Exponential Combination of a and e/g Intracellular Peptide Libraries Identifies a Selective ATF3 Inhibitor

Area of Science:

• Molecular Biology
• Protein Engineering
• Cancer Research

Background:

• Activating transcription factor 3 (ATF3) is a protein family member involved in regulating inflammatory responses.
• ATF3 plays a role in cancer progression by suppressing key inflammatory factors like interferon-γ and chemokine (C-C motif) ligand 4 (CCL4).
• Targeting ATF3 offers a potential therapeutic strategy for cancer treatment.

Purpose of the Study:

• To develop a novel library screening approach for identifying potent and selective peptide inhibitors.
• To explore large sequence spaces not typically accessible through standard intracellular screening methods.
• To derive peptide antagonists that specifically bind ATF3 and avoid self-association.

Main Methods:

• Employed a semirational library design approach combined with a protein-fragment complementation assay (PCA).
• Screened a 248,832-member library focusing on 12 amino acid positions within leucine zipper components.
• Screened a 59,049-member library probing 10 amino acid positions, followed by combining libraries to explore ~14.7 billion members.

Main Results:

• Identified a high-affinity peptide, ATF3W_aeg, that binds ATF3 with a dissociation constant (Kd) of 151 nM and a melting temperature (Tm) of 60 °C.
• ATF3W_aeg demonstrated strong disfavoring of homodimerization, indicating high specificity.
• Target specificity was predominantly driven by electrostatic interactions, offering insights into rational peptide design.

Conclusions:

• The combined exponential library screening approach is robust and accelerated for exploring vast peptide libraries.
• This method enables the derivation of potent, selective antagonists that avoid homoassociation.
• The findings provide new insights into rational peptide design for therapeutic applications, particularly in cancer.