Illuminating the "dark" kinases - effects on proteome translocation and chromatin binding

照亮“暗”激酶 - 对蛋白质组易位和染色质结合的影响

• 批准号: 10480189
• 负责人: Alexander Federation
• 金额: $ 33.39万
• 依托单位: TALUS BIOSCIENCE, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10480189
• 关键词: Acute Graft Versus Host Disease; Address; Affect; Amino Acid Sequence; Amino Acids; Binding; Biological Assay; Biology; Cell Aging; Cell Line; Cell Nucleus; Cell model; Cells; Chemicals; Chromatin; Client; Clinical; Clinical Trials; DNA Binding; DNA Repair; DNA sequencing; Data; Detection; Development; Differentiation and Growth; Disease; Disease model; Drug Targeting; Ensure; Environment; Euchromatin; FRAP1 gene; Family; Fibroblasts; Future; Gene Expression; Gene Proteins; Generations; Genes; Genome; Genomic DNA; Genomics; Goals; Heterochromatin; Human; Individual; Inflammation; Janus kinase; Link; Literature; MAP3K1 gene; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Methods; Modeling; Monitor; Myelofibrosis; Neuroblastoma; Nuclear; Nuclear Proteins; Nuclear Translocation; Oncogenic; Pathology; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phosphorylated Peptide; Phosphorylation Site; Phosphotransferases; Play; Polycythemia Vera; Population; Protein Analysis; Protein Kinase; Proteins; Proteome; Proteomics; Protocols documentation; Publishing; Refractory; Regulation; Role; Signal Pathway; Signal Transduction; Site; Small Business Innovation Research Grant; Sodium Chloride; Stat5 protein; Steroids; Stimulus; Structure; System; Talus; Technology; Testing; Therapeutic; Time; Transcription Factor Oncogene; Transcriptional Activation; United States National Institutes of Health; WI 38 cell; cellular targeting; combinatorial; drug development; drug discovery; effective therapy; experimental study; inhibitor; interest; jun Oncogene; kinase inhibitor; knock-down; member; nervous system disorder; new therapeutic target; novel; novel therapeutics; phosphoproteomics; programs; protein degradation; protein structure; response; screening services; small molecule; success; targeted cancer therapy; therapeutic target; tool; transcription factor

Technologies for DNA sequencing and protein analysis have accelerated so rapidly that generation of infor- mation now outpaces our ability to completely understand it. As a result, many novel genes and proteins have been discovered, but their functions remain unknown. The intent of the current proposal is to illuminate the function of one group of these uncharacterized (“dark”) proteins, the kinases, which are likely to have critical activities. Human cells have over 600 kinases, but the role played by a third of them is unknown. Kinases regulate all aspects of intracellular signaling, affecting everything from growth and differentiation to protein localization and DNA repair. One key activity of kinases is to activate or inhibit transcription factors (TFs), which regulate the activity of genes in the nucleus, thereby controlling the fate and activity of the cell. Many kinases therefore are targets of pharmaceutical drug development, particularly as cancer therapeutics, due to their role in regulating TFs and other key molecules. Talus has developed the ChESS-DIA technology to follow the localization of the entire population of proteins (the “proteome”) in the nucleus that are either unbound or bound to chromatin. In the current proposal, the ChESS-DIA method will be adapted to measure the effect of specific “dark” kinases on the nuclear and genomic localization of the proteome. In Aim 1 we will chemically deplete known kinases by targeted protein degradation and use ChESS-DIA to observe the effects on all TFs. The method will be optimized to ensure the results are consistent with known effects of these kinases on TFs. We will then use the optimized ChESS-DIA protocol to analyze the effect of dark kinases on TF localization. This information is critically valuable for developing novel drugs that target cellular activities of each affected TF. In Aim 2 will analyze the effects of dark kinases in more detail, by determining the exact amino acid sites on TFs that are recognized and modified (i.e., phosphorylated) by dark kinases. As in Aim 1, the ChESS-DIA technology will be tested and optimized first with known kinases, and the subsequently used to analyze understudied dark kinases. Drugs against kinases are relatively straightforward to develop, because the protein structure is well defined and accessible for interaction with small molecule drugs. In contrast, the structures of most TFs generally are difficult targets for drug development. The information obtained through this project will not only show which dark kinases are likely to be strong drug targets, but it will also be the first identification of the specific protein sequences on TFs that represent valuable drug targets. We anticipate partnering with academic and pharmaceutical partners to develop novel therapeutics to treat a variety of diseases for which there are no effective therapies.

DNA测序和蛋白质分析技术发展得如此之快，以至于信息的产生-- 现在，信息的速度已经超过了我们完全理解它的能力。因此，许多新的基因和蛋白质具有 已经被发现，但它们的功能仍不清楚。当前提案的目的是阐明 其中一组未特化的(“暗”)蛋白的功能，这类蛋白很可能具有关键的 活动。 人类细胞有600多个激酶，但其中三分之一的作用尚不清楚。蛋白水解酶调节一切 细胞内信号的各个方面，影响从生长和分化到蛋白质定位和 DNA修复。激酶的一个关键活性是激活或抑制转录因子(TF)，转录因子调节 细胞核中基因的活性，从而控制细胞的命运和活动。因此，许多激酶都是 药物开发的目标，特别是作为癌症治疗的目标，因为它们在调节 TFS和其他关键分子。 Talus已经开发了CHESS-DIA技术来跟踪整个蛋白质种群的定位 核内未与染色质结合或与染色质结合的蛋白质组。在目前的提案中， Chess-DIA方法将被用来测量特定的“暗”激酶对核和基因组的影响 蛋白质组的定位。在目标1中，我们将通过有针对性的蛋白质降解来化学耗尽已知的激酶 并用CHESS-DIA观察对所有TF的影响。该方法将进行优化，以确保结果 与已知的这些激酶对TF的影响一致。然后，我们将使用优化的CHESS-DIA协议来 分析暗蛋白激酶对转铁蛋白定位的影响。这些信息对小说创作具有重要的价值。 针对每个受影响的转铁蛋白的细胞活动的药物。在Aim 2中，我们将分析更多 细节，通过确定TF上被识别和修饰(即，磷酸化)的确切氨基酸位置 通过深色皮肤。正如在目标1中一样，CHESS-DIA技术将首先使用已知的激活器进行测试和优化， 以及随后用来分析未被充分研究的暗信号通路。 开发针对激酶的药物相对简单，因为蛋白质结构定义良好，而且 可与小分子药物相互作用。相比之下，大多数函数的结构一般都很困难 药物开发的目标。通过这个项目获得的信息不仅会显示哪些黑暗的激酶 很可能是强大的药物靶点，但它也将是第一次鉴定出特定的蛋白质序列上 代表有价值的药物靶点的TFs。我们期待着与学术和制药合作伙伴 开发新的治疗方法来治疗各种没有有效治疗方法的疾病。