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. 2008 Aug 15;3(8):486-98.
doi: 10.1021/cb800051m. Epub 2008 Jul 1.

Ligand-binding pocket shape differences between sphingosine 1-phosphate (S1P) receptors S1P1 and S1P3 determine efficiency of chemical probe identification by ultrahigh-throughput screening

Stephan C Schürer  1 , Steven J Brown, Pedro J Gonzalez-Cabrera, Marie-Therese Schaeffer, Jacqueline Chapman, Euijung Jo, Peter Chase, Tim Spicer, Peter Hodder, Hugh Rosen
Affiliations

Ligand-binding pocket shape differences between sphingosine 1-phosphate (S1P) receptors S1P1 and S1P3 determine efficiency of chemical probe identification by ultrahigh-throughput screening

Stephan C Schürer et al. ACS Chem Biol. .

Abstract

We have studied the sphingosine 1-phosphate (S1P) receptor system to better understand why certain molecular targets within a closely related family are much more tractable when identifying compelling chemical leads. Five medically important G-protein-coupled receptors for S1P regulate heart rate, coronary artery caliber, endothelial barrier integrity, and lymphocyte trafficking. Selective S1P receptor agonist probes would be of great utility to study receptor subtype-specific function. Through systematic screening of the same libraries, we identified novel selective agonist chemotypes for each of the S1P1 and S1P3 receptors. Ultrahigh-throughput screening (uHTS) for S1P1 was more effective than that for S1P3, with many selective, low nanomolar hits of proven mechanism emerging. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for subtype selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of headgroup interactions. Chemistry-space and shape-based analysis of the screening libraries in combination with the binding models explain the observed differential hit rates and enhanced efficiency for lead discovery for S1P1 versus S1P3 in this closely related receptor family.

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Figures

Figure 1
Figure 1
Representative S1P1 agonists with EC50 < 1 μM in a 2-dimensional chemistry space. EC50 values are shown for S1P1 / S1P3 (blue MLSMR, red MBHF, green SEW2871; size is scaled by S1P1 pEC50).
Figure 2
Figure 2
S1P3 agonists with EC50 < 1 μM in a 2-dimensional chemistry space. EC50 values are shown for S1P3 / S1P1 (blue MLSMR, red MBHF, size is scaled by S1P3 pEC50).
Figure 3
Figure 3
Biological activity of S1P1 and S1P3 agonists. A. S1P1-GFP internalization and ubiquitination by 6. B. Agonist induced 32P incorporation into immunoprecipitated S1P1-GFP. C. S1P3 agonist dose dependent activation of Calcium flux in S1P3/ga16 CHO cells by 20 and 28.
Figure 4
Figure 4
Comparison of the binding pocket of the S1P1 (A) and S1P3 (B) receptors with S1P and FTY720-P docked into each receptor. The main difference in the binding pocket derives from the S1P1 Leu276 and S1P3 Phe263 side chains; in S1P3 the binding pocket is narrowed by 1.5 to 2 Å compared to the same region in S1P1.
Figure 5
Figure 5
Oxadiazoles docked into the S1P1 receptor: 6 green (A), 5 yellow (B), 7 cyan (C), 8 copper (D). The S1P3 Phe263 residue interferes with these ligand poses explaining the observed selectivity.
Figure 6
Figure 6
Structure and activity of S1P3 selective dicyclohexylamides 20 and 28.
Figure 7
Figure 7
S1P3-selective dicyclohexylamides docked into the S1P3 receptor hydrophobic binding pocket: green 20 (A), copper 28 (B).
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References

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