Riboswitch Based Methyltransferase HTS Assay for 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检测依赖于SAH的酶促转化为可检测的产物，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。为了克服这一技术差距，我们建议利用自然产生的SAH结合RNA适配子或“核糖开关”的精致选择性和亲和力，这种适配子控制细菌中SAM循环基因的表达。这是2002年发现核糖开关的罗纳德·布雷克博士与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1000多个已知的候选SAH核糖开关中确定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。Bellbrook将把这些整合到适用于高通量筛选(HTS)分析的荧光SAH传感器中，并验证其是否可用于检测纯化的HMT。这将是第一个基于适配子的商业化HTS检测，它将克服非常具有挑战性的SAH/SAM识别问题，其选择性至少是抗体的10倍。通过实现对SAH的直接、高灵敏的检测，SAH核糖开关传感器(rsen-SAH)将加速筛选和分析原本难以处理的甲基转移酶靶标，从而为表观遗传药物发现的前景做出重要贡献。

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.