Harnessing the In Vitro Selection for Activity-based Proteomics and Chemical Probe Development

利用体外选择进行基于活性的蛋白质组学和化学探针开发

• 批准号: 10239098
• 负责人: Casey John Krusemark
• 金额: $ 37.61万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10239098
• 关键词: Address; Amino Acid Motifs; Area; Binding Sites; Biological; Biology; Biomedical Research; Cell physiology; Chemicals; DNA; DNA Sequence; DNA analysis; DNA sequencing; Detection; Development; Diagnosis; Disease; Enzymes; Epithelial; Family; Genetic Techniques; Genomics; Histones; In Vitro; Link; Mediating; Mesenchymal; Nature; Peptides; Pharmaceutical Chemistry; Phosphotransferases; Population; Process; Protein Kinase; Proteins; Proteome; Proteomics; Research; Research Personnel; Resistance development; Role; Serine Hydrolase; Site; System; Techniques; Technology; Transcriptional Regulation; base; design; enzyme activity; genetic analysis; inhibitor/antagonist; kinase inhibitor; lapatinib; multiplex assay; novel strategies; novel therapeutics; programs; protein protein interaction; small molecule libraries; src-Family Kinases; tool; trimethyllysine

Project Summary Developments in genetic analysis technologies, particularly DNA sequencing, have been transformative to biomedical research. In contrast to genomic information, the barrier to accessing proteomic information, particularly enzyme activity, is dramatically higher. As aberrant enzyme activities are consistently observed in disease, this information is critical for appropriate diagnosis and treatment. In addition, the ability to probe the function of enzyme activities using chemical inhibitors is critical to our understanding of biology and disease. DNA-encoded chemical libraries (DELs) have emerged as a new tool that enables medicinal chemists to capitalize on the power of genetic techniques. The in vitro selection is the key process by which the function of the synthetic molecules in a DEL are encoded with DNA sequence populations. Inspired by this transduction of functional information of abiotic molecules, this project seeks to further exploit these capabilities into new areas. Our research program centers on advancing DNA-encoded chemical approaches for the development of chemical probes and for proteome-wide enzyme activity detection. This involves developing new approaches for the synthesis and selection of DNA-encoded chemical libraries, as well as the implementation of a new, related technology for DNA-based detection of enzyme activities using the in vitro selection of DNA-encoded proteomic probes. For chemical probe development, we have chosen two target classes that involve protein:protein interactions mediated through a short linear motifs: protein kinases and histone targeting domains. We will design and build DELs that target the protein substrate site of protein kinases to develop both selective inhibitors and non-natural substrates. Present use of ATP-competitive kinase inhibitors has been significantly limited by poor selectivity. Similarly, the overlapping selectivities of peptide substrates has limited their use in activity detection. Our targets for kinase protein substrate-competitive inhibitors and substrates are the Src family of tyrosine kinases. Also, we will develop inhibitors to the trimethyllysine peptide binding site of chromodomains in the CBX family of histone targeting proteins. The homology of the chromodomains in the eight chromobox (CBX) proteins and the nature of their binding site has made the development of inhibitors difficult. Selective probes generated here will be used to the decipher roles of CBX proteins in transcriptional regulation. With regards to activity detection, we will specifically address two enzyme families: kinases and serine hydrolases. We will develop a new approach to profile the activities of protein kinases by selection of DNA-linked substrates and DNA sequencing. We will implement this approach in studies to characterize the changes in activity that occur in the development of resistance to the kinase inhibitor lapatinib and also in the cellular process of epithelial- mesenchymal transition. In the next area, we will use selection of DNA-linked, activity-based probes to assess activity of the serine hydrolase family. We will develop a high throughput, multiplexed assay system for profiling libraries of chemical inhibitors across this entire enzyme family.

项目摘要 遗传分析技术的发展，特别是DNA测序，已经改变了 生物医学研究与基因组信息相反，获取蛋白质组信息的障碍， 特别是酶的活性显著提高。由于异常的酶活性始终观察到， 疾病，这些信息对于适当的诊断和治疗至关重要。此外，探测 使用化学抑制剂研究酶活性的功能对于我们了解生物学和疾病至关重要。 DNA编码的化学文库（DELs）已经成为一种新的工具，使药物化学家能够 利用基因技术的力量。离体筛选是实现其功能的关键过程， DEL中的合成分子由DNA序列群编码。灵感来自于 该项目旨在进一步利用非生物分子的功能信息，将这些能力拓展到新的领域。 我们的研究计划集中在推进DNA编码的化学方法，用于开发 化学探针和用于蛋白质组范围的酶活性检测。这涉及开发新的方法， DNA编码的化学文库的合成和选择，以及一种新的、相关的 利用DNA编码的蛋白质组体外选择的基于DNA的酶活性检测技术 probes.对于化学探针的开发，我们选择了两个涉及蛋白质的目标类别：蛋白质 通过短的线性基序介导的相互作用：蛋白激酶和组蛋白靶向结构域。我们将设计 并构建针对蛋白激酶的蛋白底物位点的DEL，以开发选择性抑制剂， 非天然基质。目前ATP竞争性激酶抑制剂的使用受到不良的限制。 选择性类似地，肽底物的重叠选择性限制了它们在活性检测中的应用。 我们的激酶蛋白底物竞争性抑制剂和底物的目标是酪氨酸的Src家族 激酶。此外，我们还将开发CBX中染色质结构域的三甲基赖氨酸肽结合位点的抑制剂 组蛋白靶向蛋白家族。八种染色体盒蛋白中染色体结构域的同源性 并且它们的结合位点的性质使得抑制剂的开发变得困难。生成的选择性探针 本文将用于解读CBX蛋白在转录调控中的作用。关于活动 检测时，我们将专门针对两个酶家族：激酶和丝氨酸水解酶。我们将开发一个 通过选择DNA连接的底物和DNA分析蛋白激酶活性的新方法 测序我们将在研究中实施这种方法，以表征在体内发生的活性变化。 对激酶抑制剂拉帕替尼的耐药性的发展，以及上皮细胞的细胞过程， 间质转化在下一个领域，我们将使用选择DNA连接的，基于活性的探针来评估 丝氨酸水解酶家族的活性。我们将开发一个高通量，多路复用的分析系统， 整个酶家族的化学抑制剂库。