Fluorescent HTS Assay for Methyltransferases in Neurodegenerative Diseases

项目概述/摘要：甲基化是一种普遍存在的共价修饰，用于控制多种生物分子的功能，包括激素、神经递质、异种生物、蛋白质、核酸和脂质。超过50种不同的甲基转移酶（MTs）存在于人类中，它们是广泛疾病的靶标，但它们在神经退行性疾病途径中的参与具有特殊的相关性。神经递质甲基化的调节是治疗几种神经退行性疾病的一种新兴治疗策略，最著名的是帕金森病和阿尔茨海默病，DNA和蛋白质MTs也成为神经退行性疾病和中枢神经系统癌症的治疗目标。此外，一些MT家族成员参与疾病通路与其在常用处方药代谢中的作用相互交织。高选择性MT调节剂的发展显然是一个引人注目的医疗优先事项。然而，由于缺乏适应高通量筛选（HTS）的灵活的酶分析方法，实现这一目标的努力受到阻碍。目前使用的同工酶特异性测定方法依赖于放射性和繁琐的反应后处理步骤，这阻碍了将多种MT酶轻松纳入药物发现计划。为了加速鉴定选择性MT抑制剂的工作，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。该方法是基于检测不变的MT反应产物s -腺苷型同型半胱氨酸-使用竞争荧光偏振免疫分析法。I期工作包括抗体和荧光核苷酸偶联物的开发，这些抗体和偶联物能够在s -腺苷蛋氨酸存在的情况下高度选择性地检测s -腺苷同型半胱氨酸，s -腺苷蛋氨酸的不同之处在于添加了一个甲基。项目描述：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原因。为了加速发现针对这些疾病的改进疗法，我们正在开发筛选试剂盒，以找到纠正甲基转移酶蛋白故障的药物分子。

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.