| Assay Method Information | |
| | In Vitro Kinase Activity Assay |
| Description: | Table 11: The Fyn kinase inhibitory activity of representative examples of compounds of Formula (1) was evaluated by Z′-LYTE™ Kinase Assay Platform (Invitrogen). Compounds were evaluated towards active Fyn (full length active, molecular weight 87.1 kDa; Invitrogen). The enzyme (1.2 ng/μl) was incubated in a 384 low-volume microplate with a synthetic peptide-substrate, ATP (50 μM) and different inhibitor concentrations, ranging from 10−9 M up to 10−5 M final concentration. Samples representing the 0% inhibition (or total enzymatic activity) were in the presence of compound diluent (1% DMSO final in Reaction Buffer 50 mMHepes, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, pH 7.5). The kinase reaction was carried out in a total volume of 10 μl, for 60 minutes at 25° C. The 0% Phosphorylation (i.e. no ATP) and 100% Phosphorylation Assay Controls, included in each plate, allowed to calculate the percent phosphorylation achieved in the specific reaction well. Results are expressed as percentage of inhibition and the IC50 values were calculated by non-linear curve fitting using GraphPad™ Prism software (version 6 for Windows). |
| Affinity data for this assay | |
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