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: 8157509
• 负责人: Petr Kalab
• 金额: $ 5.9万
• 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/8157509
• 关键词: Running; inhibitor/antagonist; small molecule

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. We developed fluorescence resonance energy transfer (FRET)-based sensors for the RanGTP-induced importin alpha1-importin beta dissociation. These sensors were successfully tested at the NIH Chemical Genomics Center (NCGC), Rockville, MD, as applicable in quantitative highthroughput screen (qHTS) for small molecule inhibitors of the key steps involved in the Ran-regulated importin alpha1 mitotic function. Through NIH Roadmap Initiative, we received funding to perform the qHTS with a library containing 300 000 compounds. Currently, the primary screens are in preparation and will be completed this fall. This step will be followed by a collaborative effort between my lab and NCGC in which we wil will work towards developing the hits from the screen into drugs suitable for basic research and potentially for the development of cancer chemotherapeutics.

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。我们开发了基于荧光共振能量转移（FRET）的传感器用于RanGTP诱导的importin α 1-importin β解离。这些传感器在NIH化学基因组学中心（NCGC）（Rockville，MD）成功地进行了测试，适用于定量高通量筛选（qHTS），用于Ran调节的输入素α 1有丝分裂功能中涉及的关键步骤的小分子抑制剂。通过NIH路线图计划，我们获得了资金，用于使用包含30万种化合物的库进行qHTS。目前，主要屏幕正在准备中，将于今年秋季完成。这一步之后将是我的实验室和NCGC之间的合作努力，我们将致力于将筛选出的药物开发成适合基础研究的药物，并有可能用于癌症化疗药物的开发。