Development of D-box peptides to inhibit the anaphase-promoting complex/cyclosome

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• E3 ubiquitin ligases, like the anaphase-promoting complex/cyclosome (APC/C), are crucial for cell cycle regulation.
• Targeting the APC/C, particularly its activator Cdc20, is a promising strategy for anti-cancer therapies.
• Conventional inhibition methods for these large complexes are challenging, necessitating alternative approaches like peptide-based inhibitors.

Purpose of the Study:

• To design and characterize novel peptide inhibitors of Cdc20 that target the D-box motif.
• To evaluate the therapeutic potential of these peptides in inhibiting APC/C activity and inducing targeted protein degradation.
• To explore the structure-activity relationship of D-box peptides with Cdc20.

Main Methods:

• Rational design of peptides incorporating unnatural amino acids to enhance binding to Cdc20.
• Biophysical techniques including thermal-shift assays and surface plasmon resonance to confirm binding.
• Structural determination of Cdc20-peptide complexes.
• Cellular thermal shift assays (CETSA) to validate in-cell binding.
• In vitro ubiquitination assays to assess APC/C inhibition.
• Functional assays to evaluate targeted protein degradation.

Main Results:

• Developed D-box peptides that bind to Cdc20 with high affinity, confirmed by biophysical and cellular assays.
• Demonstrated that these peptides inhibit the ubiquitination activity of APC/C^Cdc20, outperforming the small-molecule inhibitor Apcin.
• Showcased the peptides' ability to function as portable degrons, driving the degradation of fused proteins.
• Observed that while inhibitory potency correlates with binding affinity, degradation efficacy is independent of binding strength, suggesting complex regulatory mechanisms.

Conclusions:

• Peptide inhibitors targeting the Cdc20 D-box motif represent a viable therapeutic strategy against APC/C-driven cancers.
• These peptides can be developed as potent inhibitors of APC/C activity.
• The study provides a foundation for utilizing these peptides for targeted protein degradation applications.
• Further research is warranted to optimize these peptides for clinical development and to fully elucidate the mechanisms of APC/C regulation.