High H3-K9 Methyltransferase Inhibitor Screen (RMI)

描述(由申请人提供)：肿瘤的发展和进展早已被认为涉及肿瘤细胞基因表达谱中的遗传和表观遗传改变。尤其重要的是肿瘤抑制基因的沉默。基因沉默的表观遗传学机制包括组蛋白去乙酰化、DMA甲基化和组蛋白甲基化。这些机制中的每一个都是相互关联、相互影响的。组蛋白去乙酰化和DMA甲基化的抑制剂都是可用的，并被用作有希望的抗癌药物。然而，直到最近，组蛋白甲基化的作用才变得明显。H3-K9甲基转移酶与肿瘤抑制基因的功能、细胞衰老和调节端粒长度有关。在乳腺癌和前列腺癌中，H3-K27甲基转移酶EZH2过度表达，是侵袭性或转移性肿瘤的一个强有力的预后因素。此外，特定的转录因子招募单个组蛋白甲基转移酶，这是其基因沉默功能所必需的。组蛋白甲基化与肿瘤和细胞生长控制之间的这些快速发展的联系表明了一个令人兴奋的新治疗靶点。然而，现有技术限制了我们快速追求这些发展的能力。值得注意的是，不存在组蛋白甲基化的特定抑制物。这项建议旨在通过开发一种简单、定量的高通量分析来消除这一限制，该方法能够单独测量每个组蛋白H3赖氨酸甲基化酶的活性。为了实现这一目标，我们的具体目标是：1)利用竞争性荧光偏振技术将现有的组蛋白甲基转移酶检测方法转化为高通量的检测方法。这一目标将把现有的技术和试剂结合起来，形成一种灵敏而快速的检测方法。该分析最初将被使用，并在NCI结构多样性化合物集上进行进一步测试，以识别用于药物发现的先导化合物。

DESCRIPTION (provided by applicant): Tumor development and progression has long been known to involve both genetic and epigenetic alterations in the gene expression profile of tumor cells. Of particular importance is the silencing of tumor suppressor genes. Epigenetic mechanisms of gene silencing include histone deacetylation, DMA methylation, and histone methylation. Each of these mechanisms are interrelated and influence each other. Inhibitors of both histone deacetylation and DMA methylation are available and are in use as promising anti-cancer agents. However, only recently has the role of histone methylation become apparent. H3-K9 methyltransferases have been linked to the function of tumor suppressor genes, cellular senescence and regulating telomere length. In both breast and prostate cancer the H3-K27 methyltransferase EZH2 is over-expressed and is a strong prognostic factor for aggressive or metastatic tumors. In addition specific transcription factors recruit individual histone methyltransferases necessary for their gene silencing function. These rapidly developing links between histone methylation and tumors and cell growth control suggest an exciting new therapeutic target. However, existing technologies limit our ability to rapidly pursue these developments. Strikingly, no specific inhibitors of histone methylation exist. This proposal seeks to eliminate this limitation by developing a simple, quantitative high throughput assay capable of individually measuring the activity of each of the histone H3 lysine methylating enzymes. To this goal the specific aims are: 1) Migrate existing histone methyltransferase assays into a high throughput format using competitive fluorescence polarization. This aim will combine existing technologies and reagents into a sensitive and rapid assay. The assay will be initially utilized and further tested on the NCI Structural Diversity set of compounds to identify lead compounds for drug discovery.