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Abstract

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 agonists chemotypes for each of the S1P1 and S1P3 receptors. uHTS for S1P1 was more effective than for S1P3, with many selective, low nanomolar hits of proven mechanism emerging for. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for sub-type selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of head-group 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 vs. S1P3 in this closely related receptor family.
Local not systemic modulation of dendritic cell S1P receptors in lung blunts virus-specific immune responses to influenza

David Marsolais, Bumsuk Hahm, Kurt H. Edelmann, Kevin B. Walsh, Miguel Guerrero, Yasuko Hatta, Yoshihiro Kawaoka, Edward Roberts, Michael B. A.Oldstone and Hugh Rosen.


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Abstract

The mechanism by which locally delivered sphingosine analogs regulate host response to localized viral infection has never been addressed. In this report, we show that intra-tracheal (i.t.) delivery of chiral sphingosine analog AAL-R or its phosphate ester inhibits the T cell response to influenza-virus infection. In contrast, neither intra-peritoneal (i.p.) delivery of AAL-R nor i.t. instillation of the non-phosphorylable stereoisomer AAL-S suppressed virus-specific T cell response, indicating that in vivo phosphorylation of AAL-R and S1P receptor modulation in lungs are essential for immunomodulation. I.t. delivery of water soluble S1P1 receptor agonist at doses sufficient to induce systemic lymphopenia did not inhibit virus-specific T cell response indicating that S1P1 is not involved in the immunosuppressive activities of AAL-R and that immunosuppression acts independently of naïve lymphocyte recirculation. Accumulation of dendritic cells (DCs) in draining lymph nodes was inhibited by i.t. but not i.p. delivery of AAL-R. Direct modulation of DCs is demonstrated by the impaired ability of virus-infected bone-marrow derived DCs treated in vitro with AAL-R to trigger in vivo T cell response after adoptive transfer to the airways. Thus, our results suggest that locally delivered sphingosine analogs induce immunosuppression by modulating S1P receptors other than S1P1 or S1P2 on dendritic cells in the lungs after influenza virus infection.

Screening Publications

  1. Stephan C. Schürer, Steven J. Brown, Pedro Gonzales Cabrera, Marie-Therese Schaeffer, Jacqueline Chapman, Euijung Jo, Peter Chase, Tim Spicer, Peter Hodder and Hugh Rosen (Accepted) Ligand-binding pocket shape differences between S1P1 and S1P3 determine efficiency of chemical probe identification by uHTS. ACS Chemical Biology.
  2. A.S. Don, H. Rosen, A fluorescent plate reader assay for ceramide kinase,
  3. Thomas Schröter, Dmitriy Minond, Amiee Weiser, Chinh Dao, Jeff Habel, Timothy Spicer, Peter Chase, Pierre Baillargeon, Louis Scampavia, Stephan Schürer, Caty Chung, Chris Mader, Mark Southern, Nick Tsinoremas, Philip LoGrasso and Peter Hodder Comparison of Miniaturized Time-Resolved Fluorescence Resonance Energy Transfer and Enzyme-Coupled Luciferase High-Throughput Screening Assays to Discover Inhibitors of Rho-Kinase II (ROCK-II).
  4. Lauer-Fields JL, Spicer TP, Chase PS, Cudic M, Burstein GD, Nagase H, Hodder P, Fields GB. (2007) Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate.

