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

项目摘要 基因分析技术的发展，特别是DNA测序，已经改变了 生物医学研究。与基因组信息相比，获取蛋白质组信息的障碍， 尤其是酶的活性，要高得多。因为持续观察到异常的酶活性 对于疾病而言，这些信息对于适当的诊断和治疗至关重要。此外，能够探测 使用化学抑制剂的酶活性的功能对于我们理解生物学和疾病是至关重要的。 DNA编码的化学库(DELs)已经成为一种新的工具，它使药物化学家能够 充分利用基因技术的力量。体外选择是决定细胞功能的关键环节。 DEL中的合成分子是用DNA序列群体编码的。灵感来自于这一转换 为了获得非生物分子的功能信息，该项目寻求将这些能力进一步开发到新的领域。 我们的研究计划集中在推进DNA编码的化学方法，以开发 化学探针和全蛋白质组酶活性检测。这涉及到开发新的方法 DNA编码化学文库的合成和选择，以及一个新的，相关的 基于DNA的酶活性检测技术--体外选择DNA编码蛋白质组 探测器。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 相互作用由一个短的线性基序介导：蛋白激酶和组蛋白靶向结构域。我们将设计 并建立以蛋白激酶的蛋白底物位置为靶点的DELs，以开发选择性抑制剂和 非天然底物。目前三磷酸腺苷竞争性激酶抑制剂的使用受到了严重的限制 选择性。同样，多肽底物的重叠选择性限制了它们在活性检测中的应用。 我们的蛋白激酶底物竞争性抑制剂和底物的靶标是酪氨酸的Src家族 激活剂。此外，我们还将开发针对CBX中染色域的三甲基赖氨酸肽结合部位的抑制剂 组蛋白靶向蛋白家族。八种色素盒(CBX)蛋白的色域同源性 而且它们结合部位的性质使抑制剂的开发变得困难。生成的选择性探针 这里将用来解释CBX蛋白在转录调控中的作用。关于活动 为了检测，我们将专门讨论两个酶家族：激酶和丝氨酸水解酶。我们将开发一种 通过选择DNA连接底物和DNA来描述蛋白激酶活性的新方法 测序。我们将在研究中采用这一方法，以表征发生在 对激酶抑制剂拉帕替尼的耐药性以及在上皮细胞过程中的耐药性- 间充质转化。在下一个领域，我们将使用选择的DNA连接的、基于活动的探针来评估 丝氨酸水解酶家族的活性。我们将开发一个高通量、多路分析的分析系统 整个酶家族的化学抑制剂库。

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.