High troughput screen for small molecule inhibitors of Ran regulated functions

背景和原理在间期，RAN GTP酶作为核质转运的关键调节因子，通过RanGTP与Importinβ超家族的NTRs相互作用来调节核孔通道的运输。核定位信号(NLS)蛋白装载在NTR接头蛋白Importinα上需要Importinα的N端与Importinβ结合。然后，Importinα-Importinβ-NLS货物复合体运输到细胞核，在那里其解离由RanGTP与Importinβ结合诱导。在有丝分裂中，在核膜解体后，由RanGTP调节的货物的动态装卸继续进行。同时作为纺锤体组装因子(SAF)的NLS货物被导入蛋白a/b结合抑制，并被周围染色体周围的RanGTP梯度局部激活。因此，RanGTP梯度作为有丝分裂纺锤体组装的调节因子具有重要作用。在人类的5个重要蛋白α-亚型中，只有导入蛋白α-1参与有丝分裂的SAF调节。有趣的是，大多数已知参与Importinα1有丝分裂调控(作用于下游或功能所需)的蛋白质都是同时被识别的癌症相关蛋白(BRCA1、HURP、hTOG、TACC、TPX2、EG5)，一些蛋白质被怀疑具有癌症有丝分裂原的功能(TPX2、Aurora A)，其中至少有一种，Aurora A(受到RanGTP-Importin Alpha1/Importinβ-TPX2依赖的激活)被认为是有希望的癌症治疗靶点(Aurora A抑制剂的II期临床试验报告)。现有的分子结构表明，importinα1-importin beta在其复合体中的界面提供了一个独特的分子表面，支持importin同型特异性化合物开发的可行性。项目摘要我们建议利用先前发表的、基于FRET的生物传感器来开发针对RanGTP调节的Importinα1功能的化合物，该传感器用于RanGTP诱导Importinα1-Importinβ的解离。为了达到这个目标，我们重新设计了这种生物传感器，以获得5倍宽的开关FRET信号幅度。接下来，我们开发了一种基于1536孔板的分析方法，用于在NIH化学基因组中心(NCGC)的小分子文库中进行定量高通量筛选(QHTS)。NCGC的验证分析表明，我们的分析足够稳健(z=0.67)，适合于筛选NCGC可用的完整化学文库。目前，我们正在开发一种改进的分析方法，以在一个屏幕上搜索针对受调控的Importinα1-Importinβ相互作用中的几个步骤的化合物。鉴定出的化合物将经过一系列二级筛选，以确定它们的分子作用机制。通过分析筛选数据实现的构效关系鉴定将指导化合物的进一步优化。我们计划分析已鉴定的化合物对癌细胞(NCI-60)有丝分裂进程和细胞生长的影响。在癌细胞中靶向importinα1/importin beta复合体的关键方面是动态抑制一组协同作用的有丝分裂激活剂，而不是完全单独抑制它们。我们预计依赖RAN的癌细胞的生长将受到抑制。同时，维持正常细胞倍性所需的Aurora A活性的残留水平也将得到保留。在未来，我们预计我们的检测策略可以用于开发专门针对所有现有进口蛋白α61472；亚型的化合物。

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.