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.

抽象的 蓬乱样激酶 (TLK) 是一种对正常细胞至关重要的核丝氨酸-苏氨酸激酶，研究很少 动物的分裂和整体生存能力。人类有两种 TLK 同工酶，TLK1 和 TLK2 (TLK1/2)，即 密切相关并被认为在基因组维护中具有冗余作用。除了在以下领域发挥关键作用之外 正常细胞生理学，TLK 失调也与人类疾病有关：TLK2 单倍体不足会导致明显的神经发育障碍，TLK1/2 上调会驱动癌细胞 增殖。尽管 TLK 在 DNA 合成过程中具有特定的作用，但激酶的其他关键功能 人们对细胞周期和对 DNA 损伤的反应知之甚少。更全面的了解 TLK1/2 功能受到以下因素的阻碍：1) 缺乏有效且特异性的小分子抑制剂 作为工具化合物，可以暂时控制激酶活性，2）直接体内知识有限 激酶的底物。该试点项目的目的是通过 X 射线解决方案来解决这些缺陷 TLK2 抑制剂和 TLK2 底物复合物的晶体结构，以及新 TLK 底物的鉴定 通过公正的屏幕。通过筛选重点激酶抑制剂库，我们确定了一组小型激酶抑制剂 分子TLK2抑制剂。我们将优化生长 TLK2 抑制剂晶体的既定条件 配合物，并解析其高分辨率结构。了解选择性底物的结构基础 靶向，我们将绘制 TLK2 与其最佳特征底物 ASF1a 之间的相互作用，并解决 TLK2 与合成和 ASF1a 衍生的肽底物复合物的结构。我们将使用一种化学品 遗传方法来识别新的 TLK2 底物。模拟敏感的 TLK2 等位基因将与 N6-一起使用 ATP--S 的替代类似物可在完整细胞核中硫代磷酸化其直接底物。胰蛋白酶 通过共价捕获和释放来分离硫代磷酸化肽，然后通过 LC-MS/MS 进行鉴定 分析。这些研究将为未来研究新的细胞功能奠定基础。 确定的底物。并为结构指导的有效和特异性抑制剂的阐述提供基础。