New Tools for the Study of O-GlcNAc Transferase in Disease

研究疾病中 O-GlcNAc 转移酶的新工具

• 批准号: 9047446
• 负责人: Sara Evelyn Schwanger Martin
• 金额: $ 5.43万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9047446
• 关键词: Active Sites; Affect; Affinity; Affinity Chromatography; Alzheimer' s Disease; Antibodies; Binding; Biological; Biological Assay; Cell Survival; Cell physiology; Cells; Characteristics; Chemicals; Complex; Crystallization; Data; Development; Diabetes Mellitus; Disease; Environment; Enzymes; Exhibits; Fluorescence; Glucose; Heart Diseases; Hour; Human; In Vitro; Investigation; Knowledge; Laboratories; Lead; Link; Malignant Neoplasms; Metabolic; Methods; Nutrient; O-GlcNAc transferase; Organism; Pathway interactions; Permeability; Post-Translational Protein Processing; Process; Proteins; Public Health; Regulation; Research; Role; Serine; Signal Pathway; Solubility; Therapeutic; Threonine; Time; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; Work; analog; aqueous; base; cellular targeting; chemical reaction; design; functional group; glycosylation; improved; inhibitor/antagonist; insight; protein function; public health relevance; research study; response; scaffold; signal processing; small molecule; small molecule inhibitor; sugar; targeted treatment; tool

 DESCRIPTION (provided by applicant): Enzymes are responsible for catalyzing all of the chemical reactions in the cell. The activity of some enzymes changes in response to levels of specific nutrients in the cell. One important and abundant nutrient is glucose. When glucose (sugar) is present in high levels in cells, it is redirected into a pathway that leads to the stimulation of an enzyme known as O-Linked N-acetylglucosamine Transferase (OGT) that is responsible for the addition of β-N-acetylglucosamine (GlcNAc) to over 1,000 proteins in the cell. The addition of these groups to target proteins can affect the localization, stability, and activity of the target proteins, as well as their interactions with other biomolecules. The misregulation of OGT has been linked to diabetes, cancer, heart disease, and Alzheimer disease. More tools are needed to enable us to study how aberrant regulation of OGT occurs and how it affects signaling processes in the cell that lead to disease. This will help us to determine if OGT could be a good candidate for therapeutics. Small molecule chemical probes are ideal for studying these signaling processes, because they can be used to study enzymes in their natural environment. Some tool compounds have already been developed to inhibit OGT, but few of them have been shown to act selectively against only OGT and to work against OGT inside cells. There is a clear need for better, more potent chemical tool compounds to study OGT. We are working to develop improved probes that are based upon a promising inhibitor, known as OSMI-1, that was developed in our laboratory, but that has some drawbacks that limit its utility. The aqueous solubility of OSMI-1 is limited, and it contains functional groups that ar known to be metabolically unstable. OSMI-1 exhibits modest levels of activity against OGT inside cells, but it causes a reduction in cell viability after 24 hours of inhibitor treatment. OSI-1 lacks sufficient potency and metabolic stability to move past use as an in vitro probe. We propose to develop improved tool compounds with higher potency against OGT in cells. To enable quantitation of potency in cells, we will develop a fluorescence-based assay for determining global O- GlcNAcylation levels. We will also work to crystallize OGT:inhibitor complexes to gain structural information that will inform on the mode of inhibition and on compound design. Finally, we will elucidate inhibitor off-targets using affinity-purification. We expect that these studies will lead to highly potent and selective tool compounds that will be valuable for studying cellular processes in which OGT participates. An improved understanding of these signaling pathways will increase our understanding of how OGT contributes to various disease states, and will help to determine if OGT could be a target for therapeutics. In accordance with the project outline above, we propose the following specific aims: Specific Aim 1- Develop potent non-covalent and covalent inhibitors of OGT beginning from the OSMI-1 scaffold. Specific Aim 2- Characterize on- and off-target cellular activity of promising small molecule OGT probes.

 描述（由适用提供）：酶负责催化细胞中的所有化学反应。某些酶的活性响应细胞中特定养分水平的响应。一种重要且丰富的养分是葡萄糖。当葡萄糖（糖）中存在高水平的细胞中时，将其重定向到一条途径中，导致刺激称为O连接的N-乙酰基葡萄糖胺转移酶（OGT），该酶负责将β-N-乙酰基葡萄糖胺（GlCNAC）添加到超过1,000个细胞中。将这些组添加到靶蛋白中可以影响靶蛋白的定位，稳定性和活性，以及​​它们与其他生物分子的相互作用。 OGT的正调与糖尿病，癌症，心脏病和阿尔茨海默氏病有关。需要更多工具来研究OGT的异常调节以及它如何影响导致疾病的细胞信号传导过程。这将有助于我们确定OGT是否可以成为治疗的好候选者。小分子化学问题是研究这些信号传导过程的理想选择，因为它们可用于研究自然环境中的酶。已经开发了一些工具化合物来抑制OGT，但是很少有人证明它们仅针对OGT起作用，并且可以针对细胞内部的OGT作用。显然需要更好，更多潜在的化学工具化合物来研究OGT。我们正在努力制定基于在我们的实验室中开发的承诺抑制剂（称为OSMI-1）的前途抑制剂的问题，但这有一些限制其效用的缺点。 OSMI-1的水溶性受到限制，并且包含已知在代谢不稳定的功能组。 OSMI-1表现出对OGT内部OGT的适度活性，但在抑制剂治疗24小时后会导致细胞活力降低。 OSI-1缺乏足够的效力和代谢稳定性，无法将其作为体外探针的使用。 我们建议开发改进的工具化合物，具有较高的细胞中OGT效力。为了实现细胞效力的定量，我们将开发基于荧光的评估，以确定全局O- Glcnacylation水平。我们还将致力于使OGT：抑制剂复合物结晶，以获取结构信息，这些信息将告知抑制和复合设计的方式。最后，我们将使用亲和力纯化阐明抑制剂脱离目标。我们预计这些研究将导致高潜力和选择性的工具化合物，这些化合物对于研究OGT参与的细胞过程很有价值。对这些信号传导途径的提高理解将提高我们对OGT如何对各种疾病状态贡献的理解，并有助于确定OGT是否可以成为治疗的目标。 根据上述项目大纲，我们提出以下特定目的：特定目的1-从OSMI-1脚手架开始的OGT的潜在非共价和共价抑制剂。特定的目标2-表征有望小分子OGT问题的靶向和脱靶细胞活性。