Figure 5. Fbxo7 FP domain small molecule inhibitor accumulates PINK1 protein.

(A) Structural analysis of the Fbxo7 FP domain. Docking study of a candidate inhibitor BC1464 within the Fbxo7-FP domain suggests hydrophilic interactions of residues GLN215, LYS235, and LYS266 with BC1464. (B) His pull down Fbxo7 protein was exposed to BC1464 at indicated concentrations. TnT-synthesized PINK1 protein was then incubated with drug-bound Fbxo7 beads overnight. The eluate was subjected to immunoblotting. The relative amounts of PINK1 detected in the pull-downs were normalized to loading and quantified; data are shown as means ± SEM (n = 3). (C) BEAS-2B cells were incubated with BC1464 or the control compound BC1465 at the indicated concentrations for 16 hours before immunoblotting. (D) BEAS-2B cells were pretreated with BC1464 or BC1465 (1 μg/mL) for 16 hours, and the cells were then incubated with CHX (40 μg/mL). The cells were collected at indicated time points for immunoblot analysis. (E) BEAS-2B cells were nucleofected with control or Fbxo7 siRNA (50 pg) and incubated for 72 hours. BC1464 was then administrated at indicated concentration for an additional 18-hour incubation. The cell lysates were subjected to immunoblotting.