Figure 1. CMPD-39 is a selective USP30 inhibitor.

(A) Chemical structure of CMPD-39. (B) DUB specificity screen (DUB profiler, Ubiquigent) with 1-100 μM CMPD-39. (C) Activity-based ubiquitin probe assay shows that CMPD-39 engages USP30 in cells at nanomolar concentrations in intact SHSY5Y cells. Samples were incubated with CMPD-39 for 2 h at the indicated concentrations, then incubated with HA-Ub-PA probe for 10 min at 37°C and immunoblotted as shown. Red arrow indicates unbound USP30; blue arrow represents probe bound USP30. (D) Inhibition of USP30 enhances the ubiquitylation of TOMM20 in YFP-PRKN over-expressing hTERT-RPE1 cells in a concentration dependent manner in response to mitophagy induction. Cells were treated for 1 h with DMSO or antimycin A and oligomycin A (AO; 1 μM each) in the absence or presence of CMPD-39 at the indicated concentrations, lysed, and analyzed by Western blotting. Black arrow indicates unmodified TOMM20, ubiquitylated species are indicated by red (mono-ubiquitylated) or blue arrow heads. Quantitation shows percentage mono-ubiquitylated.

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