ABSTRACT Existing treatments for Parkinson’s disease (PD), which affects 10 million people worldwide, primarily augment dopaminergic neurotransmission to provide symptomatic benefit. No current therapy can slow or stop the progression of PD. We propose a drug discovery platform to develop small molecules targeting the parkin-USP30 mitophagy pathway, which represents a key regulator of mitochondrial homeostasis. Converging lines of evidence from human genetics, tissue pathology and biochemical studies from sporadic PD patients, and animal model studies indicate that deficits in Mitophagy are a modifiable contributor to PD pathogenesis. Specifically, inhibition or knockdown of the deubiquitinating enzyme USP30 can enhance mitophagy in a variety of cell types and infers neuroprotection in two fruit fly models (pink1 deficiency and paraquat toxicity). However, studies examining neurodegeneration in larger animals such as rodents are an unfilled gap in knowledge that should be addressed prior to development of USP30 inhibitors as therapeutics for PD. In the proposed Aims, we will test the hypothesis that USP30 inhibitor small molecules will protect against neurodegeneration induced by a mitochondrial toxin MPTP, or a-synuclein preformed fibrils – both models with links to Parkinson’s disease and mitophagy. Our preliminary data demonstrate that we have generated a series of small molecules that potently and selectively inhibit USP30 activity in vitro, and demonstrate cellular activity without cytotoxicity in differentiated neuronal cell lines. Top compounds have good in vitro ADME properties and a lead and back up compound have been identified with excellent rat and mouse plasma and brain PK profiles which place us in a unique position to examine the proposed hypothesis. If successful, the studies proposed in this application will justify further investment toward development of our small molecules through Investigational New Drug filing (IND)-enabling studies, out-license, or partnership with big pharma or biotech for further clinical development.