Chemoselective Staudinger-induced Michael-additions to antibodies to analyze protein homeostasis in C. elegans

在这项建议中，一种新的无金属化学选择性偶联策略将被应用于修饰抗体，以分析在维持蛋白质稳态中关键相关的蛋白质-蛋白质相互作用。在这项联合工作中，Hackenberger实验室在生物正交反应工程方面的专业知识和Kirstein实验室在蛋白质动态平衡方面的专业知识将被结合起来，以识别参与蛋白质解聚的新的伴侣复合体及其在线虫中的蛋白质底物。此外，我们的目标是在体内使用抗体药物结合物作为淀粉样蛋白形成的靶向修饰剂来形成Abeta形成的纤维。随着Staudinger诱导的Michael加成反应用于抗体中半胱氨酸残基的化学选择性结合的发展，蛋白质修饰的新概念将被引入。这一新技术使两个复杂的功能构建块能够模块化和无金属耦合，而不需要中间保护基团的操作。此外，通过这种方法引入了亚膦酰胺基团，它允许在蛋白质偶合物中直接结合光或酶可裂解的键。这种新的合成工具将被用于获得用于识别未知蛋白质底物以及特定伴侣的相互作用伙伴的功能性抗体，并将进一步用于设计可光切割的抗体药物结合物，以靶向表达Abeta1-42肽的阿尔茨海默病活体动物模型中的淀粉样纤维。

In this proposal a novel metal-free chemoselective conjugation strategy will be applied to modify antibodies for the analysis of protein-protein interactions of key relevance in the maintenance of protein homeostasis. In this joint effort the expertise of the Hackenberger laboratory in the engineering of bioorthogonal reactions and the Kirstein laboratory in protein homeostasis will be combined to identify novel chaperone complexes involved in protein disaggregation and their protein substrates in C. elegans. In addition, we aim to employ antibody drug conjugates to target modifiers of amyloid formation to Abeta formed fibrils in vivo. With the development of a Staudinger-induced Michael-addition for the chemoselective conjugation of cysteine residues in antibodies a new concept for the modification of proteins will be introduced. This novel technique enables the modular and metal-free coupling of two complex functional building blocks without the need of intermediate protecting group manipulations. Additionally, a phosphonamidate moiety is introduced by this method, which allows the direct incorporation of a light- or enzymatically cleavable bond in protein conjugates. This new synthetic tool will be applied to obtain functional antibodies for the identification of unknown protein substrates as well as interaction partners of specific chaperones and will furthermore be used in the design of light-cleavable antibody drug conjugates to target amyloid fibrils in living animal models for Alzheimer's disease that express Abeta1-42 peptides.