Homogenous NAD assay for ARDT activity measurement

描述：Sirtuins和ADP核糖基转移酶（ARDTs）是两个家族的相关酶，具有重要的医学相关功能。例如，SIRT 3，一种线粒体sirtuin，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烈影响。主要需要有简单的测定来测量SIRT 3以及其他sirtuins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），并产生烟酰胺作为这些反应的副产物。不幸的是，目前用于测定这些酶的活性的方法不是最佳的，因为它们不是均质的，不是连续的，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的酶。更直接的测定将是以均相形式直接测量这些酶的产物。因此，直接检测烟酰胺外观的测定法可用于测量这些酶的活性，而与特定底物无关。利用Lucerna开发的专利传感器技术，我们将开发一种基于简单、均匀的荧光传感器的通用sirtuin/ARDT检测方法，该传感器对烟酰胺具有高度特异性。该I期SBIR应用描述了用于在SIRT 3活性测定中开发、表征和优化烟酰胺传感器的概念验证实验。对于II期，我们的测定将进行优化，以符合确保该测定可以包装并以试剂盒形式用于各种沉默调节蛋白和相关酶所需的标准。

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.