Screen for Small Molecule Inhibitors of Nuclear Protein Import

描述(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，它需要分离和协调细胞核和细胞质隔间的功能。核转运是由核粘附素(importin/exportin)超家族的核质穿梭受体介导的。核粘附素与核孔蛋白相互作用，将蛋白质和RNA转运到核膜上，并受小的GTP酶Ran调控。到目前为止，只有一类针对出口蛋白CRM1的核运输机械小分子抑制剂被描述。该项目的目标是开发和验证一种涉及通透性细胞的高通量筛选(HTS)试验，以发现更多针对核进口机械的小分子抑制剂。焦点将涉及典型的核运输受体Importin beta，以及它的两种货物，其进口涉及该受体的不同构象。该筛查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的细胞核中荧光标记的货物的积累。预计将这种化验转移到384孔格式，并自动处理所有试剂，将是直接的。由于核进口需要包括Importinβ和Ran在内的多种蛋白质-蛋白质相互作用，因此通透性细胞筛查的含量很高。尽管如此，用于确认命中和目标识别的简单协议是可行的，并将对其进行描述。在一项先导性研究中，该方法将用于从模拟多肽的小分子组合文库中筛选约5000种化合物，以评估模拟多肽的小分子文库是否可以产生有用的核运输抑制剂。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，在病理条件下可能会受到干扰。这项工作预计将提供新的药理学工具，以研究核质运输机制的功能，并提供信息，促进开发治疗人类疾病的新途径。

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.