Bypassing the Native Ubiquitination Cascade to Develop Parkin Inhibitors and Activators

  • Rice, Sarah E (PD/PI)
  • Statsyuk, Alexander Vladimirovich (Co-PD/PI)

Project: Research project

Project Details

Description

PROJECT SUMMARY: Exciting recent studies suggest that activating the E3 ubiquitin ligase Parkin may be a viable therapeutic target for Parkinson’s disease (PD)xx. Accordingly, the MJFF currently funds several projects to identify Parkin activatorsxx. Parkin inhibitors could also be valuable tools to identify proteins that antagonize Parkin activity, which themselves may be PD drug targets. A significant hurdle for identifying small molecules that alter Parkin activity is the intrinsic complexity of reconstituted native cascade ubiquitination reactions. Screening efficiency is low because small molecules must be screened against a complex native cascade reaction containing at least 5 separate components (E1, E2, and Parkin ligases, ubiquitin and ATP) in the hope of finding a selective hit for Parkin. Our laboratory has recently developed a new method to overcome this challenge. We developed a modified ubiquitin thioester probe that bypasses the need for E1, E2 and ATP in an in vitro ubiquitination reactionXX. The resulting two-component reaction recapitulates phosphorylation-dependent Parkin activation and ubiquitination of its physiological substrate Miro1. We plan to adapt this two-component reaction to create a simplified high-throughput screening (HTS) protocol for identification of small molecule Parkin activators and inhibitors. To this end, we have developed a ubiquitin C-terminal fluorescein thioester (UbFlu). UbFlu bypasses E1, E2, and ATP and releases fluorescein upon transthiolation, providing a real-time fluorescence polarization (FP) readout of Parkin activity that is amenable to HTS. Our first project goal is to verify that our simplified UbMES/UbFlu assays recapitulate the native cascade and that UbFlu accurately reports steady-state Parkin activity in real time. Our second goal is to optimize HTS using UbFlu and screen 50,000 compounds to identify Parkin activators and inhibitors. This effort will dovetail nicely with existing work by MJFF researchers to identify Parkin activators, as our new method simplifies HTS screening and validation of hit compounds.
StatusFinished
Effective start/end date3/25/163/24/17

Funding

  • The Michael J Fox Foundation for Parkinson's Research (11241)

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