Kim Yeong Sang, Nagy Katelyn, Keyser Samantha, Schneekloth John S
Chemical Biology Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
Chem Biol. 2013 Apr 18;20(4):604-13. doi: 10.1016/j.chembiol.2013.04.001.
The dynamic, posttranslational modification of proteins with a small ubiquitin-like modifier (SUMO) tag has been recognized as an important cellular regulatory mechanism relevant to a number of cancers as well as normal embryonic development. As part of a program aimed toward the identification of inhibitors of SUMO-conjugating enzymes, we developed a microfluidic electrophoretic mobility shift assay to monitor sumoylation events in real time. We disclose herein the use of this assay to identify a cell-permeable compound capable of blocking the transfer of SUMO-1 from the E2 enzyme Ubc9 to the substrate. We screened a small collection of compounds and identified an oxygenated flavonoid derivative that inhibits sumoylation in vitro. Next, we carried out an in-depth mechanistic analysis that ruled out many common false-positive mechanisms such as aggregation or alkylation. Furthermore, we report that this flavonoid inhibits a single step in the sumoylation cascade: the transfer of SUMO from the E2 enzyme (Ubc9) thioester conjugate to the substrate. In addition to having a unique mechanism of action, this inhibitor has a discrete structure-activity relationship uncharacteristic of a promiscuous inhibitor. Cell-based studies showed that the flavonoid inhibits the sumoylation of topoisomerase-I in response to camptothecin treatment in two different breast cancer cell lines, while isomeric analogs are inactive. Importantly, this compound blocks sumoylation while not affecting ubiquitylation in cells. This work identifies a point of entry for pharmacologic inhibition of the sumoylation cascade and may serve as the basis for continued study of additional pharmacophores that modulate SUMO-conjugating enzymes such as Ubc9.
用小泛素样修饰物（SUMO）标签对蛋白质进行动态的翻译后修饰，已被公认为是一种重要的细胞调节机制，与多种癌症以及正常胚胎发育相关。作为旨在鉴定SUMO缀合酶抑制剂的项目的一部分，我们开发了一种微流控电泳迁移率变动分析方法，以实时监测SUMO化事件。我们在此披露了该分析方法的用途，即鉴定一种能够阻断SUMO-1从E2酶Ubc9转移至底物的细胞可渗透化合物。我们筛选了一小批化合物，并鉴定出一种在体外抑制SUMO化的氧化黄酮衍生物。接下来，我们进行了深入的机制分析，排除了许多常见的假阳性机制，如聚集或烷基化。此外，我们报告称，这种黄酮类化合物在SUMO化级联反应中抑制单个步骤：SUMO从E2酶（Ubc9）硫酯缀合物转移至底物。除了具有独特的作用机制外，这种抑制剂还具有一种混杂抑制剂所不具备的离散结构-活性关系。基于细胞的研究表明，这种黄酮类化合物在两种不同的乳腺癌细胞系中，可抑制喜树碱处理后拓扑异构酶-I的SUMO化，而其异构体类似物则无活性。重要的是，这种化合物可阻断SUMO化，同时不影响细胞中的泛素化。这项工作确定了对SUMO化级联反应进行药理抑制的切入点，并可能为继续研究调节SUMO缀合酶（如Ubc9）的其他药效基团奠定基础。
