Selective Inhibitors of the Artemis Endonuclease

Artemis 核酸内切酶的选择性抑制剂

基本信息

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
项目类别：
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

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

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项目摘要

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）：该项目提出了一个高渗透生物化学筛选，以抑制人类核酸酶的抑制剂。寻求阿耳Nemis抑制剂的理由是，它会减慢急性lymhoblastic白血病（所有）细胞的生长，而在人类中却没有毒性。大多数患者的所有细胞表达了抗原受体基因组装的重组酶（称为RAG基因或RAG1和2）。在此重组过程中（称为V（D）J重组），创建了一个异常的DNA结构中间体，即DNA发夹。如果发夹不打开，则会出现有害的染色体断裂。人类所有具有Artemis遗传敲除的细胞的生长明显慢于其原始细胞。这与artemis是唯一可以正确打开这些发夹的人类细胞的事实一致。 Artemis是一种末端核酸酶，不仅具有发夹开口活性，而且还具有5'和3'末端核酸酶悬垂的裂解活性，这对于修复双链DNA断裂很重要。因此，如果与辐射或 DNA断裂化学疗法。该项目旨在鉴定小分子抑制剂。我们已经开发了一个可靠的高吞吐量筛选（HTS）测定法，该测定依赖于淬灭荧光DNA底物的Artemis核酸裂解，从而释放了荧光信号。该测定可以分别在384或1536井板中进行4至10个上的体积进行。在AIM 1中，我们与加利福尼亚州La Jolla的Sanford-Burnham医学研究所的Conrad Prebys化学基因组学中心（CPCCG）合作，为Artemis的小分子抑制剂进行HTS。在AIM 2中，我们使用3'端核酸酶底物测定法使用HTS生化测定法进行辅助屏幕。我们还有一个第三次测定，每天可以处理128种化合物。在AIM 3中，我们测试了特异性和一般细胞毒性。我们的所有Artemis KO细胞可用于区分具有靶向细胞毒性的化合物。我们的细胞V（d）J重组测定也可用于测试特定。该应用程序得到了USC Norris综合癌症中心和Sanford-Burnham CPCCG的经验丰富的药物发现科学家的坚定承诺的支持。 USC的异种移植物将促进该提案后的小鼠模型研究，以及针对候选化合物向临床试验运动的药物开发（实验治疗）计划。 1