Methyltransferase inhibition assay development

描述（由申请人提供）：甲基转移酶在广泛的重要过程中介导生物转化-这些过程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。甲基转移酶也在越来越多的癌细胞类型的基因组中的表观遗传改变中发挥重要作用。甲基转移酶所起的各种关键作用使其成为发现差异调节其活性的化合物的有吸引力的靶点。本提案的目标是设计一种简单的可重复的高通量酶基测定法，以促进发现抑制真核mRNA中存在的5 '-mRNA“帽”（一种序列，5'-GpppA）甲基化的试剂。在拟议的研究中，登革病毒编码的帽甲基转移酶将被用作模型酶靶标，其将部分甲基化的帽转化为完全甲基化的帽，S-腺苷甲硫氨酸作为甲基供体。在所提出的测定中，将使用荧光偏振测量荧光“替代”肽适体与靶甲基转移酶的缔合。所提出的测定是一种“混合测量”类型的测定，其基于以下原理：在离散位点处通过药物样化合物对蛋白质的扰动可以扰动结合在别处的另一种配体（替代配体）的结合。不幸的是，测量帽-甲基转移酶活性的常用测定法最适合于低通量筛选应用，因为它们：（a）使用放射性，（B）需要费力制备的底物，（c）测量需要色谱表征的产物，或者必须沉淀、过滤和计数。我们建议开发的替代配体测定：（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”）自动执行，以及（c）提供可重复的读数，该读数在几个小时内稳定。这些研究的长期目标是将本文所述的初级测定的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-蛋白质、RNA或DNA的甲基转移酶。公共卫生相关性：高通量筛选是新药发现的重要组成部分。该过程从发现/设计在细胞或酶水平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。我们建议为该过程的第一阶段开发高通量筛选，以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.