Miao Yu, T.M. Simon Tang, Lila Ghamsari, Graham Yuen, Claudio Scuoppo, Jim A. Rotolo, Barry J. Kappel and Jody M. Mason*,
{"title":"Exponential Combination of a and e/g Intracellular Peptide Libraries Identifies a Selective ATF3 Inhibitor","authors":"Miao Yu, T.M. Simon Tang, Lila Ghamsari, Graham Yuen, Claudio Scuoppo, Jim A. Rotolo, Barry J. Kappel and Jody M. Mason*, ","doi":"10.1021/acschembio.3c00779","DOIUrl":null,"url":null,"abstract":"<p >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 <i>a</i> positions to identify those that provided improved binding, with all <i>e/g</i> positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten <i>e/g</i> positions with 3 options. Similarly, during <i>e/g</i> library screening, <i>a</i> positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto <i>e/g</i> options provided. The combined <i>a/e/g</i> library represents ∼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (<i>T</i><sub>m</sub> = 60 °C; <i>K</i><sub>d</sub> = 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 <i>a/e/g</i> 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.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acschembio.3c00779","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Chemical Biology","FirstCategoryId":"99","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acschembio.3c00779","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍:
ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology.
The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies.
We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.