Interaction of substrates and inhibitors with tousled-like kinase 2

底物和抑制剂与蓬乱样激酶 2 的相互作用

• 批准号: 9813105
• 负责人: DAVID A CALDERWOOD
• 金额: $ 16.75万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2020-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813105
• 关键词: ASF1A gene; ASF1B gene; Acute; Address; Alleles; Animals; Automobile Driving; Binding; Biological Assay; Cell Cycle; Cell Nucleus; Cell Proliferation; Cell division; Cell physiology; Cells; Chemicals; Chromosome Segregation; Clinical; Complex; Crystallization; DNA Damage; DNA biosynthesis; Defect; Development; Down-Regulation; Enzymes; Funding Opportunities; Future; Gatekeeping; Genome; Goals; Growth; Histones; Human; Immune checkpoint inhibitor; Impairment; Investigation; Isoenzymes; Knowledge; Lead; Libraries; Maintenance; Malignant Neoplasms; Maps; Mitosis; Molecular Chaperones; Mutate; Neurodevelopmental Disorder; Normal Cell; Nuclear; Nucleosomes; Peptides; Phase; Phenotype; Phosphotransferases; Physiological Processes; Pilot Projects; Production; Protein-Serine-Threonine Kinases; Proteins; RNA Interference; Reporting; Research; Resolution; Roentgen Rays; Role; S Phase; Structure; Substrate Interaction; Up-Regulation; analog; anti-cancer therapeutic; cancer cell; chemical genetics; clinically relevant; design; genetic approach; high throughput screening; human disease; in vivo; inhibitor/antagonist; kinase inhibitor; knockout gene; loss of function; neoplastic cell; novel; outcome forecast; response; screening; small molecule; small molecule inhibitor; tool; tumor

ABSTRACT Tousled-like kinases (TLKs) are poorly studied nuclear serine-threonine kinases essential to proper cell division and overall viability in animals. Humans have two TLK isozymes, TLK1 and TLK2 (TLK1/2), that are closely related and thought to have redundant roles in genome maintenance. In addition to having key roles in normal cell physiology, dysregulation of TLKs has also been implicated in human disease: TLK2 haploinsufficiency causes a distinct neurodevelopmental disorder, and TLK1/2 upregulation drives cancer cell proliferation. Though TLKs have ascribed roles during DNA synthesis, other critical functions of the kinases in the cell cycle and in responses to DNA damage are poorly understood. A more complete understanding of TLK1/2 function has been hampered by 1) an absence of potent and specific small molecule inhibitors for use as tool compounds to allow temporal control of kinase activity and 2) limited knowledge of direct in vivo substrates of the kinases. The aim of this pilot project is to address these deficiencies through solution of X-ray crystal structures of TLK2-inhibitor and TLK2-substrate complexes, and by identification of new TLK substrates through unbiased screens. Screening a focused kinase inhibitor library, we have identified a set of small molecule TLK2 inhibitors. We will optimize established conditions for growing obtain crystals of TLK2-inhibitor complexes, and solve their high resolution structures. To understand the structural basis for selective substrate targeting, we will map interactions between TLK2 and its best-characterized substrate, ASF1a, and solve structures of TLK2 in complex with synthetic and ASF1a-derivied peptide substrates. We will use a chemical genetic approach to identify new TLK2 substrates. An analog-sensitive TLK2 allele will be used with N6- substituted analogs of ATP--S to thiophosphorylate its direct substrates in intact cell nuclei. Tryptic thiophosphorylated peptides will be isolated by covalent capture and release, and then identified by LC-MS/MS analysis. These studies will set the stage for future studies investigations of the cellular function of newly identified substrates. and provide a basis for structure-guided elaboration of potent and specific inhibitors.

摘要 Tousled-like kinases（TLKs）是一种研究较少的丝氨酸-苏氨酸蛋白激酶， 在动物中的分裂和整体生存能力。人类有两种TLK同工酶，TLK 1和TLK 2（TLK 1/2）， 密切相关并被认为在基因组维护中具有冗余作用。除了在以下方面发挥关键作用外， 正常细胞生理学，TLKs的失调也与人类疾病有关：TLK 2 单倍不足导致一种独特的神经发育障碍，TLK 1/2上调驱动癌细胞 增殖尽管TLKs在DNA合成中具有特定的作用，但在DNA合成中激酶的其他关键功能也是如此。 细胞周期和对DNA损伤的反应知之甚少。更完整地理解 TLK 1/2功能受到以下因素的阻碍：1）缺乏有效和特异性的小分子抑制剂 作为工具化合物，以允许激酶活性的时间控制，和2）有限的知识，直接在体内 激酶的底物。该试点项目的目的是通过解决X射线 TLK 2-抑制剂和TLK 2-底物复合物的晶体结构，并通过鉴定新的TLK底物 通过无偏见的屏幕。筛选了一个集中的激酶抑制剂库，我们已经确定了一组小的 分子TLK 2抑制剂。我们将优化已建立的生长条件，以获得TLK 2抑制剂晶体。 配合物，并解决其高分辨率结构。了解选择性底物的结构基础 靶向，我们将映射TLK 2及其最佳表征底物ASF 1a之间的相互作用，并解决 与合成的和ASF 1a衍生的肽底物复合的TLK 2的结构。我们会用一种化学物质 通过遗传学方法鉴定新的TLK 2底物。类似物敏感性TLK 2等位基因将与N6- 在完整的细胞核中，ATP-β-S的取代类似物硫代磷酸化其直接底物。胰蛋白 硫代磷酸化肽将通过共价捕获和释放分离，然后通过LC-MS/MS鉴定 分析.这些研究将为未来研究新的细胞功能奠定基础。 识别的底物。并为有效和特异性抑制剂的结构指导的制备提供基础。