Intracellular targets of paullones - Identification following affinity purification on immobilized inhibitor
Intracellular targets of paullones - Identification following affinity purification on immobilized inhibitor
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DOI：
10.1074/jbc.m202651200
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发表时间：
2002-07-12
影响因子：
4.8
通讯作者：
Meijer, L
中科院分区：
文献类型：
--
作者：
Knockaert, M;Wieking, K;Meijer, L
细胞周期蛋白依赖性激酶和糖原合成酶激酶-3（GSK-3）的许多抑制剂鉴于其对癌症和神经退行性疾病的潜在应用而被开发。其中，paullones构成了一个家庭的有效的和明显的选择性细胞周期蛋白依赖性激酶和GSK-3抑制剂。然而，它们的实际细胞内靶点仍有待确定。为了解决这个问题，我们已经将paullone，gwennpaullone固定在琼脂糖基质上。从各种细胞类型和组织的提取物中筛选与该基质相互作用的蛋白质。这种方法验证了GSK-3 α和GSK-3 β是主要的细胞内保罗酮靶点，也是线粒体而不是细胞质苹果酸脱氢酶（MDH）。线粒体MDH确实被微摩尔浓度的保罗酮抑制。线粒体MDH是墨西哥利什曼原虫中主要的保罗酮结合蛋白，保罗酮抑制寄生虫的生长。这种简单的分批亲和色谱法构成了一种简单的方法，用于识别这种特定类别的新型抗有丝分裂化合物的细胞内靶标。它揭示了一个意想不到的目标，线粒体MDH，其抑制可能参与保罗酮的药理作用。
Numerous inhibitors of cyclin-dependent kinases and glycogen synthase kinase-3 (GSK-3) are being developed in view of their potential applications against cancers and neurodegenerative disorders. Among these, paullones constitute a family of potent and apparently selective cyclin-dependent kinase and GSK-3 inhibitors. However, their actual intracellular targets remain to be identified. To address this issue we have immobilized a paullone, gwennpaullone, on an agarose matrix. Extracts from various cell types and tissues were screened for proteins interacting with this matrix. This approach validated GSK-3alpha and GSK-3beta as major intracellular paullone targets and also mitochondrial, but not cytoplasmic, malate dehydrogenase (MDH). Mitochondrial MDH was indeed inhibited by micromolar concentrations of paullones. Mitochondrial MDH was the major paullone-binding protein in the parasitic protozoon Leishmania mexicana, and paullones inhibited growth of the parasite. This simple batchwise affinity chromatography approach constitutes a straightforward method for the identification of intracellular targets of this particular class of novel anti-mitotic compounds. It has revealed an unexpected target, mitochondrial MDH, the inhibition of which may participate in the pharmacological effects of paullones.