SHetA2 interference with mortalin binding to p66shc and p53 identified using drug-conjugated magnetic microspheres.
SHetA2 interference with mortalin binding to p66shc and p53 identified using drug-conjugated magnetic microspheres.
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sheta2干扰了使用药物偶联的磁微球鉴定的玛al蛋白与p66SHC和p53的结合。
DOI：
10.1007/s10637-013-0041-x
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发表时间：
2014-06
影响因子：
3.4
通讯作者：
Berlin, K. Darrell
中科院分区：
文献类型：
--
作者：
Benbrook, Doris Mangiaracina;Nammalwar, Baskar;Long, Andrew;Matsumoto, Hiroyuki;Singh, Anil;Bunce, Richard A.;Berlin, K. Darrell
SHetA 2是一种小分子柔性杂芳维A酸（Flex-Het），具有很好的癌症预防和治疗活性。广泛的临床前测试记录了在有效剂量的25至150倍的剂量下没有SHetA 2毒性。了解介导SHetA 2作用机制的SHetA 2分子靶标对于适当设计临床试验和改进的类似物至关重要。本研究的目的是开发一种方法来鉴定癌细胞中的SHetA 2结合蛋白。合成了具有可用于连接的羟基的SHetA 2的已知代谢物，并将其缀合至用于连接至磁性微球的接头。将SHetA 2缀合的磁性微球和未缀合的磁性微球分别与来自A2780人卵巢癌细胞系的全细胞蛋白提取物的等分试样一起孵育。用蛋白质提取缓冲液洗去非特异性结合的蛋白质后，用过量的游离SHetA 2洗脱SHetA 2结合蛋白质。在两个独立的实验中，与来自未缀合的微球的洗脱液的威尔斯孔相比，在来自SHetA 2-微球的洗脱液的威尔斯孔中以差异水平存在约72 kDa的SDS凝胶带。条带（QStar）和直洗脱液（Orbitrap）的质谱分析鉴定死亡蛋白（HSPA 9）存在于来自SHetA 2-微球的洗脱液中，而不存在于来自未缀合微球的洗脱液中。免疫共沉淀实验表明，SHetA 2干扰癌细胞内的p53和p66 Src同源胶原同源物（p66 shc）的结合。Mortalin和SHetA 2冲突地调节参与细胞凋亡介导的内在凋亡的相同分子。结果验证了该方案揭示药物靶点的能力。本文的在线版本（doi：10.1007/s10637-013-0041-x）包含补充材料，可供授权用户使用。
SHetA2 is a small molecule flexible heteroarotinoid (Flex-Het) with promising cancer prevention and therapeutic activity. Extensive preclinical testing documented lack of SHetA2 toxicity at doses 25 to 150 fold above effective doses. Knowledge of the SHetA2 molecular target(s) that mediate(s) the mechanism of SHetA2 action is critical to appropriate design of clinical trials and improved analogs. The aim of this study was to develop a method to identify SHetA2 binding proteins in cancer cells. A known metabolite of SHetA2 that has a hydroxyl group available for attachment was synthesized and conjugated to a linker for attachment to a magnetic microsphere. SHetA2-conjugated magnetic microspheres and unconjugated magnetic microspheres were separately incubated with aliquots of a whole cell protein extract from the A2780 human ovarian cancer cell line. After washing away non-specifically bound proteins with the protein extraction buffer, SHetA2-binding proteins were eluted with an excess of free SHetA2. In two independent experiments, an SDS gel band of about 72 kDa was present at differential levels in wells of eluent from SHetA2-microspheres in comparison to wells of eluent from unconjugated microspheres. Mass spectrometry analysis of the bands (QStar) and straight eluents (Orbitrap) identified mortalin (HSPA9) to be present in the eluent from SHetA2-microspheres and not in eluent from unconjugated microspheres. Co-immunoprecipitation experiments demonstrated that SHetA2 interfered with mortalin binding to p53 and p66 Src homologous-collagen homologue (p66shc) inside cancer cells. Mortalin and SHetA2 conflictingly regulate the same molecules involved in mitochondria-mediated intrinsic apoptosis. The results validate the power of this protocol for revealing drug targets. The online version of this article (doi:10.1007/s10637-013-0041-x) contains supplementary material, which is available to authorized users.
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DOI：
10.1016/j.mrgentox.2012.03.009
发表时间：
2012-07-04
影响因子：
1.9
作者：
Doppalapudi, Rupa S.;Riccio, Edward S.;Davis, Zoe;Menda, Sean;Wang, Abraham;Du, Nicholas;Green, Carol;Kopelovich, Levy;Rao, Chinthalapally V.;Benbrook, Doris M.;Kapetanovic, Izet M.
通讯作者：
Kapetanovic, Izet M.
影响因子：
12.4
作者：
Liu, T.;Laurell, C.;Wiman, K. G.
通讯作者：
Wiman, K. G.
影响因子：
2.6
作者：
Kabirov, Kasim K.;Kapetanovic, Izet M.;Lyubimov, Alexander V.
通讯作者：
Lyubimov, Alexander V.
影响因子：
4.7
作者：
Moxley, Katherine Marie;Chengedza, Shylet;Mangiaracina, Doris
通讯作者：
Mangiaracina, Doris
影响因子：
5.7
作者：
Liu, Tongzu;Hannafon, Bethany;Benbrook, Doris
通讯作者：
Benbrook, Doris