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 characterization 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 in 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 to 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

“…This serves to recruit and activate Parkin, 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 76kD molecular weight range and is now reproduced with CMPD-39 or knock-out of USP30 with no additive effect (Figure 2D,F).…”

“…A further attractive feature of USP30 inhibition is that it is a non-essential gene, and this is a necessary condition for any long-term treatment option. The best characterised USP30 inhibitors to date are a series of cyanopyrrolidines, for which a consensus biochemical and cell physiological signature is beginning to emerge [5,16,17]. 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].…”

“…The best characterised USP30 inhibitors to date are a series of cyanopyrrolidines, for which a consensus biochemical and cell physiological signature is beginning to emerge [5,16,17]. 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]. We wondered if the recently published serie s of benzosulphonamide USP30 inhibitors might represent a structurally independent alternative, offering a greater dynamic range of concentration over which inhibition is achieved and specificity is retained [20].…”

“…Recent studies have reported highly related cyanopyrrolidine USP30 covalent inhibitors that recapitulate signature effects of USP30 depletion in the context of acute mitochondrial depolarisation [15][16][17]. These include enhanced TOM20 ubiquitylation, increased mitophagy and greater accumulation of phosphoubiquitin [6,[15][16][17][18].…”

“…Recent studies have reported highly related cyanopyrrolidine USP30 covalent inhibitors that recapitulate signature effects of USP30 depletion in the context of acute mitochondrial depolarisation [15][16][17]. These include enhanced TOM20 ubiquitylation, increased mitophagy and greater accumulation of phosphoubiquitin [6,[15][16][17][18]. In the case of FT385 the specificity of the drug across a panel of DUBs was high at concentrations up to 200nM but it becomes less specific at higher concentrations.…”