Michael A. Lee, Joseph S. Brown, Charlotte E. Farquhar, Andrei Loas, Bradley L. Pentelute
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引用次数: 0
摘要
尽管它们具有潜力,但用于配体发现和开发的大型合成大环文库的制备受到限制。在这里,我们生产了包含天然和非天然氨基酸的1亿元大环文库。溶液中碘的快速氧化促进了分子内近定量二硫化物的形成。在亲和选择中使用后,用二硫苏糖醇处理可以实现近定量还原,为标准串联质谱提供线性肽类似物。我们利用这些文库发现了cadherin-2和抗血凝素抗体克隆12ca5的大环结合物。初始钙粘蛋白结合肽[CBP]的构效关系研究表观解离常数(kd) = 53纳摩尔)揭示了驱动亲和力(热点)和耐突变残基(冷点)的残基。制备了两个原始的大环文库,其中这些热点和冷点由非天然氨基酸衍生。在发现和验证之后,从冷点文库中发现了高亲和力的配体,其中NCBP-4显示出更高的亲和力(K d = 29纳摩尔)。总的来说,我们期望这项工作将改善大环文库在治疗肽开发中的使用。
Affinity selection–mass spectrometry with linearizable macrocyclic peptide libraries
Despite their potential, the preparation of large synthetic macrocyclic libraries for ligand discovery and development has been limited. Here, we produce 100-million-membered macrocyclic libraries containing natural and nonnatural amino acids. Near-quantitative intramolecular disulfide formation is facilitated by rapid oxidation with iodine in solution. After use in affinity selection, treatment with dithiothreitol enables near-quantitative reduction, rendering linear peptide analogs for standard tandem mass spectrometry. We use these libraries to discover macrocyclic binders to cadherin-2 and anti-hemagglutinin antibody clone 12ca5. Structure-activity relationship studies of an initial cadherin-binding peptide [CBP; apparent dissociation constant (Kd) = 53 nanomolar] reveal residues responsible for driving affinity (hotspots) and mutation-tolerant residues (coldspots). Two original macrocyclic libraries are prepared in which these hotspots and coldspots are derivatized with nonnatural amino acids. Following discovery and validation, high-affinity ligands are discovered from the coldspot library, with NCBP-4 demonstrating improved affinity (Kd = 29 nanomolar). Overall, we expect that this work will improve the use of macrocyclic libraries in therapeutic peptide development.
期刊介绍:
Science Advances, an open-access journal by AAAS, publishes impactful research in diverse scientific areas. It aims for fair, fast, and expert peer review, providing freely accessible research to readers. Led by distinguished scientists, the journal supports AAAS's mission by extending Science magazine's capacity to identify and promote significant advances. Evolving digital publishing technologies play a crucial role in advancing AAAS's global mission for science communication and benefitting humankind.