Assay of Sphingosine 1-phosphate Transporter Spinster Homolog 2 (Spns2) Inhibitors.

The sphingosine-1-phosphate (S1P) pathway remains an active area of research for drug discovery because S1P modulators are effective medicine for autoimmune diseases such as multiple sclerosis and ulcerative colitis. As such, other nodes in the pathway can be probed for alternative therapeutic candidates. As S1P elicits its function in an 'outside-in' fashion, targeting the transporter, Spns2, which is upstream of the receptors, is of great interest. To support our medicinal chemistry campaign to inhibit S1P transport, we developed a mammalian cell-based assay. In this protocol, Spns2 inhibition is assessed by treating HeLa, U-937, and THP-1 cells with inhibitors and S1P exported in the extracellular milieu is quantified by LC-MS/MS. Our studies demonstrated that the amount of S1P in the media in inversely proportional to inhibitor concentration. The details of our investigations are described herein.