Methyltransferase inhibition assay development

描述(申请人提供)：甲基转移酶在广泛的重要过程中调节生物转化-包括神经递质和类固醇激素的生物合成，以及DNA、RNA和蛋白质的合成后修饰。甲基转移酶在越来越多的癌细胞类型中看到的基因组表观遗传变化中也发挥着重要作用。甲基转移酶扮演着不同的关键角色，这使它们成为发现以不同方式调节其活性的化合物的诱人目标。这项建议的目标是设计一种简单、可重复的高通量酶分析方法，以便于发现抑制真核基因中存在的5‘-mRNA帽的甲基化的试剂。在拟议的研究中，将使用登革病毒编码的帽-甲基转移酶作为模型酶靶标，将部分甲基化的帽转化为完全甲基化的帽，并以S-腺苷-蛋氨酸为甲基供体。在所提出的分析中，将使用荧光偏振来测量荧光“替代”肽适配子与目标甲基转移酶的关联性。建议的分析是一种“混合和测量”类型的测定，它基于这样的原理，即蛋白质在离散位置被类药物化合物干扰可以扰乱与其他地方结合的另一种配体--代理配体的结合。不幸的是，常用的测定帽-甲基转移酶活性的方法最适合于低通量的筛查应用，因为它们：(A)使用放射性，(B)需要费力地准备底物，(C)测量需要层析表征的产品，或者，必须沉淀、过滤和计数。我们建议开发的替代配基分析：(A)不需要放射性试剂，(B)可以通过简单的液体添加转移(“混合和测量”)自动实施，以及(C)提供几个小时稳定的可重复性读数。这些研究的长期目标是将这里描述的初级分析类型扩展到发现更广泛的活性化合物，包括能够修饰其他底物的甲基转移酶--蛋白质、RNA或DNA。与公共卫生相关：高通量筛查是新药发现的重要组成部分。这一过程从发现/设计在细胞或酶水平上影响生理功能的化合物开始，然后确定它们是否适合用作药物。我们建议为这一过程的第一阶段开发高通量筛查，以便发现抑制甲基转移酶的化合物，甲基转移酶是一组在中风、心脏病发作、癌症和传染病的各个方面发挥直接作用的酶。

DESCRIPTION (provided by applicant): Methyltransferases mediate biotransformations in a wide range of important processes - these include biosynthesis of neurotransmitters, and steroid hormones, as well as post-synthetic modifications of DNA, RNA, and protein. Methyltransferases also play a major role in epigenetic alterations in the genome seen in a growing list of cancer cell types. The diverse critical roles played by methyltransferases makes them attractive targets for the discovery of compounds that differentially modulate their activity. The goal of this proposal will be to design a simple reproducible high throughput enzyme-based assay to facilitate the discovery of agents that inhibit methylation of the 5'- mRNA "cap", a sequence, 5'-GpppA, present in eucaryotic mRNA's. In the proposed studies, a Dengue virus-encoded cap-methyltransferase will be used as a model enzyme target that converts the partially methylated to fully methylated cap with S-adenosyl-methionine serving as methyl donor. In the proposed assay, the association of a fluorescent "surrogate" peptide aptamer with the target methyltransferase will be measured using fluorescence polarization. The proposed assay is a "mix- and-measure" type of determination that is based on the principle that perturbation of a protein by a drug-like compound at a discrete site can perturb the binding of another ligand, the surrogate ligand, bound elsewhere. Unfortunately, commonly used assays that measure cap-methyltransferase activity are best suited for low-throughput screening applications because they: (a) use radioactivity, (b) require a substrate that is laborious to prepare, (c) measure a product that requires chromatographic characterization, or, must be precipitated, filtered, and counted. The surrogate ligand assay that we propose to develop: (a) does not require radioactive reagents, (b) can be implemented robotically with simple liquid addition transfers ("mix and measure"), and (c) gives a reproducible readout that is stable over several hours. The long-range goal of these studies is to extend the type of primary assay described herein to the discovery of a broader range of active compounds that includes methyltransferases capable of modifying other substrates - proteins, RNA, or DNA. PUBLIC HEALTH RELEVANCE: High throughput screening is an essential part of the discovery of new drugs. The process starts with the discovery/design of compounds that affect physiological functions at the cell or enzyme level, followed by determination of their suitability for use as drugs. We propose to develop high throughput screens for the first phase of this process to enable the discovery of compounds that inhibit methyltransferases, a group of enzymes that play a direct role in various aspects of stroke, heart attack, cancer, and infectious diseases.