Selective Inhibitors of the Artemis Endonuclease

描述(由申请人提供)：该项目提出了一种用于人类核酸酶抑制剂Artemis的高通量生化筛选。寻找Artemis抑制剂的理由是它会减缓人类急性淋巴细胞白血病(ALL)细胞的生长，几乎没有毒性。大多数患者的ALL细胞表达抗原受体基因组装的重组酶(称为RAG基因或RAG1和2)。在这个重组过程中(称为V(D)J重组)，产生了一种不寻常的DNA结构中间体，即DNA发夹。如果发夹没有打开，那么就会出现有害的染色体断裂。人类所有具有Artemis基因敲除的细胞生长明显慢于它们起源的野生型细胞。这与Artemis是人类细胞中唯一能正确打开这些发夹的核酸酶的事实相一致。Artemis是一种末端核酸酶，它不仅具有发夹开放活性，而且还具有5‘和3’末端核酸酶的悬垂切割活性，在双链DNA断裂的修复中起重要作用。因此，Artemis抑制剂不仅在ALL中有用，而且在其他癌症治疗中也有用，如果与放射或 DNA断裂化疗。该项目旨在确定Artemis的小分子抑制剂。我们开发了一种可靠的高通量筛选(HTS)分析方法，它依赖于Artemis对猝灭的荧光DNA底物的核裂解，从而释放荧光信号。该分析方法可分别在384孔板或1536孔板中以4~10 UP体积进行。在目标1中，我们与位于加利福尼亚州拉荷亚的Sanford-Burnham医学研究所的Conrad Prebys化学基因组学中心(CPCCG)合作，对Artemis的小分子抑制剂进行HTS。在目标2中，我们使用HTS生化分析进行二次筛选，使用3‘端核酸酶底物分析。我们还有一种三级化验方法，每天可以处理128种化合物。在目标3中，我们测试了其特异性和一般的细胞毒性。我们的人类ALL Artemis KO细胞在区分具有非靶标细胞毒性的化合物方面很有用。我们的细胞V(D)J重组分析对于检测特异性也很有用。这项申请得到了南加州大学诺里斯综合癌症中心和桑福德-伯纳姆CPCCG经验丰富的药物发现科学家的坚定承诺的支持。南加州大学的异种移植All Core将促进该提案之后的小鼠模型研究，以及将候选化合物转移到临床试验的药物开发(实验治疗)计划。1

DESCRIPTION (provided by applicant): this project proposes a high through-put biochemical screen for inhibitors of a human nuclease called Artemis. The rationale for seeking an Artemis inhibitor is that it would slow the growth of acute lymhoblastic leukemia (ALL) cells with little o no toxicity in humans. The ALL cells of most patients express the recombination enzymes for antigen receptor gene assembly (called the RAG genes or RAG1 and 2). During this recombination process (called V (D) J recombination), an unusual DNA structural intermediate is created, a DNA hairpin. If the hairpin is not opened, then a deleterious chromosome break arises. Human ALL cells that have a genetic knockout of Artemis grow significantly slower than their wild type cells of origin. This is consistent with the fact that Artemis is the only nucleasein human cells that can correctly open these hairpins. Artemis is an end nuclease which not only has hairpin opening activity, but also 5' and 3' end nuclease overhang cleavage activity, which are important in repair of double-strand DNA breaks. Therefore, Artemis inhibitors would be useful not only in ALL but also in other cancer therapies, if used in combination with radiation or DNA breakage chemotherapies. This project is directed at identifying small molecule inhibitors of Artemis. We have developed a robust high throughput screen (HTS) assay that relies on Artemis nucleolytic cleavage of a quenched fluorescent DNA substrate, thereby releasing a fluorescence signal. This assay can be done in 4 to 10 up volumes in 384 or 1536 well plates, respectively. In Aim 1, we work with the Conrad Prebys Center for Chemical Genomics (CPCCG) at the Sanford-Burnham Medical Research Institute in La Jolla, CA to carry out a HTS for small molecule inhibitors of Artemis. In Aim 2, we do a secondary screen using a HTS biochemical assay using a 3' end nuclease substrate assay. We also have a tertiary assay that can process 128 compounds per day. In Aim 3, we test the specificity and general cellular toxicity. Our human ALL Artemis KO cells are useful in discriminating compounds that have off-target cellular toxicity. Our cellular V (D) J recombination assays are useful for testing specificty as well. The application is supported by strong commitments from experienced drug discovery scientists at the USC Norris Comprehensive Cancer Center and at the Sanford-Burnham CPCCG. A Xenograft ALL Core at USC will facilitate mouse model studies subsequent to this proposal, and a Drug Development (Experimental Therapeutics) Program for movement of candidate compounds to clinical trials. 1