Chemical Approaches for Interrogating Fundamental Biomedical Processes

项目摘要 新型化学工具的开发和应用可以为以下方面提供基本见解： 生物医学过程。本建议书中概述的研究计划侧重于三个核心领域：1） 磷酸调节酶化学传感器的开发和应用，2）新的方法， 构建设计信号网络; 3）蛋白质错误折叠的基础研究。 蛋白质磷酸化在细胞信号传导中起着核心作用。因此， 维持磷酸化蛋白质组，蛋白激酶和蛋白磷酸酶，被认为是关键的药物靶标， 人类疾病目前，间接代理用于估计激酶的活性扰动， 与疾病发展相关的磷酸酶。尽管这些代理很有用，但它们不提供直接的 测量酶活性，导致激酶和磷酸酶活性的不准确估计。因此，在本发明中， 清楚地了解激酶和磷酸酶活性扰动在发育过程中的作用， 缺乏人类疾病的进展。因此，迫切需要开发和应用 直接量化人类疾病状态中激酶和磷酸酶活性的化学工具。在这一提议中， 我们将利用磷酸化敏感的氨基酸来构建一组激酶和磷酸酶， 活性探针，并应用此面板开发人类过程中信号变化的纵向活性曲线 疾病进展。 信号蛋白的精细调节活性对于正常生理是必不可少的。事实上， 信号蛋白的活性对人类疾病的发展至关重要。不幸的是， 仍然缺乏一种统一的方法来同时调节多种信号蛋白的活性， 系统来模拟人类疾病。为了满足这一关键需求，我们将利用分裂蛋白 为了定义用于微调信令节点的活动的标准化部分的词典， 活细胞从长远来看，这套混合和匹配的部分将用于模拟人类疾病状态， 确定潜在的药物靶点。 最后，我们的实验室将研究蛋白质错误折叠的基本方面， 被认为是许多人类疾病状态的中心病理机制。目前缺乏 评估生命系统中蛋白质错误折叠和聚集的方法产生了对 开发新的方法来弥补这一差距。通过利用一种新的基于发光的检测方法 对于蛋白质的错误折叠和聚集，我们的实验室将评估蛋白质的分子决定因素， 在活细胞中聚集。此外，我们将利用这种方法来鉴定能够干扰 蛋白质聚集

Project Summary The development and application of novel chemical tools can provide fundamental insights into biomedical processes. The research program outlined in this proposal focuses on three core areas: 1) the development and application of chemosensors for phospho-regulatory enzymes, 2) new methodologies for constructing designer signaling networks, and 3) fundamental studies of protein misfolding. Protein phosphorylation plays a central role in cellular signaling. Consequently, the enzymes that maintain the phosphoproteome, protein kinases and protein phosphatases, are considered key drug targets in human disease. Currently, indirect proxies are used to estimate activity perturbations of kinases and phosphatases associated with disease development. Although useful, these proxies do not provide a direct measure of enzymatic activity, leading to inaccurate estimates of kinase and phosphatase activity. As a result, a clear understanding of the role of kinase and phosphatase activity perturbations during the development and progression of human disease is lacking. Therefore, there is a critical need for the development and application of chemical tools to directly quantify kinase and phosphatase activity in human disease states. In this proposal, we will leverage a phosphorylation-sensitive amino acid to construct a panel of kinase and phosphatase activity probes and apply this panel to develop longitudinal activity profiles of signaling changes during human disease progression. The finely tuned activity of signaling proteins is essential for normal physiology. Indeed, perturbations in the activity of signaling proteins are central to the development of human disease. Unfortunately, the field still lacks a unified approach to modulating the activity of multiple signaling proteins simultaneously in living systems in order to model human disease. To address this critical need, we will leverage split-protein reassembly in order to define a lexicon of standardized parts for fine-tuning the activity of signaling nodes in living cells. In the long-term, this set of mix-and-match parts will be utilized to model human disease states and identify potential drug targets. Lastly, our laboratory will investigate the fundamental aspects of protein misfolding, which is now recognized as a central pathological mechanism in numerous human disease states. The current lack of approaches to assess protein misfolding and aggregation in living systems has created a critical need for the development of novel methodologies to address this gap. By leveraging a novel, luminescence-based assay for protein misfolding and aggregation our laboratory will assess the molecular determinants of protein aggregation in living cells. In addition, we will utilize this approach to identify molecules capable interfering with protein aggregation.