Tools for the understudied kinase PNK3

用于研究中的激酶 PNK3 的工具

• 批准号: 9813666
• 负责人: Jarrod A. Marto
• 金额: $ 17.8万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813666
• 关键词: Affinity; Affinity Chromatography; Autoimmune Process; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; CRISPR/Cas technology; Cell Proliferation; Cell Survival; Cells; Clinical; Clinical Trials; Complement; Computer Simulation; Cysteine; Data; Development; Dose; Drug Targeting; Elements; Encephalopathies; Epitopes; Family; Funding; Future; Genes; Genetic; Genome; Growth Disorders; Hand; Head; Human; In Vitro; Information Resources Management; Kidney Failure; Libraries; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Methods; PC3 cell line; Peptides; Pharmaceutical Chemistry; Pharmacology; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Play; Positioning Attribute; Prostate; Prostatic Neoplasms; Protein Kinase; Proteins; Proteome; Proteomics; Publications; Publishing; Reagent; Reporting; Research Design; Research Personnel; Resources; Role; Scanning; Signal Pathway; Small Interfering RNA; Solid Neoplasm; Structure; Techniques; The Cancer Genome Atlas; Therapeutic; Time; United States National Institutes of Health; Work; analog; base; cancer therapy; cellular targeting; chemoproteomics; developmental disease; feeding; functional genomics; improved; inhibitor/antagonist; kinase inhibitor; knowledge base; malignant breast neoplasm; member; mutant; nervous system disorder; novel; outreach; overexpression; phosphoproteomics; response; small molecule; small molecule inhibitor; tool

ABSTRACT The NIH consortium focused on Illuminating the Druggable Genome (IDG) has identified 134 kinases (the `Dark Kinome') as members of a slightly larger group of biologically important proteins which are understudied. PKN3 (protein kinase N3) is an IDG-eligible member of the Dark Kinome. As of October 2018, Pharos lists only 23 publications for this kinase, and only a subset of these provide biochemical or functional data for PKN3 at the gene or protein level. Nonetheless, data from the Cancer Genome Atlas (TCGA) project and other func- tional studies highlight PKN3 as a potential drug target. There are currently no published, selective small mole- cule inhibitors for PKN3. In preliminary studies we developed and validated a first-in-class covalent inhibitor of PKN3. We went on to use this compound in elegant phosphoproteomic studies to identify potential PKN3 substrates. In parallel we used our well-established platform for tandem affinity purification followed by mass spectrometry (TAP-MS) to identify potential PKN3 protein binding partners. In this proposal we will extend these studies in order to pro- vide key biochemical characterization data for PKN3. Our study plan has two specific aims. Aim 1 is to interrogate signaling pathways relevant to PKN3, identify potential substrates and protein binding partners. We will use our well-established methods for phosphoproteomics along with our PKN3- focused reagents to identify PKN3-dependent signaling pathways as well as potential direct protein substrates. In parallel we will identify PKN3 protein binding partners by tandem affinity purification followed by mass spec- trometry (TAP-MS). Aim 2 is to utilize medicinal chemistry to optimize cellular potency and target selectivity of JZ128. We will diversify the structural elements of JZ128 to improve to create analogs with improved potency. Analogs will be screened in our in vitro PKN3 kinase assay, with the most promising candidates analyzed by CITe-Id to ver- ify selectivity for PKN3.

抽象的 专注于阐明可药物基因组 (IDG) 的 NIH 联盟已鉴定出 134 种激酶（ “暗激酶组”）作为一组稍大的生物学重要蛋白质的成员，该蛋白质组尚未得到充分研究。 PKN3（蛋白激酶 N3）是符合 IDG 资格的暗激酶组成员。截至 2018 年 10 月，Pharos 仅列出 有关该激酶的 23 篇出版物，其中只有一部分提供了 PKN3 的生化或功能数据，网址为 基因或蛋白质水平。尽管如此，来自癌症基因组图谱（TCGA）项目和其他功能的数据 一些研究强调 PKN3 是一个潜在的药物靶点。目前还没有发表的、选择性的小摩尔 PKN3 的 Cule 抑制剂。 在初步研究中，我们开发并验证了一种一流的 PKN3 共价抑制剂。我们继续 在优雅的磷酸化蛋白质组学研究中使用该化合物来识别潜在的 PKN3 底物。与此同时，我们 使用我们完善的串联亲和纯化平台和质谱 (TAP-MS) 鉴定潜在的 PKN3 蛋白结合伴侣。在本提案中，我们将扩展这些研究，以促进 请参阅 PKN3 的关键生化特征数据。我们的学习计划有两个具体目标。 目标 1 是询问与 PKN3 相关的信号通路，识别潜在的底物和蛋白质 具有约束力的伙伴。我们将使用我们成熟的磷酸蛋白质组学方法以及我们的 PKN3- 专注于鉴定 PKN3 依赖性信号传导途径以及潜在的直接蛋白质底物的试剂。 同时，我们将通过串联亲和纯化和质谱鉴定 PKN3 蛋白结合伴侣。 旋转测量法（TAP-MS）。 目标 2 是利用药物化学来优化 JZ128 的细胞效力和靶点选择性。我们 将使 JZ128 的结构元件多样化，以改进创造具有更高效力的类似物。类似物将 在我们的体外 PKN3 激酶测定中进行筛选，通过 CITe-Id 分析最有希望的候选者以验证 确定 PKN3 的选择性。