鞘氨醇激酶 (SphK) 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，与多种疾病相关，包括癌症和感染。为此，开发了许多 SphK 抑制剂。尽管已描述了选定药物的脱靶效应，但 SphK 抑制剂主要用于研究，而不监测对鞘脂组的影响。我们现在研究了七种常用的 SphK 抑制剂（5c、ABC294640（opaganib）、DMS、K145、PF-543、SLM6031434 和 SKI-II）对 Chang、HepG2 和 HUVEC 细胞中选定鞘脂谱的影响。虽然我们观察到 DMS、PF-543、SKI-II 和 SLM6031434 预期 (dh)S1P 减少，但 5c 几乎没有显示任何效果。值得注意的是，对于据报道对 SphK2 具有特异性的 K145 和 ABC294640，我们观察到跨细胞系的 dhS1P 和 S1P 呈剂量依赖性强烈增加。可以排除 SphK1 的代偿作用，因为这一观察也在 SphK1 缺陷的 HK-2 细胞中进行。此外，我们观察到所有测试的抑制剂对二氢神经酰胺去饱和酶 (DEGS) 活性的影响，正如之前针对 ABC294640 和 SKI-II 所指出的那样。在其他机制研究中，我们更详细地研究了 ABC294640 和 K145 短期细胞处理后 dhS1P 和 S1P 的大量增加。我们发现这两种化合物都会影响鞘脂从头合成，以 3-酮二氢鞘氨醇还原酶和 DEGS 作为其靶标。我们的研究强调了在机制研究中使用 SphK 抑制剂时监测细胞鞘脂谱的紧迫性，因为测试的七种 SphK 抑制剂中没有一种没有意外的靶向和/或脱靶效应。版权所有 © 2024 作者。由爱思唯尔公司出版。保留所有权利。

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), DMS, K145, PF-543, SLM6031434 and SKI-II) on profiles of selected sphingolipids in Chang, HepG2 and HUVEC cells. While we observed the expected (dh)S1P reduction for DMS, 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 (DEGS) 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 DEGS as their targets. Our study emphasizes the urgency of monitoring cellular sphingolipid profiles when SphK inhibitors are used in mechanistic investigations, as none of the seven SphK inhibitors tested was free of unexpected on-target and/or off-target effects.Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.