Figure 2.

GSK3 small molecule inhibitors bind at the ATP binding pocket and have similar residence times. To test for binding at the ATP pocket, HEK293T cells were transiently transfected with either NanoLuc-GSK3α or NanoLuc-GSK3β plasmids and the following day treated for 2 h with the inhibitor compounds in a 10-point dose response curve (range: 3 nM–30 μM) and the NanoBRET Target Engagement Kinase Tracer-8. (A–C) Our results show competitive binding of inhibitors AZ1080 (A), BRD0705 (B), and compound 1 (C) with the kinase tracer indicating displacement at the kinase ATP pocket. Each data point represents the mean ± SEM from four biological replicates within a single run of the assay. mBU: milliBRET Units (please see Methods). To investigate compound residence time, cells were transiently transfected with NanoLuc plasmids and the following day kinase inhibitors were added at a concentration of 10× the previously identified IC₅₀. After 2 h of treatment, media with compound was removed and Tracer-6908 (Promega) was added. The BRET signal was measured every 5 min for a total of 3 h after tracer addition. (D,E) There was no difference in the residence times between any of the inhibitors and either GSK3α (D) or GSK3β (E). Each data point represents the mean ± SEM from eight biological replicates within a single run of the assay.