Figure 2.
Assessment of potency and cell permeability of the selected compounds. (A) Inhibition of PARK7 at 0.25 and 1 μM final concentration of 5 compounds designed from structural modification and top 10 compounds from HTS, determined using the PARK7 FP assay.15 (B) Fluorescent probe labeling of PARK7 remaining activity after inhibitor treatment to investigate cellular engagement. HEK293T cells were treated with the indicated compounds for 24 h. After cell lysis and incubation with the fluorescent PARK7 probe JYQ-92 for 1 h, the samples were analyzed by SDS-PAGE, fluorescence scanning, and immunoblot against PARK7 and β-actin. β-Actin was used as a loading control. (C) Target engagement of compounds 84, 336, and 340 in HEK293T cells in a dose–response manner. HEK293T cells were incubated with the compounds at the indicated final concentrations for 24 h, prior to cell lysis and incubation with PARK7 probe JYQ-92. The samples were analyzed by SDS-PAGE, fluorescence scanning, and immunoblot against PARK7 and β-actin. β-Actin was used as a loading control.