High troughput screen for small molecule inhibitors of Ran regulated functions

Ran 调节功能的小分子抑制剂的高通量筛选

• 批准号: 8349210
• 负责人: Petr Kalab
• 金额: $ 6.32万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349210
• 关键词: Aneuploidy; BRCA1 gene; Basic Science; Binding; Bioinformatics; Biological; Biological Assay; Breast Cancer Cell; Calcium; Cancer Cell Growth; Cell Cycle; Cell Cycle Stage; Cell division; Cells; Chemicals; Clinical Trials; Complex; Data; Dependence; Development; Dissociation; Embryo; Energy Transfer; Fibroblasts; Funding; Genes; Genomics; Goals; Guanosine Triphosphate Phosphohydrolases; HMMR gene; Hela Cells; Human; Importins; Individual; Interphase; Link; Malignant Neoplasms; Mediating; Mitosis; Mitotic; Mitotic spindle; Monitor; Mus; Normal Cell; Nuclear Envelope; Nuclear Import; Nuclear Pore Complex; Pathway interactions; Performance; Phase; Protein Dephosphorylation; Publishing; RNA Interference; Reporter; Reporting; Role; Running; Screening procedure; Secure; Structure-Activity Relationship; TACC3 gene; Testing; Therapeutic; United States National Institutes of Health; Work; alpha Karyopherins; aurora-A kinase; base; beta Karyopherins; cancer cell; high throughput screening; human STK6 protein; inhibitor/antagonist; karyopherin alpha 2; kinase inhibitor; mRNA decapping; mutant; nucleocytoplasmic transport; overexpression; prevent; sensor; small molecule; small molecule libraries; survivin; tissue/cell culture; tumor

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.

在有丝分裂过程中受RAN GTP酶调控的许多纺锤体组装因子(SAF)有多种被广泛认识和深入研究(但通常不是很好)的与癌症的联系：BRCA1、HURP、TPX2、Aurora A、TACC3、Survivin、Rhamm、CDK11。此外，在许多人类肿瘤中，RAN的水平高度增加，与非癌症来源的细胞不同，肿瘤来源的细胞的生长受到RAN RNAi的抑制。可以说，最具代表性的可能将RAN与癌症联系起来的机制是RanGTP、Importinβ和Importin Alpha1，它们通过与TPX2结合来调节Aurora A的激活。从其与Importinα1-Importinβ的抑制复合体中释放TPX2需要RanGTP。不含Importin的TPX2与Aurora A结合，保护Aurora A不被去磷酸化，从而支持Aurora A对许多有丝分裂靶点的持续激活。30多种Aurora A激酶抑制剂正在被开发为潜在的癌症治疗药物，其中几种进入了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，杂合子小鼠发生显著更多的肿瘤数量和非整倍体。因此，完全抑制Aurora A可能会导致从头开始的非整倍体和癌症。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RAN调节的有丝分裂途径具有类似的作用，它们的作用可能足够强，以防止显示出对陡峭有丝分裂RanGTP梯度的依赖的细胞的有丝分裂。2010年，我们从NIH Roadmap Initiative获得了竞争性的R03资金，以进行两个HTS格式定量筛选(QHTS)筛选，用于RAN调节的importinα1/importin beta相互作用的小分子抑制剂。2011年5月，在马里兰州罗克维尔的NIH化学基因组中心(NCGC)完成了对34万种化合物的初步筛选，每种化合物有5种不同的浓度。在分析A中，我们筛选了Importinα1-Importinβ结合的抑制剂，在分析B中，我们筛选了RanGTP诱导的Importinα和Importinβ解离的抑制剂。经过生物信息学分析和确认筛选，最初的3500次点击被缩小到最高质量候选抑制剂的子集：在Assay A中有104个，在Assay B中有242个候选抑制剂。在初级点击中挑选樱桃使这组值得进一步分析的抑制剂增加到大约1000个。目前，我们正致力于正交二次化学发光筛选，以确定特定的作用抑制剂。我们还在筛选候选抑制剂的生物活性，我们正在监测它们对组织培养细胞有丝分裂纺锤体组装的影响，以及对钙调节的NFAT-GFP核运输报告的核进口的影响。目前，我们在NCI的实验室正在以低通量格式优化二次分析，然后我们计划在NCGC以更高的吞吐量格式进行二次分析，并使用完整的1000种候选抑制剂。我们的目标是鉴定具有预期生物活性的具有特定作用的抑制性化合物。这些数据是为项目第二年获得R03资金所必需的，在该项目中，我们将专注于最佳识别的抑制剂(S)，并分析它们的作用机理，并基于结构-活性关系分析优化它们的性能。