Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches

Abstract

Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.

Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy

“…Here, we now show that this defect can be restored by a specific inhibitor of USP30, providing further encouragement for preclinical development of these compounds. Some overlapping results have recently been reported for a set of cyanopyrrolidine inhibitors (17).…”

“…This serves to recruit and activate PRKN, setting off a cascade of ubiquitylation at the mitochondrial surface ( 25 ). USP30 inhibition with cyanopyrrolidine inhibitors has the effect of modestly enhancing the PINK1 dependent accumulation of pUb on mitochondria ( 16 , 17 ). In SHSY5Y cells this is most apparent between the 38 and 76 kD molecular weight range and is now reproduced with CMPD-39 or KO of USP30 with no additive effect ( Fig 2E and G ).…”

“…A further attractive feature of USP30 inhibition is that it is a nonessential gene, and this is a necessary condition for any long-term treatment option. The best characterized USP30 inhibitors to date are a series of cyanopyrrolidines, for which a consensus biochemical and cell physiological signature is beginning to emerge ( 15 , 16 , 17 ). One limitation of the use of these compounds is that they become less specific at higher concentrations and this can become problematic if the cellular uptake and target engagement efficiency is unknown ( 15 , 17 ).…”

“…Recent studies have reported highly related cyanopyrrolidine covalent USP30 inhibitors that recapitulate signature effects of USP30 depletion in the context of acute mitochondrial depolarization ( 15 , 16 , 17 ). These include enhanced TOMM20 ubiquitylation, increased mitophagy and greater accumulation of pUb (phosphoSer65 ubiquitin) ( 6 , 15 , 16 , 17 , 18 ).…”

“…Recent studies have reported highly related cyanopyrrolidine covalent USP30 inhibitors that recapitulate signature effects of USP30 depletion in the context of acute mitochondrial depolarization ( 15 , 16 , 17 ). These include enhanced TOMM20 ubiquitylation, increased mitophagy and greater accumulation of pUb (phosphoSer65 ubiquitin) ( 6 , 15 , 16 , 17 , 18 ). In the case of FT385, the specificity of the drug across a panel of DUBs (deubiquitylases) was high at concentrations up to 200 nM but it becomes less specific at higher concentrations.…”