BACKGROUND
Targeted protein degradation (TPD) is an alternative approach to conventional paradigms (e.g. small molecule inhibitors) that target disease. In this technique, a PROteolysis TArgeting Chimera (PROTAC) or a molecular glue is used to initiate the degradation process of the targeted protein. While these two molecular classes are mechanistically distinct, they both enable degradation by recruiting an E3 ligase, an enzyme which enables the transfer of the ubiquitin tag needed for proteasome-mediated degradation. One obstacle to the development of these treatment modalities is that the two E3 ligases largely used with this approach only work with some proteins. Thus, identification of new E3 ligases can increase the disease targeting space of targeted protein degradation therapeutics.
TECHNOLOGY
Researchers at the University of Toronto have identified a collection of human proteins that can act as degraders, or even stabilizers, of target proteins involved in disease. They used a genome-wide screen (Figure 1) to evaluate 19,000 open reading frames (ORFs) for their potency and potential as targets for PROTACs or molecular glues. Several known E3 ligases were identified thus validating the screen, while several new and promising candidates were also discovered.
Figure 1. Targeted protein degradation and stabilization screen. Binding of the EGFP antibody (vhhGFP) to the EGFP fusion, enables proximity-dependent ubiquitination by the ORF enzyme that results in degradation of the EGFP fusion and a decrease in green fluorescence. Alternatively, if the ORF protein is a stabilizer, green fluorescence increase. The ORF region was varied between 19,000 different open reading frames.
COMPETITIVE ADVANTAGE
- Significantly more potent degraders than current gold standards (CRBN, VHL) were identified
- e.g. Candidate #1 ~ 85% degradation, while CRBN ~ 20% degradation
- Insensitivity to induced complex geometry (i.e. C- vs N-terminal attachment)
- Potency demonstrated across:
- Multiple targets
- Cell compartments
APPLICATIONS
- Targeted protein degradation or stabilization
INTELLECTUAL PROPERTY STATUS
- Provisional patent filed (April, 2022)
PROJECT STATUS
Proof-of-concept experiment have been conducted. A screen of 19,000 unique open reading frames was conducted to identify protein degraders and stabilizers. Cell compartment specific potency was analyzed for several top candidates, along with the orientational effect on efficacy of the point-of-attachment (N- vs C-terminal). Finally, results were benchmarked to the two commonly used E3 ligases, CRBN and VHL.
