High troughput screen for small molecule inhibitors of Ran regulated functions

许多纺锤体组装因子（SAFs）在有丝分裂过程中受到Ran GTPase的调节，它们与癌症有着各种广泛认识和深入研究（但通常不太了解）的联系：BRCA1， HURP, TPX2, Aurora A, TACC3, survivin， RHAMM, cdk11。此外，Ran的水平在许多人类肿瘤中高度增加，与非癌源性细胞不同，癌源性细胞的生长受到Ran RNAi的抑制。可能将Ran与癌症联系起来的最具特征的机制是RanGTP， importin β和importin α 1通过与TPX2结合来调节Aurora A的激活。RanGTP是TPX2从其与输入蛋白α 1-输入蛋白β的抑制复合体中释放所必需的。无进口蛋白的etpx2结合Aurora A，保护其免于去磷酸化，从而支持Aurora A激酶对其许多有丝分裂靶点的持续活性。超过30种Aurora A激酶抑制剂正在被开发作为潜在的癌症治疗药物，其中一些已进入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，杂合小鼠的肿瘤和非整倍体的数量明显增加。因此，完全抑制Aurora A可能潜在地诱导新生非整倍体和癌症。相反，我们建议开发抑制剂，通过RanGTP梯度阻止TPX2/Aurora A途径的局部激活，从而潜在地减缓Aurora A的激活和有丝分裂进程。由于这些抑制剂可能对许多其他由ran调控的有丝分裂途径具有类似的作用，因此它们的作用可能足够强，可以阻止依赖于有丝分裂陡峭的RanGTP梯度的细胞中的有丝分裂。2010年，我们获得了NIH路线图倡议的竞争性R03资助，用于携带两种定量HTS格式筛选（qHTS），用于检测ran调节的进口蛋白α 1/进口蛋白β相互作用的小分子抑制剂。2011年5月，位于马里兰州Rockville的NIH化学基因组学中心（NCGC）在34万种化合物中完成了5种不同浓度的初步筛选。在试验A中，我们筛选了进口蛋白α 1-进口蛋白β结合抑制剂，在试验B中筛选了rangtp诱导的进口蛋白α与进口蛋白β分离抑制剂。经过生物信息学分析和确认筛选，最初的3500个主要命中点被缩小到最高质量的候选抑制剂子集：试验a中的104个和试验b中的242个候选抑制剂。在主要命中点中进行筛选，将值得进一步分析的抑制剂组增加到大约1000个。目前，我们正在进行正交二次化学发光筛选，以确定特异性作用的抑制剂。筛选候选抑制剂的生物活性也在进行中，我们正在监测它们对组织培养细胞中有丝分裂纺锤体组装的影响，以及对钙调节的NFAT- GFP核转运报告基因核输入的影响。二级分析目前正在NCI的实验室以低通量格式进行优化，然后我们计划在NCGC以更高的通量格式携带全部1000个候选抑制剂。我们的目标是确定特异性抑制化合物，显示预期的生物活性。该数据是为该项目第二年获得R03资助所必需的，我们将重点关注已确定的最佳抑制剂，并根据结构-活性关系分析分析其作用机制并优化其性能。

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.