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的表观遗传调节与多种疾病有关，包括癌症、糖尿病和炎症，高通量筛选组蛋白甲基转移酶(HMT)抑制剂是密集的药物发现工作的一个领域。然而，现有的HMT酶分析方法存在重大缺陷，这些都减缓了对这些新兴靶点治疗潜力的探索。检测特定的甲基化事件可能非常复杂，在大多数情况下，检测所有HMT反应的不变产物S同型半胱氨酸将是首选的。然而，HMT是非常差的催化剂，许多对SAM的要求非常低-这些因素的组合对基于SAH的分析方法产生了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，因为它需要一种能够区分SAH和S-腺苷蛋氨酸的试剂，这两种物质只有一个甲基不同。现有的SAH检测在很大程度上依赖于SAH的酶促转化为可检测的产物，并且固有地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。缺乏合适的分析试剂正在拖延，在某些情况下阻碍了对潜在治疗靶点的筛选。为了克服这一技术差距，我们正在使用微生物SAH感测RNA适配子，或“核糖开关”，它以纳摩尔亲和力和精致的选择性结合SAH。在第一阶段，我们建立了这种方法的关键技术可行性，证明了SAH与核糖开关的结合可以在不破坏亲和力或选择性的情况下转化为荧光偏振(FP)和时间分辨Förster共振能量转移(TRRET)信号。为了实现这一点，我们将核糖开关分成两半，这样SAH结合就可以诱导一个三聚体复合体的组装；这种修饰大大提高了核糖开关的灵敏度、选择性和稳定性 信号。我们使用被称为AptaFluor SAH的分离式适配子分析来检测几种HMT产生的SAH，其水平比目前检测的灵敏度极限低几倍。在第二阶段，我们将利用适体和纳米颗粒技术的最新进展，使基于FP-和TRRET的新型分析方法适用于工业HTS，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。此外，我们将开发一种超灵敏的类似ELISA法的方法来检测生物样品中的HMT活性，方法是使用一种创新的分裂适体邻近连接方法。通过在均相中直接、高灵敏度地检测SAH，FP和TRRET AptaFluor SAH检测将为抑制剂发现和领先优化提供一个通用的HMT分析平台，并允许追求其他难以处理的目标。固相AptaFluor SAH分析将有助于发现生物标记物，并为HMT靶向治疗的临床开发开发配套的诊断分析。总而言之，这些进展将加快筛选新的HMT靶点，并以表观遗传学为基础开发治疗癌症、糖尿病和其他疾病的小分子药物。