Selective Inhibitors of the Artemis Endonuclease

描述（由申请人提供）：该项目提出了一种高通量的生物化学筛选方法，用于筛选一种名为Artemis的人类核酸酶抑制剂。寻找Artemis抑制剂的理由是，它可以减缓急性白血病（ALL）细胞的生长，对人体几乎没有毒性。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RAG 1和2）。在这个重组过程（称为V（D）J重组）中，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。具有Artemis基因敲除的人类ALL细胞生长明显慢于其野生型起源细胞。这与Artemis是人类细胞中唯一能够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，它不仅具有发夹打开活性，而且具有5'和3'末端核酸酶突出端切割活性，这在双链DNA断裂的修复中是重要的。因此，Artemis抑制剂不仅可用于ALL，而且可用于其他癌症治疗，如果与放射或化疗联合使用的话。 DNA断裂化疗。该项目旨在鉴定Artemis的小分子抑制剂。我们已经开发了一种稳健的高通量筛选（HTS）测定，其依赖于Artemis对淬灭的荧光DNA底物的溶核裂解，从而释放荧光信号。该测定可以分别在384或1536孔板中以4至10倍体积进行。在目标1中，我们与加利福尼亚州拉霍亚的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生物化学测定法进行二次筛选，所述HTS生物化学测定法使用3'端核酸酶底物测定法。我们还有一个三级检测，每天可以处理128种化合物。在目标3中，我们测试了特异性和一般细胞毒性。我们的人类ALL Artemis KO细胞可用于区分具有脱靶细胞毒性的化合物。我们的细胞V（D）J重组检测也可用于检测特异性。该申请得到了USC Norris综合癌症中心和Sanford-Burnham CPCCG经验丰富的药物发现科学家的大力支持。南加州大学的异种移植ALL核心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