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 TLK是一种对正常细胞至关重要的核丝氨酸-苏氨酸激酶，研究甚少 动物的分裂和总体生存能力。人类有两种TLK同工酶，TLK1和TLK2(TLK1/2)，它们是 密切相关的，被认为在基因组维护中有多余的作用。除了在以下方面担任关键角色外 正常的细胞生理，TLKs的失调也与人类疾病有关：TLK2 单倍体功能不全导致一种明显的神经发育障碍，TLK1/2上调驱动癌细胞 扩散。尽管TLKs在DNA合成过程中发挥了作用，但在DNA合成过程中，TLKs的其他关键功能 对细胞周期和对DNA损伤的反应知之甚少。更全面地了解 TLK1/2的功能受到以下因素的阻碍：1)缺乏有效和特异的小分子抑制剂 作为工具化合物允许在时间上控制激酶活性和2)体内直接的有限知识 激动酶的底物。该试验项目目的是通过解决X光来解决这些缺陷 TLK2-抑制剂和TLK2-底物络合物的晶体结构及其新底物的鉴定 通过不带偏见的屏幕。筛选一个有焦点的激酶抑制物库，我们已经鉴定出一组小分子 TLK2分子抑制剂。我们将优化已建立的生长条件，获得TLK2-抑制剂的晶体 并解决了它们的高分辨结构。了解选择性底物的结构基础 作为目标，我们将绘制TLK2与其特征最好的底物ASF1a之间的相互作用图，并解决 TLK2与合成的和ASF1a衍生的多肽底物的复合体的结构。我们将使用一种化学物质 用遗传学方法鉴定新的TLK2底物。对模拟敏感的TLK2等位基因将用于N6- 三磷酸腺苷--S的取代类似物在完整的细胞核中将其直接底物硫代磷酸化。胰酶 硫代磷酸化多肽将通过共价捕获和释放来分离，然后用LC-MS/MS鉴定 分析。这些研究将为未来研究新生的细胞功能奠定基础。 已确定的底物。并为结构导向的有效和特异的抑制剂的精制提供了基础。