Potent inhibitors of the sphingosine-1-phosphate transporter SPNS2 as immunosuppressive agents

Sphingosine-1-phosphate (S1P) is a pleiotropic signaling molecule involved in several biological pathways and plays an essential role in immune system function. S1P is predominantly produced intracellularly by one of two sphingosine kinase isoforms from ATP and the lipid sphingosine. S1P is transported from the cells where it is capable of binding to one of five S1P-specific G-protein coupled receptors (S1P1-5). S1P is excreted via a transmembrane transport protein, spinster homolog 2 (SPNS2), which is prominent in endothelial cells. This extrusion of S1P coupled with brisk intracellular degradation generates an S1P gradient with concentrations highest in blood and areas of inflammation and lowest in lymph tissue. Efficient lymphocyte egress from lymph nodes depends on the higher S1P concentration of lymph. Given the role S1P plays in lymphocyte trafficking, targeting the S1P pathway has applications in the treatment of various diseases such as kidney fibrosis and multiple sclerosis. Herein, we report the structure-activity relationship studies on the first documented SPNS2 inhibitor. We identified compound SLF1081851 as an SPNS2 inhibitor with an IC50 value of 900 nM in vitro. Administration of SLF1081851 into mice resulted in peripheral blood lymphopenia, which phenocopies Spns2 knock-out mice. Extensive investigations on the warhead region of the pharmacophore revealed SLF80821178 with an IC50 of 70 nM, the most potent SPNS2 inhibitor to date. The results from this SAR also indicate that the SPNS2 binding site has a preference for hydrophilic functionalities that act as both hydrogen bond donors and acceptors.