Chemical Glycobiology Tool Development: LYTACs

项目摘要 这是R37 GM 058867的更新申请，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。在下一个赠款期，我们将集中精力， 用于靶向降解细胞外蛋白的化学生物学平台。靶向蛋白降解 诸如蛋白水解靶向嵌合体（PROTAC）的平台现在已经被公认为是 解决典型的“不可用”蛋白质。然而，规范的PROTAC方法涉及操纵 细胞溶质蛋白质降解机制，因此从根本上限于具有可配体的靶点。 胞质结构域。这一要求排除了大多数分泌的和细胞表面膜相关的蛋白质， 据估计，它包含了40%的蛋白质编码基因，是癌症、衰老相关疾病、 疾病和自身免疫性疾病。因此，最近人们对新方法的兴趣激增， 靶向降解细胞外蛋白，特别关注利用内体-溶酶体 通路本文提出的工作重点是我们认为是该领域的领先技术。 我们开发了“溶酶体靶向嵌合体”（LYTACs），其将感兴趣的蛋白质通过 阳离子非依赖性甘露糖-6-磷酸受体（CI-M6 PR）的结合。LYTAC包含 结合元件（例如，抗体或小分子配体），所述抗体或小分子配体对细胞外靶蛋白特异， 涉及接合CI-M6 PR的甘露糖-6-磷酸（M6 P）类似物。受体内源性转运 在N-聚糖残基上标记有M6 P帽的溶酶体酶通过循环到达其目的地细胞器 在核内体、细胞表面和高尔基复合体之间连续地存在。CI-M6 PR已被利用来 递送用于治疗溶酶体贮积症的治疗性酶。然而，在我们工作之前， 溶酶体递送系统尚未被考虑作为靶向降解的媒介物。 在初步工作中，我们使用生物正交化学方法来缀合结合蛋白质的配体或抗体， 对合成CI-M6 PR聚合物感兴趣。我们证明了可溶性细胞外蛋白和 膜结合的细胞表面蛋白可以被LYTAC靶向降解。这些初步研究 为扩展该计划奠定基础，使其包括对LYTAC范围和机制的基础研究 以及转化治疗应用。本项目的具体目标是（1）合成 均质LYTAC和优化结构，用于体外和体内应用，（2）表征 LYTAC可蛋白质组，以及（3）将LYTAC应用于涉及可溶性和细胞表面的治疗模型 膜结合靶点。

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.