Development of Light-activatable Probes and Dual-Action Inhibitors of Nek2 Kinase

项目摘要/摘要 Nek2是一种丝氨酸/酪氨酸氨基转移酶，定位于中心体，在中心体有丝分裂中发挥重要作用 析取。Nek2功能异常与多种人类疾病有关，包括高度 浸润性癌症。尽管如此，它的许多细胞功能仍然鲜为人知。这主要是由于 缺乏功能研究所需的选择性小分子探针。例如，时间和空间 与有丝分裂进入和退出相关的Nek2激活窗口没有被很好地定义。尽管它已经被证明 动粒定位蛋白MAD2是Nek2激酶的生理底物，在 动粒(和微管)曾被检测到。Nek2活动是否受其他机构的监管 有丝分裂酶，如Aurora A和CDc1，也仍然是一个推测的问题。在本申请的目标1中， 我们计划开发一种针对Nek2的、基于活性的小分子生物传感器探针，并展示其 利用精细的时间和空间控制来研究这种激酶的未被证实的功能。 具体地说，我们将(I)开发高效和选择性的基于荧光的NeK2生物传感器(KCAT/KM≥106-107M- 1s-1)；我们的工作假设是，一旦被Nek2激酶磷酸化，生物传感器探针将表现出强大的 荧光增强(~300-800%)(II)构建可光激活的Nek2生物传感器类似物 并展示了它在监测活体着丝粒的Nek2激酶活性方面的应用(作为原理证明) 细胞。拟议的研究具有创新性，因为它将导致开发第一个高度选择性和 高效的小分子生物传感器的Nek2激酶活性与强大的荧光读数。此外，Nek2 在三阴性乳腺癌(TNBC)-一种高度转移性癌症-其 目前还没有针对性的治疗方法。在我们的实验室中，我们使用了完整的动物体内模型，我们已经展示了 Nek2激酶可以与其他癌基因协同促进肿瘤转移。在这项研究中，我们还发现 一种来自EGFR靶向候选药物的新的Nek2药效团。由于Nek2和Nek2的表达 38%的TNBC肿瘤中EGFR显著升高，我们假设一种抑制剂能够 同时抑制Nek2和EGFR激酶活性将是TNBC的有效策略 干预。为了验证这一假设，在目标2中，我们建议开发一类新的铅化学试剂， 抑制TNBC中这两个靶癌基因的功能。一旦确定了先导化合物，我们就会 评估它们在TNBC细胞系中的疗效。通过同时抑制两个互补靶点nek2和 EGFR，我们的目标是开发一种先导化合物，解决肿瘤的复杂性和可塑性。最后， 在此提出的项目将为我们的本科生提供在该领域的直接实践研究经验。 药物化学、计算建模、酶学和细胞生物学。此外，它将显著地 改善以本科生为主的皇后学院的研究环境。

PROJECT SUMMARY/ABSTRACT Nek2, a ser/thr kinase, localizes to centrosomes and plays a crucial role in mitosis during centrosome disjunction. Aberrant Nek2 function has been linked to a wide variety of human diseases, including highly invasive cancers. Despite this, many of its cellular function remain poorly understood. This is primarily due to the lack of selective small molecule probes needed for functional studies. For example, temporal and spatial windows of Nek2 activation with regards to mitotic entry and exit are not well defined. Although it has been shown that a kinetochore-localizing protein Mad2 is a physiological substrate of Nek2 kinase, no Nek2 `activity' at the kinetochore (and microtubule) has ever been detected. Whether Nek2 activity is subject of regulation by other mitotic kinases, such as Aurora A and Cdc1, also remains a matter of speculation. In Aim 1 of this application, we plan to develop a Nek2-specific, activity-based, small molecule biosensor probe and demonstrate its utilizations in investigating the undocumented function of this kinase with exquisite temporal and spatial control. Specifically, we will (i) Develop efficient and selective fluorescence-based Nek2 biosensors (kcat/Km ≥ 106-107 M- 1s-1); our working hypothesis is that upon phosphorylation by Nek2 kinase, biosensor probes will exhibit robust fluorescence enhancement (~300-800%) (ii) Construct photoactivatable analogues of developed Nek2 biosensor and demonstrate its utilization (as proof-of-principle) in monitoring Nek2 kinase activity at the kinetochore in live cells. The proposed study is innovative because it will result in development of FIRST highly selective and efficient small molecule biosensor of Nek2 kinase activity with a robust fluorescence readout. Furthermore, Nek2 kinase is notably overabundant in Triple Negative Breast Cancer (TNBC) – a highly metastatic cancer for which currently no targeted therapeutics exists. In our laboratory using a whole animal in-vivo model, we have shown that Nek2 kinase can cooperate with other oncogenes to promote metastasis. In this study, we also discovered a new Nek2 pharmacophore derived from EGFR-targeting drug candidates. Since the expression of both Nek2 and EGFR is strongly elevated in 38 percent of TNBC tumors, we hypothesize that an inhibitory agent capable of suppressing both Nek2 and EGFR kinase activities simultaneously will be an effective strategy for TNBC intervention. To test this hypothesis, in Aim 2 we propose to develop a new class of lead chemical agents that inhibit the function of both of these target oncogenes in TNBC. Once the lead compounds are identified, we will evaluate their efficacy in TNBC cell lines. By simultaneously inhibiting two complementary targets Nek2 and EGFR, our goal is to develop a lead compound that addresses the tumors' complexity and plasticity. Finally, the project proposed herein will provide our undergraduate students a direct hands-on research experience in area of medicinal chemistry, computational modeling, enzymology, and cell biology. Additionally, it will significantly enhance the research environment of Queens College, a primarily undergraduate institution.