High troughput screen for small molecule inhibitors of Ran regulated functions

背景和基本原理在间期，Ran GTdR作为一个关键的调节剂， RanGTP介导的核孔通道的核质转运 与importin beta超家族的NTR相互作用。核定位装载 NTR衔接蛋白输入素α上的信号（NLS）蛋白货物需要 输入蛋白α与输入蛋白β结合的N末端。输入素α-输入素β-NLS 货物复合物然后运输到细胞核，在那里它被RanGTP诱导解离 与输入蛋白β结合。在有丝分裂中，动态的RanGTP调节的 在核膜被拆卸后，货物继续。NLS货物同时 作为纺锤体组装因子（SAF），被输入素a/B结合抑制， 由染色体周围的RanGTP梯度激活。因此，RanGTP梯度具有 作为有丝分裂纺锤体组装的调节器的重要作用。在5个进口中 在人类中，只有输入素α 1已知参与 有丝分裂SAF调节。有趣的是，大多数已知参与 输入素α 1有丝分裂调节（作用于下游或功能所需）位于 同时识别的癌症相关蛋白（BRCA 1，HURP，hTOG，TACC，TPX 2，Eg 5），有些是 怀疑作为癌症有丝分裂原（TPX 2，Aurora A）发挥作用，其中至少有一种，Aurora A （受到RanGTP-输入蛋白α 1/输入蛋白β-TPX 2依赖性激活）被认为是 作为有前途的癌症治疗靶点（Aurora A抑制剂的II期临床试验， 报告）。现有的分子结构表明，输入素α的界面 1-在它们的复合物中，importin beta提供了独特的分子表面， 进口同种型特异性化合物开发的可行性。项目概要我们建议 开发特异性靶向RanGTP调节的输入素α 1功能的化合物， 先前发表并充分表征的用于RanGTP诱导输入的基于FRET的生物传感器 α 1 -输入蛋白β解离。为了实现这一目标，我们重新设计了这个biosenso，以获得 5倍宽的开-关FRET信号幅度。接下来，我们开发了一个1536孔板 用于小分子文库中定量高通量筛选（qHTS）的测定方案 NIH化学基因组学中心（NCGC）NCGC的验证试验表明， 该测定法足够稳健（z= 0.67）并且适合于筛选整套化学品 图书馆在NCGC。目前，我们正在开发一种改良版的测定法， 寻找靶向调节的输入蛋白α 1-输入蛋白β中的几个步骤的化合物 在一个屏幕上互动。将对鉴定的化合物进行一系列 二次筛选以确定其分子作用机制。进一步优化 化合物将由结构-活性关系鉴定指导， 屏幕数据分析。我们计划分析所鉴定的化合物对 有丝分裂进程和癌源细胞（NCI-60）的细胞生长。的关键方面 importin α 1/importin β复合物靶向癌细胞将是动态抑制的 一组合作作用的有丝分裂激活剂，与其完整的 个体抑制我们预计依赖于Ran的癌细胞的生长 将被压制。与此同时，残留的极光A活性水平， 正常细胞中维持倍性所需的蛋白质将被保留。在未来，我们期待 我们的检测策略可以用来开发化合物， 分别靶向所有现有的输入素α同种型。

Background and rationale In the interphase, Ran GTPase acts as a key regulator of the nucleo-cytoplasmic transport through the nuclear pore channel which is mediated by RanGTP interaction with NTRs of the importin beta superfamily. The loading of nuclear localization signal (NLS) protein cargos on the NTR adaptor protein importin alpha requires that the N-terminus of importin alpha binding to importin beta. The importin alpha-importin beta-NLS cargo complex then transports to the nucleus where its dissociation is induced by RanGTP binding to importin beta. In mitosis, the dynamic RanGTP-regulated loading and unloading of cargos continues after the nuclear envelope is disassembled. NLS cargos that at the same time serve as spindle assembly factors (SAFs) are inhibited by importin a/b binding and locally activated by RanGTP gradient surrounding chromosomes. Thus, the RanGTP gradient has an essential role as the regulator of mitotic spindle assembly. Out of 5 importins alpha isoforms in humans, only importin alpha 1 is known to participate in mitotic SAF regulation. Interestingly, majority of proteins that are known to be involved in importin alpha1 mitotic regulation (acting downstream or required for function) are at the same time recognized cancer-related proteins (BRCA1, HURP, hTOG, TACC, TPX2, Eg5), some are suspected to function as cancer mitogens (TPX2, Aurora A) and at least one of them, Aurora A (subjected to RanGTP- importin alpha1/importin beta- TPX2- dependent activation) is considered as promising cancer treatment target (phase II clinical trials for Aurora A inhibitors were reported). The available molecular structures suggest that the interface of importin alpha 1-importin beta in their complex provides a unique molecular surface, supporting the feasibility of importin isotype-specific compound development. Project summary We propose to develop compounds specifically targeting RanGTP-regulated function of importin alpha 1 using previously published and well characterized FRET-based biosensor for RanGTP-induced importin alpha 1 - importin beta dissociation. Towards this goal, we redesigned this biosenso to obtain a 5 times wider on-off FRET signal amplitude. Next, we developed a 1536-well plate- based assay protocol for quantitative high throughput screening (qHTS) in small molecule libraries of the NIH Chemical Genomics Center (NCGC). The validation assays at NCGC suggested that our assay is sufficiently robust (z= 0.67) and suitable for screening the complete set of chemical libraries available at NCGC. Presently we are developing a modified version of the assay to search for compounds targeting several steps in the regulated importin alpha 1- importin beta interaction in one screen. The identified compounds will be subjected to a battery of secondary screens to define their molecular mechanism of action. Further optimization of the compounds will be guided by the structure-activity relationship identification achieved by the analysis of the screen data. We plan to analyze the effects of the identified compounds on mitosis progression and cell growth of cancer-derived cells (NCI-60). The key aspect of importin alpha1/importin beta complex targeting in cancer cells would be dynamic suppression of a group of cooperatively acting mitotic activators, as compared to their complete individual inhibition. We expect that the growth of cancer cells that are dependent on Ran would be suppressed. At the same time the residual levels of Aurora A activity which is required for ploidy maintenance in normal cells would be preserved. In the future, we expect that the strategy of our assay could be used to develop compounds that would specifically target all existing importin alpha isoforms separately.