Yan Kejia, Miskolzie Mark, Banales Mejia Fernando, Peng Chuanhao, Ekanayake Arunika I, Atrazhev Alexey, Cao Jessica, Maly Dustin J, Derda Ratmir
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
Graduate Program in Biological Physics, Structure and Design, University of Washington, Seattle, Washington 98195, United States.
J Am Chem Soc. 2025 Jan 8;147(1):789-800. doi: 10.1021/jacs.4c13561. Epub 2024 Dec 19.
Genetically encoded libraries (GEL) are increasingly being used for the discovery of ligands for "undruggable" targets that cannot be addressed with small molecules. Foundational GEL platforms like phage-, yeast-, ribosome-, and mRNA-display have enabled the display of libraries composed of 20 natural amino acids (20AA). Unnatural amino acids (UAA) and chemical post-translational modification (cPTM) expanded GEL beyond the 20AA space to yield unnatural linear, cyclic, and bicyclic peptides. The standard operating procedure incorporates UAA and cPTM into a "naive" library with 10-10 compounds and uses a chemically upgraded library in multiple rounds of selection to discover target-binding hits. However, such an approach uses zero knowledge of natural peptide-receptor interactions that might have been discovered in selections performed with 20AA libraries. There is currently no consensus regarding whether "zero-knowledge" naive libraries or libraries with pre-existing knowledge can offer a more effective path to discovery of molecular interactions. In this manuscript, we evaluated the feasibility of discovery of macrocyclic and bicyclic peptides from "nonzero-knowledge" libraries. We approach this problem by late-stage chemical reshaping of a preselected phage-displayed landscape of 20AA binders to NS3aH1 protease. The reshaping is performed using a novel multifunctional C-symmetric linchpin, 3,5-bis(bromomethyl)benzaldehyde (termed KYL), that combines two electrophiles that react with thiols and an aldehyde group that reacts with N-terminal amine. KYL diversified phage-displayed peptides into bicyclic architectures and delineated 2 distinct sequence populations: (i) peptides with the HXDMT motif that retained binding upon bicyclization and (ii) peptides without the HXDMT motif that lost binding once chemically modified. The same HXDMT family can be found in traditional selections starting from the naive KYL-modified library. Our report provides a case study for discovering advanced, chemically upgraded macrocycles and bicycles from libraries with pre-existing knowledge. The results imply that other selection campaigns completed in 20AA space, potentially, can serve for late-stage reshaping and as a starting point for the discovery of advanced peptide-derived ligands.
基因编码文库（GEL）越来越多地用于发现小分子无法作用的“不可成药”靶点的配体。噬菌体展示、酵母展示、核糖体展示和mRNA展示等基础GEL平台能够展示由20种天然氨基酸（20AA）组成的文库。非天然氨基酸（UAA）和化学翻译后修饰（cPTM）将GEL扩展到20AA空间之外，以产生非天然的线性、环状和双环肽。标准操作程序将UAA和cPTM纳入一个含有10-10种化合物的“原始”文库，并在多轮筛选中使用化学升级文库来发现与靶点结合的命中物。然而，这种方法没有利用在使用20AA文库进行的筛选中可能发现的天然肽-受体相互作用的任何知识。目前对于“零知识”原始文库或具有先验知识的文库是否能提供更有效的分子相互作用发现途径尚无共识。在本手稿中，我们评估了从“非零知识”文库中发现大环和双环肽的可行性。我们通过对预先选择的展示20AA与NS3aH1蛋白酶结合物的噬菌体展示景观进行后期化学重塑来解决这个问题。重塑过程使用一种新型多功能C对称关键分子3,5-双（溴甲基）苯甲醛（称为KYL），它结合了两个与硫醇反应的亲电试剂和一个与N端胺反应的醛基。KYL将噬菌体展示的肽多样化成双环结构，并确定了2个不同的序列群体：（i）具有HXDMT基序的肽，在双环化后保留结合能力；（ii）没有HXDMT基序的肽，一旦化学修饰就失去结合能力。在从原始KYL修饰文库开始的传统筛选中也能发现相同的HXDMT家族。我们的报告提供了一个从具有先验知识的文库中发现先进的、化学升级的大环和双环化合物的案例研究。结果表明，在20AA空间中完成的其他筛选活动可能可用于后期重塑，并作为发现先进的肽衍生配体的起点。
