| Assay Method Information | |
| | In Vitro Assay |
| Description: | Activity on human GSK-3β was assessed using the following methods (according to Meijer et al., Chem. Biol., 2003-10:1255-1266).In a first screening assay, compounds were tested in duplicate at a concentration of 10 μM.Human recombinant enzyme GSK-3β was incubated for 90 minutes at 22° C. in the presence of compounds or vehicle in a reaction buffer containing ATP plus 100 nM unphosphorylated specific substrate peptide (Ulight-CFFKNIVTPRTPPPSQG K-amide). Substrate phosphorylation was measured by LANCE technology (PerkinElmer, Conn., USA).The results, reported in the following Table 4, are expressed as a percentage of inhibition of control specific activity obtained in the presence of the test compounds (as % inhibition at 10 μm).In a second assay, the same compounds were assayed at five concentrations ranging from 100 μM to 10 nM with ten-fold dilutions in duplicate. Compounds 1 to 15 were tested using the same first assay, compounds 16 to 41 were tested in another assay based on the binding and displacement of AlexaFluor 647 labeled, ATP-competitive Kinase inhibitor scaffold using LanthaScreen TR-FRET technology Eu Kinase assay packet according to manufacturer's instruction (Life Technologies, Italy). The results of the two assays are comparable. |
| Affinity data for this assay | |
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