Discovery of a Selective Agent that Activates PON1 Enzymatic Activity

描述(申请人提供)：动脉粥样硬化是西方社会导致死亡的头号原因。因此，寻找新的更有效的抗动脉粥样硬化治疗方法对于挽救许多人的生命至关重要。PON1是一种血清酶，已被认为对动脉粥样硬化的发展起到保护作用。这项研究的长期目标是测试一种假设，即通过选择性激活剂增强PON1的催化活性将在疾病的小鼠模型中减轻动脉粥样硬化。这一目标的成功可能为新的治疗策略提供概念验证数据。这项研究提案的具体目标是检验这样一种假设，即可以发现增强PON1酶活性的适配子。具体目标1是生成一种基于机理的活性探针(AP)，该探针在催化作用下共价修饰PON1，从而将活性酶标记为生物素。特异性目标2是分离PON1激活适配子，特异性目标3是鉴定适配子。将通过一种新的正选择方案分离出激活PON1催化活性的适体，该方案包括将纯化的PON1暴露在随机DNA文库中，然后是有限浓度的AP。因此，限制AP的竞争将被建立，这将导致由结合适体激活的催化活性更高的PON1分子的优先生物素标记。结合的DNA将通过使用链霉亲和素结合的珠子捕获生物素化的PON1来分离。该DNA将通过PCR扩增，转化为单链DNA，并在较低浓度的AP下重复选择周期。因此，当与PON1结合时具有增强活性的适体将在每一轮选择中优先分离。此外，对探针的竞争将导致选择具有最大PON1激活活性的适体。通过该方案分离的适配子将表征在有和没有重组高密度脂蛋白颗粒的情况下激活PON1的效力和有效性，其中三种不同的底物代表PON1的三种底物类别。这些数据将有助于制备和优先选择适配子作为未来体外和体内实验的工具，以确定激活PON1催化活性是否会增强PON1的抗动脉粥样硬化活性。动脉粥样硬化是西方社会导致死亡的头号原因。这项研究建议的重点是生成研究工具，以评估预防动脉粥样硬化的新治疗策略。

DESCRIPTION (provided by applicant): Atherosclerosis is the number one cause of mortality in Western societies. Therefore, finding new and more effective anti-atherogenic therapies is critical to saving many lives. PON1 is a serum enzyme that has been implicated in playing a protective role against development of atherosclerosis. The long-term objective of this research is to test the hypothesis that enhancement of PON1 catalytic activity by a selective activating agent will attenuate atherosclerosis in a mouse model of the disease. Success in this objective may provide proof-of-concept data for a novel therapeutic strategy. The specific goal of this research proposal is to test the hypothesis that aptamers can be discovered that enhance PON1 enzymatic activity. Specific aim 1 is to generate a mechanism-based activity probe (AP) that covalently modifies PON1 upon catalysis and thus labels active enzyme with biotin. Specific aim 2 is to isolate PON1 activating aptamers and specific aim 3 is to characterize the aptamers. Aptamers will be isolated that activate PON1 catalytic activity through a novel positive selection scheme involving exposure of purified PON1 to a random DNA library followed by a limiting concentration of AP. Thus, a competition for limiting AP will be established which will result in preferential biotin labeling of catalytically more active PON1 molecules activated by a bound aptamer. The bound DNA will be isolated by capturing biotinylated PON1 using streptavidin conjugated beads. This DNA will be amplified by PCR, converted to single-stranded DNA and the selection cycle repeated with a lower concentration of AP. Thus, aptamers that have enhancing activity when bound to PON1 will be preferentially isolated with each round of selection. In addition, the competition for probe will result in selection for aptamers with the greatest PON1 activation activity. The aptamers isolated by this scheme will be characterized for potency and efficacy in activating PON1 with and without reconstituted HDL particles with three different substrates representing the three substrate classes for PON1. These data will aid in preparing and prioritizing aptamers as tools for future in vitro and in vivo experiments to determine whether activating PON1 catalytic activity will enhance the antiatherogenic activities of PON1. Atherosclerosis is the number one cause of mortality in Western societies. This research proposal focuses on generating research tools to evaluate a novel therapeutic strategy to prevent atherosclerosis.