High troughput screen for small molecule inhibitors of Ran regulated functions

在有丝分裂期间由Ran GT3调节的许多纺锤体组装因子（SAF）具有各种公认的和深入研究的（但通常不太清楚）与癌症的联系：BRCA 1、HURP、TPX 2、Aurora A、TACC 3、生存素、RHAMM、cdk 11。此外，Ran的水平在许多人类肿瘤中高度增加，并且与非癌症来源的细胞不同，癌症来源的细胞的生长被Ran RNAi抑制。可以说，可能将Ran与癌症联系起来的最佳表征机制是RanGTP、输入蛋白β和输入蛋白α 1，它们通过与TPX 2结合来调节Aurora A的活化。RanGTP是TPX 2从其与输入素α 1-输入素β的抑制复合物中释放所必需的。无输入蛋白的TPX 2结合极光A，保护它免于去磷酸化，从而支持持续的极光A激酶活性，作用于许多有丝分裂靶点。目前正在开发30多种Aurora A激酶抑制剂作为潜在的癌症治疗药物，其中几种已进入I期和II期临床试验。然而，Aurora A基因的缺失在胚胎中是致命的，并且杂合子小鼠发展出显著更高数量的肿瘤和非整倍体。因此，Aurora A的完全抑制可能潜在地诱导从头非整倍性和癌症。相反，我们建议开发抑制剂，这将防止通过RanGTP梯度局部激活TPX 2/Aurora A通路，从而可能减缓Aurora A激活和有丝分裂进程。因为这样的抑制剂可能对许多其他Ran调节的有丝分裂途径具有类似的作用，所以它们的作用可能足够强以防止显示对陡峭的有丝分裂RanGTP梯度的依赖性的细胞中的有丝分裂。2010年，我们从NIH路线图计划获得了竞争性R 03资金，用于进行两次定量HTS格式筛选（qHTS），以筛选Ran调节的输入蛋白α 1/输入蛋白β相互作用的小分子抑制剂。2011年5月在NIH化学基因组学中心（NCGC）（Rockville，MD）完成了340 000种化合物（每种化合物5种不同浓度）的初步筛选。在试验A中，我们筛选输入蛋白α 1-输入蛋白β结合的抑制剂，在试验B中筛选RanGTP诱导的输入蛋白α与输入蛋白β解离的抑制剂。在生物信息学分析和确认筛选后，将3500个主要命中的初始集合缩小到最高质量候选抑制剂的子集：试验A中的104个和试验B中的242个候选抑制剂。在主要命中中挑选樱桃将这组值得进一步分析的抑制剂增加到约1000。目前，我们正致力于正交二级化学发光筛选，以确定特异性作用的抑制剂。还在进行候选抑制剂的生物活性的筛选，其中我们正在监测它们对组织培养细胞中的有丝分裂纺锤体组装和对钙调节的NFAT-GFP核转运报告子的核输入的影响。二级检测目前正在我们NCI实验室以低通量形式进行优化，然后我们计划在NCGC以更高通量的形式进行，其中包含整套1000种候选抑制剂。我们的目标是确定特异性作用的抑制性化合物，显示预期的生物活性。这些数据是确保R 03项目第二年资金所必需的，我们将专注于最佳识别的抑制剂，并分析其作用机制，并基于结构-活性关系分析优化其性能。

A number of the spindle assembly factors (SAFs) which are regulated by Ran GTPase during mitosis have variety of well recognized and intensively studied (but often not well understood) connections to cancer: BRCA1, HURP, TPX2, Aurora A, TACC3, survivin, RHAMM, cdk11. Also the levels of Ran are highly increased in many human tumors and unlike in non-cancer derived cells, the growth of cancer-derived cells is inhibited by Ran RNAi. Arguably the best characterized mechanism potentially linking Ran to cancer is the RanGTP, importin beta and importin alpha1 regulating the activation of Aurora A through its binding to TPX2. RanGTP is required for the release of TPX2 from its inhibitory complex with importin alpha1-importin beta. The importin-freeTPX2 binds Aurora A, protecting it from dephosphorylation and thus supporting sustained Aurora A kinase activity towards is many mitotic targets. More than 30 Aurora A kinase inhibitors are being developed as potential cancer therapeutics and several of them entered phase I and II clinical trials. However, the deletion of Aurora A gene is lethal in embryos and the heterozygous mice develop significantly higher number of tumors and aneuploidy. Complete inhibition of the Aurora A could therefore potentially induce de novo aneuploidy and cancer. Instead, we propose developing inhibitors which would prevent local activation of TPX2/Aurora A pathway by RanGTP gradient, thus potentially decelerating Aurora A activation and mitosis progression. Because such inhibitors would likely have similar effect on many other Ran-regulated mitotic pathways, their effect could be sufficiently strong to prevent mitosis in cells displaying dependence on steep mitotic RanGTP gradient. In 2010 we obtained competitive R03 funding from NIH Roadmap Initiative to carry two quantitative HTS format screens (qHTS) screens for small molecule inhibitors of Ran-regulated importin alpha1/importin beta interaction. The primary screens among 340 000 compounds, each at 5 different concentrations were completed in May 2011 at NIH Chemical Genomics Center (NCGC), Rockville, MD. In Assay A, we were screening for inhibitors of importin alpha1- importin beta binding and in Assay B for inhibitors of RanGTP-induced dissociation of the importin alpha from importin beta. After bioinformatics analysis and confirmation screens, the initial set of 3500 primary hits was narrowed down to a subset of highest quality candidate inhibitors: 104 in Assay A and 242 candidate inhibitors in Assay B. Cherry picking among the primary hits increased this set of inhibitors worthy further analysis to approximately 1000. Currently we are working on orthogonal secondary chemiluminescence screens to identify specifically acting inhibitors. In progress are also screens for biological activity of the candidate inhibitors where we are monitoring their effects on mitotic spindle assembly in tissue culture cells and on nuclear import of calcium-regulated NFAT- GFP nuclear transport reporter. The secondary assays are currently being optimized in a low throughput format in our lab at NCI and we then plan to carry them at a higher throughput format at NCGC with the entire set of 1000 candidate inhibitors. Our objective is to identify specifically acting inhibitory compounds which display the expected biological activity. This data is required to secure R03 funding for the second year of the project where we will focus on the best identified inhibitor(s) and analyze their mechanism of action and optimize their performance based on structure-activity relationship analysis.