Figure 3.

PRT062607 and related compounds inhibit human PINK1 in organello and in cells. (a) In organello ubiquitin kinase assays with accumulated HsPINK1 and tetraubiquitin (Ub₄). Reactions were analyzed by immunoblotting against phospho-ubiquitin (pUb). 100 μM of compounds were used in the assay. Olaparib (PARP inhibitor) was used as a small molecule negative control (n = 1). (b) Same as in (a), with different inhibitors at 100 μM. Formation of pUb is significantly impaired at alpha = 0.05. ns at P > 0.05, * at P ≤ 0.05, ** at P ≤ 0.01, *** at P ≤ 0.001 and **** at P ≤ 0.0001 (n = 2). The Ponceau staining of the above gel is shown as loading control. (c) IC₅₀ generated from HeLa S3 cells transfected with endogenous HA-tagged HsPINK1 treated for 4 h with 20 µM CCCP and PRT062607 (1.8 nM, 5.5 nM, 16 nM, 49 nM, 150 nM, 440 nM, 1.3 μM, 4.0 μM, 12 μM and 36 μM). Reaction was performed twice as biological replicates. Reactions were analyzed by immunoblotting against phospho-ubiquitin (pUb). The 100% activity baseline was measured as no PRT062607 with CCCP treatment. Immunoblots against blots HA and TOM40 are shown as loading controls. (d) Dopaminergic (DA) neurons were treated with PRT062607 (or DMSO) for 48 h, and then with 20 µM CCCP (or DMSO) for 2 h (n = 1). Samples were analyzed by immunoblotting. Cleavage of Opa1 is used to monitor mitochondrial depolarization, and VCL is a loading control.