Development of a high-throughput screening-compatible assay for the discovery of inhibitors of the AF4-AF9 interaction using AlphaScreen technology.
Development of a high-throughput screening-compatible assay for the discovery of inhibitors of the AF4-AF9 interaction using AlphaScreen technology.
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DOI：
10.1089/adt.2012.495
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发表时间：
2013-05
影响因子：
1.8
通讯作者：
V. G. Watson;Katherine M. Drake;Yu Peng;A. Napper
中科院分区：
文献类型：
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作者：
V. G. Watson;Katherine M. Drake;Yu Peng;A. Napper
混合谱系白血病 (MLL) 基因的重排主要发生在儿童白血病病例中，通常是预后不良的预测因素。这些染色体重排导致 MLL 蛋白与 60 多种蛋白伴侣之一融合。 MLL 融合通过激活癌基因表达而成为白血病的有效诱导剂；因此，针对这种转录激活功能可能会阻止 MLL 重排 (MLL-R) 白血病。含有最常见融合蛋白 MLL-AF4 的白血病细胞系需要 AF4 与转录因子 AF9 直接相互作用才能生存和自我更新；破坏这种与细胞穿透性 AF4 衍生肽的相互作用会导致细胞死亡，这表明 AF4-AF9 相互作用可能成为新型 MLL-R 白血病治疗的可行靶标。在这里，我们描述了使用 AlphaScreen 技术开发高通量筛选 (HTS) 测定法来检测 AF4-AF9 结合的非肽抑制剂。该测定经济实惠，仅需要在小体积 384 孔板中使用低纳摩尔浓度的生物素化 AF4 衍生肽和 FLAG 标记的 AF9。 Z' 因子为 0.71，信号背景比为 21.3，表明该测定方法稳健，并且通过竞争性 AF4 衍生肽证明了对抑制的敏感性。 Nemours 和 Broad Institute 的两个试点筛选包含 5,680 种化合物，作为 HTS 的验证。使用包含生物素化FLAG肽的反筛选排除测定假象。这是第一个报道的 HTS 兼容测定，用于识别抑制 MLL 融合配偶体关键结合相互作用的化合物，这里提供的结果证明适合筛选高密度、低体积板格式的大型化学库。
Rearrangements of the mixed-lineage leukemia (MLL) gene occur predominately in pediatric leukemia cases and are generally predictors of a poor prognosis. These chromosomal rearrangements result in fusion of the protein MLL to one of more than 60 protein partners. MLL fusions are potent inducers of leukemia through activation of oncogene expression; therefore, targeting this transcriptional activation function may arrest MLL-rearranged (MLL-R) leukemia. Leukemic cell lines harboring the most common fusion protein, MLL-AF4, require the direct interaction of AF4 with the transcription factor AF9 to survive and self-renew; disrupting this interaction with a cell-penetrating AF4-derived peptide results in cell death, suggesting that the AF4-AF9 interaction could be a viable target for a novel MLL-R leukemia therapy. Here we describe the use of AlphaScreen technology to develop a high-throughput screening (HTS) assay to detect nonpeptidic inhibitors of AF4-AF9 binding. The assay is economical, requiring only low nanomolar concentrations of biotinylated AF4-derived peptide and FLAG-tagged AF9 in low-volume 384-well plates. A Z'-factor of 0.71 and a signal-to-background ratio of 21.3 showed the assay to be robust, and sensitivity to inhibition was demonstrated with competing AF4-derived peptides. Two pilot screens comprising 5,680 compounds served as validation for HTS at Nemours and the Broad Institute. Assay artifacts were excluded using a counterscreen comprising a biotinylated FLAG peptide. This is the first reported HTS-compatible assay to identify compounds that inhibit a key binding interaction of an MLL fusion partner, and the results presented here demonstrate suitability for screening large chemical libraries in high-density, low-volume plate formats.