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Sphingosine-1-phosphate (S1P) is a bioactive metabolite with pleiotropic effects on multiple cellular processes in health and disease. Responses elicited by S1P are a result of binding to five specific G-protein–coupled receptors. We have developed multiple assays to systematically study the downstream signaling of these receptors, including early events such as direct receptor activation (GTPγS) as well as more distal events such as S1P 1 receptor degradation. Employing such assays, we have characterized and compared multiple S1P 1 agonists that are in clinical development including FTY720, BAF312, CS-0777, and other molecules from the S1P 1 patent literature. Our parallel assessment has allowed us to compare their potency against S1P 1 , their selectivity against the four other S1P receptors, as well as species cross-reactivity. We note that all of the compounds studied signal in an identical manner through S1P 1 , leading to receptor degradation.

The crystal structure of human p56lck SH2 domain in complex with an inhibitor containing the singly chargedp-(carboxymethyl)phenylalanine residue (cmF) as a phosphotyrosine (Tyr(P) or pY) replacement has been determined at 1.8 A resolution. The binding mode of the acetyl-cmF-Glu-Glu-Ile (cmFEEI) inhibitor is very similar to that of the pYEEI inhibitor, confirming that the cmFEEI inhibitor has a similar mechanism of SH2 domain inhibition despite its significantly reduced potency. Observed conformational differences in the side chain of the cmF residue can be interpreted in terms of maintaining similar interactions with the SH2 domain as the Tyr(P) residue. The crystal structure of the free p56lck SH2 domain has been determined at 1.9 A resolution and shows an open conformation for the BC loop and an open phosphotyrosine binding pocket, in contrast to earlier studies on the srcSH2 domain that showed mostly closed conformation. The structural information presented here suggests that the carboxymethyl-phenylalanine residue may be a viable Tyr(P) replacement and represents an attractive starting point for the design and development of SH2 domain inhibitors with better pharmaceutical profiles.

One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR ζ ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon ζ ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of ζ chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein–phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein–phosphopeptide complex relative to the protein alone.