Department of Pharmacology and Toxicology, Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany.
Institute of Pharmacology, Inselspital, INO-F, University of Bern, Bern, Switzerland.
J Lipid Res. 2024 Oct;65(10):100631. doi: 10.1016/j.jlr.2024.100631. Epub 2024 Aug 23.
Sphingosine kinases (SphKs), enzymes that produce the bioactive lipids dihydrosphingosine 1-phosphate (dhS1P) and sphingosine 1-phosphate (S1P), are associated with various diseases, including cancer and infections. For this reason, a number of SphK inhibitors have been developed. Although off-target effects have been described for selected agents, SphK inhibitors are mostly used in research without monitoring the effects on the sphingolipidome. We have now investigated the effects of seven commonly used SphK inhibitors (5c, ABC294640 (opaganib), N,N-dimethylsphingosine, K145, PF-543, SLM6031434, and SKI-II) on profiles of selected sphingolipids in Chang, HepG2, and human umbilical vein endothelial cells. While we observed the expected (dh)S1P reduction for N,N-dimethylsphingosine, PF-543, SKI-II, and SLM6031434, 5c showed hardly any effect. Remarkably, for K145 and ABC294640, both reported to be specific for SphK2, we observed dose-dependent strong increases in dhS1P and S1P across cell lines. Compensatory effects of SphK1 could be excluded, as this observation was also made in SphK1-deficient HK-2 cells. Furthermore, we observed effects on dihydroceramide desaturase activity for all inhibitors tested, as has been previously noted for ABC294640 and SKI-II. In additional mechanistic studies, we investigated the massive increase of dhS1P and S1P after short-term cell treatment with ABC294640 and K145 in more detail. We found that both compounds affect sphingolipid de novo synthesis, with 3-ketodihydrosphingosine reductase and dihydroceramide desaturase as their targets. Our study indicates that none of the seven SphK inhibitors tested was free of unexpected on-target and/or off-target effects. Therefore, it is important to monitor cellular sphingolipid profiles when SphK inhibitors are used in mechanistic studies.
鞘氨醇激酶（SphKs）是产生生物活性脂质二氢鞘氨醇 1-磷酸（dhS1P）和鞘氨醇 1-磷酸（S1P）的酶，与包括癌症和感染在内的各种疾病有关。因此，已经开发了许多 SphK 抑制剂。尽管已经描述了一些选定的药物的脱靶效应，但 SphK 抑制剂主要用于研究而不监测对鞘脂谱的影响。我们现在研究了七种常用的 SphK 抑制剂（5c、ABC294640（opaganib）、N,N-二甲基鞘氨醇、K145、PF-543、SLM6031434 和 SKI-II）对 Chang、HepG2 和人脐静脉内皮细胞中选定鞘脂的影响。虽然我们观察到 N,N-二甲基鞘氨醇、PF-543、SKI-II 和 SLM6031434 预期的（dh）S1P 减少，但 5c 几乎没有效果。值得注意的是，对于 K145 和 ABC294640，两者都被报道为 SphK2 的特异性抑制剂，我们观察到跨细胞系的 dhS1P 和 S1P 剂量依赖性强烈增加。SphK1 的代偿作用可以排除，因为这一观察结果也在 SphK1 缺陷型 HK-2 细胞中观察到。此外，我们还观察到所有测试的抑制剂对二氢神经酰胺去饱和酶活性的影响，正如之前在 ABC294640 和 SKI-II 中所指出的那样。在进一步的机制研究中，我们更详细地研究了 ABC294640 和 K145 短期处理细胞后 dhS1P 和 S1P 的大量增加。我们发现这两种化合物都影响鞘脂从头合成，其靶标是 3-酮二氢鞘氨醇还原酶和二氢神经酰胺去饱和酶。我们的研究表明，在所测试的七种 SphK 抑制剂中，没有一种没有意外的靶标和/或脱靶效应。因此，当 SphK 抑制剂在机制研究中使用时，监测细胞鞘脂谱非常重要。
