Sphingosine kinase type 2 inhibition elevates circulating sphingosine 1-phosphate

Abstract

Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator involved in numerous cellular and physiological functions. Notable among these are cell survival and migration as well as lymphocyte trafficking. S1P, which exerts its effects via five G protein coupled receptors (S1P1-5), is formed by the action of two sphingosine kinases (SphKs). While SphK1 is the more intensively studied isotype, SphK2 is unique in it nuclear localization and has been reported to oppose some of the actions ascribed to SphK1. While several scaffolds of SphK1 inhibitors have been described, there is a scarcity of selective SphK2 inhibitors that are necessary to evaluate the downstream effects of inhibition of this isotype. Herein we report a cationic amphiphilic small molecule that is a selective SphK2 inhibitor. In the course of characterizing this compound in wild type and SphK null mice we discovered that administration of the inhibitor to wild type mice resulted in a rapid increase in blood S1P, which is in contrast to our SphK1 inhibitor that drives circulating S1P levels down. Using a cohort of F2 hybrid mice, we confirmed, compared to wild type mice, that circulating S1P levels were higher in SphK2 null mice and lower in SphK1 null mice. Thus both SphK1 and SphK2 inhibitors recapitulate the blood S1P levels observed in the corresponding null mice. Moreover, circulating S1P levels mirror SphK2 inhibitor levels providing a convenient biomarker of target engagement.

Sources, metabolism, and regulation of circulating sphingosine-1-phosphate

“…These differences are likely related to the protocol used to isolate plasma. In order to prevent S1P Pharmacological inhibition or knockout of SPHK1 decreases the S1P level in whole blood and plasma by approximately 50%, suggesting that both SPHK isoforms are equally important in generation of the blood pool of this sphingolipid ( 77,78 ). Unexpectedly, genetic or pharmacological inhibition of SPHK2 results in a striking increase in plasma and blood cell S1P concentration ( 77,79 ).…”

S1P Signaling and De Novo Biosynthesis in Blood Pressure Homeostasis

“…One caveat to be considered is that while circulating levels of S1P are reduced in Sphk2 2/2 / Sphk1 1/2 mice (Pappu et al, 2007;Camerer et al, 2009) and in Sphk1 2/2 mice (Allende et al, 2004), circulating levels of S1P are actually increased in Sphk2 2/2 mice (Olivera et al, 2007;Kharel et al, 2012), possibly due to overcompensation by SPHK1 (Olivera et al, 2007).…”

Sphingosine Kinase 2 Inhibition and Blood Sphingosine 1-Phosphate Levels

“…Mice with no functional SphK alleles die in utero, with no detectable S1P (Mizugishi et al, 2005). We have previously documented that the results of these genetic manipulations can be recapitulated with small-molecule inhibitors of SphKs in that administration of an SphK1 inhibitor to wild-type mice or rats decreases blood S1P Patwardhan et al, 2015), whereas administration of an SphK2 inhibitor to mice raises blood S1P levels (Kharel et al, 2012).…”

“…Curiously, ablation of the other SphK gene, Sphk2, in the germ line results in mice with substantially higher blood steady-state S1P levels than corresponding wild-type mice (Mizugishi et al, 2005;Zemann et al, 2006;Sensken et al, 2010;Kharel et al, 2012). Mice with no functional SphK alleles die in utero, with no detectable S1P (Mizugishi et al, 2005).…”