Riboswitch Based Methyltransferase HTS Assay for Epigenetic Drug Discovery

基于核糖开关的甲基转移酶 HTS 测定用于表观遗传药物发现

基本信息

批准号：
8646158
负责人：
Robert G Lowery
金额：
$ 22.49万
依托单位：
BELLBROOK LABS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-15 至 2016-01-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8646158
关键词：
Affinity Agreement Antibodies Antineoplastic Agents Area Bacteria Binding Biochemical Bioinformatics Biological Assay Biology Cellular biology Chemicals Collaborations Coupled DNA DNA Sequence Rearrangement Detection Developmental Biology Diabetes Mellitus Discrimination Disease Drug Targeting Energy Transfer Enzymatic Biochemistry Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Epigenetic Process Event Exhibits Fluorescence Resonance Energy Transfer Gene Expression Regulation Genes In Vitro Inflammation Ligands Malignant Neoplasms Methods Methylation Methyltransferase Modification Molecular Biology Nucleosomes Oligonucleotides Peptides Property RNA Binding Reaction Reagent Recombinants Recycling Regulation S-Adenosylhomocysteine S-Adenosylmethionine Signal Transduction Structure Temperature Testing Therapeutic Time Universities Variant aptamer assay development base catalyst drug candidate drug discovery high throughput screening histone methyltransferase improved inhibitor/antagonist knowledge base methyl group microbial molecular recognition public health relevance screening sensor stem success thermophilic organism

项目摘要

DESCRIPTION (provided by applicant): Epigenetic regulation of gene expression via methylation has been implicated in diverse diseases including cancer, diabetes and inflammation, and high throughput screening for histone methyltransferase (HMT) inhibitors is an area of intense drug discovery effort. However, there are significant shortcomings with existing HMT enzyme assay methods, and these are slowing exploration of the therapeutic potential of these emerging targets. Detection of specific methylation events can be quite complicated, and detection of S-adenosylhomocysteine (SAH), the invariant product of all HMT reactions, would be preferred in most cases. However, HMTs are very poor catalysts and many have very low SAM requirements - a combination of factors that creates very stringent sensitivity requirements for SAH-based assay methods. Moreover, direct detection of SAH is a very challenging molecular recognition problem as it requires a reagent capable of discriminating between SAH and S- adenosylmethionine (SAM), which differ by a single methyl group. The available SAH assays - which rely on enzymatic conversion of SAH to a detectable product - are inherently prone to interference from screening compounds and lack the sensitivity needed for detection of some methyltransferases. To overcome this technical gap, we propose to leverage the exquisite selectivity and affinity of naturally occurring SAH-binding RNA aptamers, or "riboswitches", that control the expression of SAM recycling genes in bacteria. This is a collaborative effort between Dr. Ronald Breaker, who discovered riboswitches in 2002, and BellBrook Labs. Dr. Breaker will use a bioinformatics approach to identify candidate SAH riboswitches with suitable properties from more than 1,000 that are known and characterize the SAH/SAM binding properties of the most promising candidates. BellBrook will incorporate these into a fluorescent SAH sensor suitable for high-throughput screening (HTS) assays and validate it for detection of purified HMTs. This will be the first commercial HTS assay based on an aptamer, and it will overcome the very challenging SAH/SAM discrimination problem with at least 10- fold greater selectivity than has been possible with antibodies. By enabling direct, highly sensitive detection of SAH, the SAH riboswitch sensor (rSen-SAH), will accelerate the screening and profiling of otherwise intractable methyltransferase targets, and thereby make an important contribution to the promising field of epigenetic drug discovery.

描述（由申请人提供）：通过甲基化对基因表达的表观遗传调节与包括癌症，糖尿病和炎症在内的多种疾病有关，以及对组蛋白甲基转移酶（HMT）抑制剂的高吞吐量筛查是强烈的药物发现工作的领域。但是，现有的HMT酶测定方法存在很大的缺点，并且这些方法正在放缓对这些新兴靶标的治疗潜力的探索。在大多数情况下，检测特定的甲基化事件可能非常复杂，并且所有HMT反应的不变产物S-腺苷硬同星半胱氨酸（SAH）的检测将是优选的。但是，HMT是非常差的催化剂，许多SAM需求非常低 - 对基于SAH的测定方法产生非常严格的灵敏度要求的组合。此外，直接检测SAH是一个非常具有挑战性的分子识别问题，因为它需要能够区分SAH和S-腺苷甲硫代氨酸（SAM）的试剂，该试剂通过单个甲基的不同。可用的SAH测定方法 - 依赖于SAH到可检测的产品的酶促转化 - 本质上容易受到筛选化合物的干扰，并且缺乏检测某些甲基转移酶所需的灵敏度。为了克服这一技术差距，我们建议利用自然存在的SAH结合RNA适体或“核糖开关”的精致选择性和亲和力，以控制细菌中SAM回收基因的表达。这是Ronald Breaker博士在2002年发现Riboswitches和Bellbrook Labs之间的合作努力。 Breaker博士将使用一种生物信息学方法来识别候选SAH核糖开关，具有来自1,000多个已知和表征最有前途的候选人的SAH/SAM结合特性的合适特性。 Bellbrook将将它们结合到适合高通量筛选（HTS）测定的荧光SAH传感器中，并验证其以检测纯化的HMT。这将是基于适体的第一个商业HTS分析，它将克服非常具有挑战性的SAH/SAM歧视问题，至少10倍的选择性比抗体具有更大的选择性。通过启用对SAH的直接，高度敏感的检测，SAH核糖开关传感器（RSEN-SAH）将加速筛选和分析原本可怕的甲基转移酶靶标，从而对表观遗传药物发现的有前途的领域做出重要贡献。