Lee Michael A, Brown Joseph S, Farquhar Charlotte E, Loas Andrei, Pentelute Bradley L
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Sci Adv. 2025 Mar 21;11(12):eadr1018. doi: 10.1126/sciadv.adr1018. Epub 2025 Mar 19.
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 [; apparent dissociation constant () = 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 demonstrating improved affinity ( = 29 nanomolar). Overall, we expect that this work will improve the use of macrocyclic libraries in therapeutic peptide development.
尽管具有潜力，但用于配体发现和开发的大型合成大环文库的制备一直受到限制。在此，我们制备了包含天然和非天然氨基酸的亿级大环文库。通过在溶液中用碘快速氧化促进了近乎定量的分子内二硫键形成。在用于亲和选择后，用二硫苏糖醇处理可实现近乎定量的还原，生成用于标准串联质谱分析的线性肽类似物。我们使用这些文库发现了与钙黏蛋白-2和抗血凝素抗体克隆12ca5结合的大环化合物。对初始钙黏蛋白结合肽[；表观解离常数（）=53纳摩尔]的构效关系研究揭示了驱动亲和力的残基（热点）和耐受突变的残基（冷点）。制备了两个原始大环文库，其中这些热点和冷点用非天然氨基酸进行了衍生化。经过发现和验证，从冷点文库中发现了高亲和力配体，其显示出亲和力得到了改善（=29纳摩尔）。总体而言，我们预计这项工作将改进大环文库在治疗性肽开发中的应用。
