High-content phenotypic screen to identify small molecule enhancers of Parkin-dependent ubiquitination and mitophagy.

Mitochondrial dysfunction and aberrant mitochondrial homeostasis are key aspects of Parkinson's disease (PD) pathophysiology. Mutations in PINK1 and Parkin proteins lead to autosomal recessive PD, suggesting that defective mitochondrial clearance via mitophagy is key in PD etiology. Accelerating the identification and/or removal of dysfunctional mitochondria could therefore provide a disease-modifying approach to treatment. To that end, we performed a high-content phenotypic screen (HCS) of ∼125,000 small molecules to identify compounds that positively modulate mitochondrial accumulation of the PINK1-Parkin-dependent mitophagy initiation marker p-Ser65-Ub in Parkin haploinsufficiency (Parkin +/R275W) human fibroblasts. Following confirmatory counter-screening and orthogonal assays, we selected compounds of interest that enhance mitophagy-related biochemical and functional endpoints in patient-derived fibroblasts. Identification of inhibitors of the ubiquitin-specific peptidase and negative regulator of mitophagy USP30 within our hits further validated our approach. The compounds identified in this work provide a novel starting point for further investigation and optimization.

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

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.

Targeting mitophagy in Parkinson's disease.

The genetics and pathophysiology of Parkinson's disease (PD) strongly implicate mitochondria in disease aetiology. Elegant studies over the last two decades have elucidated complex molecular signaling governing the identification and removal of dysfunctional mitochondria from the cell, a process of mitochondrial quality control known as mitophagy. Mitochondrial deficits and specifically reduced mitophagy are evident in both sporadic and familial PD. Mendelian genetics attributes loss-of-function mutations in key mitophagy regulators PINK1 and Parkin to early-onset PD. Pharmacologically enhancing mitophagy and accelerating the removal of damaged mitochondria are of interest for developing a disease-modifying PD therapeutic. However, despite significant understanding of both PINK1-Parkin-dependent and -independent mitochondrial quality control pathways, the therapeutic potential of targeting mitophagy remains to be fully explored. Here, we provide a summary of the genetic evidence supporting the role for mitophagy failure as a pathogenic mechanism in PD. We assess the tractability of mitophagy pathways and prospects for drug discovery and consider intervention points for mitophagy enhancement. We explore the numerous hit molecules beginning to emerge from high-content/high-throughput screening as well as the biochemical and phenotypic assays that enabled these screens. The chemical and biological properties of these reference compounds suggest many could be used to interrogate and perturb mitochondrial biology to validate promising drug targets. Finally, we address key considerations and challenges in achieving preclinical proof-of-concept, including in vivo mitophagy reporter methodologies and disease models, as well as patient stratification and biomarker development for mitochondrial forms of the disease.

Ethyl-for-methyl substitution enhances the subtype specificity of mecamylamine analogues.

The synthesis of novel mecamylamine analogues is described in which one, two or three of the methyl groups of mecamylamine have been systematically replaced with ethyl groups. Assessment of the compounds highlights that simple ethyl for methyl changes changes to the parent structure can dramatically enhance activity and selectivity towards either the α4ß2 (at the expense of α3ß4) or the α3ß4 (at the expense of α4ß2) nicotinic acetylcholine receptor sub-type as compared to the parent compound.

A new synthesis and preliminary evaluation of some analogues of mecamylamine - a compound with anti-addiction properties.

A new synthesis of mecamylamine - a known anti-hypertensive drug with anti-addictive properties is described. The new route allowed access to two novel analogues whose activity at two nicotinic acetylcholine receptor subtypes was assessed.