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: 9266793
• 负责人: Robert G Lowery
• 金额: $ 39.02万
• 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 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9266793
• 关键词: Affinity; Antineoplastic Agents; Area; Binding; Biochemical; Biological; Biological Assay; Chemicals; Chemistry; Complex; Detection; Development; Diabetes Mellitus; Disease; Drug Targeting; Elements; Energy Transfer; Enzyme-Linked Immunosorbent Assay; Enzymes; Epigenetic Process; Event; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Freezing; Gene Expression Regulation; Immobilization; Industrialization; Inflammation; Lead; Ligation; Malignant Neoplasms; Methods; Methylation; Methyltransferase; Modification; Performance; Phase; Production; Quantum Dots; RNA; Reaction; Reagent; Research Personnel; Resort; S-Adenosylhomocysteine; S-Adenosylmethionine; Sampling; Signal Transduction; Site; Solid; Technology; Therapeutic; Time; aptamer; assay development; base; biomarker development; biomarker discovery; catalyst; clinical assay development; commercialization; companion diagnostics; diagnostic assay; drug discovery; epigenetic drug; epigenetic regulation; high throughput screening; histone methyltransferase; improved; inhibitor/antagonist; innovation; methyl group; microbial; molecular recognition; nanomolar; nanoparticle; novel; prevent; public health relevance; scale up; screening; sensor; small molecule; stability testing; targeted treatment; therapeutic target

 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 rely largely on enzymatic conversion of SAH to a detectable product, and are inherently prone to interference from screening compounds and lack the sensitivity needed for detection of some methyltransferases. The lack of suitable assay reagents is delaying and in some cases preventing the screening of potential therapeutic targets. To overcome this technical gap, we are using microbial SAH-sensing RNA aptamers, or "riboswitches", that bind SAH with nanomolar affinity and exquisite selectivity. In Phase I, we established the critical technical feasibility for this approach by showing that SAH binding to a riboswitch can be transduced into fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals without disrupting affinity or selectivity. To achieve this, we split the riboswitch into two halves, such that SAH binding induces assembly of a trimeric complex; this modification vastly improved the sensitivity, selectivity and stability of the signaling. We used the split aptamer assays, called AptaFluor SAH, to detect SAH produced by several HMTs at levels several-fold below the sensitivity limit for current assays. In Phase II we will leverage recent advances in aptamer and nanoparticle technologies to make the novel FP- and TR-FRET based assays suitable for industrial HTS, validate them extensively for inhibitor screening and profiling with HMTs, and establish stability and manufacturing aspects required for commercialization. In addition, we will develop an ultrasensitive ELISA-like assay for detecting HMT activity in biological samples using an innovative split aptamer proximity ligation method. By enabling direct, highly sensitive detection of SAH in homogenous the FP and TR-FRET AptaFluor SAH assay will provide a universal HMT assay platform for inhibitor discovery and lead optimization and allow pursuit of otherwise intractable targets. The solid phase AptaFluor SAH assay will enable discovery of biomarkers and development of companion diagnostic assays for clinical development of HMT targeted therapies. Taken together these developments will accelerate screening of new HMT targets and development of small molecule drugs for cancer, diabetes and other diseases with an epigenetic basis.

 描述（由申请人提供）：通过甲基化对基因表达进行表观遗传调控与包括癌症、糖尿病和炎症在内的多种疾病有关，而组蛋白甲基转移酶（HMT）抑制剂的高通量筛选是药物发现工作的一个重点领域。现有的 HMT 酶测定方法存在显着的缺点，这些缺点减缓了对这些新兴靶点的治疗潜力的探索，特定甲基化事件的检测可能相当复杂，并且检测这些新兴靶点的治疗潜力可能会相当复杂。 S-腺苷高半胱氨酸 (SAH) 是所有 HMT 反应的不变产物，在大多数情况下是首选。然而，HMT 是非常差的催化剂，而且许多催化剂的 SAM 要求非常低 - 这些因素的结合对 HMT 产生了非常严格的灵敏度要求。此外，基于 SAH 的检测方法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，两者的区别在于。现有的 SAH 检测很大程度上依赖于 SAH 酶促转化为可检测的产物，并且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，因此缺乏合适的检测试剂。为了克服这一技术差距，我们正在使用微生物 SAH 传感 RNA 适体或“核糖开关”，以纳摩尔亲和力结合 SAH，并在某些情况下阻止潜在治疗靶点的筛选。在第一阶段，我们通过证明 SAH 与核糖开关的结合可以转变成荧光偏振 (FP) 和时间分辨福斯特共振能量转移 (TR-FRET) 信号而不会破坏亲和力或为了实现这一点，我们将核糖开关分成两半，这样 SAH 结合就会诱导三聚体复合物的组装；这种修饰极大地提高了灵敏度、选择性和稳定性。 我们使用称为 AptaFluor SAH 的分离适体检测来检测多种 HMT 产生的 SAH，其水平比当前检测的灵敏度极限低几倍。适合工业 HTS 的新型 FP 和 TR-FRET 测定法，广泛验证它们用于抑制剂筛选和 HMT 分析，并建立商业化所需的稳定性和制造方面。开发一种超灵敏的类似 ELISA 的检测方法，使用创新的分割适体邻近连接方法检测生物样品中的 HMT 活性。通过在同质中直接、高灵敏度地检测 SAH，FP 和 TR-FRET AptaFluor SAH 检测将提供一个通用的 HMT 检测平台。固相 AptaFluor SAH 测定将能够发现生物标志物并开发用于 HMT 靶向临床开发的伴随诊断测定。总而言之，这些进展将加速新 HMT 靶点的筛选以及针对癌症、糖尿病和其他具有表观遗传学基础的疾病的小分子药物的开发。