Abstract
Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions, including lymphocyte trafficking and vascular development, by activating G protein-coupled receptors for S1P, namely S1P
1
through S1P
5
. Here we map the S1P
3
binding pocket with a novel allosteric agonist (CYM-5541), an orthosteric agonist (S1P), and a novel bitopic antagonist (SPM-242). With a combination of site-directed mutagenesis, ligand competition assay, and molecular modeling, we concluded that S1P and CYM-5541 occupy different chemical spaces in the ligand binding pocket of S1P
3
. CYM-5541 allowed us to identify an allosteric site where Phe263 is a key gate-keeper residue for its affinity and efficacy. This ligand lacks a polar moiety and the novel allosteric hydrophobic pocket permits S1P
3
selectivity of CYM-5541 within the highly similar S1P receptor family. On the other hand, a novel S1P
3
-selective antagonist, SPM-242, in the S1P
3
pocket occupies the ligand binding spaces of both S1P and CYM-5541, showing its bitopic mode of binding. Therefore, our coordinated approach with biochemical data and molecular modeling, based on our recently published S1P
1
crystal structure data in a highly conserved set of related receptors with a shared ligand, provides a strong basis for the successful optimization of orthosteric, allosteric, and bitopic modulators of S1P
3
.
