A high-throughput screen for inhibitors of Plk1-interacting checkpoint helicase (PICH)

项目总结/摘要 最近的研究表明，PIK 1相互作用的检查点解旋酶（PICH）是一种潜在的癌症治疗剂 目标PICH在有丝分裂中对染色体分离的保真度具有关键作用， 是后期超细DNA桥（UFB）的频率增加，这导致多核化， 染色体丢失其他在有丝分裂中具有关键功能的蛋白质，以及精确染色体定位所需的蛋白质 分离，已被证明是癌症药物的有效靶点。例如，靶向微管的药物 动力学重要的是，发现PICH在乳腺癌细胞中过表达，并且PICH的缺失是乳腺癌细胞中的一个重要因素。 发现对三阴性乳腺癌细胞具有极强的毒性，而不影响匹配的正常组织。 由于PICH在一系列癌症中过度表达，因此可能许多癌症类型需要PICH 生存的功能。因此，小分子PICH抑制剂具有改善患者预后的潜力， 癌症治疗然而，尚未鉴定出PICH的抑制剂。在这里，我们建议识别选择性 PICH抑制剂，并确定其对一系列癌症类型的细胞毒性，其中PICH过度- 表达。由于PICH是dsDNA依赖性ATP酶，因此其具有适合于高通量分析的活性。 使用不同的小分子文库筛选（HTS）抑制剂。我们已经建立了生物化学 活性PICH的纯化，我们已经设计了策略，并产生了工具， 有效地进行二次筛选，以验证真正的PICH抑制剂， 消除非特异性抑制剂。将确定PICH抑制的细胞后果， 表征对正常细胞和癌细胞的影响。细胞毒性研究将确定哪些癌症类型可以 被抑制剂选择性地杀死，我们将确定所需的剂量。结果将是有选择性的 小分子PICH抑制剂，将是有价值的研究工具，并将是先导化合物， 在动物癌症模型研究之前开发最佳有效分子。

Project Summary/Abstract Recent studies have shown that PIk1-interacting checkpoint helicase (PICH) is a potential cancer therapeutic target. PICH has critical functions in mitosis for the fidelity of chromosome segregation and without PICH there is an increased frequency of Ultra-Fine DNA Bridges (UFBs) in anaphase, which results in multi-nucleation and chromosome loss. Other proteins with key functions in mitosis, and that are required for accurate chromosome segregation, have proven to be effective targets of cancer drugs. For example, drugs that target microtubule dynamics. Importantly, PICH was found to be over-expressed in breast cancer cells and depletion of PICH was found to be extremely toxic to triple negative breast cancer cells, while not affecting matched normal tissue. Because PICH is over-expressed in a range of cancers, it is likely that many cancer types require PICH function for survival. Therefore, small molecule PICH inhibitors have potential for improved patient outcomes in cancer therapy. However, no inhibitors of PICH have been identified. Here we propose to identify selective PICH inhibitors and determine their cytotoxicity towards a range of cancer types in which PICH is over- expressed. Since PICH is a dsDNA-dependent ATPase, it has an activity suitable for high-throughput screening (HTS) for inhibitors using diverse small molecule libraries. We have established biochemical purification of active PICH and we have devised strategies and generated the tools that will allow primary hits from the HTS to be efficiently subjected to secondary screening, to validate bona fide PICH inhibitors and to eliminate non-specific inhibitors. The cellular consequences of PICH inhibition will be determined to fully characterize the effects on normal and cancer cells. Cytotoxicity studies will determine which cancer types can be selectively killed by the inhibitors and we will define the dosages required. The outcomes will be selective small molecule PICH inhibitors that will be valuable research tools and will be lead compounds for development of optimally potent molecules ahead of studies in animal cancer models.