Exponential Combination of a and e/g Intracellular Peptide Libraries Identifies a Selective ATF3 Inhibitor
Exponential Combination of a and e/g Intracellular Peptide Libraries Identifies a Selective ATF3 Inhibitor
复制标题
DOI：
10.1021/acschembio.3c00779
复制
发表时间：
2024-02-27
影响因子：
4
通讯作者：
Mason，Jody M.
中科院分区：
文献类型：
--
作者：
Yu，Miao;Tang，T. M. Simon;Mason，Jody M.
激活转录因子3(ATF3)是一个激活转录因子/环磷酸腺苷(CAMP)反应元件结合蛋白(CREB)家族成员。它通过抑制干扰素-γ和趋化因子(C-C基序)配体4(CCl_4)等关键炎症因子的表达，被认为是癌症进展的重要调节因子。在这里，我们描述了一种新的文库筛选方法，该方法先探测单个亮氨酸拉链组件，然后将它们组合在一起，搜索通常不能用于细胞内筛选的指数级更大的序列空间。为此，我们采用了两种单独的半分子文库设计方法，并使用蛋白质片段互补分析(PCA)进行筛选。首先，一个248,832名成员的文库探索了所有五个位置上的12个氨基酸位置，以确定那些提供了更好结合的位置，所有的LE/G位置固定为Q，给所提供的文库选项施加了选择压力。接下来，一个由59,049名成员组成的图书馆用3个选项调查了所有Tene/g位置。类似地，在GING/g文库筛选期间，异位被锁定在一般可结合的序列模式(Aiaia)中，弱有利于亮氨酸拉链形成，同时将选择压力施加到所提供的TOE/g选项上。该文库代表ATF3W_AEG，与∼结合亲和力高(Tm=60°C；Kd=151 nm)，但强烈不利于均二聚。此外，ATF3W_AEG比成分PCA HITS有显著的改善，发现靶向特异性主要是由静电相互作用驱动的。组合EDA/e/G指数文库筛选方法为探索更大的多肽文库提供了一个强大的、加速的平台，从而衍生出有效但选择性强的拮抗剂，避免了同源关联，为合理的多肽设计提供了新的见解。
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 fiveapositions to identify those that provided improved binding, with alle/gpositions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all tene/gpositions with 3 options. Similarly, duringe/glibrary screening,apositions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure ontoe/goptions provided. The combineda/e/glibrary 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 combineda/e/gexponential 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.