Ligand-Binding Pocket Shape Differences between Sphingosine 1-Phosphate (S1P) Receptors S1P 1 and S1P 3 Determine Efficiency of Chemical Probe Identification by Ultrahigh-Throughput Screening

Department of Scientific Computing, The Scripps Research Institute, La Jolla, California and Jupiter, Florida, USA.
ACS Chemical Biology (Impact Factor: 5.33). 08/2008; 3(8):486-98. DOI: 10.1021/cb800051m
Source: PubMed


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.

Download full-text


Available from: Stephan Schurer, Feb 04, 2014
  • Source
    • "th an antagonist sphingolipid analog ML - 056 ( W - 146 ) ( Hanson et al . , 2012 ) ( Fig . 6 ) . Unique features of the S1P1 receptor were not predictable from the extensive and rigorous analysis by mutagen - esis previously performed ( Fujiwara et al . , 2007 , 2005 ; Inagaki et al . , 2005 ; Jo et al . , 2005 ; Parrill et al . , 2000a , 2000b ; Schurer et al . , 2008 ; Wang et al . , 2001 ) . The extracellular face of S1P1 receptor is tightly structured ( Hanson et al . , 2012 ) where the N - terminus a - helix folds over the top of the receptor to block access to the binding pocket from the aqueous phase . This static view of the receptor suggests that S1P might require an alternate route into the "
    [Show abstract] [Hide abstract]
    ABSTRACT: The cytokine storm is an aggressive immune response characterized by the recruitment of inflammatory leukocytes and exaggerated levels of cytokines and chemokines at the site of infection. Here we review evidence that cytokine storm directly contributes to the morbidity and mortality resulting from influenza virus infection and that sphingosine-1-phosphate (S1P) receptor agonists can abort cytokine storms providing significant protection against pathogenic human influenza viral infections. In experiments using murine models and the human pathogenic 2009 influenza viruses, S1P1 receptor agonist alone reduced deaths from influenza virus by over 80% as compared to lesser protection (50%) offered by the antiviral neuraminidase inhibitor oseltamivir. Optimal protection of 96% was achieved by combined therapy with the S1P1 receptor agonist and oseltamivir. The functional mechanism of S1P receptor agonist(s) action and the predominant role played by pulmonary endothelial cells as amplifiers of cytokine storm during influenza infection are described.
    Full-text · Article · Jan 2013 · Virology
  • Source
    • "Phosphorylation of ERK-1/2 and Akt, two signaling pathways downstream of S1P receptors, was then assessed following stimulation with the S1P3 specific agonist Compound 20, a dicyclohexylamide molecule which showed an agonistic activity on S1P3 (EC50 = 350 nM), while not activating any other S1P receptor (EC50s>40 µM) [32]. Primary rat astrocytes were pre-incubated with 100 ng/ml LPS for 12 h in serum-free medium and then stimulated with the S1P3 agonist (Compound 20, 10 µM) for 20 min. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The sphingosine kinase 1 (SphK1)/sphingosine1-phosphate (S1P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P(3)) signaling and SphK1 in activated rat astrocytes has not been defined. Using immunohistochemistry we observed the upregulation of S1P(3) and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P(3) and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S1P(3) agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P(3) upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P(3) signaling compared to S1P(1) signaling was demonstrated by the use of selective S1P(3) or S1P(1) agonists. In summary, our data demonstrate that the SphK1/S1P(3) signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e.g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL1.
    Preview · Article · Aug 2011 · PLoS ONE
  • Source
    • "High-throughput screening (HTS) of an available chemical library showed that SEW2871 acted as an in vivo active heterocyclic S1P1 selective agonist81, 82 and compound 26 was synthesized as a potent 3,5-diphenyl-12,4-oxadiazole S1P1 agonist83. Later, using ultra-HTS, 3,5-diaryloxadiaxole (CYM5181) and dicyclohexylamide were found to be selective agonists for S1P1 and S1P3, respectively84. Using computational modeling, CYM-5442 was developed as an S1P1 selective agonist that was more potent than CYM518185. AUY954, an aminocarboxylate analogue of FTY720, was also introduced as an S1P1 selective agonist86. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Previous studies on lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) using various approaches have shown that both the molecules can act as intercellular signaling molecules. The discovery of the Edg subfamily of G-protein-coupled receptors (GPCRs) (later renamed LPA(1-3) and S1P(1-5)) for these molecules has opened up a new avenue for pathophysiological research on lysophospholipids. Genetic and molecular studies on lysophospholipid GPCRs have elucidated pathophysiological impacts and roles in cellular signaling pathways. Recently, lysophospholipid GPCR genes have been used to develop receptor subtype-selective agonists and antagonists. The discovery of FTY720, a novel immune modulator, along with other chemical tools, has provided a means of elucidating the functions of each lysophospholipid GPCR on an organ and the whole body level. This communication attempts to retrospectively review the development of agonists and antagonists for lysophospholipid GPCRs, provide integrated information on pharmacological tools for lysophospholipid GPCR signaling, and speculate on future drug development.
    Full-text · Article · Sep 2010 · Acta Pharmacologica Sinica
Show more