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

项目总结/摘要 Nek 2是一种丝氨酸/苏氨酸激酶，定位于中心体，在中心体有丝分裂中起着重要作用 析取Nek 2功能异常与多种人类疾病有关，包括高度 侵袭性癌症尽管如此，它的许多细胞功能仍然知之甚少。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 关于有丝分裂进入和退出的Nek 2激活窗口没有很好地定义。尽管已经证明 一个激动素定位蛋白Mad 2是Nek 2激酶的生理底物，在该区域没有Nek 2“活性”。 动粒（和微管）已经被检测到。Nek 2活动是否受其他组织的监管 有丝分裂激酶如Aurora A和Cdc 1也仍然是推测的问题。在本申请的目标1中， 我们计划开发一种Nek 2特异性的、基于活性的小分子生物传感器探针，并证明其 利用精确的时间和空间控制来研究这种激酶的未记录功能。 具体而言，我们将（i）开发高效和选择性的基于荧光的Nek 2生物传感器（kcat/Km ≥ 106-107 M-1）。 1 s-1）;我们的工作假设是，在Nek 2激酶磷酸化后，生物传感器探针将表现出鲁棒性， 荧光增强（~300-800%）（ii）构建开发的Nek 2生物传感器的光活化类似物 并证明其在监测活的小鼠中动粒处Nek 2激酶活性中的用途（作为原理证明）。 细胞拟议的研究是创新的，因为它将导致发展第一高选择性， Nek 2激酶活性的高效小分子生物传感器，具有强大的荧光读数。此外，Nek 2 激酶在三阴性乳腺癌（TNBC）中显著过量，三阴性乳腺癌是一种高度转移性癌症， 目前不存在靶向治疗剂。在我们的实验室中，使用整个动物体内模型，我们已经表明 Nek 2激酶可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， 一个新的Nek 2药效团来自EGFR靶向候选药物。由于Nek 2的表达 EGFR在38%的TNBC肿瘤中强烈升高，我们假设能够抑制EGFR的抑制剂 同时抑制Nek 2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，我们提出开发一类新的铅化学剂， 抑制TNBC中这两种靶致癌基因的功能。一旦确定了先导化合物，我们将 评估它们在TNBC细胞系中的功效。通过同时抑制两个互补靶点Nek 2和 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 本文提出的项目将为我们的本科生提供一个直接的实践研究经验，在该领域 药物化学、计算建模、酶学和细胞生物学。此外，它将大大 加强皇后学院的研究环境，主要是本科院校。

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.