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

GalNAc 型 O-连接糖基化小分子抑制剂的发现

• 批准号: 9763582
• 负责人: Jennifer J Kohler
• 金额: $ 25.43万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9763582
• 关键词: Acetylgalactosamine; Affinity; Anabolism; Asthma; Behavior; Binding; Biochemical; Biological Assay; Biological Process; Biophysics; Cell-Cell Adhesion; Cells; Chemicals; Collaborations; Collection; Complex; Core Facility; Data; Development; Disease; Dose; Environment; Enzymes; Epithelial Cells; Event; Family; Fibroblast Growth Factor; Genetic Diseases; Genetic Transcription; Glycoconjugates; Glycolipids; Glycoproteins; Grant; Human Genetics; In Vitro; Integrins; Kinetics; Lead; Libraries; Link; Lung diseases; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Mammalian Cell; Mass Spectrum Analysis; Measures; Modification; Molecular; Monitor; Mucins; Mucous body substance; N-Acetylgalactosaminyltransferases; Natural Products; Neoplasm Metastasis; Non-Small-Cell Lung Carcinoma; Organism; Outcome; Oxygen; Pathology; Peptides; Pharmaceutical Preparations; Pharmacology; Play; Polysaccharides; Process; Production; Protein Biosynthesis; Protein Glycosylation; Proteins; Reaction; Research; Role; Serine; Signal Transduction; Structure; Structure-Activity Relationship; Testing; Threonine; Universities; Zavesca; base; cancer cell; drug discovery; enzyme substrate; experimental study; extracellular; glycosylation; glycosyltransferase; high throughput screening; in vitro activity; inhibitor/antagonist; innovation; novel therapeutic intervention; novel therapeutics; pharmacophore; polypeptide; pre-clinical; respiratory; small molecule; small molecule inhibitor; sugar nucleotide; therapy development; tool; tumor progression

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.

许多细胞外和分泌的蛋白质都用聚糖进行翻译后修饰， 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来启动糖基化。 GalNAc 型 O 连接糖基化在多种生物过程中发挥功能作用 包括粘蛋白组装、发育信号传导、人类遗传疾病、细胞间粘附 事件和癌症进展。尽管发挥着重要作用，但对 GalNAc 型 O 连接糖基化的功能不完整，部分原因是其不足 可用于探测和控制这种修改的工具。为了应对这一挑战，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) 酶家族，负责添加 GalNAc 将残基连接到蛋白质上以启动 GalNAc 型 O-连接聚糖生物合成。我们的主要 HTS 测定是一种基于质谱的测定，使用纯化的 ppGalNAcT1 酶和粘蛋白- 衍生肽作为糖基化底物。我们将筛选 330,000 个化合物集合 来自 UT 西南高温超导核心设施，包括商业化合物和天然化合物 产品分数。对 ppGalNAcT 表现出有效且剂量依赖性抑制的化合物 体外将评估细胞中 ppGalNAcT 抑制作用。我们还将使用基于平板的测定 测试命中分子是否抑制呼吸道上皮细胞粘蛋白分泌，这是一个过程 这取决于 GalNAc 型 O 连接聚糖。显示基于细胞的候选抑制剂 将通过旨在测试作用机制的一系列检测进一步分析活性 和抑制的选择性。结构-活性关系（SAR）分析将进行 顶级化合物。少数高性能抑制剂将接受完整的分子研究 表征，包括动力学机制分析、诱导转录分析 和糖基化变化，测量 ppGalNAcT 结合的亲和力，结构表征 抑制剂-ppGalNAcT 复合物的研究，以及概念验证实验测试对 肺癌细胞的迁移行为。在授予期结束时，我们的目标是确定一个 或更多药效团，对 ppGalNAcT 家族提供有效和选择性的抑制 体外和细胞内。除了用作学术研究的化学探针外，这些 化合物可能有潜力作为新治疗方法的先导分子 治疗癌症，例如胰腺癌和非小细胞肺癌（NSCLC），以及 呼吸道疾病，包括哮喘，其特征是粘液分泌过多。