 compound concentrations in singlicate with the HotSpot™ assay.
Screening formats: 2 data point projects with 1 concentration in duplicates and 5 to 10-doses response curves.
Assays: Protein kinases are screened via radiometric HotSpot™ assay. Lipid kinases are screened via ADP-Glo (Promega) assay. The assay protocol for HotSpot™ assay is available here.
ATP concentration: 1uM or 10 uM or apparent ATP-Km up to 100 uM
Controls: No inhibitor (DMSO vehicle) control. For every kinase, we include the screening of one kinase-specific control compound in 10-dose IC50 format.
Timelines: Panel screening is performed twice per month. Reports are sent 2 to 3 weeks after the project start.
Screening dates: Upcoming profiling dates are shown here.
Report: The raw data, % enzyme activity and control compound IC50 values will be reported in Excel format. Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.
Mapper: The results of the selectivity of compounds can be graphically presented with our mapper tool.
Compound requirements: In brief, for kinase panel profiling 100 ul of a 10 mM DMSO stock or solid material is needed. Shipping information can be found in our FAQs.
Screening formats: 2 data point projects are performed with 1 concentration in duplicates or 2 concentrations in singlicates. IC50 projects are performed with 6 concentrations in singlicate.
Assays: Protein kinases are screen via radiometric ³³PanQinase™ assay. Lipid kinases are screened via ADP-Glo™ (Promega) assay. The assay protocol for the ³³PanQinase™ assay is available here.
ATP concentrations: apparent ATP-Km
Controls: DMSO only as full enzymatic activity control.
Timelines: Panel screening is performed once per month or on-demand. Reports are sent 10 business days after the project start.
Screening dates: Upcoming profiling dates are shown here.
Report: A detailed report will be provided including assay conditions, target, and substrate information. The raw data and % enzyme activity will be reported in Excel format including IC50 values and curve fitting, if applicable. Compound selectivity on wild type protein kinases will be graphically presented with the mapper tool.
Compound requirements: For testing with 1 concentration in duplicate, 1700 µl of a 100x stock solution is required. For testing with 2 concentrations in singlicate, 850 µl of a 100x stock solution of each concentration is required. For IC50 testing, 1000 µl of 100x stock solution is required. Solid material is also accepted.
Example of an FLT3 inhibitor with ³³PanQinase™ assay: IC50 values of 320 wild type protein kinases are shown with values of lower than 10-4. IC50 value determination for selectivity profiling helps assess differences in the potency of a given compound and allows a precise judgment on the selectivity of kinase inhibitors.
Only the targets with the lowest IC50 values are named.
IC50 based profiling is designed to:
- Obtain an accurate guiding parameter for optimization of kinase selectivity during the medicinal chemical optimization phase
- Ensure that observed functional effects of a kinase inhibitor are not due to the inhibition of off-target-kinases and to identify potential adverse side effects of the drug
- Provide accurate comparison to a reference compound