S1P3 Agonist Primary HTS and Confirmation Assays
Sphingosine 1-phosphate (S1P) influences heart rate  , coronary artery caliber, endothelial integrity, lung epithelial integrity  and lymphocyte recirculation  - through five related high affinity G-protein coupled receptors . Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection, but more ..
BioActive Compounds: 62
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Grant Proposal Number: 1 R03 MH076533-01
The biology of S1P receptor subtypes:
Sphingosine 1-phosphate (S1P) influences heart rate  , coronary artery caliber, endothelial integrity, lung epithelial integrity  and lymphocyte recirculation  - through five related high affinity G-protein coupled receptors . Inhibition of lymphocyte recirculation by nonselective 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 unavailability of selective agonists or antagonists for the 5 receptor subtypes. Separation of receptor subtype usage for control of endothelial and epithelial integrity will allow the identification of selective immunosuppressive S1P3 receptor agonists and antagonists that could be of use in the control of cardiac function and the prevention of Adult Respiratory Distress Syndrome . S1P receptor subtype selective agonists and antagonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P1 would suggest that selective tools for the resolution of function across this broad lipid receptor family is now possible ,.
Selective chemical probes of S1P3:
S1P3 receptor subtype plays a critical role in cardiac rhythm and lung epithelial barrier function. S1P1 and S1P3 are coexpressed in some cells, especially endothelium. The association of a dose-dependent bradycardia with administration of the relatively non-selective receptor FTY720 in man led us to study the lymphopenic and heart rate responses that associated with S1P1 and S1P3. Induction of lymphopenia in homozygous S1P3-/- mice was indistinguishable from wild-type mice  , with no statistically significant difference in the depth of lymphopenia at 5 hours between the S1P1-selective agonist SEW2871 and the S1P1,3,4 and 5 active prodrug AAL-(R), which is phosphorylated to its active form AFD-(R). Deletion of S1P3 therefore did not affect the S1P receptor agonist-induced inhibition of lymphocyte recirculation. We then tested the ability of the non-selective S1P receptor agonist AFD-(R) for the induction of heart rate changes in conscious mice by ECG analysis. Wild-type mice showed a significant maximal sinus bradycardia (-41.5 + 2.0%; p = 0.0001 by ANOVA) sustained for over 5 hours in response to the administration of AFD(R) or a structurally unrelated non-selective S1P3 agonist. AFD administration in S1P3-deletant mice was statistically equivalent to administration of vehicle alone in wild-type mice, and no bradycardia was seen. We tested the S1P1-selective agonist SEW2871 at a dose of 10 mg/kg that induced full lymphopenia for bradycardia and found no induction of bradycardia in either wild-type or S1P3-/- mice and it was indistinguishable from vehicle alone. Non-selective S1P receptor agonists therefore have effects upon both lymphocyte recirculation and heart rate. The use of SEW2871 together with the S1P3-deletant mice shows that S1P1 and S1P3 appear to have mutually exclusive roles: activation of S1P1 is sufficient to control lymphocyte numbers and plays no discernable role in control of sinus rhythm, whereas S1P3 regulates sinus rhythm and not lymphocyte recirculation. Agonists and antagonists of S1P3 may be useful probes of cardiac function in vivo.
In lung, S1P3 is exclusively expressed upon pulmonary epithelium and activation of S1P3 results in rapid dissolution of epithelial tight junctions, measured by both ZO-1 and claudin degradation  . This produces acute development of paracellular gaps and pulmonary edema that is reversed in the S1P3-deltant mouse. S1P3 antagonists would be of great utility in the assessment of mechanisms of ARDS.
1. Sanna, M. G. et al. Sphingosine 1-phosphate (S1P) receptor subtypes S1P(1) and S1P(3), respectively, regulate lymphocyte recirculation and heart rate. Journal of Biological Chemistry 279, 13839-13848 (2004).
2. Forrest, M. et al. Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes. Journal of Pharmacology and Experimental Therapeutics 309, 758-768 (2004).
3. Gon, Y. et al. S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung barrier integrity and is potentiated by TNF. PNAS 102, 9270-5 (2005).
4. Wei, S. H. et al. Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses. Nat. Immunol. 6, 1228-1235 (2005).
5. Jo, E. et al. S1P1-Selective In Vivo-Active Agonists from High- Throughput Screening: Off-the-Shelf Chemical Probes of Receptor Interactions, Signaling, and Fate. Chemistry & Biology 12, 703-715 (2005).
6. Alfonso, C., McHeyzer-Williams, M. & Rosen, H. CD69 down-modulation and inhibition of thymic egress by short and long-term selective chemical agonism of S1P1 receptors. Eur. J. Immunol. 36 (2006).
