Discovery of In Vivo Active Sphingosine-1-phosphate Transporter (Spns2) Inhibitors

Abstract

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that interacts with five G-protein-coupled receptors (S1P1-5) to regulate cellular signaling pathways. S1P export is facilitated by Mfsd2b and spinster homologue 2 (Spns2). While mouse genetic studies suggest that Spns2 functions to maintain lymph S1P, Spns2 inhibitors are necessary to understand its biology and to learn whether Spns2 is a viable drug target. Herein, we report a structure–activity relationship study that identified the first Spns2 inhibitor 16d (SLF1081851). In vitro studies in HeLa cells demonstrated that 16d inhibited S1P release with an IC50 of 1.93 μM. Administration of 16d to mice and rats drove significant decreases in circulating lymphocyte counts and plasma S1P concentrations, recapitulating the phenotype observed in mice made deficient in Spns2. Thus, 16d has the potential for development and use as a probe to investigate Spns2 biology and to determine the potential of Spns2 as a drug target.

2-Aminobenzoxazole Derivatives as Potent Inhibitors of the Sphingosine-1-Phosphate Transporter Spinster Homolog 2 (Spns2)

“…Further, 33p was found to exert this effect at 16 h post administration in a dose-dependent manner (Figure B). These results are consistent with the phenotype that is observed with Spns2-knockout mice suggesting in vivo target engagement. , However, neither 32p nor 33p administration resulted in a significant decrease in plasma [S1P] (Figure C), which is unlike our experience with our first-generation Spns2 inhibitor, SLF1081851 . As mentioned previously, the literature regarding genetically modified, Spns2-deficient mice is contradictory on this point.…”

“…We initially focused our studies on the linker region of the pharmacophore . The fusion of phenyl and oxadiazole moieties produces attractive 5,6-bicyclic ring systems that enforce conformational rigidity and polarity.…”

“…We screened the putative inhibitors for Spns2 inhibition at either a 1 or 0.3 μM concentration (Table ) using a mouse Spns2 HeLa cell-based S1P release assay . Our initial studies focused on the polar head group by comparing alkyl and cycloalkyl amines with various lengths and ring sizes.…”

“…Our initial studies focused on the polar head group by comparing alkyl and cycloalkyl amines with various lengths and ring sizes. Decyl tails were investigated first since they were shown to have favorable potency as we have previously reported . Starting with a benzoxazole linker, a homologation study of linear alkyl amines from 2- to 5-carbon ( 15a – d ) showed that the 3-carbon linker ( 15b ) was preferred increasing the inhibitory activity from 0 to 40%.…”

Sphingosine 1-phosphate signaling in perivascular cells enhances inflammation and fibrosis in the kidney

“…Because our observations suggested that S1P exported via Spns2 is an initial step in the inside-out signaling of S1P in kidney perivascular cells to enhance inflammatory response, Spns2 may be a drug target candidate. To explore this idea, we used a small-molecule Spns2 inhibitor [SLF1081851 ( 50 ) and fig. S17A] that evokes dose-dependent peripheral blood lymphocytopenia and a decrease in plasma S1P in mice (fig.…”