Sphingosine 1-Phosphate Analogs as Receptor Antagonists

Abstract

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokes a variety of cell and tissue responses via a set of cell surface receptors. The recent development of S1P receptor agonists, led by the immunomodulatory pro-drug FTY720, has revealed that S1P signaling is an important regulator of lymphocyte trafficking. With the twin goals of understanding structureactivity relationships of S1P ligands and developing tool compounds to explore S1P biology, we synthesized and tested numerous S1P analogs. We report herein that a subset of our aryl amide-containing compounds are antagonists at the S1P 1 and S1P 3 receptors. The lead compound in the series, VPC23019, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S1P 1 and S1P 3 receptors. The structureactivity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S1P 3 receptor. These new chemical entities will enable further understanding of S1P signaling and provide leads for further S1P receptor antagonist development. Sphingosine 1-phosphate (S1P)1 is a lysophospholipid mediator that evokes a variety of cellular responses by stimulation of five members of the endothelial cell differentiation gene receptor family. The endothelial cell differentiation gene receptors are G-protein coupled receptors that, upon stimulation, propagate second messenger signals via activation of heterotrimeric G-protein ␣ subunits and ␤-␥ dimers. Ultimately, this S1P-driven signaling results in cell survival, increased cell migration, and, often, mitogenesis. The recent development of agonists targeting S1P receptors has provided insight regarding the role of this signaling system in physiologic homeostasis. For example, the immunomodulator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol), which is a pan S1P receptor agonist following phosphorylation, revealed that S1P tone influences lymphocyte trafficking (1-4). The utility of an S1P receptor agonist was unexpected; indeed, prior speculation focused on the potential (as yet unrealized) for S1P antagonists as anti-angiogenic agents.Recent findings also suggest a physiological influence for S1P in the vasculature.Although not yet explored in detail, it has been hypothesized that S1P may exert anti-inflammatory actions on endothelial cells through its release from high density lipoprotein (5). A recent study found that S1P inhibited tumor necrosis factor ␣-mediated monocyte-endothelial cell adhesion.2 Furthermore, an S1P 1 receptor antagonist described herein blocked the anti-inflammatory action of S1P, thereby providing evidence that this effect maps to the S1P 1 receptor. If verified, these results would expand the role of the S1P 1 receptor to include influencing monocyte extravasation and would further highlight how the development of S1P receptor-specific compounds is expanding our understanding of the biology of this important signaling system.To further characterize ...

Asymmetric Synthesis of Conformationally Constrained Fingolimod Analogues—Discovery of an Orally Active Sphingosine 1-Phosphate Receptor Type-1 Agonist and Receptor Type-3 Antagonist

“…The parameter measured is membrane-bound GTP[γ-35 S] (see methods for details). 21 (b) Concentration effect curve for 2-P and 2-P in the presence of 1 μM 3a-P-3d-P. The parameter measured is calcium mobilization (see methods for details).…”

“…The parameter measured is calcium mobilization (see methods for details). 21 The images shown are representative of three independent experiments. 2 and 3a-3d were incubated with homogenates of HEK293T cells, wherein mouse Sphk2 expression had been forced.…”

Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes

“…Unfortunately, the use of genetically manipulated mice is not an option for these types of studies, as knockout of the S1P 1 receptor subtype is embryonically lethal (34). As an alternative approach, the population of S1P 1 receptors on the ventricular myocyte was blocked by pretreatment with the antagonist VPC23019 (12). Subsequent application of S1P in these myocytes from wild-type mice was used to study the contributions of S1P 3 to cell shortening.…”

Sphingosine 1-phosphate chemical biology

“…3) with a phenyl amide functionality. In the course of performing an SAR analysis of this scaffold, a subset of molecules was found in GTPγ[ 35 S] binding assays to be inverse agonists at the S1P1 and S1P3 receptors [56]. While most molecules in the phenyl amide series are S1P receptor agonists (or inactive), when the 'tail' is no longer than eight carbon atoms, meta (1,3) or ortho (1,2) to the amide linker, and the amino carbon is in the R configuration, the molecules are S1P1/3 receptor antagonists.…”

“…While most molecules in the phenyl amide series are S1P receptor agonists (or inactive), when the 'tail' is no longer than eight carbon atoms, meta (1,3) or ortho (1,2) to the amide linker, and the amino carbon is in the R configuration, the molecules are S1P1/3 receptor antagonists. The lead molecule in the series, VPC23019, behaves as a competitive antagonist and exhibits Ki values of about 25 and 300 nanoM at the S1P type 1 and type 3 receptors, respectively [56].…”