Figure 2. TOPK is a specific target of ADA-07.

A, computational docking model of ADA-07 with TOPK. B, ADA-07 binds with TOPK in an ATP-competitive manner. Active TOPK (200 ng) was incubated with different concentrations of ATP (0, 10 or 100 μM) and then mixed with ADA-07-conjugated Sepharose 4B beads or Sepharose 4B beads. Proteins were pulled down and analyzed by Western blot. Data are representative of 3 independent experiments that gave similar results. ADA-07 binds to either TOPK (C) or MEK1/2 (D) ex vivo. Lysates from HaCaT cells (500 μg) were incubated with ADA-07-conjugated Sepharose 4B beads or Sepharose 4B beads alone and the pulled-down proteins were analyzed by Western blot. Data are representative of 3 independent experiments that gave similar results. ADA-07 inhibits TOPK kinase (E) activity but not MEK1 kinase (F) activity in vitro. Active TOPK (200 ng) was mixed with ADA-07 (0, 0.5, 1, 3, or 5 μM) or HI-032 (TOPK inhibitor, 10 μM) and then incubated with a [γ-³²P] ATP mixture. Similarly, MEK1 (200 ng) was mixed with ADA-07 (0, 0.5, 1, 3, 5, or 10 μM) or PD098059 (MEK1 inhibitor, 5 μM) and then incubated with a [γ-³²P] ATP mixture. For E and F, the results are visualized by autoradiography and Coomassie blue staining serves as a loading control. Data are representative of 3 independent experiments that gave similar results.