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（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测定主要依赖于SAH到可检测产物的酶促转化，并且固有地易于受到来自筛选化合物的干扰，并且缺乏检测一些甲基转移酶所需的灵敏度。缺乏合适的测定试剂正在延迟并且在某些情况下阻止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传感RNA适体，或“核糖开关”，结合SAH与纳摩尔亲和力和精致的选择性。在第一阶段，我们通过证明SAH与核糖开关的结合可以被转换成荧光偏振（FP）和时间分辨的Förster共振能量转移（TR-FRET）信号，而不会破坏亲和力或选择性，建立了这种方法的关键技术可行性。为了实现这一点，我们将核糖开关分成两半，使得SAH结合诱导三聚体复合物的组装;这种修饰极大地提高了检测的灵敏度、选择性和稳定性。 信号。我们使用称为AptaFluor SAH的分裂适体测定法来检测由几种HMT产生的SAH，其水平低于当前测定法的灵敏度限度几倍。在第二阶段，我们将利用适体和纳米颗粒技术的最新进展，使新的FP和TR-FRET为基础的测定适用于工业HTS，验证他们广泛的抑制剂筛选和分析与HMT，并建立稳定性和商业化所需的生产方面。此外，我们将开发一种超灵敏的ELISA样测定法，用于使用创新的分裂适体邻近连接方法检测生物样品中的HMT活性。通过实现SAH的直接、高灵敏度检测，FP和TR-FRET AptaFluor SAH测定将为抑制剂发现和先导物优化提供通用HMT测定平台，并允许追求其他难以处理的靶标。固相AptaFluor SAH检测将能够发现生物标志物并开发用于HMT靶向治疗临床开发的伴随诊断检测。总之，这些发展将加速筛选新的HMT靶点，并开发用于癌症，糖尿病和其他具有表观遗传基础的疾病的小分子药物。

 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.