NOVEL HIGH THROUGHPUT SCREEN TO IDENTIFY REGULATORS OF UBIQUITIN LIGASE ACTIVITY

Protein ubiquitination, a multi-step process utilizing a cascade of three enzyme classes (E1, E2 and E3), tightly regulates cellular protein turnover. It plays a key role in cellular proliferation and inflammation and is associated with a multitude of diseases, including cancer, metabolic disorders, and muscle and nerve degeneration.

Dr. Pan and colleagues developed a novel high throughput functional screen to identify small molecule inhibitors of the ubiquitin system. They reconstituted the enzymatic cascade in vitro, using E1, E2 and E3 together with fluorescently labeled ubiquitin (Ub). Formation of lysine 48 linked di-ubiquitin (diUb) chains is detected via fluorescence spectroscopy as a direct measure of enzymatic activity, confirmed by separation and visualization of diUb by conventional gel electrophoresis and imaging.

This spectroscopy based high throughput assay allows screening small molecule libraries for antagonists and agonists of E1, E2 and E3 activity. As a proof of principle, a Cdc34 E2 inhibitor CC0651 was shown to block FRET in a dose-dependent manner.

Current Development Status

• The reconstituted in vitro reaction is currently performed in a 384-well format. (Z=0.6)

Applications

• Screening for antagonists and agonists of E1, E2 and E3 activity
• Amendable for modification to screen for antagonists and agonists of regulators such as Nedd8
• Extended utility to screen for antagonists and agonists of the E3-substrate complexes
• Test ubiquitin conjugating and ligase activity of newly identified proteins
• Quantify E2 and E3 activity in real-time

Advantages

• Assay measures formation of di-ubiquitin chains in real-time
• Assay specifically measures lysine 48 linked ubiquitin chains, which regulate protein turnover in vivo
• Assay is amenable to 384-well or greater format (high throughput screening)

Publications

• Not available

Patent Status

• Not yet filed

Contact

Jeanne Farrell, PhD
Business Development Director
Mount Sinai Innovation Partners | Icahn School of Medicine at Mount Sinai
Phone: 646.605.7314