Introduction
Lipids form the largest group of small molecules in living cells, yet we possess only a limited number of tools with which to understand their functions. S1P binds the S1P1 receptor, among others, initiating a variety of downstream signals (Fig. 1). However, researchers still do not have a detailed understanding of this relatively new signaling cascade, partially because of complications arising from the presence of additional S1P receptors. To allow us to gain insight into this system, a standard set of tools must be made available, including agonists and antagonists. Ideally, these tools should function in a receptor subtype– specific manner. S1P itself acts as a nonselective agonist. The oxadiazole compound SEW2871 (5-(4-phenyl-5-trifluoromethyl-thiophen-2-yl)-3-(3-fluoromethylphenyl)-1,2,4-oxadiazole; Fig. 1a) is more specific, predominantly activating the type 1 receptor isoform S1P1 (ref. 1). Although receptor activation can be investigated using these compounds, receptor inhibition has been harder to study. In this issue, Sanna et al. report a new inhibitor, R-3-amino-4-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146; Fig. 1a), that switches the phosphate ester found in the S1P structure to an isosteric C-phosphonate moiety2. The application of this molecule in a substantial number of in vivo experiments provides new insight into the role of S1P/S1P1 signaling.
Figure 1: Intracellular signaling of S1P via its S1P1 receptor.
(a) Structures of S1P, its agonist SEW2871 and the new antagonist W146. (b) Binding of S1P or the specific S1P1 agonist SEW2871 to the S1P receptor type 1 (S1P1) results in the activation or regulatory modification of intracellular signaling molecules. For instance, both phospholipase C (PLC) and the 
isoform of PI3K are activated via G proteins8. (c) This signaling concert triggers, among many other events, crucial physiological responses in target cells of the hematopoietic and cardiovascular systems. AC, adenylate cyclase; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase.
Katie Ris
Full size image (65 KB)S1P acts as an extracellular messenger for lymphocytes and thereby influences the function of primary and secondary lymphoid organs, such as the thymus, lymph nodes and Peyer's patches, as well as the endothelia of the vascular system. Its signal is transduced by a family of five G protein–coupled receptors3. G protein action is initiated by S1P binding and triggers protein kinase B (Akt) and the MAP kinase pathway, which, in concert with increased calcium levels, eventually results in physiological cell responses. Internalization of the ligand-bound receptor seems to negatively regulate the pathway.
S1P agonists are used as immunosuppressant drugs, predominantly because of their ability to downregulate lymphocyte emigration from lymphoid organs. This effect is attributed to a single S1P receptor (S1P1). Other receptor subtypes seem to be important in the cardiovascular system, and their activation leads to bradycardia and other unwanted side effects1. Although investigators tested S1P analogs, including phosphonates, as early as 1974 (ref. 4), useful antagonists have been discovered only recently5, 6. However, these initial compounds still have only limited utility in vivo because of either low potency or fast metabolism. The switch from phosphate to phosphonate that is performed in the creation of W146 overcomes rapid degradation when the compound is applied systemically. Interestingly, synthetic efforts indicate that the stereochemistry of the 3-amino group is crucial for effective antagonism. In fact, the S enantiomer is sufficiently inactive to serve as a useful control compound.
The present biological study is an extension of recent work in which the S1P-specific agonist SEW2871 was instrumental in showing that transendothelial migration of lymphocytes from the lymph node into the lymphatic system is negatively regulated by the S1P/S1P1 receptor pathway6. Sanna et al. have demonstrated that the new antagonist W146 reverses this inhibition of lymphocyte egress and that the agonist-reduced velocity of T lymphocytes in the medulla region is markedly increased after intravenous injection of W146. Given that they observed no effect on T cells in the cortex region, lymphocyte arrest seems to be restricted to medullary egress alone. The pronounced effect of S1P on endothelial barrier function has been previously described for blood vessels and other endothelia. Thus the authors were able to show that W146 not only reverses the effects of the agonist SEW1871 but also enhances capillary leakage, which suggests that the precise regulation of S1P concentrations is crucial for basal capillary integrity. Similarly, vascular administration of W146 substantially increased basal pulmonary leakage from the vasculature into the alveolar space. All of these physiologically relevant effects were demonstrated in vivo.
These responses show the physiological consequences of S1P-S1P1 interaction, but how are the signals from the S1P1 receptor mediated within a cell? A good deal is already known about the events immediately following receptor occupation (Fig. 1b)7, 8. Building on this knowledge, Sanna et al. have also shown that the S1P antagonist inhibits agonist-induced MAP kinase and Akt phosphorylation. In addition, S1P receptor internalization, one of the mechanisms used to regulate S1P signaling, was fully prevented by the antagonist.
These results add substantially to our understanding of S1P1 signaling. However, up to now current datasets have been sparse and have had limited temporal resolution. Comprehensive imaging of downstream events in real time and (if possible) in entire organs should be the next step. Another unresolved area in S1P signaling is triggering of physiological events downstream of standard intracellular signaling (Fig. 1c). This concerns protein expression profiles in particular. Receptor-specific agonists and antagonists will be crucial tools for addressing these questions.
Will W146 become a drug candidate? Because of its adverse effect on capillary integrity, at first glance a systemic application seems unlikely. However, as the S1P-S1P1 receptor system is fine-tuned at comparably low (subsaturating) S1P concentrations, reversible and cautious administration of antagonist might in principle be possible. This could become particularly important in light of the fact that S1P is essential for tumor-associated angiogenesis as well as tumor cell proliferation8, 9.
The present results are only a starting point on the way to unraveling S1P signaling. Monitoring of intracellular signaling events downstream of receptor occupation using live cell imaging will improve our understanding of the ways in which cells deal with sensitive equilibria that are pivotal for organ function. Photoactivatable agonists and antagonists may be of particular value for this purpose. This future work will help to identify targets suitable for drug development. At the same time, the process of optimizing small molecules to a certain level of specificity and efficacy will need to be cultivated, and this will require close collaboration between chemists and biologists. Finally, the optimized compounds need to be made available for use in further in vivo studies such as that of Sanna et al.2.
