Design of molecular tools to study protein dynamics in living cells

梅纳尔实验室的研究性质是化学和神经生物学的结合。我们试图回答有关神经元如何与相邻细胞（星形胶质细胞）通信的基本问题。该提案描述了我们为实现这一目标而必须采取的化学步骤。我们化学生物学研究计划的长期目标是创建一个化学工具箱来研究蛋白质，以补充生物学家目前使用的遗传方法。具体而言，我们将：（1）设计化学工具来标记活细胞中的蛋白质，（2）应用这些探针来了解衰老大脑中突触消除的分子机制。我们的多学科研究计划整合了合成化学，分子和细胞生物学，细胞成像和电生理学技术。该提案的一个中心主题是修改自然界进化的天然产物，并将其用作靶向特定蛋白质的分子探针。为了更好地理解神经退行性变的机制，科学家需要一套新的工具来研究分子水平上的蛋白质-蛋白质相互作用。利用化学手段监测蛋白质而不进行基因改造将大大加快对健康和疾病中重要细胞过程的研究。目前研究蛋白质动力学的遗传方法存在明显的缺点，例如，蛋白质的表达或活性经常受到干扰，必须为每种生物体重新设计新的载体。化学家在设计可以在实验时应用的小分子方面具有独特的优势。这确保我们观察到正常的蛋白质活性。我们提出的方法的成功实施将有助于解决化学和生物学前沿的基本科学问题。这样的分子也将是用于制药工业中的活细胞测定的有吸引力的工具。预期成果该研究提案的重点是小分子的合成，这将提供新的手段来可视化，标记和影响特定的蛋白质在其天然环境中。具体而言，我们希望这项研究将提供化学生物学社区与新的试剂，以实现蛋白质中赖氨酸残基的无痕标记。修饰天然产物的策略的广泛适用性将证明可以从任何配体创建新工具来研究活细胞中的相关蛋白质。虽然我们在本文中关注神经元和星形胶质细胞之间的细胞通信，但我们将用于研究钙级联的一套分子工具将适用于任何钙活性组织，如心脏细胞和肌肉。最后，我们在生理条件下了解化学反应的方法也应该导致基础知识，以帮助药物化学家设计新的药物与共价连接（一个新兴的概念，可以提高药物效率几倍）。

Nature of the Research Research in the Menard lab lies at the interface of chemistry and neurobiology. We seek to answer fundamental questions about how neurons communicate with their neighbouring cells, the astrocytes. This proposal describes the chemical steps we must take to achieve this goal. A long-term goal of our Chemical Biology research program is to create a chemical toolbox to study proteins that will complement the current genetic methods used by biologists. Specifically, we will: (1) design chemical tools to label proteins in live cells, and (2) apply these probes to understand the molecular mechanisms of synapse elimination in the aging brain. Our multidisciplinary research program integrates techniques of synthetic chemistry, molecular and cell biology, cell imaging, and electrophysiology. A central theme to this proposal is to modify natural products that have been evolved by Nature and use them as molecular probes to target specific proteins. Significance To better understand the mechanisms of neurodegeneration, scientists need new sets of tools to investigate protein-protein interactions at the molecular level. Having access to chemical means to monitor proteins without genetic modification would greatly accelerate the study of important cellular processes in health and disease. Current genetic methods to study protein dynamics suffer from significant drawbacks, e.g., protein expression or activity is often perturbed, and new vectors must be re-engineered for each organism. Chemists are uniquely positioned to design small molecules that can be applied at the time of experiment. This ensures we observe normal protein activity. The successful implementation of our proposed methods will help unravel fundamental scientific questions at the frontiers of chemistry and biology. Such molecules will also be attractive tools for live cell assays in the pharmaceutical industry. Expected Outcomes This research proposal centers on the synthesis of small molecules that will offer novel means to visualize, label, and influence specific proteins in their native environment. Specifically, we expect this research will afford the chemical biology community with new reagents to achieve the traceless labeling of lysines residues in proteins. The broad applicability of the strategy of modifying natural products will demonstrate that new tools can be created from any ligand to study its associated protein in live cells. While we focus herein on cellular communication between neurons and astrocytes, the set of molecular tools we will use to study the calcium cascade will be applicable to any calcium-active tissue, such as heart cells and muscles. Finally, our approach to learn about chemical reactivity under physiological conditions should also lead to fundamental knowledge to help medicinal chemists design new drugs with covalent linkers (an emerging concept that can increase drug effiency several-fold).