High troughput screen for small molecule inhibitors of Ran regulated functions

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。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Importinα1-Importinβ解离。这些传感器在马里兰州罗克维尔的NIH化学基因组中心(NCGC)成功地进行了测试，适用于RAN调节的Importinα1有丝分裂功能所涉及的关键步骤的小分子抑制剂的定量高通量筛选(QHTS)。通过NIH Roadmap Initiative，我们获得了执行QHTS的资金，并建立了一个包含30万个化合物的图书馆。目前，主要屏幕正在准备中，将于今年秋天完成。这一步之后，我的实验室将与NCGC合作，我们将努力将屏幕上的热门药物开发成适合基础研究的药物，并可能用于癌症化疗药物的开发。

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.