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Sphingosine 1-phosphate (S1P) levels are significantly higher in blood and lymph than in tissues. This S1P concentration difference is necessary for proper lymphocyte egress from secondary lymphoid tissue and to maintain endothelial barrier integrity. Studies with mice lacking either sphingosine kinase (SphK) type 1 and 2 indicate that these enzymes are the sole biosynthetic source of S1P, but they play different roles in setting S1P blood levels. We have developed a set of drug-like SphK inhibitors, with differing selectivity for the two isoforms of this enzyme. Although all SphK inhibitors tested decrease S1P when applied to cultured U937 cells, only those inhibitors with a bias for SphK2 drove a substantial increase in blood S1P in mice and this rise was detectable within minutes of administration of the inhibitor. Blood S1P also increased in response to SphK2 inhibitors in rats. Mass-labeled S1P was cleared more slowly after intravenous injection into SphK2 inhibitortreated mice or mice lacking a functional SphK2 gene; thus, the increased accumulation of S1P in the blood appears to result from the decreased clearance of S1P from the blood. Therefore, SphK2 appears to have a function independent of generating S1P in cells. Our results suggest that differential SphK inhibition with a drug might afford a method to manipulate blood S1P levels in either direction while lowering tissue S1P levels.

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that acts as a ligand for five G-protein coupled receptors (S1P1–5) whose downstream effects are implicated in a variety of important pathologies including sickle cell disease, cancer, inflammation, and fibrosis. The synthesis of S1P is catalyzed by sphingosine kinase (SphK) isoforms 1 and 2, and hence, inhibitors of this phosphorylation step are pivotal in understanding the physiological functions of SphKs. To date, SphK1 and 2 inhibitors with the potency, selectivity, and in vivo stability necessary to determine the potential of these kinases as therapeutic targets are lacking. Herein, we report the design, synthesis, and structure–activity relationship studies of guanidine-based SphK inhibitors bearing an oxadiazole ring in the scaffold. Our studies demonstrate that SLP120701, a SphK2-selective inhibitor (Ki = 1 μM), decreases S1P levels in histiocytic lymphoma (U937) cells. Surprisingly, homologation with a single methylene unit between the oxadiazole and heterocyclic ring afforded a SphK1-selective inhibitor in SLP7111228 (Ki = 48 nM), which also decreased S1P levels in cultured U937 cells. In vivo application of both compounds, however, resulted in contrasting effect in circulating levels of S1P. Administration of SLP7111228 depressed blood S1P levels while SLP120701 increased levels of S1P. Taken together, these compounds provide an in vivo chemical toolkit to interrogate the effect of increasing or decreasing S1P levels and whether such a maneuver can have implications in disease states.

Rationale: Emerging evidence has suggested that sphingosine 1-phosphate (S1P), a bioactive metabolite of sphingolipids, may play an important role in the pathophysiological processes of cerebral hypoxia and ischemia. However, the influence of S1P on cerebral hemodynamics and metabolism remains unclear.Material and Methods: Uniquely capable of high-resolution, label-free, and comprehensive imaging of hemodynamics and oxygen metabolism in the mouse brain without the influence of general anesthesia, our newly developed head-restrained multi-parametric photoacoustic microscopy (PAM) is well suited for this mechanistic study. Here, combining the cutting-edge PAM and a selective inhibitor of sphingosine kinase 2 (SphK2) that can increase the blood S1P level, we investigated the role of S1P in cerebral oxygen supply-demand and its neuroprotective effects on global brain hypoxia induced by nitrogen gas inhalation and focal brain ischemia induced by transient middle cerebral artery occlusion (tMCAO).Results: Inhibition of SphK2, which increased the blood S1P, resulted in the elevation of both arterial and venous sO2 in the hypoxic mouse brain, while the cerebral blood flow remained unchanged. As a result, it gradually and significantly reduced the metabolic rate of oxygen. Furthermore, pre-treatment of the mice subject to tMCAO with the SphK2 inhibitor led to decreased infarct volume, improved motor function, and reduced neurological deficit, compared to the control treatment with a less potent R-enantiomer. In contrast, post-treatment with the inhibitor showed no improvement in the stroke outcomes. The failure for the post-treatment to induce neuroprotection was likely due to the relatively slow hemodynamic responses to the SphK2 inhibitor-evoked S1P intervention, which did not take effect before the brain injury was induced.Conclusions: Our results reveal that elevated blood S1P significantly changes cerebral hemodynamics and oxygen metabolism under hypoxia but not normoxia. The improved blood oxygenation and reduced oxygen demand in the hypoxic brain may underlie the neuroprotective effect of S1P against ischemic stroke.

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