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（由适用提供）：通过甲基化对基因表达的表观遗传调节已在包括癌症，糖尿病和注射（包括组蛋白甲基转移酶（HMT）抑制剂的高吞吐量筛选）中实施，是强烈的药物发现努力的领域。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是一个非常挑战的分子识别问题，因为它需要能够区分SAH和S-腺苷甲硫氨酸（SAM）的试剂，该试剂通过单个甲基的不同。可用的SAH分析很大程度上依赖于SAH向可检测产物的酶促转化，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。潜在的治疗靶标。为了克服这一技术差距，我们使用的是微生物SAH传感RNA适体或“核糖开关”，该核能与纳摩尔亲和力和独家选择性结合SAH。在第一阶段，我们通过表明SAH与核糖开关的结合可以转移到荧光极化（FP）和时间分析的Försterresonance Energy传递（TR-FRET）信号而不破坏亲和力或选择性的情况下，确定了这种方法的关键技术可行性。为了实现这一目标，我们将核糖开关分为两半，因此SAH结合会诱导三聚体复合物的组装。这种修改极大地提高了灵敏度，选择性和稳定性 信号传导。我们使用了称为aptafluor SAH的分裂的Aptertn分析，以检测由几个HMT产生的SAH，低于当前测定的灵敏度极限的水平。在第二阶段，我们将利用Apattern和Nanoparpicle技术的最新进展，使新型的FP和TR-FRET测定法适用于工业HTS，对它们进行广泛验证以使用HMT进行抑制剂筛查和分析，并建立商业化所需的稳定性和制造方面。此外，我们将开发一种超敏感的ELISA样测定法，用于使用创新的分裂放大器接近结扎方法在生物样品中检测HMT活性。通过启用对同质的直接，高度敏感的SAH检测，FP和TR-FRET APTAFLUOR SAH分析将为抑制剂发现和铅优化提供通用的HMT测定平台，并允许追求其他棘手的目标。固相的APTAFLUOR SAH分析将使生物标志物和开发伴侣诊断测定法，以用于HMT靶向疗法的临床开发。这些发展将加速筛查新的HMT靶标，并以表观遗传基础的癌症，糖尿病和其他疾病的小分子药物开发。