Fan Shihui, Li Jie, Zhuang Jie, Zhou Qingtong, Mai Yiting, Lin Bingni, Wang Ming-Wei, Wu Chuanliu
The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.
Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
J Am Chem Soc. 2025 Feb 12;147(6):4821-4832. doi: 10.1021/jacs.4c12808. Epub 2024 Dec 17.
Many peptide hormones adopt long α-helical structures upon interacting with their cognate receptors but often exhibit flexible conformations when unbound. Strategies that can stabilize long α-helices without disrupting their binding to receptors are still lacking, which hinders progress in their biological applications and drug development. Here, we present an approach that combines rational design with library screening to create and identify a unique disulfide-directed multicyclic peptide (DDMP) scaffold, which could effectively stabilize N-terminally extendable α-helices while displaying exceptional efficiency in disulfide pairing and oxidative folding. This DDMP scaffold was then utilized for stabilizing the α-helical structure of glucagon-like peptide-1 (GLP-1), resulting in a potent GLP-1 receptor (GLP-1R) agonist with a significantly improved α-helicity and proteolytic stability. By incorporating external α-helices into the DDMP scaffold, we can effectively preserve the native N-terminal α-helical structures while allowing for extensive evolution of the C-terminal disulfide-rich domain for enhancing target binding, as demonstrated by the generation of the DDMP-stabilized GLP-1 (g1:Ox). The cryo-electron microscopy structure of the g1:Ox-GLP-1R in complex with heterotrimeric G reveals the molecular basis for the potent binding between g1:Ox and GLP-1R. Specifically, the DDMP moiety establishes additional interactions with the extracellular domain of GLP-1R, which are absent in the case of GLP-1. Thus, this work offers a novel and effective approach for engineering therapeutic peptides and other peptide α-helices, ensuring that both the N- and C-terminal regions remain essential for target recognition and activation.
许多肽激素在与它们的同源受体相互作用时会形成长的α-螺旋结构，但在未结合时通常表现出灵活的构象。目前仍缺乏能够稳定长α-螺旋而不破坏其与受体结合的策略，这阻碍了它们在生物学应用和药物开发方面的进展。在此，我们提出了一种将理性设计与文库筛选相结合的方法，以创建并鉴定一种独特的二硫键导向多环肽（DDMP）支架，该支架可以有效地稳定N端可延伸的α-螺旋，同时在二硫键配对和氧化折叠方面表现出卓越的效率。然后，这种DDMP支架被用于稳定胰高血糖素样肽-1（GLP-1）的α-螺旋结构，从而产生了一种强效的GLP-1受体（GLP-1R）激动剂，其α-螺旋度和蛋白水解稳定性得到了显著提高。通过将外部α-螺旋整合到DDMP支架中，我们可以有效地保留天然的N端α-螺旋结构，同时允许富含二硫键的C端结构域进行广泛的进化以增强与靶点的结合，如DDMP稳定的GLP-1（g1:Ox）的产生所证明的那样。g1:Ox与异源三聚体G复合物的冷冻电子显微镜结构揭示了g1:Ox与GLP-1R之间强效结合的分子基础。具体而言，DDMP部分与GLP-1R的胞外结构域建立了额外的相互作用，而在GLP-1的情况下则不存在这些相互作用。因此，这项工作为工程化治疗性肽和其他肽α-螺旋提供了一种新颖有效的方法，确保N端和C端区域对于靶点识别和激活仍然至关重要。
