Fig 2: Effect of TIM-098a on AAK1, NAKs, and CaMKK activities. (a) Protein kinase activities of His-tagged AAK1 catalytic domain (25–396) were measured at 30 °C for 20 min using 100 μM [γ-32P]ATP in the presence of various concentrations of TIM-098a or absence as described in the “Methods”. AAK1 activities are quantitated and expressed as a percentage of the average value in the absence of the compound. Results represent duplicate experiments. Chemical structure of TIM-098a is indicated (insert). (b) IC50 value of TIM-098a against NAKs activities. Activities of NAKs, including AAK1, GAK, BIKE and STK19, were measured in the presence of various concentrations of TIM-098a in a solution described in (a) and the “Methods”. IC50 values were estimated from duplicate experiments (see Supplemental Information S3). (c) Activities of CaMKKα/1 (left panels) and β/2 (right panels) were measured at 30 °C for 10 min in the presence of 2.6 µM TIM-063 or TIM-098a or absence (−) via immunoblot analysis using anti-phospho-CaMKIα (pThr177) antibody (upper panels) as described in the “Methods”. CaMKK activities are quantitated by densitometric scanning of the immunoreactive band and expressed as a percentage of the average value in the absence of the compound (−). Results are represented as the mean ± the standard deviation indicated by error bars from triplicate experiments. Molecular mass markers (kDa) are indicated in the left lanes of immunoblot panels. Statistical differences are marked as **p < 0.05; n.s., not significant vs. CaMKK activities in the absence of compounds (−). (d) Energy minimized (Macromodel) Glide top scored docking poses of (A) TIM-063 and (B) TIM-098a with AAK1. (C) Overlay of (A) and (B).

Fig 3: Effect of TIM-098a on AAK1 overexpression-reduced early endosome formation. HeLa cells were transfected with an expression vector containing His-tagged AAK1 (1–863). After 42 h of culture, cells were incubated with 10 µM TIM-098a-containing medium or without (DMSO) for 6 h. (a) Cells were fixed and immunostained with anti-AAK1 antibody (red) and anti-EEA1 antibody (green), followed by nuclear staining with Hoechst 33342 (blue). The right panels show the merged images. Scale bars = 10 µm (b) The number of EEA1 vesicles were quantified in 54–169 cells per condition using the ImageJ software38 as described in the “Methods”. The data are represented as the mean ± the standard deviation indicated by error bars. Statistical differences are marked as **p < 0.05; n.s., not significant vs. untransfected cells without TIM-098a treatment.

Fig 4: Characterization of TIM-063 derivatives as AAK1 inhibitors. (a) Protein kinase activities of His-tagged AAK1 catalytic domain (25–396) were measured at 30 °C for 20 min using 100 μM [γ-32P]ATP in the presence of 26 µM TIM-063 or 2.6 µM of its derivatives (TIM-055 − TIM-106) or absence as described in the “Methods”. AAK1 activities are quantitated and expressed as a percentage of the average value in the absence of the compound (–). Results represent duplicate experiments. (b) Protein kinase activities of CaMKKα/1 (left panel) and β/2 (right panel) were measured at 30 °C for 20 min using 100 µM [γ-32P]ATP, and 2 mM CaCl2 with 6 µM CaM in the presence of 2.6 µM TIM-063 or its derivatives (TIM-088 − TIM-106) or absence as described in the “Methods”. CaMKK activities are quantitated and expressed as a percentage of the average value in the absence of the compound (−). Results represent duplicate experiments.

Fig 5: Identification and characterization of AAK1 as a TIM-063–interactant. (a) Protocol for identification of TIM-063–interactants using TIM-063–immobilized sepharose (TIM-127-sepharose12) combined with mass spectrometry analysis. Mouse cerebrum extracts were applied onto TIM-127-sepharose, and after extensive washing of resin, TIM-063–interactants were eluted by the addition of 100 µM TIM-063 containing buffer, followed by identification via mass spectrometry analysis as described in the “Methods”. (b) Eluates from TIM-127-sepharose (TIM-127–S) or a control sepharose without the inhibitor (Control–S) as described in (a) were analyzed by immunoblotting with either anti-CaMKKβ/2 antibody (upper panel), anti-AAK1 antibody (middle panel), or anti-ERK1/2 antibody (bottom panel) together with mouse cerebrum extracts (Input). (c) Extracts from COS-7 cells expressing His-tagged AAK1 wild type (1–863) or His-tagged AAK1 catalytic domain (25–396) were incubated with either TIM-127-sepharose or control sepharose, and after extensive washing of resin, TIM-063–interactants were eluted by the addition of 100 µM TIM-063–containing buffer, followed by immunoblot analysis with an anti-AAK1 antibody as described in the “Methods”. An arrow and asterisk indicate His-tagged AAK1 wild type (1–863) and His-tagged AAK1 catalytic domain (25–396), respectively. (d) Phosphorylation of GST-AP2µ2 (145–162) at Thr156 by the His-tagged AAK1 catalytic domain (25–396) for various time periods (0–120 min) was detected by immunoblot analysis with an anti-phospho-AP2µ2 (pThr156) antibody (insert), quantitated by densitometric scanning of the immunoreactive bands, and expressed as a percentage of the value at 120 min of the reaction. An asterisk indicates phospho-Thr156 in GST-AP2µ2 (145–162). (e) Inhibition of AAK1 activity by TIM-063 in a dose-dependent manner. Phosphorylation of GST-AP2µ2 (145–162) by His-tagged AAK1 catalytic domain (25–396) was measured in the presence or absence of various concentrations of TIM-063 at 30 °C for 20 min using 100 µM [γ-32P]ATP as described in the “Methods”. AAK1 activities are expressed as a percentage of the average value in the absence of the compound. Results represent duplicate experiments. Chemical structure of TIM-063 is indicated (insert). Molecular mass markers (kDa) are indicated in the left lanes of immunoblot panels.