| Assay Method Information | |
| | PIKfyve Enzyme Inhibition Assay |
| Description: | For each of the compounds prepared in Examples 1 to 33, the PIKfyve enzyme inhibitory activity was measured by using a QS S Assis PIKFYVE (PIP5K3)_ADP-Glo™ Kit (Cat. #11-118) made by Carna Bioscience. 5 μL of each of human-derived PIKfyve kinase, PI(3)P, and ATP solution was added to a 384-well plate (such that final concentrations of each solution are 375 ng/ml, 10 μm, and 2 μm, respectively). The compound to be evaluated underwent serial dilution according to the set concentrations, and 5 μL of the diluted solution was added to each well and caused a reaction for 60 minutes. MgCl2 was mixed in ADP-Glo™ solution (Promega, Cat. #V9102) to a final concentration of 100 mM, and 20 μL of the mixed solution was added to each well and caused a reaction at room temperature for 40 minutes. 40 μL of kinase detection solution was added to each well and caused a reaction at room temperature for 40 minutes. Then, the luminescence signals were detected by using a Synergy™ NEO microplate analyzer. Well to which no enzyme was added were used as a negative control, whereas wells to which no drug was added were used as a positive control, so as to calculate the enzyme inhibitory ability (%) of drugs. The 50% inhibitor concentration (IC50) values were calculated by using a GraphPad Prism software. |
| Affinity data for this assay | |
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