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

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).

Crystal structure of a lipid G protein-coupled receptor.

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P(1)-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor.

Full pharmacological efficacy of a novel S1P1 agonist that does not require S1P-like headgroup interactions.

Strong evidence exists for interactions of zwitterionic phosphate and amine groups in sphingosine-1 phosphate (S1P) to conserved Arg and Glu residues present at the extracellular face of the third transmembrane domain of S1P receptors. The contribution of Arg(120) and Glu(121) for high-affinity ligand-receptor interactions is essential, because single-point R(120)A or E(121)A S1P(1) mutants neither bind S1P nor transduce S1P function. Because S1P receptors are therapeutically interesting, identifying potent selective agonists with different binding modes and in vivo efficacy is of pharmacological importance.

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

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.

Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo.

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung.

Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses.

Sphingosine 1-phosphate type 1 (S1P(1)) receptor agonists cause sequestration of lymphocytes in secondary lymphoid organs by a mechanism that is not well understood. One hypothesis proposes that agonists act as 'functional antagonists' by binding and internalizing S1P(1) receptors on lymphocytes; a second hypothesis proposes instead that S1P(1) agonists act on endothelial cells to prevent lymphocyte egress from lymph nodes. Here, two-photon imaging of living T cells in explanted lymph nodes after treatment with S1P(1) agonists or antagonists has provided insight into the mechanism by which S1P(1) agonists function.

S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate.

The essential role of the sphingosine 1-phosphate (S1P) receptor S1P(1) in regulating lymphocyte trafficking was demonstrated with the S1P(1)-selective nanomolar agonist, SEW2871. Despite its lack of charged headgroup, the tetraaromatic compound SEW2871 binds and activates S1P(1) through a combination of hydrophobic and ion-dipole interactions. Both S1P and SEW2871 activated ERK, Akt, and Rac signaling pathways and induced S1P(1) internalization and recycling, unlike FTY720-phosphate, which induces receptor degradation.

S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung barrier integrity and is potentiated by TNF.

Pulmonary pathologies including adult respiratory distress syndrome are characterized by disruption of pulmonary integrity and edema compromising respiratory function. Sphingosine 1-phosphate (S1P) is a lipid mediator synthesized and/or stored in mast cells, platelets, and epithelial cells, with production up-regulated by the proinflammatory cytokines IL-1 and TNF. S1P administration via the airways but not via the vasculature induces lung leakage.

alpha-Synuclein-synaptosomal membrane interactions: implications for fibrillogenesis.

alpha-Synuclein exists in two different compartments in vivo-- correspondingly existing as two different forms: a membrane-bound form that is predominantly alpha-helical and a cytosolic form that is randomly structured. It has been suggested that these environmental and structural differences may play a role in aggregation propensity and development of pathological lesions observed in Parkinson's disease (PD). Such effects may be accentuated by mutations observed in familial PD kindreds.

Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate.

Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P(1) knock-out and the unavailability of selective agonists or antagonists.

Defective membrane interactions of familial Parkinson's disease mutant A30P alpha-synuclein.

alpha-Synuclein (alpha-Syn) is an abundant presynaptic protein of unknown function, which has been implicated in the pathogenesis of Parkinson's disease. Alpha-Syn has been suggested to play a role in lipid transport and synaptogenesis, and growing evidence suggests that alpha-Syn interactions with cellular membranes are physiologically important. In the current study, we demonstrate that the familial Parkinson's disease-linked A30P mutant alpha-Syn is defective in binding to phospholipid vesicles in vitro as determined by vesicle ultracentrifugation, circular dichroism spectroscopy, and low-angle X-ray diffraction.