Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• The ubiquitin-proteasome system (UPS) is crucial for protein homeostasis and cell survival.
• Inhibition of the UPS induces proteotoxic stress, a validated strategy for cancer treatment.
• Current UPS-targeting drugs face resistance due to mutations in binding sites.

Purpose of the Study:

• To identify and validate alternative druggable sites within the UPS.
• To develop a novel therapeutic strategy targeting the E1 enzyme of the UPS.
• To overcome resistance mechanisms associated with existing UPS inhibitors.

Main Methods:

• Identification of an interaction site between E2 and E1 enzymes.
• Design and synthesis of stapled peptides mimicking the E2 alpha-1 helix.
• In vitro assays to assess E1 inhibition and disruption of ubiquitination.

Main Results:

• Stapled peptides successfully bound to a pocket in the E1 ubiquitin-fold domain.
• Peptide binding induced conformational changes in E1, inhibiting ubiquitin thiotransfer.
• This disruption effectively blocked E2 ubiquitin charging and cellular ubiquitination.

Conclusions:

• A novel E1-targeting strategy using stapled peptides was developed.
• This approach offers a distinct mechanism to inhibit the UPS, bypassing existing resistance pathways.
• This study provides a blueprint for developing new anti-cancer therapeutics targeting the UPS.