Chemical Glycobiology Tool Development: LYTACs

项目总结 这是R37 GM058867的续订应用，它支持我们在化学领域的基础工作 自1999年以来，开发了糖生物学工具。在下一个授权期内，我们将集中力量制定新的 定向降解胞外蛋白的化学生物平台。蛋白质靶向降解 像蛋白水解靶向嵌合体(PROTAC)这样的平台现在已经被公认为是有效的策略 解决经典的“不能下药的”蛋白质。然而，规范的PROTAC方法涉及对 一种胞浆蛋白降解机制，因此从根本上仅限于具有可配基的靶标 胞液结构域。这一要求排除了大多数分泌和细胞表面膜相关蛋白， 据估计，它们包含40%的蛋白质编码基因，是与衰老相关的癌症的关键因素 疾病和自身免疫性疾病。因此，最近出现了对新方法的兴趣激增 有针对性地降解细胞外蛋白质，特别是利用内酶体-溶酶体 路径。这里提出的工作重点是我们认为是这一领域的领先技术。 我们开发了“溶酶体靶向嵌合体”(LYTAC)，通过 阳离子非依赖性甘露糖-6-磷酸受体(CI-M6PR)的结合。LYTAC包括一个 胞外靶蛋白特异性结合元件(如抗体或小分子配体)，共轭 涉及与CI-M6PR结合的甘露糖-6-磷酸(M6P)类似物。受体内源性转运 N-糖链残基上M6P标记的溶酶体酶循环至其目的细胞器 在内体、细胞表面和高尔基复合体之间持续存在。CI-M6PR已被利用来 提供治疗溶酶体储存障碍的治疗性酶。但是，在我们的工作之前，这个 溶酶体递送系统没有被认为是一种靶向降解的载体。 在初步工作中，我们使用生物正交化学方法来偶联与蛋白质结合的配体或抗体。 对合成CI-M6PR客户的兴趣。我们证明了可溶性胞外蛋白和 膜结合的细胞表面蛋白可以被LYTAC降解。这些初步研究 为计划的扩展做好准备，包括对LYTAC范围和机制的基础研究 以及转化性治疗应用。本项目的具体目标是：(1)综合 用于体外和体内应用的均质LYTAC和优化结构，(2)表征 以及(3)将LYTAC应用于涉及可溶性和细胞表面的治疗模型 膜结合靶标。

PROJECT SUMMARY This is a renewal application of R37 GM058867 which has supported our foundational efforts in chemical glycobiology tool development since 1999. In the next granting period we will focus our efforts on a new chemical biology platform for targeted degradation of extracellular proteins. Targeted protein degradation platforms such as proteolysis targeting chimeras (PROTACs) are now well-established as powerful strategies to address canonically “undruggable” proteins. However, canonical PROTAC approaches involve manipulation of a cytosolic protein degradation machinery and therefore are fundamentally limited to targets with ligandable cytosolic domains. This requirement excludes most secreted and cell-surface membrane-associated proteins, which are estimated to comprise 40% of protein-encoding genes and are key agents in cancer, aging-related diseases, and autoimmune disorders. Thus, there has been a recent surge of interest in new approaches for targeted degradation of extracellular proteins, with a particular focus on harnessing the endosome-lysosome pathway. The work proposed herein focuses on what we believe to be a leading technology in this space. We developed “lysosome targeting chimeras” (LYTACs) that direct proteins of interest to lysosomes via engagement of the cation-independent mannose-6-phosphate receptor (CI-M6PR). LYTACs comprise a binding element (e.g., an antibody or small molecule ligand) specific to the extracellular target protein, conjugated to mannose-6-phosphate (M6P) analogs that engage CI-M6PR. The receptor endogenously transports lysosomal enzymes marked with M6P caps on N-glycans residues to their destination organelle by cycling continuously between endosomes, the cell surface, and the Golgi complex. CI-M6PR has been exploited to deliver therapeutic enzymes for treatment of lysosomal storage disorders. However, prior to our work, this lysosome delivery system had not been contemplated as a vehicle for targeted degradation. In preliminary work we used bioorthogonal chemistries to conjugate ligands or antibodies that bind a protein of interest to synthetic CI-M6PR engagers. We demonstrated that both soluble extracellular proteins and membrane-bound cell-surface proteins can be targeted for degradation by LYTACs. These preliminary studies set the stage for expansion of the program to include fundamental studies of LYTAC scope and mechanism as well as translational therapeutic applications. The Specific Aims of this project are to (1) synthesize homogeneous LYTACs and optimize structures for in vitro and in vivo applications, (2) characterize the LYTACable proteome, and (3) apply LYTACs in therapeutic models that involve soluble and cell-surface membrane-bound targets.