| Assay Method Information | |
| | JAK1, JAK2, and JAK3 Enzymatic Assays |
| Description: | The JAK1/JAK2 potency ratio, defined as the inverse ratio of IC50 values for JAK1 over JAK2, is about at least 30, and in some cases 50 to 85 or more. The JAK1/JAK3 potency ratio, defined as the inverse ratio of IC50 values for JAK1 over JAK3, is about at least 3, and in some cases 40 to 70 or more.The assay results demonstrate significantly lower IC50 values for JAK1 inhibition, which shows the selectivity of compounds of Formula I for JAK1 over JAK2 and JAK3, and provide a basis for determining whether a more selective biochemical profile will translate into an improved clinical profile by sparing the JAK2 or JAK-dependent cellular pathways.The JAK enzymatic assay used the following reagents.JAK1 (BPS Bioscience, San Diego, CA Cat.No. 40449)JAK2 (Carna Bio, USA, Inc. Natick, MA, Cat.No. 08-045)JAK3 (Carna Bio, USA, Inc. Natick, MA, Cat.No. 08-046)TYK2 (Carna Bio, USA, Inc. Natick, MA, Cat.No 08-147)Peptide FAM-P22 (GL Biochem, Cat. No. 112393)Peptide FAM-Pep D (GL Biochem, Cat No.358783)Peptide FAM-P30 (GL Biochem, Cat. No. 263631)ATP (Millipore Sigma, Cat. No. A7699-1G, CAS No. 987-65-5)DMSO (Millipore Sigma, Cat. No. D2650)EDTA (Millipore Sigma, Cat. No. E5134, CAS No. 60-00-4)Brij-35 [Sigma, Cat. No. B4184]96-well plate (Corning, Corning, NY, Cat. No. 3365)384-well plate (Corning, Corning, NY, Cat. No. 3573)Oclacitinib [HUIFEIChem (WuXi) PharmaTech Co. Ltd., Batch No.: D0228-W-0814-1]Tofacitinib (Med Chem Express, Monmouth Junction, NJ, Cat. No. HY-40354)Filgotinib [MCE, Cat. No. HY-18300]Preparation of assay plate. Each sample or reference compound was diluted to 50× the desired highest inhibitor concentration with DMSO. Each sample or reference compound was serially diluted using a 96-well source plate so that there were 10 concentrations for testing. 10 μL from each well of the source plate was transferred to a 96-well intermediate plate. To each well of the intermediate plate was added 90 μL of the 1× kinase buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35) and the intermediate plates were shaken on a shaker for 10 min. 5 μL from each well of the 96-well intermediate plate was transferred to a 384-well plate.Kinase reaction. 10 μL of 2.5× enzyme solution (([enzyme in 1× kinase base buffer) was added to the respective well of the assay plate The assay plate was incubated at room temperature for 10 minutes. 10 μL of 2.5× peptide solution (FAM-labeled peptide and ATP in 1× kinase base buffer) was added to the respective well of the assay plate. The kinase reaction proceeded for 28° C. for 60 min followed by the addition of 25 μL of stop buffer. The data was collected using the CALIPER program. The conversion data from the CALIPER program was converted to inhibition values as follows: % inhibition=(maximum conversion)/(max−min)*100. The term “max” refers to the DMSO control and “min” stands for low control. Curve-fitting of the data was performed using Xlfit excel add-in version 5.4.0.8 to obtain IC50 values. |
| Affinity data for this assay | |
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