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。某些酶的活性随细胞中特定营养素的水平而变化。一种重要而丰富的营养素是葡萄糖。当葡萄糖（糖）在细胞中以高水平存在时，它被重定向到一种途径，该途径导致称为O-连接N-乙酰葡糖胺转移酶（OGT）的酶的刺激，该酶负责将β-N-乙酰葡糖胺（GlcNAc）添加到细胞中的1，000多种蛋白质中。将这些基团添加到靶蛋白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的失调与糖尿病、癌症、心脏病和阿尔茨海默病有关。我们需要更多的工具来研究OGT的异常调节是如何发生的，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是否可能成为治疗的良好候选药物。小分子化学探针是研究这些信号传导过程的理想选择，因为它们可以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，但是很少有工具化合物显示出仅选择性地针对OGT起作用并且针对细胞内的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。我们正在努力开发改进的探针，该探针基于我们实验室开发的一种有前途的抑制剂OSMI-1，但它有一些限制其实用性的缺点。OSMI-1的水溶性有限，并且其含有已知代谢不稳定的官能团。OSMI-1对细胞内的OGT表现出中等水平的活性，但在抑制剂处理24小时后导致细胞活力降低。OSI-1缺乏足够的效力和代谢稳定性，无法作为体外探针使用。 我们建议开发具有更高效力的改进的工具化合物来对抗细胞中的OGT。为了能够定量细胞中的效价，我们将开发一种基于荧光的测定法，用于测定总体O-GlcNAc酰化水平。我们还将努力结晶OGT：抑制剂复合物，以获得结构信息，这将有助于抑制模式和化合物设计。最后，我们将阐明抑制剂脱靶使用亲和纯化。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细胞过程中，OGT参与。对这些信号通路的更好理解将增加我们对OGT如何影响各种疾病状态的理解，并将有助于确定OGT是否可以成为治疗靶点。 根据上述项目概述，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。具体目标2-表征有前景的小分子OGT探针的靶细胞活性和脱靶细胞活性。