Probe Publications

  1. David Marsolais, Bumsuk Hahm, Kurt H. Edelmann, Kevin B. Walsh, Miguel Guerrero, Yasuko Hatta, Yoshihiro Kawaoka, Edward Roberts, Michael B. A. Oldstone and Hugh Rosen. Local not systemic modulation of dendritic cell S1P receptors in lung blunts virus-specific immune responses to influenza. Molecular Pharmacology (2008) 74: doi: 10.1124/mol.108.048769
  2. Feng Y, Cameron MD, Frackowiak B, Griffin E, Lin L, Ruiz C, Schröter T, LoGrasso P. Structure-activity relationships, and drug metabolism and pharmacokinetic properties for indazole piperazine and indazole piperidine inhibitors of ROCK-II. :
  3. Nakai, R., C.M. Salisbury, H. Rosen, and B.F. Cravatt. 2008. Ranking the selectivity of PubChem screening hits by activity-based protein profiling: MMP13 as a case study.
  4. Roth, J., F. Madoux, P. Hodder, and W.R. Roush. 2008. Synthesis of Small Molecule Inhibitors of the Orphan Nuclear Receptor Steroidogenic Factor-1 (NR5A1) Based on Isoquinolinone Scaffolds.
  5. Lauer-Fields, J.L., D. Minond, P.S. Chase, P.E. Baillargeon, S.A. Saldanha, R. Stawikowska, P. Hodder, and G.B. Fields. 2008. High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate.
  6. Rosen, H., P. Gonzalez-Cabrera, D. Marsolais, S. Cahalan, A.S. Don, and M.G. Sanna. 2008. Modulating tone: the overture of S1P receptor immunotherapeutics. Immuno Rev In Press:
  7. Tsukada, Y.T., M.G. Sanna, H. Rosen, and R.A. Gottlieb. 2007. S1P1-selective agonist SEW2871 exacerbates reperfusion arrhythmias.
  8. Guha, R., and S. Schurer. 2008. Utilizing high throughput screening data for predictive toxicology models: protocols and application to MLSCN assays.
  9. Madoux, F., X. Li, P. Chase, G. Zastrow, M. Cameron, J. Conkright, P. Griffin, S. Thacher, and P. Hodder. 2008. Potent, selective and cell penetrant inhibitors of SF-1 by functional uHTS.
  10. Dai SY, Chalmers MJ, Bruning J, Bramlett KS, Osborne HE, Montrose-Rafizadeh C, Barr RJ, Wang Y, Wang M, Burris TP, Dodge JA, Griffin PR. Prediction of the tissue-specificity of selective estrogen receptor modulators by using a single biochemical method.
  11. Niessen, F., F. Schaffner, C. Furlan-Freguia, R. Pawlinski, G. Bhattacharjee, J. Chun, C.K. Derian, P. Andrade-Gordon, H. Rosen, and W.Ruf. 2008. Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation.
  12. Gong Q, Ou Q, Ye S, Lee WP, Cornelius J, Diehl L, Lin WY, Hu Z, Lu Y, Chen Y, Wu Y, Meng YG, Gribling P, Lin Z, Nguyen K, Tran T, Zhang Y, Rosen H, Martin F, Chan AC. (2005) Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy.
  13. Rosen H. (2005) Chemical approaches to the lysophospholipid receptors.
  14. Martinez X, Kreuwel HTC, Redmond WL, Trenney R, Hunter K, Rosen H, Sarvetnick N, Wicker LS, Sherman LA. (2005) CD8+ T cell tolerance in NOD mice is restored by insulin dependent diabetes (Idd) resistance alleles.
  15. Jo E, Sanna MG, Gonzalez-Cabrera PJ, Thangada S, Tigy G, Osborne DA, Hla T, Parrill AL, Rosen H. (2005) S1P1-selective in vivo-active agonist from high throughput screening: Off-the-shelf chemical probes of receptor interactions, signaling and fate.
  16. Sanna, G., Liao, J., Jo, E., Alfonso, C., Ahn, M., Peterson, M., Webb, B., Lefebvre, S., Chun, J., Gray, N., Rosen, H. (2004) Spingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate.
  17. Rosen, H. & Goetzl, E.J. Sphingosine 1-phosphate and its receptors: An autocrine and paracrine network.
  18. Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH, Parker I, Cahalan MD. (2005) Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses.
  19. Alfonso C, McHeyzer-Williams MG, Rosen H (2006) CD69 down-modulation and inhibition of thymic egress by short and long-term selective chemical agonism of S1P1 receptors
  20. Sanna MG, Wang SK, Gonzalez-Cabrera P, Don A, Marsolais D, Jo E, Cheng WC, Wong CH, Rosen H. (2006) Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo.
  21. Chun, J. and H. Rosen (2006). "Lysophospholipid receptors as potential drug targets in tissue transplantation and autoimmune diseases.
  22. Rosen H, Sanna MG, Cahalan S, Gonzalez-Cabrera P (2007) Tipping the gatekeeper: S1P regulation of stromal barrier function.
  23. Gonzalez-Cabrera P, Hla T, Rosen H (2007) Mapping pathways downstream of sphingosine 1-phosphate subtype 1 by differential chemical perturbation and proteomics.
  24. Don A, Martinez-Lamenca C, Webb WR, Proia RL, Roberts E, Rosen H (2007) Essential requirement for sphingosine kinase 2 in a sphingolipid apoptosis pathway activated by FTY720 analogs.
  25. Lee H-K, Brown SJ, Rosen H, Tobias PS (2007) Application of beta-lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors.