PROJECT SUMMARY/ABSTRACT The proposed Phase I research is designed to establish the technical/scientific merit and feasibility of developing first/best-in-class, USP30 inhibitors for the treatment of Parkinson’s disease (PD), an age- associated neurodegenerative disorder second only to Alzheimer’s disease (AD) in prevalence. No therapy that can slow or stop the progression of PD currently exists. Instead, treatments for PD, which affects 10 million people worldwide, merely augment dopaminergic neurotransmission to provide symptomatic benefit. To address this unmet need, Vincere Biosciences has initiated a platform to develop small molecules targeting the parkin-USP30 ubiquitination pathway, which represents a key regulator of mitochondrial homeostasis, as a means of slowing disease progression. Converging lines of evidence – human pharmacology, genetics, tissue pathology and animal model studies – indicate that deficits in mitochondrial quality control pathways underlie PD pathogenes. While parkin, an E3 ubiquitin ligase, drives mitophagy by adding ubiquitin chains to proteins on damaged mitochondria, USP30 removes these chains to inhibit clearance of the damaged mitochondria, thus acting as the yin to parkin’s yang. Of note, functional genomic studies in mammalian cells and flies have validated USP30 as a key target of mitochondrial quality control. While mitochondrial abnormalities have long been implicated in sporadic PD, compelling scientific rationale also now exists for restoring mitochondrial health in AD. By inhibiting USP30, we aim to indirectly enhance parkin’s downstream signaling, thereby increasing mitophagy and restoring mitochondrial homeostasis. In so doing, we will test the hypothesis that USP30 inhibitors promote the clearance of damaged mitochondria, thereby attenuating the pathogenic cascade associated with PD pathogenesis. We have identified several hit compounds that potently inhibit USP30 activity in vitro, and demonstrate cellular activity without cytotoxicity in human primary fibroblast cells. Moreover, our exciting preliminary data indicate that we have rigorous flow scheme assays and starting chemical scaffolds in place to deliver: two distinct lead series with IP potential for lead optimization (Aim 1); and, up to 12 optimized compounds for further in vivo pharmacokinetic (PK) and target modulation/efficacy assessment and preclinical development (Aim 2). In the proposed Phase I studies, we will answer the following technical questions: 1) Can we identify potent and patentable USP30 inhibitors with sufficient selectivity; that, 2) Induce mitophagy in human cells without cytotoxicity and effects of basal mitochondrial membrane potential; and, 3) Display desired in vitro ADME properties engineered to enable in vivo proof of concept studies and preclinical development in Phase II? A future Phase II will carry these molecules through in vivo PK, PK-Pharmacodynamic (PK-PD) research, and preclinical development (Investigati...