HTS Assay Development for Modulators of a Nuclear Import/Export Switch

描述(申请人提供)：最近对小鼠和人类的遗传学研究表明，通过钙调神经磷酸酶和NFAT发出的信号可能会受到调节，影响多种疾病过程，包括糖尿病、心脏病、骨质疏松症、精神分裂症、免疫缺陷和自身免疫。此外，我们最近已经表明，许多生物过程对NFATc蛋白的核占位水平非常敏感。这些发现提出了NFATc核占位的小分子调节剂可能是有价值的治疗药物的可能性。当钙离子进入激活磷酸酶钙调神经磷酸酶时，NFAT信号被启动，进而使NFATc蛋白去磷酸化，导致其核进入。在细胞核中，NFATc蛋白与核伙伴蛋白(NFATn‘s)组装在DNA上，形成活跃的转录复合体。NFATc蛋白被Dyrk1a和GSK3激酶连续磷酸化后活跃地从细胞核输出。快速的核进出口过程受蛋白质N端一个未知的变构开关调控，该开关调节NFATc蛋白与核进出口机制之间的交互作用。对这一开关的微小修改可以改变核的占有率，并引起显著的表型变化。在过去，变构开关一直是有效的药物和小分子靶标。因此，这种开关是小分子干预的理想场所。我们将开发一种新的核进出口开关小分子调节剂的分析方法。我们建议开发高通量筛选(HTS)方法，针对NFATc蛋白的构象变化和细胞质到核转位所需的关键蛋白质-蛋白质相互作用。我们将分析的蛋白质-蛋白质相互作用是NFATc转录因子家族与核粘蛋白？2(Importin？，KPNA2)和Exportin(CRM1，XPO1)之间的相互作用。 本申请中提出的第一个特定目标的目标是建立一种体外多分析物时间分辨荧光(TRF)分析方法，该方法可以定量评估重组NFATc蛋白与核进出口机制的相互作用。我们建议的第二个具体目标是优化和自动化高通量筛选(HTS)的这一检测方法，并在中试筛选中使用它们来发现钙调神经磷酸酶/NFAT信号的小分子调节器。TRF分析将由两个基于细胞的二次分析补充，在这两个二次分析中，靶标是在比生化一次分析更生理的背景下进行筛选的。二次检测将用于确定NFATc胞浆到核转位和NFAT依赖转录的小分子调节剂的特异性和有效性。我们的目标是为高温超导环境优化这些二次检测。我们将以定量的高通量格式使用药理活性化合物库(LOPAC)中的1,280种化合物来识别技术问题，并确认更大规模高通量筛选的可行性。如果这项应用的目标实现，未来与MLSCN合作实施HTS检测应该会发现调节NFAT转录活性的新化合物。活性化合物会 是研究这一关键信号通路的有价值的化学工具，并有可能在各种疾病过程中发挥作用。考虑到在以下情况下需要新的治疗方法 钙调神经磷酸酶/NFAT信号转导是一个重要的靶点，通过所提出的方法确定的活性化合物将很有可能刺激额外的药物开发。

DESCRIPTION (provided by applicant): Recent genetic studies in mice and humans have indicated that signaling through calcineurin and NFAT might be modulated to affect a wide variety of disease processes including diabetes, heart disease, osteoporosis, schizophrenia, immune deficiency and autoimmunity. Moreover we have recently shown that many biologic processes are exquisitely sensitive to the level of nuclear occupancy of NFATc proteins. These findings raise the possibility that small molecule modulators of NFATc nuclear occupancy might be valuable therapeutics. NFAT signaling is initiated when Ca2+ entry activates the phosphatase calcineurin, which in turn dephosphorylates NFATc proteins leading to their nuclear entry. In the nucleus NFATc proteins assemble on DNA with nuclear partner proteins (NFATn's) and form active transcription complexes. NFATc proteins are actively exported from the nucleus after sequential phosphorylation by the kinases Dyrk1a and GSK3. The process of rapid nuclear import and export is regulated by an uncharacterized allosteric switch in the N-terminus of the protein, which regulates the alternate interaction between NFATc proteins and the nuclear import and export machinery. Minor modifications of this switch can change nuclear occupancy and induce dramatic phenotypic changes. In the past allosteric switches have been effective drug and small molecule targets. Hence, this switch represents an ideal site for intervention by small molecules. We will develop a new assay for small molecule modulators of the nuclear import/export switch. We propose to develop high throughput screening (HTS) assays that target the conformational change and the key protein-protein interaction required for cytoplasmic-to-nuclear translocation of NFATc proteins. The protein-protein interactions, which we will assay are the interaction between the NFATc family of transcription factors and the karyopherin ?2 (importin?, KPNA2) and exportin (Crm1, XPO1). The objective of the first specific aim proposed in this application is to develop an in vitro multianalyte time resolved fluorescence (TRF) assay, which allows a quantitative assessment of the interaction of recombinant NFATc protein with the nuclear import and export machinery. The goal of the second specific aim of our proposal is to optimize and automate this assay for high-throughput screening (HTS) and use them in the pilot screen to discover small molecule modulators of calcineurin/NFAT signaling. The TRF assay will be complemented by two cell-based secondary assays, where the target is screened in a more physiological context than in the biochemical primary assays. The secondary assays will be used to establish the specificity and efficacy of small molecule modulators of NFATc cytoplasmic-to-nuclear translocation and NFAT dependent transcription. We are aiming at optimizing these secondary assays also for a HTS environment. We will use the 1,280 compounds in the library of pharmacologically active compounds (LOPAC) in a quantitative high throughput format to identify technical problems and confirm the feasibility of a larger scale high throughput screen. If the aims of this application are achieved, future implementation of the HTS assays in collaboration the MLSCN should identify novel compounds, which modulate NFAT transcriptional activity. Active compounds will be valuable chemical tools to study this critical signaling pathway and have the potential to be active in a variety of disease processes. Given the need for new therapeutic approaches in diseases where calcineurin /NFAT signaling is an important target, active compounds identified by the proposed assay would have a high probability of stimulating additional pharmaceutical development.