7. Mandala, S. et al. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296, 346-349 (2002).
8. Rosen, H. Chemical approaches to the lysophospholipid receptors. Prostaglandins & other Lipid Mediators 77, 179-84 (2005).
9. Rosen, H. & Liao, J. Y. Sphingosine 1-phosphate pathway therapeutics: a lipid ligand-receptor paradigm. Current Opinion in Chemical Biology 7, 461-468 (2003).
10. Goetzl, E. J. & Rosen, H. Regulation of immunity by lysosphingolipids and their G protein - coupled receptors. Journal of Clinical Investigation 114, 1531-1537 (2004).
Sphingosine Receptor, S1P3, Adult Respiratory Distress Syndrome, Agonist, HTS, Confirmation, 1536, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN
A cell line containing the human S1P3 receptor as well as the beta-lactamase (BLA) reporter-gene under control of the nuclear factor of activated T-cells (NFAT) promoter was used to measure S1P3 agonism. If the S1P3 receptor was stimulated by agonist, transcription of the NFAT-BLA gene occurred via a G-alpha-16 protein coupled signaling cascade. The amount of BLA activity was proportional to the concentration of agonist. BLA activity was measured with a fluorescent BLA substrate.
The entire campaign was run with S1P as the positive control. In this assay, S1P had a 50% effective concentration (EC50) of approximately 200 nM. All data reported was normalized on a per-plate basis to wells that contained cells in the presence of 1 micromolar S1P (i.e. 100% activation). The primary HTS assay was conducted in 1536-well format. All compounds were tested once at a 4.5 micromolar final concentration.
A mathematical algorithm was used to determine nominally active compounds in the primary screen. Two values were calculated: (1) the average percent activity of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater %activation than the cutoff parameter was declared active. In addition, wells with fluorescence values that could be attributed to artifact were also chosen for further follow-up.
For the confirmation assay, all compounds were tested in triplicate in identical conditions to the primary HTS assay.
The results of the potency assay will be published in a future submission.
For all assays, a Chinese Hamster Ovary (CHO) cell line stably transfected with human S1P3 receptor, nuclear factor of activated T-cell-beta lactamase (NFAT-BLA) reporter construct and the G-alpha-16 pathway coupling protein was used.
Cells were cultured in T-175 sq cm Corning flasks (part 431080) at 37 deg C and 95% RH. The growth media consisted of Dulbecco's Modified Eagle's Media (Invitrogen, part 11965-092) containing 10% v/v heat inactivated bovine growth serum (Hyclone, part SH30541.03), 0.1 mM NEAA (Invitrogen, part 1114-050), 1 mM Sodium Pyruvate (Invitrogen, part 11360-070), 25 mM HEPES (Invitrogen, part 15630-080), 5 mM L-Glutamine (Invitrogen, part 25030-081), 2 mg/mL Geneticin (Invitrogen, part 10131-027), 0.2 mg/mL Hygromycin B (Invitrogen, part 10687-010) and 1x penicillin-streptomycin (Invitrogen, part 15140-122).
Prior to assay, cells were suspended to a concentration of 1 million/milliliter in phenol red free Dulbecco's Modified Eagle's Media (Invitrogen, part 21063-029) containing 0.5% charcoal/dextran treated fetal bovine serum (Hyclone, part SH30068.03), 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, and 5 mM L-Glutamine.
The assay began by dispensing 5 microliters of cell suspension to each test well of a 1536 well plate. The cells were then allowed to incubate in the plates overnight at 37 deg C in 5% CO2. The next day, 25 nL of test compound or DMSO control was added. The S1P positive control was also added to the appropriate control wells to a final concentration of 1 micromolar. Plates were then incubated at 37 deg C in 5% CO2 for 4 hrs. After the incubation, 1 microliter/well of the GeneBLAzer's fluorescent substrate mixture (Invitrogen, LiveBLAzer, part K1085), prepared according to the manufacturer's protocol and containing 200 mM probenicid (Sigma, part P8761) was added. After 2 hours of incubation at room temperature, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength 405 nm and emission wavelengths of 535 nm & 460 nm. Each channel of raw data was corrected by subtracting "background" (i.e. wells containing media and substrate only) before the ratio of 460 nm/535 nm fluorescence emission was calculated. Percent activation was calculated from the median ratio of the positive control after subtracting the basal signal ratio from the sample well and the positive control.
Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on the microtiter plate, compounds that non-specifically inhibit or enhance beta-lactamase activity, and compounds that quench or emit fluorescence.
Data Table (Concise)