High troughput screen for small molecule inhibitors of Ran regulated functions

背景和原理在间期，Ran GTPase是核-胞质通过核孔通道运输的关键调节剂，该通道是由RanGTP与输入蛋白β超家族的ntr相互作用介导的。核定位信号（NLS）蛋白货物在NTR接头蛋白输入蛋白α上的装载要求输入蛋白α的n端与输入蛋白β结合。然后，输入蛋白α -输入蛋白β - nls货物复合体转运到细胞核，在那里，RanGTP与输入蛋白结合诱导其解离。在有丝分裂中，核包膜被分解后，rangtp调控的装载和卸载仍在继续。同时作为纺锤体组装因子（SAFs）的NLS货物通过输入a/b结合被抑制，并被染色体周围的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器具有重要作用。在人类的5种进口蛋白α &#61472；亚型中，已知只有进口蛋白α 1参与有丝分裂SAF调节。有趣的是，大多数已知参与输入α - 1有丝分裂调节（作用于下游或功能所需）的蛋白质同时也是公认的癌症相关蛋白（BRCA1， HURP, hTOG， TACC, TPX2, Eg5），一些被怀疑作为癌症有丝分裂原（TPX2, Aurora A），其中至少有一种，Aurora A（受RanGTP- importin α 1/importin β - TPX2依赖性激活）被认为是有希望的癌症治疗靶点（Aurora A抑制剂的II期临床试验报道）。现有的分子结构表明，在它们的复合物中输入蛋白α - 1-输入蛋白β的界面提供了一个独特的分子表面，支持了输入蛋白同型特异性化合物开发的可行性。我们建议利用先前已发表并已充分表征的基于fret的生物传感器，开发特异性靶向rangtp调控的进口蛋白α 1功能的化合物，用于rangtp诱导的进口蛋白α 1 -进口蛋白β解离。为了实现这一目标，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，我们在NIH化学基因组学中心（NCGC）的小分子文库中开发了一种基于1536孔板的定量高通量筛选（qHTS）检测方案。NCGC的验证试验表明，我们的分析具有足够的鲁棒性（z= 0.67），适合筛选NCGC提供的全套化学文库。目前，我们正在开发一种改进版本的检测方法，以在一个屏幕上搜索针对受调节的输入蛋白α 1-输入蛋白β相互作用的几个步骤的化合物。鉴定出的化合物将进行一系列的二次筛选，以确定它们的分子作用机制。通过对筛选数据的分析获得的结构-活性关系鉴定将指导化合物的进一步优化。我们计划分析鉴定的化合物对癌源细胞（NCI-60）有丝分裂进程和细胞生长的影响。癌细胞中输入蛋白α 1/输入蛋白β复合物靶向的关键方面是动态抑制一组协同作用的有丝分裂激活因子，而不是完全的单个抑制。我们预计依赖Ran的癌细胞的生长将受到抑制。同时，正常细胞中维持倍性所需的Aurora A活性的残余水平将被保留。在未来，我们期望我们的分析策略可以用于开发能够单独针对所有现有的进口α &#61472；同种异构体的化合物。

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.