Discovery of small molecule inhibitors of GalNAc-type O-linked glycosylation

许多细胞外和分泌的蛋白质在翻译后被多聚糖修饰， 包括N-乙酰半乳糖胺(GalNAc)-O型连接的多糖。这种常见的蛋白质形式 糖基化是通过将GalNAc加到丝氨酸或苏氨酸残基的氧原子上来启动的。 GalNAc-型O-连接糖基化在多种生物过程中发挥作用 包括粘蛋白组装、发育信号、人类遗传疾病、细胞-细胞黏附 事件和癌症进展。尽管扮演着至关重要的角色，但机械地理解 GalNAc-型O-连接糖基化的功能不完全，部分原因是不充分 可用于探测和控制这一修改的工具。为了迎接这一挑战，我们将携带 开展高通量筛选(HTS)活动，以发现抑制 多肽GalNAc转移酶(PpGalNAcT)家族，负责添加GalNAc 残基到蛋白质，启动GalNAc-类型O-连接的糖的生物合成。我们的主要HTS 检测是一种使用纯化的ppGalNAcT1酶和粘蛋白- 衍生的多肽作为糖基化底物。我们将筛选包含33万个化合物的收藏 来自德克萨斯大学西南高温超导核心设施，包括商业化合物和天然化合物 乘积分数。显示出对ppGalNActs的有效和剂量依赖的抑制的化合物 将在体外评估ppGalNAcT对细胞的抑制作用。我们还将使用平板化验 为了测试HIT分子是否抑制呼吸道上皮细胞的粘蛋白分泌，这是一个过程 这取决于GalNAc-型O-连接的糖链。以细胞为基础展示的候选抑制剂 将通过一系列旨在测试作用机制的分析来进一步分析活动 以及抑制的选择性。将进行结构-活性关系(SAR)分析 顶级化合物。少数高效的抑制剂将受到全分子的影响 表征包括动力学机制的分析、诱导转录的图谱 和糖基化变化，测定ppGalNAcT结合的亲和力，结构表征 抑制剂-ppGalNAcT复合体的作用，并进行了概念验证实验，测试了对 肺癌细胞的迁移行为。在授权期结束时，我们的目标是确定一个 或更多提供对ppGalNAcT家族的有效和选择性抑制的药效团 在体外和细胞内。除了用作学术研究的化学探针外，这些 化合物有可能成为新的治疗方法的先导分子。 治疗癌症，如胰腺癌和非小细胞肺癌，以及 呼吸道疾病，包括哮喘，以粘液过多为特征。

Many extracellular and secreted proteins are post-translationally modified with glycans, including N-acetylgalactosamine (GalNAc)-type O-linked glycans. This common form of protein glycosylation is initiated by addition of GalNAc to oxygen atoms of serine or threonine residues. GalNAc-type O-linked glycosylation plays functional roles in diverse biological processes including mucin assembly, developmental signaling, human genetic disorders, cell-cell adhesion events, and cancer progression. Despite playing essential roles, mechanistic understanding of the functions of GalNAc-type O-linked glycosylation is incomplete, due in part to the inadequacy of tools available to probe and control this modification. To meet this challenge, we will carry out a high-throughput screening (HTS) campaign to discover small molecules that inhibit the polypeptide GalNAc-transferase (ppGalNAcT) family of enzymes, responsible for adding GalNAc residues to proteins to initiate GalNAc-type O-linked glycan biosynthesis. Our primary HTS assay is a mass spectrometry-based assay using purified ppGalNAcT1 enzyme and a mucin- derived peptide as a glycosylation substrate. We will screen the 330,000-compound collection from the UT Southwestern HTS core facility, including commercial compounds and natural product fractions. Compounds that show potent and dose-dependent inhibition of ppGalNAcTs in vitro will be evaluated for ppGalNAcT inhibition in cells. We will also use a plate-based assay to test whether hit molecules inhibit mucin secretion from respiratory epithelial cells, a process that depends on GalNAc-type O-linked glycans. Candidate inhibitors that display cell-based activity will be further analyzed by a cascade of assays aimed at testing the mechanism of action and selectivity of inhibition. Structure-activity relationship (SAR) analysis will be performed on top compounds. A handful of high-performing inhibitors will be subjected to a full molecular characterization including analysis of kinetic mechanism, profiling of induced transcriptional and glycosylation changes, measuring affinity of ppGalNAcT binding, structural characterization of the inhibitor-ppGalNAcT complex, and a proof-of-concept experiment testing effects on the migratory behavior of lung cancer cells. At the end of the granting period, we aim to identify one or more pharmacophores that provide potent and selective inhibition of the ppGalNAcT family in vitro and in cells. In addition to their utility as chemical probes for academic research, these compounds may have the potential to serve as lead molecules for new therapeutic approaches to treat cancers, such as pancreatic cancer and non-small cell lung cancer (NSCLC), and respiratory disease, including asthma, that are characterized by mucus overproduction.