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

ACS Chem Biol. 2024 Mar 15;19(3):753-762. doi: 10.1021/acschembio.3c00779. Epub 2024 Feb 27.

Abstract

Activating transcription factor 3 (ATF3) is an activation transcription factor/cyclic adenosine monophosphate (cAMP) responsive element-binding (CREB) protein family member. It is recognized as an important regulator of cancer progression by repressing expression of key inflammatory factors such as interferon-γ and chemokine (C-C motif) ligand 4 (CCL4). Here, we describe a novel library screening approach that probes individual leucine zipper components before combining them to search exponentially larger sequence spaces not normally accessible to intracellular screening. To do so, we employ two individual semirational library design approaches and screen using a protein-fragment complementation assay (PCA). First, a 248,832-member library explored 12 amino acid positions at all five a positions to identify those that provided improved binding, with all e/g positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten e/g positions with 3 options. Similarly, during e/g library screening, a positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto e/g options provided. The combined a/e/g library represents ∼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (Tm = 60 °C; Kd = 151 nM) while strongly disfavoring homodimerization. Moreover, ATF3W_aeg is notably improved over component PCA hits, with target specificity found to be driven predominantly by electrostatic interactions. The combined a/e/g exponential library screening approach provides a robust, accelerated platform for exploring larger peptide libraries, toward derivation of potent yet selective antagonists that avoid homoassociation to provide new insight into rational peptide design.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factor 3* / metabolism
  • Cyclic AMP Response Element-Binding Protein / chemistry
  • Cyclic AMP Response Element-Binding Protein / genetics
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Gene Expression Regulation
  • Peptide Library*
  • Peptides / metabolism

Substances

  • Peptide Library
  • Activating Transcription Factor 3
  • Cyclic AMP Response Element-Binding Protein
  • Peptides