DEVELOPMENT OF INHIBITORS OF THE DNA METHYLATION PROCESS

主要目标是发现和开发靶向DNA甲基化过程的抗癌药物。DNA甲基化改变和异常表观遗传基因沉默与许多类型癌症的进展完全相关。在许多癌症的进展期间增加的DNA甲基转移酶活性导致“肿瘤抑制基因”、DNA修复基因和细胞-细胞粘附基因的表达丧失。DNA甲基化过程是药物干预的有效靶点，因为异常甲基化的基因可以被特定的药物去甲基化，从而重新激活基因并恢复健康状态。我们最近发现了超过50个小分子抑制剂Dnmt-1，主要的DNA胞嘧啶甲基转移酶，使用高通量筛选纯化的酶。在这个I期提案中，描述了两种方法，它们利用了独特和创新的基于细胞的测定。这些细胞具有编码绿色荧光蛋白（GFP）的表观遗传学沉默基因。我们的目标是通过干扰沉默机制来重新激活表达。将用已经被鉴定为Dnmt-1抑制剂的小分子进行“二次筛选”。“初级筛选”将扩大对干扰沉默机制任何地方的药物的搜索。证明能够刺激GFP再表达的药物将被应用于充分表征的癌细胞模型系统，以在将化合物进入小动物研究之前提供进一步的概念证明。拟议的商业应用：DNA甲基化途径抑制剂对许多形式的癌症的治疗和逆转有很大的希望。我们估计，美国800万非皮肤癌病例中有500万涉及甲基化异常。由于该提案的目的是开发新型抗癌化合物，因此在该提案获得资助后的几年内，人类应用不会取得成果。

The primary objective is to discover and develop anti-cancer drugs that target the DNA methylation process. DNA methylation changes and abnormal epigenetic gene silencing correlate perfectly with the advancement of many types of cancer. An increased DNA methyltransferase activity during the progression of many cancers leads to the loss of expression of "tumor suppressor", DNA repair and cell- cell adhesion genes. The DNA methylation process is a validated target for drug intervention, because aberrantly methylated genes can be demethylated by specific drugs that reactivate genes and restore a healthy condition. We recently discovered over 50 small molecule inhibitors of Dnmt-1, the major DNA cytosine methyltransferase, using a high throughput screen with purified enzyme. In this Phase I proposal, two methods are described that utilize a unique and innovative cell-based assay. The cells have an epigenetically silenced gene encoding the green fluorescent protein (GFP). We aim to reactivate expression by interfering with the silencing mechanism. A `secondary screen' will be done with small molecules already identified as inhibitors of Dnmt-1. A `primary screen' will broaden the search to drugs that interfere anywhere in the silencing mechanism. Drugs that prove capable of stimulating re-expression of GFP will be applied to well characterized cancer cell model systems to provide further proof of concept before entering the compounds into small animal studies. PROPOSED COMMERCIAL APPLICATIONS: DNA methylation pathway inhibitors hold great promise for the treatment and reversal of many forms of cancer. We estimate that 5 million of the 8 million non-skin cancers cases in the US involve methylation abnormalities. Because the aims of this proposal are to develop novel anti-cancer compounds, human applications will not come to fruition for a several of years after this proposal is funded.