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

许多细胞外和分泌的蛋白质是用聚糖后修饰的， 包括N-乙酰半乳糖胺（GalNAc）型O-连接聚糖。这种常见的蛋白质形式 通过将GalNAc添加到丝氨酸或苏氨酸残基的氧原子上来引发糖基化。 GalNAc型O-连接糖基化在多种生物学过程中起着重要作用 包括粘蛋白组装、发育信号传导、人类遗传疾病、细胞-细胞粘附 事件和癌症进展。尽管扮演着重要的角色， GalNAc型O-连接的糖基化的功能是不完整的，部分原因是 可以探测和控制这种变化的工具。为了迎接这一挑战，我们将 开展高通量筛选（HTS）活动，以发现抑制 多肽GalNAc-转移酶（ppGalNAcT）家族的酶，负责添加GalNAc 残基与蛋白质结合以启动GalNAc型0-连接聚糖生物合成。我们的主要HTS 测定是使用纯化的ppGalNAcT 1酶和粘蛋白的基于质谱的测定。 衍生肽作为糖基化底物。我们将筛选330，000个化合物集合 从UT西南HTS核心设施，包括商业化合物和天然 产物馏分。显示ppGalNAcTs的有效和剂量依赖性抑制的化合物 将在体外评价ppGalNAcT在细胞中的抑制。我们还将使用基于平板的测定法 为了测试hit分子是否抑制呼吸道上皮细胞的粘蛋白分泌， 这取决于GalNAc型O-连接聚糖。显示基于细胞的候选抑制剂 活性将通过旨在测试作用机制的级联测定法进一步分析 和抑制的选择性。将进行构效关系（SAR）分析， 顶级化合物。一些高性能的抑制剂将受到全分子 表征包括动力学机制分析、诱导转录的谱分析、 和糖基化变化，测量ppGalNAcT结合的亲和力，结构表征 和概念验证实验测试对细胞的影响。 肺癌细胞的迁移行为。在资助期结束时，我们的目标是确定一个 提供ppGalNAcT家族的有效和选择性抑制的药效团 在体外和细胞中。除了作为学术研究的化学探针， 化合物可能有潜力作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（NSCLC），和 呼吸道疾病，包括哮喘，其特征是粘液分泌过多。

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.