| Assay Method Information | |
| | LanthaScreen TR-FRET Assay |
| Description: | A LanthaScreen kinase activity assay was conducted to assess WEE1 kinase inhibitory activity for compounds of this disclosure. LanthaScreen Eu time-resolved fluorescence resonance energy transfer (TR-FRET) kinase binding assays (Invitrogen) were performed in 384-well, low-volume plates (Corning) using recombinant WEE1 kinase, Kinase Tracer 178 and LanthaScreen Eu-anti-GST antibody (Invitrogen). Assays were performed at 25° C. in a reaction mixture consisting of 5 μL serially diluted inhibitor solution, 5 μL Kinase Tracer 178 solution, and 5 μL kinase/antibody solution. All reagents were prepared as solutions in 1× kinase buffer A (Invitrogen) at 3× final desired concentration. Inhibitor solutions were prepared such that final DMSO concentrations did not exceed 0.5%, which was shown to have no effect on kinase activity. Inhibitors were assayed in the final concentration range of 0.04 nM to 10 μM. Kinase Tracer 178 was used at a final concentration of 150 nM and the antibody and kinase were used at final concentrations of 3 nM and 5 nM, respectively. All reagents were incubated together for 1 hour at room temperature and read using a PerkinElmer Envision 2104 Multilabel Reader enabled for TR-FRET (Excitation=340 nm; Tracer emission=665 nm; Antibody emission=615 nm; Delay=100 μs; Integration=200 μs). Emission ratios (665 nm/615 nm) were determined for each inhibitor concentration and the data analyzed usinga non-linear regression analysis of the log dose-response curve todetermine IC50 values. |
| Affinity data for this assay | |
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