Small molecules targeting the NEDD8·NAE protein-protein interaction.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Ubiquitination regulates protein homeostasis, with some pathways modulated by the NEDDylation cascade.
• The NEDDylation cascade involves a critical protein-protein interaction (PPI) between NEDD8 and its activating enzyme, NAE.
• Current therapeutic strategies target NAE's ATP-binding site, but disrupting the NEDD8·NAE PPI presents a novel inhibition approach.

Purpose of the Study:

• To evaluate chemotype designs for their potential to disrupt the NEDD8·NAE PPI using the Exploring Key Orientations (EKO) method.
• To synthesize and test a library of compounds targeting the NEDD8·NAE interaction.
• To identify a first-in-class inhibitor of the NEDD8·NAE PPI.

Main Methods:

• Utilized Exploring Key Orientations (EKO) to assess chemotype designs for PPI disruption.
• Performed solid-phase synthesis to create a targeted library of 24 compounds.
• Employed fluorescence polarization, cell-based immunoblotting, and cytotoxicity assays to evaluate compound activity.

Main Results:

• Identified a hit compound with a K_i of 6.4 ± 0.3 μM for NAE binding, inhibiting NEDDylation.
• Demonstrated suppression of E1-3 complex formation and induced cytotoxicity in K562 leukemia cells via apoptosis.
• Observed accumulation of NEDD8 in cells, indicating downstream pathway inhibition.

Conclusions:

• A novel chemotype was identified that disrupts the NEDD8·NAE PPI, representing a first-in-class inhibitor for NEDDylation.
• The EKO method proved effective in evaluating user-defined chemotypes for PPI inhibitor discovery.
• This study provides a new avenue for targeting protein homeostasis dysregulation.