Development of a haspin kinase assay for high throughput drug screening

描述(由申请人提供)：在细胞增殖过程中，适当控制有丝分裂对维持基因组的稳定性至关重要，而基因组的不稳定可能直接导致癌症的产生。因此，研究调节有丝分裂的机制对于了解癌症是如何发展的，以及发现预防和治疗这种疾病的新方法至关重要。有丝分裂也是癌症治疗的重要靶点。最近，新的选择性抗有丝分裂药物，如极光激酶抑制剂，在临床前实验中显示出巨大的前景，现在人们对寻找有丝分裂的新药物靶点产生了极大的兴趣。我们最近发现了一种新的有丝分裂组蛋白激酶，haspin，它在不同的真核生物中具有同源物。Haspin基因在增殖期细胞中表达，但在非增殖期细胞中不表达。在有丝分裂过程中，haspin与浓缩的染色体结合，特别是在着丝粒上，并负责组蛋白H3中Thr-3的磷酸化。Haspin也存在于有丝分裂中心体。重要的是，haspin RNA干扰导致中期染色体错位和纺锤体缺陷，阻止正常有丝分裂的完成。这些研究将haspin添加到调节有丝分裂染色体动态和纺锤体活动的精选激酶组中，并提供了第一个迹象，表明haspin与极光激酶一样，可能是癌症治疗的合适靶点。然而，haspin在有丝分裂中作用的进一步研究以及haspin作为抗癌药物靶点的验证目前受到缺乏该激酶的特定小分子抑制剂的限制。为了鉴定haspin的小分子抑制物，我们将建立一种适合高通量筛选化学文库的体外haspin激酶检测方法。在目标1中，我们将在大肠杆菌或杆状病毒系统中生产具有功能的全长重组haspin，用于筛选试验。在目标2中，我们将建立和优化haspin的均相时间分辨荧光蛋白测定法。还描述了使用基于分离的方法的替代策略。在目标3中，我们概述了从筛选过程中确认HITS的方法，并开发了二次筛选来评估这些化合物在体外和细胞中的抑制特性和功能效果。Haspin抑制剂将为研究细胞分裂的基本生物学提供一种新的方法，并将产生使用现有技术无法获得的见解。此外，这些抑制剂将提供一个很好的方法来验证haspin作为癌症治疗的靶点，它们可能会直接用作化疗药物。

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.