化学拓扑为制备具有天然结构和完整功能的治疗性蛋白质生物缀合物提供了独特的维度。然而拓扑的影响仍然难以捉摸。在这里,设计,合成,和三种拓扑结构中治疗性蛋白质生物缀合物的表征(即,tadpole,大环,和八字),已报告。选择干扰素α2b(IFN)和白蛋白结合结构域(ABD)作为用于生物缀合和概念验证的模型蛋白。这些拓扑同工型的生物合成通过使用SpyTag-SpyCatcher化学和/或分裂内含肽介导的连接在细胞中的直接表达来实现拓扑多样化。用LC-MS的组合技术证明了相应的拓扑结构,SDS-PAGE,和受控的蛋白水解消化。虽然这些拓扑同工型的性质在大多数情况下是相似的,八字生物结合物,f8-IFN-ABD,表现出最佳的热稳定性和抗聚集特性以及延长的半衰期和增强的肿瘤保留相对于t形对照,tadp-IFN-ABD,和大环控制,c-IFN-ABD,展示了相当大的拓扑效应。这项工作扩展了蛋白质的拓扑多样性,并证明了利用化学拓扑来实现蛋白质疗法中多功能整合之外的功能益处的潜在优势。
Chemical topology provides a unique dimension for making therapeutic protein bioconjugates with native structure and intact function, yet the effects of topology remain elusive. Herein, the design, synthesis, and characterization of therapeutic protein bioconjugates in three topologies (i.e., tadpole,
macrocycle, and figure-of-eight), are reported. The interferon α2b (IFN) and albumin binding domain (ABD) are selected as the model proteins for bioconjugation and proof-of-concept. The biosynthesis of these topological isoforms is accomplished via direct expression in cells using SpyTag-SpyCatcher chemistry and/or split-intein-mediated ligation for topology diversification. The corresponding topologies are proven with combined techniques of LC-MS, SDS-PAGE, and controlled proteolytic digestion. While the properties of these topological isoforms are similar in most cases, the figure-of-eight-shaped bioconjugate, f8-IFN-ABD, exhibits the best thermal stability and anti-aggregation properties along with prolonged half-life and enhanced tumor retention relative to the tadpole-shaped control, tadp-IFN-ABD, and the macrocyclic control, c-IFN-ABD, showcasing considerable topological effects. The work expands the topological diversity of proteins and demonstrates the potential advantages of leveraging chemical topology for functional benefits beyond multi-function integration in protein therapeutics.