Homogenous NAD assay for ARDT activity measurement

用于 ARDT 活性测量的均质 NAD 测定

• 批准号: 8735970
• 负责人: Karen Wu
• 金额: $ 8.72万
• 依托单位: LUCERNA, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-18 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8735970
• 关键词: ADP Ribose Transferases; Affect; Appearance; Basic Science; Binding; Biological Assay; Characteristics; Collaborations; Development; Diabetes Mellitus; Drug Industry; Ensure; Enzymatic Biochemistry; Enzymes; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Inflammation; Legal patent; Malignant Neoplasms; Measurement; Measures; Metabolic syndrome; Metabolism; Methods; Mitochondria; Niacinamide; Nicotinamide adenine dinucleotide; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Peptides; Performance; Phase; Process; RNA; Reaction; Relative (related person); Research; Sampling; Signal Transduction; Sirtuins; Small Business Innovation Research Grant; Spinach - dietary; System; Technology; Testing; Time; aptamer; base; commercialization; drug discovery; enzyme activity; interest; novel; public health relevance; research study; screening; sensor; tool

DESCRIPTION: Sirtuins and ADP ribosyltransferases (ARDTs) are two families of related enzymes that have important medically relevant functions. For example, SIRT3, a mitochondrial sirtuin, controls mitochondrial function, and thereby exerts a strong effect on metabolism, obesity, type 2 diabetes, and metabolic syndrome. There is a major need to have simple assays to measure the activity of SIRT3, as well as other sirtuins and their close relatives, the ARDTs. Each of these enzymes uses nicotinamide adenine dinucleotide (NAD), and generates nicotinamide as a byproduct of these reactions. Unfortunately, current methods for assaying the activity of these enzymes are not optimal because they are not homogeneous, are not continuous, lack sensitivity, and often not generalizable to screening all of the diverse enzymes that generate nicotinamide. A more straightforward assay would be to directly measure the products of these enzymes in a homogeneous format. Therefore, assays that directly detect the appearance of nicotinamide would be useful for measuring the activity these enzymes, regardless of the specific substrate. Using a patented sensor technology developed by Lucerna, we will develop a universal sirtuin/ARDT assay based on a simple, homogeneous fluorescence sensor that is highly specific for nicotinamide. This Phase I SBIR application describes proof-of-concept experiments for developing, characterizing, and optimizing our nicotinamide sensors in an assay for SIRT3 activity. For Phase II, our assay will be optimized to comply with standards required for ensuring that this assay can be packaged and used in a kit format for a variety of sirtuins and related enzymes.

描述：Sirtuins和ADP核糖基转移酶（ARDTS）是两个具有重要医学相关功能的相关酶家族。例如，线粒体sirtuin Sirt3控制线粒体功能，从而对代谢，肥胖，2型糖尿病和代谢综合征产生强大影响。主要需要进行简单的测定，以测量SIRT3的活动，以及其他Sirtuins及其近亲Ardts。这些酶中的每一种都使用烟酰胺腺嘌呤二核苷酸（NAD），并生成烟酰胺作为这些反应的副产品。不幸的是，当前分析这些酶活性的方法不是最佳的，因为它们不是均匀的，不连续的，缺乏灵敏度，并且通常无法推广用于筛选产生烟酰胺的所有多种酶。更直接的测定方法是直接以均匀格式直接测量这些酶的产物。因此，直接检测烟酰胺的外观的测定对于测量这些酶的活性都是有用的，无论特定底物如何。使用卢塞纳（Lucerna）开发的专利传感器技术，我们将基于一种简单，均匀的荧光传感器开发通用的Sirtuin/ARDT分析，该传感器对烟酰胺高度特定。此阶段I SBIR应用描述了概念验证实验，用于在SIRT3活性测定中开发，表征和优化我们的烟酰胺传感器。对于第二阶段，我们的测定法将进行优化，以符合确保可以以套件格式包装和使用该测定法的标准，以用于各种Sirtuins和相关酶。