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: 9232005
• 负责人: Sara Evelyn Schwanger Martin
• 金额: $ 5.71万
• 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/9232005
• 关键词: Active Sites; Affect; Affinity; Affinity Chromatography; Alpha Cell; Alzheimer' s Disease; Antibodies; Asses; 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; N-Acetylglucosaminyltransferases; Nutrient; O-GlcNAc transferase; Organism; Pathway interactions; Permeability; Post-Translational Protein Processing; Process; Proteins; Public Health; Regulation; Research; Role; Serine; Signal Pathway; Signal Transduction; Solubility; Threonine; Time; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; Work; analog; aqueous; base; cellular targeting; chemical reaction; design; experimental study; functional group; glycosylation; improved; inhibitor/antagonist; insight; protein function; public health relevance; response; scaffold; small molecule; small molecule inhibitor; sugar; targeted treatment; therapeutic candidate; 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(申请人提供)：酶负责催化细胞中的所有化学反应。某些酶的活性会随着细胞中特定营养物质的水平而变化。葡萄糖是一种重要而丰富的营养物质。当葡萄糖(糖)在细胞中处于高水平时，它被重定向到一条导致刺激O-连接N-乙酰氨基葡萄糖转移酶(OGT)的途径，该酶负责将β-N-乙酰氨基葡萄糖(GlcNAc)添加到细胞中的1,000多种蛋白质中。将这些基团加入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的错误调控与糖尿病、癌症、心脏病和阿尔茨海默病有关。需要更多的工具来使我们能够研究OGT的异常调控是如何发生的，以及它如何影响细胞中导致疾病的信号传递过程。这将帮助我们确定OGT是否可能是一个很好的治疗学候选药物。小分子化学探针是研究这些信号过程的理想选择，因为它们可以用来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，但很少有工具化合物被证明只对OGT起选择性作用，并在细胞内对OGT起作用。显然需要更好、更有效的化学工具化合物来研究OGT。我们正在努力开发基于我们实验室开发的一种有前途的抑制剂OSMI-1的改进探针，但这存在一些缺陷，限制了其用途。OSMI-1在水中的溶解度是有限的，它含有已知的代谢不稳定的官能团。OSMI-1在细胞内表现出适度的抗OGT活性，但在抑制剂治疗24小时后，它会导致细胞存活率下降。OSI-1缺乏足够的效力和代谢稳定性，不能作为体外探针使用。 我们建议开发改进的工具化合物，在细胞内具有更高的对抗OGT的效力。为了能够量化细胞中的效力，我们将开发一种基于荧光的分析方法来确定全球O-GlcN酰化水平。我们还将致力于使OGT：抑制剂复合体结晶，以获得有关抑制模式和化合物设计的结构信息。最后，我们将利用亲和纯化来阐明抑制物的脱靶作用。我们希望这些研究将导致高度有效和选择性的工具化合物，这将是有价值的研究OGT参与的细胞过程。对这些信号通路的更好的了解将增加我们对OGT如何导致各种疾病状态的理解，并将有助于确定OGT是否可能成为治疗的靶点。 根据上述项目大纲，我们提出了以下具体目标：具体目标1-从OSMI-1支架开始开发有效的OGT非共价和共价抑制剂。特定目标2-表征有希望的小分子OGT探针的靶上和靶外细胞活性。