Department of Pathology and Lab Medicine, University of Rochester Medical Center, Rochester, New York 14642, United States.
Biochemistry. 2011 Dec 6;50(48):10431-41. doi: 10.1021/bi200962u. Epub 2011 Nov 10.
The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.
锌指蛋白 EVI1 与急性髓系白血病的发生有因果关系，用小分子治疗药物抑制其功能可能为表达 EVI1 的白血病提供有效的治疗方法。本文描述了一种吡咯-咪唑聚酰胺的开发，以特异性阻断 EVI1 与 DNA 的结合。我们首先通过对 EVI1 和 t(3;21)(q26;q22)-衍生的 RUNX1-MDS1-EVI1 (RME) 蛋白的结构-功能研究，确定了白血病发生的必需结构域，结果表明通过两个锌指结构域(ZF1，包含手指 1-7)中的第一个结合到同源基序 GACAAGATA 的 DNA 结合对于转化活性是必需的。为了通过 ZF1 抑制 DNA 结合，我们合成了一种吡咯-咪唑聚酰胺 1，设计用于结合 GACAAGATA 基序的亚位点，从而阻断 EVI1 结合。DNase I 足迹和电泳迁移率变动分析显示聚酰胺 1 与 GACAAGATA 基序之间存在特异性和高亲和力相互作用。在使用带有染色体嵌入的 tet 诱导型 EVI1-VP16 的 NIH-3T3 衍生细胞系和 EVI1 反应性报告基因进行的 CAT 报告基因测定中，聚酰胺 1 完全阻断了 EVI1 反应性报告基因的活性。用聚酰胺 1 处理过表达 EVI1 的白血病细胞系的生长也受到抑制，而缺乏 EVI1 的对照细胞系则没有。最后，在连续平板克隆形成测定中，聚酰胺 1 减弱了 RME 的集落形成。这些研究提供了证据表明，一种细胞穿透性小分子可以有效地阻断致癌转录因子的活性，并为剖析 EVI1 在白血病发生中的关键功能提供了有价值的工具。
