Development of a haspin kinase assay for high throughput drug screening

开发用于高通量药物筛选的 haspin 激酶测定法

• 批准号: 7256902
• 负责人: JONATHAN M HIGGINS
• 金额: $ 22.6万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-07 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7256902
• 关键词: Antineoplastic Agents; Baculoviruses; Biological Assay; Biology; Cell Proliferation; Cell division; Cells; Cellular Assay; Centromere; Centrosome; Chemicals; Chromosomes; Defect; Development; Development Plans; Disease; Drug Delivery Systems; Escherichia coli; Eukaryota; Eukaryotic Cell; Fluorescence; Generations; Genome Stability; Genomic Instability; Haspin; Histone H3; Homologous Gene; Human; In Vitro; Inhibitory Concentration 50; Insecta; Length; Malignant Neoplasms; Messenger RNA; Metaphase; Mitosis; Mitotic; Mitotic Chromosome; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Preclinical Drug Evaluation; Process; Production; Proliferating; Property; Protamine Kinase; RNA Interference; Recombinants; Screening procedure; System; Technology; Time; Validation; aurora kinase; base; cancer therapy; direct application; high throughput screening; inhibitor/antagonist; insight; interest; kinase inhibitor; novel; novel strategies; pre-clinical; prevent; research study; small molecule; small molecule libraries

DESCRIPTION (provided by applicant): Proper control of mitosis is critical to maintain the stability of the genome during cell proliferation, and genomic instability may contribute directly to the generation of cancer. Study of mechanisms that regulate mitosis, therefore, is critical to understand how cancer develops, and to discover new ways to prevent and treat the disease. Mitosis is also an important target for cancer therapy. Recently, new selective anti-mitotic drugs such as aurora kinase inhibitors have shown great promise in pre-clinical experiments, and there is now immense interest in identifying new drug targets in mitosis. We have recently discovered a novel mitotic histone kinase, haspin, that has homologs in diverse eukaryotes. Human haspin mRNA is expressed in proliferating but not non-proliferating cells. During mitosis, haspin associates with condensed chromosomes, particularly at centromeres, and is responsible for phosphorylation of Thr-3 in histone H3. Haspin is also found at mitotic centrosomes. Importantly, haspin RNA interference causes misalignment of metaphase chromosomes and spindle defects, preventing completion of normal mitosis. These studies add haspin to the select group of kinases that regulate mitotic chromosome dynamics and spindle activity and provide the first indication that haspin, like the aurora kinases, might be a suitable target for cancer therapy. Further study of haspin action in mitosis and validation of haspin as a cancer drug target are currently limited, however, by the lack of specific small molecule inhibitors of the kinase. To identify small molecule inhibitors of haspin, we will develop an in vitro haspin kinase assay suitable for high-throughput screening of chemical libraries. In Aim 1 we will produce, in E. coli or the baculovirus system, functional full-length recombinant haspin for use in screening assays. In Aim 2 we will develop and optimize a homogenous time-resolved fluorescence kinase assay for haspin. An alternative strategy using a separation-based approach is also described. In Aim 3, we outline assays to confirm hits from the screening process and develop secondary screens to assess the inhibitory properties and functional effects of these compounds in vitro and in cells. Haspin inhibitors will provide a new approach to investigate the basic biology of cell division and will yield insights that cannot be obtained using existing technology. Furthermore, such inhibitors will provide an excellent way to validate haspin as a target for cancer treatment, and they might find direct application as chemotherapeutic drugs.

描述（由申请人提供）：正确控制有丝分裂对于维持细胞增殖过程中基因组的稳定性至关重要，基因组不稳定性可能直接有助于癌症的产生。因此，研究调节有丝分裂的机制对于了解癌症的发展方式并发现预防和治疗疾病的新方法至关重要。有丝分裂也是癌症治疗的重要靶标。最近，新的选择性抗溶毒药物（例如Aurora激酶抑制剂）在临床前实验中表现出了很大的希望，现在对鉴定有丝分裂的新药物靶标具有巨大的兴趣。我们最近发现了一种新型有丝分裂组蛋白激酶Haspin，该激酶具有多种真核生物的同源物。人haspin mRNA在增殖但不增殖细胞中表达。在有丝分裂期间，Haspin与冷凝的染色体相关，尤其是在丝粒中，并负责组蛋白H3中THR-3的磷酸化。在有丝分裂的中心体中也发现了haspin。重要的是，Haspin RNA干扰会导致中期染色体和纺锤体缺陷的未对准，从而阻止正常有丝分裂的完成。这些研究为调节有丝分裂染色体动力学和主轴活性的精选激酶增加了HASPIN，并提供了第一个指示，即Haspin（如Aurora激酶）可能是癌症治疗的合适靶标。然而，由于缺乏特异性小分子抑制剂，目前，进一步研究了Haspin在有丝分裂和验证Haspin作为癌症药物靶靶标的有促有丝分裂和验证中的进一步研究。为了鉴定Haspin的小分子抑制剂，我们将开发一种体外HASPIN激酶测定法，适合于化学文库的高通量筛选。在AIM 1中，我们将在大肠杆菌或杆状病毒系统中生产功能性的全长重组HASPIN，以用于筛选分析。在AIM 2中，我们将开发并优化Haspin的同质时间分辨荧光激酶测定。还描述了使用基于分离方法的替代策略。在AIM 3中，我们概述了确认筛选过程中的命中并开发次级筛选以评估这些化合物在体外和细胞中的抑制特性和功能效应。 Haspin抑制剂将提供一种新方法来研究细胞分裂的基本生物学，并会产生无法使用现有技术获得的见解。此外，这种抑制剂将为验证Haspin作为癌症治疗的靶标提供一种绝佳的方法，并且他们可能会直接应用作为化学治疗药物。