Homogenous NAD assay for ARDT activity measurement

Sirtuins和ADP核糖基转移酶(ARDTs)是两个相关的酶家族，具有重要的医学相关功能。例如，SIRT3，一种线粒体sirtuin，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。主要需要有简单的分析方法来测量SIRT3以及其他sirtuin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)，并在这些反应中产生烟酰胺。不幸的是，目前测定这些酶活性的方法并不是最理想的，因为它们不是均匀的，不连续的，缺乏敏感性，而且往往不能推广到筛选产生烟酰胺的所有不同的酶。一种更直接的分析方法是以均一的形式直接测量这些酶的产物。因此，无论底物是什么，直接检测烟酰胺外观的方法都有助于测量这些酶的活性。使用Lucerna开发的专利传感器技术，我们将开发一种通用的sirtuin/ARDT检测方法，该方法基于一种简单、均一的荧光传感器，该传感器对烟酰胺具有高度特异性。这个第一阶段的SBIR应用描述了用于开发、表征和优化我们的烟酰胺传感器的概念验证实验，以测试SIRT3的活性。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sirtuins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.