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

描述（由申请人提供）：最近在小鼠和人类中的遗传研究表明，通过钙调神经磷酸酶和NFAT的信号传导可能被调节，影响多种疾病的过程，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，我们最近表明，许多生物过程对NFATc蛋白的核占用水平非常敏感。这些发现提出了NFATc核占用的小分子调节剂可能是有价值的治疗方法的可能性。当Ca2+进入激活磷酸酶钙调磷酸酶时，NFAT信号被启动，这反过来使NFATc蛋白去磷酸化，导致它们进入核。在细胞核中，NFATc蛋白与核伴侣蛋白（NFATn's）在DNA上组装并形成活性转录复合物。NFATc蛋白在被Dyrk1a和GSK3激酶连续磷酸化后，被积极地从细胞核中输出。快速的核进出口过程是由蛋白质n端一个未知的变构开关调节的，该开关调节NFATc蛋白与核进出口机制之间的交替相互作用。该开关的微小修改可以改变核占用并诱导显着的表型变化。在过去，变构开关一直是有效的药物和小分子靶标。因此，这个开关代表了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测定方法。我们建议开发针对NFATc蛋白细胞质到核易位所需的构象变化和关键蛋白相互作用的高通量筛选（HTS）检测。我们要分析的蛋白-蛋白相互作用是NFATc家族转录因子和核丝蛋白之间的相互作用。2 (importin吗?（KPNA2）和出口蛋白（Crm1, XPO1）。

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.