Discovery of a Selective Agent that Activates PON1 Enzymatic Activity

发现激活 PON1 酶活性的选择性试剂

• 批准号: 7500845
• 负责人: John Scott
• 金额: $ 9.86万
• 依托单位: NORTH CAROLINA CENTRAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-25 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7500845
• 关键词: Affect; Antiatherogenic; Arylesterase; Atherosclerosis; Attenuated; Binding; Biological Assay; Biomedical Research; Biotin; Catalysis; Class; Condition; Coupled; DNA; DNA Binding; DNA Library; Data; Development; Disease; Drug Industry; Enzyme Activation; Enzymes; Funding; Future; Galactosidase; Goals; Grant; High Density Lipoproteins; Hydrolysis; In Vitro; Insecticides; Label; Length; Libraries; Life; Measures; Mediating; Molecular; Mus; Numbers; Oligonucleotides; Paraoxon; Play; Polymerase Chain Reaction; Population; Positioning Attribute; Procedures; Protocols documentation; Publications; Research; Research Proposals; Role; Scheme; Serum; Single-Stranded DNA; Societies; Specificity; Standards of Weights and Measures; Streptavidin; Structure; Testing; Western Blotting; Work; analog; aptamer; base; career; concept; desire; enzyme activity; in vivo; magnetic beads; mortality; mouse model; mutant; novel; novel therapeutics; particle; phenylacetate; prevent; programs; reconstitution; research study; size; success; tool

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.

描述（由申请人提供）：动脉粥样硬化是西方社会第一大死因。因此，寻找新的、更有效的抗动脉粥样硬化疗法对于挽救许多生命至关重要。 PON1 是一种血清酶，具有防止动脉粥样硬化发展的保护作用。这项研究的长期目标是测试以下假设：通过选择性激活剂增强 PON1 催化活性将减轻小鼠模型中的动脉粥样硬化。这一目标的成功可能为新型治疗策略提供概念验证数据。本研究计划的具体目标是检验可以发现增强 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），该探针在催化下共价修饰PON1，从而用生物素标记活性酶。具体目标 2 是分离 PON1 激活适体，具体目标 3 是表征适体。通过一种新颖的正选择方案，将纯化的 PON1 暴露于随机 DNA 文库，然后加入限制浓度的 AP，从而分离出激活 PON1 催化活性的适体。因此，将建立限制 AP 的竞争，这将导致由结合适体激活的催化活性更高的 PON1 分子优先生物素标记。使用链霉亲和素缀合珠捕获生物素化的 PON1 来分离结合的 DNA。该 DNA 将通过 PCR 进行扩增，转化为单链 DNA，并用较低浓度的 AP 重复选择循环。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被分离。此外，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体将表征在使用和不使用代表 PON1 的三种底物类别的三种不同底物的重构 HDL 颗粒的情况下激活 PON1 的效力和功效。这些数据将有助于准备适体并优先考虑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的第一大死因。该研究计划的重点是生成研究工具来评估预防动脉粥样硬化的新治疗策略。