Fig 1: SGC-AAK1-1 Is a Potent and Specific Inhibitor of AAK1 and BMP2K(A) Chemical structures of the AAK1/BMP2K chemical probe SGC-AAK-1, the related AAK1/BMP2K inhibitor 25A, and the negative control compound 34A that has a similar chemical structure but does not inhibit AAK1 or BMP2K.(B) SGC-AAK-1 selectively bound AAK1 and BMP2K with a more than 30-fold difference in Ki in a TR-FRET binding displacement assay over the related kinases GAK and STK16. Sixteen-point dose-response curves were measured in duplicate.(C) SGC-AAK1-1 has good selectivity over the human kinome. SGC-AAK1-1 was used at 1 µM concentration and tested against over 400 wild-type human kinases. KD determination was completed for all kinases that exhibited >85% inhibition and/or were observed as common off-targets for this structural class. Kinases for which SGC-AAK1-1 has a KD < 100 nM are marked with a large red circle and 100 nM < KD < 1.0 µM with a smaller red circle.(D) ITC confirmed a KD of 120 nM for binding of SGC-AAK1-1 to AAK1.(E) A co-crystal structure of BMP2K (2.41 Å) bound to 25A revealed the binding mode of the inhibitor in the ATP site. This structure revealed five key hydrogen bonds and revealed the orientation of the central hydrophobic portion of the inhibitor with respect to the binding pocket.(F and G) NanoBRET cellular target engagement assays in HEK293T cells showed that 25A (F) and SGC-AAK1-1 (G) both entered cells and directly bound to AAK1 and displaced a fluorescent tracer molecule from the ATP site. Cells were treated with serially diluted inhibitors in the presence of four different concentrations of a NanoBRET tracer molecule, and IC50 values were calculated from a linear interpolation of the IC50 values at each tracer concentration to obtain the predicted IC50 in the absence of tracer. Measurements were made with three technical replicates.(H) Binding of SGC-AAK1-1 to BMP2K in cells was weaker than to AAK1 with IC50 > 1 µM. Displacement of the same fluorescent tracer molecule required increased concentrations of SGC-AAK1-1, revealing an elevated IC50 value for binding to BMP2K versus AAK1 in the cellular target engagement assay.(I) SGC-AAK1-1 inhibited the phosphorylation of the AP2M1 subunit at Thr156 in HEK293T cells in a concentration-dependent manner or with 10 µM 34A. HEK293T cells were treated with inhibitors for 2 hr before western blot analysis.Error bars represent SE. See also Figure S1 and Tables S2, S3, S4, and S5.