Fluorescent HTS Assay for Methyltransferases in Neurodegenerative Diseases

描述(申请人提供)：项目摘要/摘要：甲基化是一种普遍存在的共价修饰，用于控制各种生物分子的功能，包括激素、神经递质、外源生物、蛋白质、核酸和脂质。人类体内存在50多种不同的甲基转移酶(MTS)酶，它们针对的疾病范围很广，但它们参与神经退行性疾病的途径具有特殊的相关性。调节神经递质甲基化是一种新兴的治疗策略，用于治疗几种神经退行性疾病，最明显的是帕金森氏病和阿尔茨海默病，DNA和蛋白质MTS也是神经退行性疾病和中枢神经系统癌症的靶点。此外，一些MT家族成员在疾病途径中的参与与他们在代谢常用处方药方面的作用交织在一起。开发高选择性的MT调节剂显然是医学上的当务之急。然而，由于缺乏适用于高通量筛选(HTS)的灵活的酶分析方法，实现这一目标的努力正受到阻碍。目前使用的同工酶特异性分析方法依赖于放射性和繁琐的反应后处理步骤，这阻碍了将不同的MT酶轻松地整合到药物发现计划中。为了加快识别选择性MT抑制剂的努力，我们建议开发一种通用的催化分析方法，使使用一套检测试剂筛选MT家族的所有成员成为可能。该方法是基于使用竞争性荧光偏振免疫分析检测不变的MT反应产物S-腺苷同型半胱氨酸。第一阶段的工作包括开发抗体和荧光核苷酸结合物，使在S-腺苷蛋氨酸存在下能够高选择性地检测S-腺苷同型半胱氨酸，S-腺苷蛋氨酸的唯一不同之处在于添加了一个甲基。项目简介：甲基转移酶家族中几种蛋白质的异常行为可能是导致阿尔茨海默病和帕金森病等神经退行性疾病的一个可能原因。为了加速发现这些疾病的改进治疗方法，我们正在开发筛查试剂盒，以寻找纠正故障甲基转移酶蛋白的药物分子。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids and lipids. More than 50 distinct methyltransferase (MTs) enzymes are present in humans, and they are being targeted for a broad range of diseases, but their involvement in neurodegenerative disease pathways is of special relevance. Modulation of neurotransmitter methylation is an emerging therapeutic strategy for the treatment of several neurodegenerative diseases, most notably Parkinson's and Alzheimer's diseases, and DNA and protein MTs are also being targeted for neurodegenerative diseases and cancers of the CNS. Moreover, the involvement of some MT family members in disease pathways is intertwined with their role in metabolizing commonly prescribed drugs. The development of highly selective MT modulators is clearly a compelling medical priority. However, efforts to achieve this are being hampered by a lack of flexible enzyme assay methods adaptable to high throughput screening (HTS). The current use of isozyme-specific assay methods that rely on radioactivity and cumbersome post reaction processing steps is preventing facile incorporation of diverse MT enzymes into pharmaceutical drug discovery programs. To accelerate efforts to identify selective MT inhibitors we propose to develop a universal catalytic assay that will enable screening of all members of the MT family using a single set of detection reagents. The method is based on detection of the invariable MT reaction product S-adenosyl homocysteine-using a competitive fluorescence polarization immunoassay. The Phase I work includes development of antibodies and fluorescent nucleotide conjugates that enable highly selective detection of S-adenosyl homocysteine in the presence of S-adenosyl methionine, which differs only in the addition of a methyl group. Project Narrative: Aberrant behavior by several proteins in the methyltransferase family is a possible cause of debilitating neurodegenerative disorders like Alzheimer's disease and Parkinson's disease. To accelerate the discovery of improved therapies for these diseases, we are developing screening kits to find drug molecules that correct the malfunctioning methyltransferase proteins.