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 生物医学过程。本提案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发展和应用，2)新的检测方法 构建设计的信号网络；3)蛋白质错误折叠的基础研究。 蛋白质磷酸化在细胞信号转导中起着核心作用。因此，这些酶 维持磷蛋白质组，蛋白激酶和蛋白磷酸酶，被认为是关键的药物靶点 人类疾病。目前，间接指标被用来估计激酶的活性扰动和 与疾病发展相关的磷酸酶。尽管这些代理很有用，但它们并不提供直接的 对酶活性的测量，导致对酶活性和磷酸酶活性的不准确估计。结果, 清楚地了解在发育和发育过程中激酶和磷酸酶活性扰动的作用。 人类疾病的进展是缺乏的。因此，迫切需要对其进行开发和应用 直接量化人类疾病状态下的激酶和磷酸酶活性的化学工具。在这份提案中， 我们将利用一种对磷酸化敏感的氨基酸来构建一组激酶和磷酸酶。 活性探测和应用该面板来开发人类在 疾病的发展。 信号蛋白的微调活性对于正常的生理来说是必不可少的。的确，扰动 在信号的活动中，蛋白质是人类疾病发展的中心。不幸的是，这个领域 仍然缺乏一种在生活中同时调节多个信号蛋白活性的统一方法 系统，以模拟人类疾病。为了满足这一关键需求，我们将利用分裂蛋白 重新组装，以定义用于微调信令节点活动的标准化部件词典 活细胞。从长远来看，这组混合匹配部分将被用于模拟人类疾病状态和 确定潜在的毒品目标。 最后，我们的实验室将研究蛋白质错误折叠的基本方面，这是现在 被认为是许多人类疾病状态的中心病理机制。目前缺乏的 评估生命系统中蛋白质错误折叠和聚集的方法已经产生了对 开发新的方法来解决这一差距。通过利用一种新的、基于发光的分析 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识别能够干扰 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.