Targeting Trafficking Defects in Parkinson's Disease: A Chemical Biology Approach to Modulating Protein Networks

Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by defective proteostasis mechanisms that induce neuronal toxicity. A common pathological hallmark of PD is the impairment of intracellular protein trafficking, which disrupts the essential movement of cargo between neuronal organelles. While phenotypic screens have identified a select number of small-molecule modulators that are efficacious at restoring some of these pathways, the molecular targets, pharmacophore, and mechanisms of action (MOA) for many remain poorly characterized. The Lindquist laboratory identified a molecular scaffold, N-arylbenzimidazole (NAB), that was efficacious in alleviating multiple phenotypic markers of α-synuclein toxicity in a Rsp5-dependent manner (yeast homologue of Nedd4-1). The McCafferty lab discovered that the lead compound, NAB2, induces small but phenotypically relevant changes in the ubiquitylome under α-synuclein toxicity and identified potential NAB2 targets in wild-type cells without toxicity. Despite the genetic validation of the E3 ubiquitin ligase Nedd4-1 in regulating PD proteostasis, previous biophysical characterization and chemoprotomics of the NAB target are inconclusive and conflicting. To resolve these discrepancies, a series of biochemical and biophysical analyses was performed to determine whether Nedd4-1 was the putative target of the NAB scaffold. Herein, a method was developed for recombinant expression and purification of Nedd4-1, which was used to biophysically characterize the NAB2-Nedd4-1 interaction by surface plasmon resonance (SPR) and microscale thermophoresis (MST). These analyses revealed no direct interaction between NAB2 and recombinant Nedd4-1, suggesting that NAB2’s MOA may be indirect or complex-mediated. To further our understanding of the NAB pharmacophore, a robust, facile synthetic route was developed to generate 14 NAB derivatives. Evaluation in a C. elegans model induced with neurodegeneration by 6-hydroxydopamine (6-OHDA) identified 12 neuroprotective compounds, with two derivatives displaying superior neuroprotection over NAB2. To facilitate mechanistic studies, an inducible α-synuclein toxicity model was developed in S. cerevisiae and the human neuroblastoma SH-SY5Y cell lines. Photoaffinity labeling derivatives of NAB were synthesized, evaluated for neuroprotection in C. elegans, and used in the developed PD model in SH-SY5Y cells. The chemoproteomics identified 36 significant protein hits, 11 of which are central to protein folding and trafficking. Notably, eight of these targets possess yeast homologs, and five are functionally linked to Rsp5, further delineating a conserved NAB MOA. Finally, Nedd4-1 molecular constructs were developed for TurboID-based proximity labeling and Flag-EGFP-fusion-based proteoform analysis to map the Nedd4-1 interactome and post-translational modifications. Cumulatively, this work refines the molecular framework of the NAB-mediated neuroprotection and provides a toolkit for targeting specific trafficking networks in PD.