Development of a haspin kinase assay for high throughput drug screening

描述（由申请人提供）：有丝分裂的适当控制对于维持细胞增殖期间基因组的稳定性至关重要，基因组的不稳定性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如何发展以及发现预防和治疗该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，如极光激酶抑制剂已显示出巨大的前景，在临床前实验，现在有巨大的兴趣，在有丝分裂中确定新的药物靶点。我们最近发现了一种新的有丝分裂组蛋白激酶，haspin，在不同的真核生物中有同源物。人haspin mRNA在增殖而非非增殖细胞中表达。在有丝分裂过程中，haspin与浓缩的染色体，特别是在着丝粒，并负责组蛋白H3中Thr-3的磷酸化。Haspin也存在于有丝分裂的中心体。重要的是，haspin RNA干扰导致中期染色体的错位和纺锤体缺陷，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力学和纺锤体活性的选择性激酶组中，并首次表明haspin，如极光激酶，可能是癌症治疗的合适靶点。然而，由于缺乏激酶的特异性小分子抑制剂，目前对haspin在有丝分裂中的作用的进一步研究和对haspin作为癌症药物靶点的验证受到限制。为了鉴定haspin的小分子抑制剂，我们将开发一种适合于高通量筛选化学文库的体外haspin激酶测定法。在目标1中，我们将产生，在E。大肠杆菌或杆状病毒系统，用于筛选测定的功能性全长重组haspin。在目标2中，我们将开发和优化haspin的均相时间分辨荧光激酶测定法。还描述了使用基于分离的方法的替代策略。在目标3中，我们概述了用于确认筛选过程中的命中的测定，并开发了二次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的方法来研究细胞分裂的基本生物学，并将产生使用现有技术无法获得的见解。此外，这样的抑制剂将提供一个很好的方式来验证haspin作为癌症治疗的目标，他们可能会发现作为化疗药物的直接应用。

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.