Homogenous NAD assay for ARDT activity measurement

用于 ARDT 活性测量的均质 NAD 测定

• 批准号: 8591776
• 负责人: Karen Wu
• 金额: $ 21.18万
• 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 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8591776
• 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 (ARDT) 是两个相关酶家族，具有重要的医学相关功能。例如，SIRT3（一种线粒体去乙酰化酶）控制线粒体功能，从而对新陈代谢、肥胖、2 型糖尿病和代谢综合征产生强烈影响。非常需要简单的检测方法来测量 SIRT3 以及其他去乙酰化酶及其近亲 ARDT 的活性。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物。不幸的是，目前测定这些酶活性的方法并不是最佳的，因为它们不均质、不连续、缺乏灵敏度，并且通常不能推广到筛选产生烟酰胺的所有不同酶。更直接的测定是以均质形式直接测量这些酶的产物。因此，无论特定底物如何，直接检测烟酰胺外观的测定对于测量这些酶的活性都是有用的。利用 Lucerna 开发的专利传感器技术，我们将开发一种基于简单、均匀的烟酰胺高度特异性荧光传感器的通用 Sirtuin/ARDT 检测方法。该 I 期 SBIR 应用描述了在 SIRT3 活性测定中开发、表征和优化我们的烟酰胺传感器的概念验证实验。对于第二阶段，我们的检测将进行优化，以符合确保该检测可以包装并以试剂盒形式用于各种去乙酰化酶和相关酶所需的标准。