New Chemical Strategies for the Modification and Control of Endogenous Proteins in Living Cells

梅纳德实验室的研究性质在于化学和神经生物学的交界处。我们试图回答有关神经元如何与其邻近的细胞--星形胶质细胞--通信的基本问题。这项建议描述了我们为实现这一目标必须采取的第一步。我们化学生物学研究计划的一个长期目标是创建一个化学工具箱来研究蛋白质，这将补充生物学家目前使用的遗传方法。具体地说，我们将：(1)设计化学工具来标记活细胞中的蛋白质，以及(2)应用这些探针来了解老化大脑中突触消除的分子机制。我们的多学科研究项目融合了合成化学、分子和细胞生物学、细胞成像和电生理学等技术。这项提议的一个中心主题是修改自然进化的天然产品，并将它们用作针对特定蛋白质的分子探针。为了更好地了解神经退化的机制，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。如果能够利用化学手段监测蛋白质，而无需基因改造，将极大地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着明显的缺陷，例如，蛋白质的表达或活性经常受到干扰，并且必须为每个生物体重新设计新的载体。化学家在设计可在实验时应用的小分子方面具有独特的地位。这确保了我们观察到正常的蛋白质活动；此外，在神经生物学中，由于大脑受体跨物种的保守性，分子可以很容易地从一个有机体转移到另一个有机体。我们提议的方法的成功实施将有助于揭开化学和生物学前沿的基本科学问题。这些分子也将是制药业活体细胞分析的有吸引力的工具。预期结果这项研究计划的中心是小分子的合成，这将提供新的方法来可视化、标记和影响特定蛋白质在其自然环境中。具体地说，我们希望这项研究将为化学生物界提供新的试剂，以实现蛋白质中赖氨酸残留物的无痕迹标记。天然产物修饰策略的广泛适用性将证明，可以从任何配体创建新的工具来研究其在活细胞中的相关蛋白质。虽然我们在这里关注的是神经元和星形胶质细胞之间的细胞通讯，但我们将用来研究钙级联的一套分子工具将适用于任何钙活性组织，如心肌细胞和肌肉。最后，我们将采取的系统方法来了解生理条件下的化学反应，也应该导致基础知识，以帮助药物化学家设计具有共价连接物的新药(一个新兴的概念，可以将药物效率提高几倍)。我们实验室的加拿大学生和研究人员将获得包括化学、分子生物学和神经生物学在内的跨学科研究的独特培训。虽然化学生物学领域发展迅速，但我们带来的专业知识在加拿大是独一无二的。鉴于治疗神经退行性疾病的迫切需求，我们实验室培养的学生将是国家和国际层面上备受追捧的科学家。预计通过这些研究获得的知识将为如何开发治疗或预防神经退行性疾病的药物提供宝贵的见解。

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 first 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; moreover, in neurobiology, the molecules can easily be transferred from one organism to another due to the conserved nature of brain receptors across species. 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, the systematic approach we will take 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). Benefits to Canadians Students and researchers in our lab will gain a unique training in interdisciplinary research including chemistry, molecular biology and neurobiology. While the field of chemical biology is growing rapidly, the expertise we bring is unique in Canada. In view of the pressing needs to treat neurodegenerative diseases, the students trained in our lab will be highly sought-after scientists at the national and international level. It is anticipated that the knowledge gained through these studies will provide valuable insight into how to develop pharmaceutical agents to treat or prevent neurodegenerative diseases.