Selective Inhibitors of the Artemis Endonuclease

Artemis 核酸内切酶的选择性抑制剂

• 批准号: 8420339
• 负责人: MICHAEL R LIEBER
• 金额: $ 3.98万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-06 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8420339
• 关键词: Acute; Acute Lymphocytic Leukemia; Adverse effects; Antigen Receptors; Biochemical; Biological; Biological Assay; Cells; Cellular Assay; Chemicals; Chemotherapy-Oncologic Procedure; Chromosomal Breaks; Clinical Trials; DNA; DNA Double Strand Break; DNA Structure; Development; Enzymes; Experimental Drug Development; Fluorescence; Gel; Genes; Genetic; Genetic Recombination; Genomics; Goals; Growth; Human; In Vitro; Inhibitory Concentration 50; Intercalating Agents; Knock-out; Lymphoid; Lymphoid Cell; Lymphoma; Magic; Malignant Neoplasms; Medical Research; Movement; Patients; Pharmaceutical Preparations; Process; Proteins; Radiation; Receptor Gene; Research Institute; Roentgen Rays; Scientist; Signal Transduction; Somatic Cell; Specificity; Structure; T-Lymphocyte; Testing; Therapeutic; Therapeutic Agents; Time; Topoisomerase-II Inhibitor; Toxic effect; Tumor stage; USC/Norris Comprehensive Cancer Center and Hospital; V(D)J Recombination; Work; Xenograft procedure; artemis; base; cancer therapy; cell type; cellular targeting; chemotherapy; drug discovery; endonuclease; experience; high throughput screening; inhibitor/antagonist; killings; leukemia; mouse model; neoplastic cell; nuclease; programs; repaired; scale up; screening; small molecule

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

描述（由申请人提供）：该项目提出了一种高通量生化筛选方法，用于筛选称为 Artemis 的人类核酸酶抑制剂。寻找 Artemis 抑制剂的理由是，它可以减缓急性淋巴细胞白血病 (ALL) 细胞的生长，并且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 RAG1 和 2）。在这个重组过程（称为 V (D) J 重组）中，会产生一种不寻常的 DNA 结构中间体，即 DNA 发夹。如果发夹没有打开，则会出现有害的染色体断裂。具有 Artemis 基因敲除的人类 ALL 细胞的生长速度明显慢于其原始野生型细胞。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这一事实是一致的。 Artemis 是一种末端核酸酶，不仅具有发夹打开活性，还具有 5' 和 3' 末端核酸酶悬垂切割活性，这对于修复双链 DNA 断裂非常重要。因此，Artemis 抑制剂不仅可用于治疗 ALL，而且如果与放射或放射治疗联合使用，也可用于其他癌症治疗。 DNA 断裂化疗。该项目旨在鉴定 Artemis 的小分子抑制剂。我们开发了一种强大的高通量筛选 (HTS) 测定法，该测定法依赖于 Artemis 对淬灭荧光 DNA 底物的溶核切割，从而释放荧光信号。该测定可分别在 384 或 1536 孔板中以 4 至 10 倍体积进行。在目标 1 中，我们与位于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，针对 Artemis 小分子抑制剂开展 HTS。在目标 2 中，我们使用 HTS 生化检测和 3' 末端核酸酶底物检测进行二次筛选。我们还有三级检测，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 重组测定也可用于测试特异性。该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 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