Screen for Small Molecule Inhibitors of Nuclear Protein Import

描述（由申请人提供）：蛋白质和rna在细胞核和细胞质之间的运输是真核细胞的一个基本过程，这是分离和协调细胞核和细胞质室功能所必需的。核转运是由核丝蛋白（输入/输出蛋白）超家族的核质穿梭受体介导的。核丝蛋白与核孔蛋白相互作用，通过核膜转运蛋白质和RNA货物，并受小GTPase Ran的调节。到目前为止，仅报道了一类以输出蛋白Crm1为靶点的核转运机制小分子抑制剂。该项目的目标是开发和验证涉及渗透细胞的高通量筛选（HTS）试验，以发现针对核进口机制的其他小分子抑制剂。重点将涉及典型的核转运受体进口β，以及其进口涉及该受体不同构象的两种货物。屏幕将使用自动数据采集和处理来测量在96孔板中培养的渗透细胞细胞核中荧光标记的货物的积累。预计将该分析转移到384孔格式，机器人处理所有试剂，将是直截了当的。由于核输入需要多种蛋白质相互作用，包括输入β和Ran，因此渗透细胞筛选的含量很高。尽管如此，验证命中和目标识别的直接协议是可行的，并将进行描述。在一项初步研究中，该试验将用于筛选拟肽小分子组合文库中的大约5000种化合物，以评估拟肽文库是否能产生有用的核转运抑制剂。蛋白质和rna在细胞核和细胞质之间的运输对所有人类细胞的正常功能至关重要，并且在病理条件下可能会受到干扰。这项工作有望为研究核-细胞质转运机制的功能提供新的药理学工具，并为促进人类疾病治疗新途径的发展提供信息。

DESCRIPTION (provided by applicant): Trafficking of proteins and RNAs between the nucleus and the cytoplasm is a fundamental process of eukaryotic cells, which is required to segregate and coordinate the functions of the nuclear and cytoplasmic compartments. Nuclear transport is mediated by nucleocytoplasmic shuttling receptors of the karyopherin (importin/exportin) superfamily. Karyopherins interact with nucleoporins to translocate protein and RNA cargoes across the nuclear envelope, and are regulated by the small GTPase Ran. Up to now, only one class of small molecule inhibitors of the nuclear transport machinery has been described, which targets the exportin Crm1. The goal of this project is to develop and validate a high-throughput screening (HTS) assay involving permeabilized cells, to discover additional small molecule inhibitors that target the nuclear import machinery. The focus will involve the prototypical nuclear transport receptor importin beta, and two of its cargoes whose import involves different conformations of this receptor. The screen will use automated data acquisition and processing to measure the accumulation of fluorescently labeled cargoes in the nucleus of permeabilized cells, which are cultured in 96-well plates. It is expected that transfer of this assay to a 384-well format, with robotic handling of all reagents, will be straightforward. Since nuclear import requires multiple protein-protein interactions involving importin beta and Ran, the permeabilized cell screen is high content. Nonetheless, straightforward protocols for validation of hits and target identification are feasible and will be described. In a pilot study, the assay will be used to screen approximately 5000 compounds from a combinatorial library of peptidomimetic small molecules, to evaluate whether peptidomimetic libraries can yield useful inhibitors of nuclear transport. Transport of protein and RNAs between the nucleus and the cytoplasmic is critical to the proper functioning of all human cells, and can be perturbed in pathological conditions. This work is expected to provide new pharmacological tools to investigate the functioning of the nuclear- cytoplasmic transport machinery, and to contribute information that will promote the development of new avenues for treatment of human diseases.