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。我们开发了基于荧光共振能量转移（FRET）的传感器，用于rangtp诱导的输入蛋白α 1输入蛋白β解离。这些传感器在马里兰州Rockville的NIH化学基因组学中心（NCGC）成功进行了测试，适用于定量高通量筛选（qHTS），用于检测涉及ran调节的输入蛋白α - 1有丝分裂功能的关键步骤的小分子抑制剂。我们现正申请拨款，以一个含有30万个化合物的文库进行qHTS。从长远来看，我们将努力将筛选出来的药物开发成适合癌症治疗的抗有丝分裂药物。

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. We are currently applying for funding to perform the qHTS with a library containing 300 000 compounds. In a longer term, we will work towards developing the hits from the screen into antimitotic drugs suitable for cancer treatment.