Received for publication June 20, 2008.
Revised August 15, 2008.
Accepted for publication August 15, 2008.

Full pharmacological efficacy of a novel S1P₁ agonist that does not require S1P-like head-group interactions

Abstract

Strong evidence exists for interactions of zwitterionic phosphate and amine groups in Sphingosine-1 phosphate (S1P) to conserved R and E residues present at the extracellular face of transmembrane-3 (TM3) of S1P receptors. The contribution of R¹²⁰ and E¹²¹ for high affinity ligand-receptor interactions is essential, as single-point R¹²⁰A or ¹²¹A S1P₁ mutants neither bind S1P nor transduce S1P function. Because S1P receptors are therapeutically interesting, identifying potent selective agonists with different binding modes and in vivo efficacy is of pharmacological importance. Here we describe a modestly water-soluble highly-selective S1P₁ agonist (CYM-5442) that does not require R¹²⁰ or E¹²¹ residues for activating S1P₁-dependent p42/p44 MAPK phosphorylation, which defines a new hydrophobic pocket in S1P₁. CYM-5442 is a full agonist in vitro for S1P₁ internalization, phosphorylation and ubiquitination. Importantly, CYM-5442 was a full agonist for induction and maintenance of S1P₁-dependent lymphopenia, decreasing B-lymphocytes by 65% and T-lymphocytes by 85% of vehicle. Induction of CYM-5442 lymphopenia was dose and time-dependent, requiring serum concentrations in the 50 nM range. In vitro measures of S1P₁ activation by CYM-5442 were non-competitively inhibited by a specific S1P₁ antagonist (W146), competitive for S1P, FTY720-P and SEW2871. In addition, lymphopenia by CYM-5442 was reversed by W146 administration or upon pharmacokinetic agonist clearance. Pharmacokinetics in mice also indicated that CYM-5442 partitions significantly in central nervous tissue. These data show that CYM-5442 activates S1P₁-dependent pathways in vitro and to levels of full efficacy in vivo through a hydrophobic pocket, separable from the orthosteric site of S1P binding that is headgroup dependent.

Key words: Gi family, Sphingolipids, Sequestration/Internalization, Func. analysis receptor/ion channel mutants, Angiogenesis