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.2012 Dec 21;7(12):1975-83.
doi: 10.1021/cb300392z. Epub 2012 Sep 14.

Novel selective allosteric and bitopic ligands for the S1P(3) receptor

Affiliations

Novel selective allosteric and bitopic ligands for the S1P(3) receptor

Euijung Jo et al. ACS Chem Biol..

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. However, 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).

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Figures

Figure 1
Figure 1
Structures of S1P3-selective agonist and antagonist. (A) CYM-5541 (N,N-dicyclohexyl(5-cyclopropylisoxazol-3-yl)carboxamide). (B) SPM-242 ((+)-2-amino-4-(2-chloro-4-((3-hydroxyphenyl)thio)phenyl)-2-(hydroxymethyl)butyl dihydrogen phosphate).
Figure 2
Figure 2. Ligand-induced ERK phosphorylation in WT S1P3 Jump-In stable cell lines (Mean ± SEM; n=3), revealing CYM-5541 is a full agonist of S1P3
Targetable Jump-In™ TI™ CHO-K cell lines were used to integrate a single copy of S1P3 in a site-specific manner. WT Jump-In stable cell lines were stimulated with increasing concentrations of either S1P or CYM-5541.
Figure 3
Figure 3. Radioligand binding and competition
WT, W256L, and F263L Jump-In stable cell lines were incubated with 0.1 nM [33P]S1P in the presence of increasing concentrations of (A) CYM-5541, (B) S1P, and (C) SPM-242. [33P]S1P binding was competitively reversed with S1P and SPM-242 in all cell lines tested whereas CYM-5541 was unable to compete for [33P]S1P binding (Mean ± SEM; n=3).
Figure 4
Figure 4. Receptor mutagenesis and ligand-induced ERK phosphorylation
(A) ERK phosphorylation assay revealed that F263L mutation shifted CYM-5541-induced receptor activation but not S1P-induced activation. (B) W256L mutation did not affect either S1P- or CYM-5541-induced receptor activation (Mean ± SEM; n=3). Data is a representative of four independent experiments.
Figure 5
Figure 5. SPM-242, an S1P3-selective antagonist, is a bitopic antagonist
(A) SPM-242 shifted the dose-response curve of S1P and CYM-5541 (ERK phosphorylation assay). (B) SPM-242 (concentrations ranging from 10−9.5 to 10−7 M) shifted the dose-response curve of CYM-5541 in a dose-dependent manner (NFAT β–lactamase reporter assay) (Mean ± SEM; n=3).
Figure 6
Figure 6. Visualization of the receptor binding pocket by homology modeling and docking. Three dimensional plot of S1P3 binding to (A) S1P, (B) CYM-5541, and (C and D) both
(A) S1P headgroup interacting with R114 and E115. (B) CYM-5541 in the hydrophobic pocket. (C) S1P and CYM-5541 co-docked to S1P3. In the presence of S1P, the pocket opens up in the lower hydrophobic region adjusting CYM-5541 (after 5ns MD optimization). (D) Top view from the extracellular surface with helix orientation identical to the other panels. Detailed modeling and docking procedures are described in Methods section. (E) Calcium response assay upon co-application of S1P and CYM-5541 to S1P3-CHO cells. When both S1P and CYM-5541 were added to S1P3-CHO cells, calcium release response was increased, indicating their additive responses (Mean ± SEM; n=9).
Figure 6
Figure 6. Visualization of the receptor binding pocket by homology modeling and docking. Three dimensional plot of S1P3 binding to (A) S1P, (B) CYM-5541, and (C and D) both
(A) S1P headgroup interacting with R114 and E115. (B) CYM-5541 in the hydrophobic pocket. (C) S1P and CYM-5541 co-docked to S1P3. In the presence of S1P, the pocket opens up in the lower hydrophobic region adjusting CYM-5541 (after 5ns MD optimization). (D) Top view from the extracellular surface with helix orientation identical to the other panels. Detailed modeling and docking procedures are described in Methods section. (E) Calcium response assay upon co-application of S1P and CYM-5541 to S1P3-CHO cells. When both S1P and CYM-5541 were added to S1P3-CHO cells, calcium release response was increased, indicating their additive responses (Mean ± SEM; n=9).
Figure 6
Figure 6. Visualization of the receptor binding pocket by homology modeling and docking. Three dimensional plot of S1P3 binding to (A) S1P, (B) CYM-5541, and (C and D) both
(A) S1P headgroup interacting with R114 and E115. (B) CYM-5541 in the hydrophobic pocket. (C) S1P and CYM-5541 co-docked to S1P3. In the presence of S1P, the pocket opens up in the lower hydrophobic region adjusting CYM-5541 (after 5ns MD optimization). (D) Top view from the extracellular surface with helix orientation identical to the other panels. Detailed modeling and docking procedures are described in Methods section. (E) Calcium response assay upon co-application of S1P and CYM-5541 to S1P3-CHO cells. When both S1P and CYM-5541 were added to S1P3-CHO cells, calcium release response was increased, indicating their additive responses (Mean ± SEM; n=9).
Figure 6
Figure 6. Visualization of the receptor binding pocket by homology modeling and docking. Three dimensional plot of S1P3 binding to (A) S1P, (B) CYM-5541, and (C and D) both
(A) S1P headgroup interacting with R114 and E115. (B) CYM-5541 in the hydrophobic pocket. (C) S1P and CYM-5541 co-docked to S1P3. In the presence of S1P, the pocket opens up in the lower hydrophobic region adjusting CYM-5541 (after 5ns MD optimization). (D) Top view from the extracellular surface with helix orientation identical to the other panels. Detailed modeling and docking procedures are described in Methods section. (E) Calcium response assay upon co-application of S1P and CYM-5541 to S1P3-CHO cells. When both S1P and CYM-5541 were added to S1P3-CHO cells, calcium release response was increased, indicating their additive responses (Mean ± SEM; n=9).
Figure 6
Figure 6. Visualization of the receptor binding pocket by homology modeling and docking. Three dimensional plot of S1P3 binding to (A) S1P, (B) CYM-5541, and (C and D) both
(A) S1P headgroup interacting with R114 and E115. (B) CYM-5541 in the hydrophobic pocket. (C) S1P and CYM-5541 co-docked to S1P3. In the presence of S1P, the pocket opens up in the lower hydrophobic region adjusting CYM-5541 (after 5ns MD optimization). (D) Top view from the extracellular surface with helix orientation identical to the other panels. Detailed modeling and docking procedures are described in Methods section. (E) Calcium response assay upon co-application of S1P and CYM-5541 to S1P3-CHO cells. When both S1P and CYM-5541 were added to S1P3-CHO cells, calcium release response was increased, indicating their additive responses (Mean ± SEM; n=9).
Figure 7
Figure 7. Visualization of the orthosteric, allosteric, and bitopic ligand interactions based upon the biochemistry, mutagenesis, homology modeling, and molecular dynamic simulations. Dual inhibition of SPM-242 is illustrated
(A) S1P (green) and SPM-242 (cyan) overlap in the S1P3 binding pocket. (B) CYM-5541 (green) and SPM-242 (cyan) overlap in the S1P3 binding pocket.
Figure 7
Figure 7. Visualization of the orthosteric, allosteric, and bitopic ligand interactions based upon the biochemistry, mutagenesis, homology modeling, and molecular dynamic simulations. Dual inhibition of SPM-242 is illustrated
(A) S1P (green) and SPM-242 (cyan) overlap in the S1P3 binding pocket. (B) CYM-5541 (green) and SPM-242 (cyan) overlap in the S1P3 binding pocket.
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References

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