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

• 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
• 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

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多种疾病（包括癌症、糖尿病和炎症）有关，组蛋白甲基转移酶（HMT）抑制剂的高通量筛选是一个密集的药物发现领域。然而，现有的HMT酶测定方法存在显著的缺点，并且这些缺点减缓了对这些新兴靶点的治疗潜力的探索。特异性甲基化事件的检测可能相当复杂，并且在大多数情况下优选检测所有HMT反应的不变产物S-腺苷高半胱氨酸（SAH）。然而，HMT是非常差的催化剂，并且许多具有非常低的SAM要求-这些因素的组合对基于SAH的测定方法产生非常严格的灵敏度要求。此外，SAH的直接检测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-腺苷甲硫氨酸（SAM）的试剂，其区别在于单个甲基。可用的SAH测定-其依赖于SAH到可检测产物的酶促转化-固有地易于受到来自筛选化合物的干扰，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，我们建议利用天然存在的SAH结合RNA适体或“核糖开关”的精致选择性和亲和力，其控制细菌中SAM回收基因的表达。这是2002年发现核糖开关的罗纳德布雷克博士和贝尔布鲁克实验室的合作成果。Breaker博士将使用生物信息学方法从1，000多个已知的候选SAH核糖开关中识别具有合适特性的候选SAH核糖开关，并表征最有希望的候选SAH/SAM结合特性。贝尔布鲁克将把这些结合到一个适合高通量筛选（HTS）检测的荧光SAH传感器中，并验证它用于检测纯化的HMT。这将是第一个基于适体的商业HTS检测，它将克服非常具有挑战性的SAH/SAM区分问题，其选择性比抗体高至少10倍。通过实现SAH的直接、高灵敏度检测，SAH核糖开关传感器（rSen-SAH）将加速筛选和分析否则难以处理的甲基转移酶靶点，从而为有前途的表观遗传药物发现领域做出重要贡献。