Discovery of a Selective Agent that Activates PON1 Enzymatic Activity

描述（由申请人提供）：动脉粥样硬化是西方社会死亡的头号原因。因此，寻找新的和更有效的抗动脉粥样硬化治疗是挽救许多生命的关键。PON1是一种血清酶，在防止动脉粥样硬化的发展中起保护作用。本研究的长期目标是验证一种假设，即通过一种选择性活化剂增强PON1的催化活性将减轻该疾病小鼠模型中的动脉粥样硬化。这一目标的成功可能为一种新的治疗策略提供概念验证数据。本研究计划的具体目标是验证可以发现增强PON1酶活性的适体的假设。具体目标1是生成一种基于机制的活性探针（AP），该活性探针在催化作用下共价修饰PON1，从而用生物素标记活性酶。特异性目的2是分离PON1激活适配体，特异性目的3是表征适配体。通过一种新的正选择方案，将纯化的PON1暴露于随机DNA文库，然后限制AP浓度，将分离出激活PON1催化活性的适配体。因此，将建立限制AP的竞争，这将导致被结合的适配体激活的催化活性更强的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珠捕获生物素化的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，与PON1结合时具有增强活性的适体将在每轮选择中优先分离。此外，对探针的竞争将导致对具有最大PON1激活活性的适体的选择。通过该方案分离的适体将对具有和不具有三种不同底物（代表PON1的三种底物类别）的重组HDL颗粒激活PON1的效力和有效性进行表征。这些数据将有助于准备和优先考虑适配体，作为未来体外和体内实验的工具，以确定激活PON1的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的头号死亡原因。本研究计划的重点是产生研究工具来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.