Dose Response Cell-Based Assay for Agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Purchased Analogues
Name: Dose Response Cell-Based Assay for Agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Purchased Analogues ..more
BioActive Compounds: 2
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Hugh Rosen, TSRI
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1 R03 MH076533-01
Grant Proposal PI: Germana Sanna
External Assay ID: S1P3_AG_BLA_384_EC50_Purchased_Analogues
Name: Dose Response Cell-Based Assay for Agonists of the Sphingosine 1-Phosphate Receptor 3 (S1P3): Purchased Analogues
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 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. The success of a chemical approach for S1P1 suggests that selective tools for the resolution of function across this broad lipid receptor family is now possible ,.
Selective chemical probes of S1P3:
The S1P3 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. The effect of SEW2871 was indistinguishable from vehicle alone. Non-selective S1P receptor agonists therefore have effects upon both lymphocyte recirculation and heart rate. Data from SEW2871 studies together with studies on S1P3-deletant mice show that S1P1 and S1P3 appear to have mutually exclusive roles: S1P1 activation is sufficient to control lymphocyte numbers, with no discernable role in sinus rhythm control, whereas S1P3 regulates sinus rhythm but not lymphocyte recirculation. Agonists and antagonists of S1P3 may be useful probes of cardiac function in vivo.
In lung, S1P3 is expressed exclusively 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-deletant mouse.
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, EDG3, Adult Respiratory Distress Syndrome, agonist, HTS, GPCR, dose response, 384, purchased analogues, beta-lactamase, BLA, reporter gene, fluorescence, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN.
The purpose of this assay is to determine dose response curves for purchased structural analogues of a S1P3 agonist (SID 7967985) identified as active in a previous set of experiments entitled, "S1P3 Agonist Dose-Response Potency Assay" (PubChem AID 439). In this assay, a CHO cell line containing human S1P3 and the beta-lactamase (BLA) reporter-gene under control of the nuclear factor of activated T-cells (NFAT) promoter was used to measure S1P3 agonism by test compound. Stimulation of S1P3 by agonist induces transcription of NFAT-BLA via a G-alpha16 protein coupled signaling cascade, and an increase in BLA activity. BLA activity is measured using a fluorescent BLA substrate. As designed, a compound that acts as a S1P3 agonist will increase well fluorescence. Compounds were tested in quadruplicate in 384-well plates using ten 1:3 serial dilutions, starting at a nominal test concentration of 10 micromolar.
Cells were cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) containing 10% v/v heat inactivated bovine growth serum 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 2 mg/mL Geneticin, 0.2 mg/mL Hygromycin B and 1x penicillin-streptomycin. Prior to the start of the assay, cells were suspended at a concentration of 1 million/mL in phenol red-free DMEM supplemented as above, except with 0.5% charcoal/dextran-treated fetal bovine serum and no antibiotics.
The assay was started by dispensing 10 ul of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO2. The next day, 50 nL of test compound in DMSO, DMSO alone, or S1P (1 uM final nominal concentration) was added to the appropriate wells. After 4 hours of incubation, 2 ul/well of the GeneBLAzer fluorescent substrate mixture, prepared according to the manufacturer's protocol and containing 6 mM Probenicid, was added to all wells. The plates were then incubated for 2 hours at room temperature. Plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 590 nm and 460 nm.
To normalize assay data, values measured from the probe's fluorescence emission wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = I460 nm/I590 nm
where I represents the measured fluorescence emission intensity at the enumerated wavelength
Percent activation was calculated from the median ratio as follows:
% Activation = ((Ratio_Test_Compound - Median_Ratio_Low_Control) / (Median_Ratio_High_Control - Median_Ratio_Low_Control))*100
Test compound is defined as wells containing test compound
Low_Control is defined as wells containing DMSO
High_Control is defined as wells containing S1P
For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal concentration-response curve with adjustable baseline was fitted using Assay Explorer software by MDL. The reported EC50 values are generated from fitted curves by solving for the X-intercept at the 50% activity level of the Y-intercept value. In cases where the highest concentration tested (10 uM) did not result in 50% activation, the EC50 was determined manually as > 10 uM. Compounds with EC50 values of greater than 10 uM were considered inactive. Compounds with EC50 values equal to or less than 10 uM were considered active. The activity score is reported as normalized EC50 for samples with EC50 of less than 10 uM and as zero for samples with EC50 greater than or equal to 10 uM.
Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
List of reagents:
Dulbecco's Modified Eagle's Media (Invitrogen, part 11965-092)
Bovine Growth Serum (Hyclone, part SH30541.03)
NEAA (Invitrogen, part 1114-050)
Sodium Pyruvate (Invitrogen, part 11360-070)
HEPES (Invitrogen, part 15630-080)
L-Glutamine (Invitrogen, part 25030-081)
Geneticin (Invitrogen, part 10131-027)
Penicillin-Streptomycin antibiotic mix (Invitrogen part 15140-122)
Dulbecco's Modified Eagle's Media (Invitrogen, part 21063-029)
Charcoal/dextran-treated Fetal Bovine Serum (Hyclone, part SH30068.03)
S1P (Biomol, part SL140-0001)
Fatty Acid Free BSA (JHR, part 85041)
GeneBLAzer Fluorescent Substrate Mixture (Invitrogen, part K1085)
Probenecid (Sigma, part P8761)
384-well Plates (Greiner, part 788092)
T175 Flasks (Corning, part 431080)
Due to the increasing size of the MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis to wells that contained cells in the presence of 1 uM S1P (i.e. 100% activation). In this assay, S1P had a 50% effective concentration (EC50) of approximately 200 nM. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate NFAT or BLA activity, and compounds that quench or emit fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Active compounds of this assay fall into the activity score range of 88 to 100 and all inactive compounds have activity score of zero.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: CHO
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)