Discovery and evaluation of inhibitors of human sphingosine kinase.
Discovery and evaluation of inhibitors of human sphingosine kinase.
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发表时间：
2003-09
期刊：
影响因子：
11.2
通讯作者：
K. French;Randy S. Schrecengost;Brian D. Lee;Zhuang Yan;Staci N Smith;Justin L Eberly;Jong K. Yun
中科院分区：
文献类型：
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作者：
K. French;Randy S. Schrecengost;Brian D. Lee;Zhuang Yan;Staci N Smith;Justin L Eberly;Jong K. Yun
鞘磷脂代谢酶控制细胞内生物活性脂水平的动态平衡，包括促凋亡化合物神经酰胺和增殖化合物1-磷酸鞘氨醇。越来越多的证据表明，鞘氨醇激酶(SK)在调节肿瘤生长中起着关键作用，SK可作为癌基因发挥作用。尽管SK对细胞增殖很重要，但由于目前缺乏这种酶的非脂类抑制剂，SK的药理抑制是一种未经测试的治疗癌症的方法。为了进一步评估SK在人类肿瘤中的参与程度，我们分析了从肿瘤组织和正常邻近组织制备的成对样本中SK的RNA水平。与同一患者的正常组织相比，SK RNA在多种实体瘤中的表达显著升高。为了鉴定和评价SK的抑制剂，建立了重组人SK与谷胱甘肽S转移酶融合的中通量分析方法，并用于筛选合成化合物的文库。我们发现了一些新型的人SK抑制剂，并对其中几个具有代表性的化合物进行了详细的表征。这些化合物在亚摩尔到微摩尔浓度下表现出活性，使它们比任何其他已报道的SK抑制剂更有效，并且与一系列人类脂质和蛋白激酶相比，它们对SK具有选择性。动力学研究表明，这些化合物不是三磷酸腺苷结合位点的竞争性抑制剂。SK抑制剂对一组肿瘤细胞系具有抗增殖作用，包括由于P-糖蛋白或多药耐药表型1过表达而具有多药耐药表型的肿瘤细胞系，并被证明在完整细胞中抑制内源性人SK活性。此外，每种抑制物在诱导细胞凋亡的同时，也伴随着肿瘤细胞的细胞毒作用。建立了SK的一系列极光抑制剂的合成方法，并发现一种典型的二羟基极光在体内具有中等的抗肿瘤活性，对小鼠没有明显的毒性。这些化合物是具有体内抗肿瘤活性的SK的非脂类抑制剂的第一个例子，因此为进一步开发这一重要分子靶点的抑制剂提供了线索。
Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of bioactive lipids, including the proapoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate. Accumulating evidence indicates that sphingosine kinase (SK) plays a pivotal role in regulating tumor growth and that SK can act as an oncogene. Despite the importance of SK for cell proliferation, pharmacological inhibition of SK is an untested means of treating cancer because of the current lack of nonlipid inhibitors of this enzyme. To further assess the involvement of SK in human tumors, levels of RNA for SK in paired samples of cDNA prepared from tumors and normal adjacent tissue were analyzed. Expression of SK RNA was significantly elevated in a variety of solid tumors, compared with normal tissue from the same patient. To identify and evaluate inhibitors of SK, a medium throughput assay for recombinant human SK fused to glutathione S-transferase was developed, validated, and used to screen a library of synthetic compounds. A number of novel inhibitors of human SK were identified, and several representative compounds were characterized in detail. These compounds demonstrated activity at sub- to micromolar concentrations, making them more potent than any other reported SK inhibitor, and were selective toward SK compared with a panel of human lipid and protein kinases. Kinetic studies revealed that the compounds were not competitive inhibitors of the ATP-binding site of SK. The SK inhibitors were antiproliferative toward a panel of tumor cell lines, including lines with the multidrug resistance phenotype because of overexpression of either P-glycoprotein or multidrug resistance phenotype 1, and were shown to inhibit endogenous human SK activity in intact cells. Furthermore, each inhibitor induced apoptosis concomitant with tumor cell cytotoxicity. Methods for the synthesis of a series of aurone inhibitors of SK were established, and a prototypical dihydroxyaurone was found to have moderate antitumor activity in vivo in the absence of overt toxicity to the mice. These compounds are the first examples of nonlipid inhibitors of SK with in vivo antitumor activity and so provide leads for additional development of inhibitors of this important molecular target.