The sphingosine kinase 2 inhibitors ABC294640 and K145 elevate (dihydro)sphingosine 1-phosphate levels in various cells
Sphingosine kinases (SphKs), which are enzymes responsible for producing the bioactive lipids dihydrosphingosine 1-phosphate (dhS1P) and sphingosine 1-phosphate (S1P), are linked to various diseases, including cancer and infections. Consequently, several SphK inhibitors have been developed. Although some of these agents have shown off-target effects, SphK inhibitors are often used in research without assessing their impact on the sphingolipidome. In our study, we examined the effects of seven widely used SphK inhibitors—5c, ABC294640 (opaganib), DMS, K145, PF-543, SLM6031434, and SKI-II—on the profiles of selected sphingolipids in Chang, HepG2, and HUVEC cells. As expected, DMS, PF-543, SKI-II, and SLM6031434 reduced (dh)S1P levels, while 5c had minimal impact. Notably, K145 and ABC294640, both considered specific for SphK2, caused significant dose-dependent increases in dhS1P and S1P across the cell lines. These effects were not due to compensatory actions by SphK1, as they were also observed in SphK1-deficient HK-2 cells. Additionally, all inhibitors tested affected dihydroceramide desaturase (DEGS) activity, consistent with previous findings for ABC294640 and SKI-II. Further mechanistic studies revealed that the substantial increase in dhS1P and S1P following short-term treatment with ABC294640 and K145 was due to their influence on sphingolipid de novo synthesis, specifically targeting 3-ketodihydrosphingosine reductase and DEGS. Our study highlights the critical need to monitor cellular sphingolipid profiles when using SphK inhibitors in mechanistic research, as none of the seven inhibitors tested were free from unexpected on-target or off-target effects.