Development of Potent Inhibitors of the Sphingosine-1-Phosphate Transporter Spns2 for the Treatment of Multiple Sclerosis

Sphingosine-1-phosphate (S1P) is an amino-alcohol signaling molecule produced from the intracellular phosphorylation of the lipid sphingosine. Despite possessing several identified intracellular targets, the predominant signaling functionality of S1P is derived from its activation of membrane-bound G-protein coupled receptors (GPCRs). The binding of S1P to these receptors (S1P1-5) is closely associated with immune cell development and recruitment. As such, the modulation of S1P-related pathways is of particular interest for the development of immunomodulating agents.

To reach its native GPCRs, S1P must be released from the cell. This process is facilitated by the transmembrane transport protein Spinster homolog 2 (Spns2) in most vertebrates. Studies in murine species have demonstrated that the protein plays a key role in directing immune cell chemotaxis and the progression of autoimmune diseases. Consequently, Spns2 represents an attractive target for the pharmaceutical induction of immunosuppression. While several drugs that act through the modulation of S1P receptor signaling have received FDA approval for the treatment of autoimmune disorders (fingolimod, siponimod, ozanimod, and ponesimod), they typically manifest on-target cardiovascular side-effects. Therefore, the development of novel Spns2 inhibitors is a prudent alternative approach to achieve S1P-mediated lymphopenia.

In this dissertation, the design, synthesis, and activities of highly potent Spns2 inhibitors are disclosed. These structures spanned several scaffolds and culminated in the discovery of a phenylurea derivative 4.11i. In vitro assessment of 4.11i demonstrated that the compound possessed an IC50 value of 92 nM, making it the most potent inhibitor of Spns2 disclosed to date. Intraperitoneal administration of 4.11i (10 mg/kg dose) into mice reduced circulating lymphocyte counts and impaired the progression of experimental autoimmune encephalomyelitis (a murine model of multiple sclerosis). Taken together, these data validated the target of 4.11i in vivo and represented the first reported instance of Spns2 inhibition as a viable multiple sclerosis treatment. Additional work is currently being undertaken to further improve in vivo activity and pharmacokinetic properties of 4.11i.