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，用于人类核酸酶抑制剂Artemis。寻找Artemis抑制剂的基本原理是，它可以减缓急性淋巴母细胞白血病（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重组酶（称为RAG基因或RAG1和rag2）。在这个重组过程（称为V (D) J重组）中，一种不寻常的DNA结构中间体被创造出来，即DNA发夹。如果发夹不打开，就会产生有害的染色体断裂。具有阿尔忒弥斯基因敲除的人类ALL细胞的生长速度明显慢于其野生型细胞。这与阿尔忒弥斯是人类细胞中唯一能正确打开这些发夹的核酸酶的事实是一致的。Artemis是一种末端核酸酶，不仅具有发夹开口活性，还具有5‘和3’端核酸酶悬垂裂解活性，在DNA双链断裂的修复中起重要作用。因此，阿尔忒弥斯抑制剂不仅对急性淋巴细胞性白血病有用，而且如果与放疗或药物联合使用，对其他癌症治疗也有用

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