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死亡率的第一原因。因此，发现新的，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，与动脉粥样硬化发育发挥了保护作用。这项研究的长期目的是检验以下假设：在该疾病的小鼠模型中，选择性激活剂对PON1催化活性的增强将减轻动脉粥样硬化。该目标的成功可能会为新型治疗策略提供概念验证数据。这项研究建议的具体目标是检验可以发现适体增强PON1酶活性的假设。具体目的1是生成基于机制的活性探针（AP），该探针在催化后共价修改PON1，从而用生物素标记活性酶。特定的目标2是隔离PON1激活适体，而特定的目标3是表征适体。可以通过一种新型的阳性选择方案来分离适体，从而激活PON1催化活性，涉及将纯化的PON1暴露于随机DNA文库中，然后限制AP的浓度。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PON1分子的优先生物素标记。通过使用链霉亲和素共轭珠捕获生物素化的PON1，将分离结合的DNA。该DNA将通过PCR扩增，转换为单链DNA，而选择周期则以较低浓度的AP重复。因此，与PON1结合时具有增强活性的适体将在每一轮选择中优先隔离。此外，探针的竞争将导致选择具有最大PON1激活活性的适体。该方案分离的适体将以有效性和有效性为特征，在有和没有重构的HDL颗粒的情况下激活PON1，具有三个不同的底物，代表PON1的三个底物类。这些数据将有助于准备和优先选择适体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的抗透明性活性。动脉粥样硬化是西方社会死亡率的头号原因。该研究建议着重于生成研究工具，以评估一种新的治疗策略，以防止动脉粥样硬化。