Dose Response Assays for S1P1 Agonists and Agonism Potentiators - Potentiator Assay 60K MLSMR
Sphingosine 1-phosphate (S1P) influences heart rate (1,2), coronary artery caliber, endothelial integrity, lung epithelial integrity (3) and lymphocyte recirculation (1,4-6) through five related high affinity G-protein coupled receptors (7). Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produce clinical immunosuppression preventing transplant rejection, but is more ..
BioActive Compounds: 266
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: The Scripps Research Institute, TSRI
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1 R03 MH076533-01
The biology of S1P receptor subtypes
Sphingosine 1-phosphate (S1P) influences heart rate (1,2), coronary artery caliber, endothelial integrity, lung epithelial integrity (3) and lymphocyte recirculation (1,4-6) through five related high affinity G-protein coupled receptors (7). Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produce 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(3). S1P receptor subtype selective agonists and antagonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and 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 (8,9).
S1P1 receptor subtype plays a critical role in lymphoid and lung endothelial barrier function. S1P1 and S1P3 are coexpressed in some cells, especially endothelium. Agonists of S1P1 would be of interest in the enhancement of endothelial barriers and therefore potentially for the treatment of multiple sclerosis, transplant rejection and adult respiratory distress syndrome(10).
A cell line containing the human S1P1 receptor as well as the beta-lactamase (BLA) reporter-gene under control of the cyclic AMP response element (CRE) promoter was used to measure S1P1 activation. Since the S1P1 receptor is a member of Gi/o protein coupled receptor family, agonism was measured by adding test compounds in the presence of a forskolin challenge. Through stimulation of adenylate cyclase, forskolin increases the production of cAMP and therefore the transcription of the CRE-BLA reporter gene. S1P1 agonists would abrogate this effect; similarly agonism potentiators would increase the potency of an S1P1 agonist. Therefore the amount of BLA activity was inversely proportional to the concentration of agonist or agonism potentiators in the presence of an S1P1 agonist. BLA activity was measured with a fluorescent BLA substrate.
As previously reported, to discover both S1P1 agonists and agonism potentiators, the primary uHTS campaign was run in #potentiator# mode, described as follows. All compounds were tested in the presence of the EC20 (2 nM) of the S1P1-selective agonist SEW2871 and the EC80 (4 micromolar) of forskolin. The EC20 of SEW2871 was used in order to identify both S1P1 agonists and agonism potentiators. All data reported was normalized on a per-plate basis to wells that contained the EC100 of SEW2871 (1 micromolar) and the EC80 of forskolin.
The primary HTS assay was conducted in 1536-well format. All compounds were tested once at a 10 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 this experiment, 508 compounds that passed hit selection criteria were tested to determine individual EC50 values. The compounds were #hit-picked# at a 10 millimolar concentration in DMSO and further serially diluted nine times at three fold-dilutions for a total of 10 different compound concentrations.
The assay was conducted in 1536-well format. Each compound dilution series was assayed in triplicate. As with the primary run, SEW-2871 was used as the positive control. All data reported was normalized on a per-plate basis to wells that contained cells in the presence of 1 micromolar SEW-2871 (i.e. 100% agonist) Vs wells that contained forskolin only (0% agonist).
Dose-response curves were plotted and fitted to a four parameter equation describing a sigmoidal concentration-response curve with adjustable baseline using Assay Explorer software by MDL. The reported EC50 values are generated from fitted curves by solving for x-intercept at the 50% activity level of Y-intercept. In cases where the highest concentration tested (95 micromolar) did not result in > 50% inhibition or where no curve fit was achieved, the EC50 was determined manually depending on the observed inhibition at the individual concentrations. Compounds with EC50 values of greater than 10 micromolar were considered inactive, compounds with EC50 equal to less than 10 micromolar are considered active.
In addition to determining the Ec50 values for the #potentiator assay#, Ec50 values were also determined for the S1P1 assay in #Agonist# mode as well as counterscreened for EC50 values in the parental CHO Cre Bla cell line. These three assay results are submitted as individual publications. For the S1P1 agonist assay execution, all parameters are identical to the #potentiator# assay except test wells only include forskolin devoid of the Ec20 concentration of SEW 2871. For the parental CHO Cre Bla cell counterscreen assay all parameters were again identical to the #potentiator assay except this cell line doesn#t express S1P1 and was tested in the presence of 4uM forskolin only. Ec50#s for these two assays were plotted using the same parameters as above. For the agonist assay, data was normalized to the 1micromolar SEW-2871 Vs forskolin only and for the parental cell counterscreen, data was normalized to untreated cells Vs forskolin treated cells.
The activity score was calculated based on pEC50 values for compounds for which an exact EC50 value was calculated and based on the observed pEC50 range, specifically the maximum lower limit of the pEC50 value as calculated from the lowest concentration for which greater than 50% inhibition is observed. This results in a conservative estimate of the activity score for compounds for which no exact EC50 value is given while maintaining a reasonable rank order of all compounds tested.
 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).
 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).
 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).
 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).
 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).
 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).
 Mandala, S. et al. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296, 346-349 (2002).
 Rosen, H. Chemical approaches to the lysophospholipid receptors. Prostaglandins & other Lipid Mediators 77, 179-84 (2005).
 Rosen, H. & Liao, J. Y. Sphingosine 1-phosphate pathway therapeutics: a lipid ligand-receptor paradigm. Current Opinion in Chemical Biology 7, 461-468 (2003).
 Sanna, M.G., Wang S-K et al. Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo Nature Chemical Biology. 3: Published online: 9 July 2006 | doi:10.1038/nchembio804.
For these dose response assays, one of two cell lines was used. The S1P-1 "potentiator" and agonist assays used a Chinese Hamster Ovary (CHO) cell line stably transfected with human S1P1 receptor (pcDNA3.1-S1P1) and a cyclic AMP response element (CRE) reporter construct. The "parental" counter screen dose response assay used a CHO Cre Bla cell line (Invitrogen part#K1129).
All cells were cultured in T-175 cm2 Corning flasks (part#431080) at 37 degrees 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), and 1x penicillin-streptomycin (Invitrogen part#15140-122).For the parental cell line no selection antibiotic was used but for the S1P1 transformed cells 2 mg/ml Geneticin (Invitrogen part#10131-027) was used.
Prior to assay, cells were suspended to a concentration of 1.25 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, 25mM HEPES, and 5mM L-Glutamine.
The assay began by dispensing 4 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 C in 5% CO2. The next day, 48 nL of test compound or DMSO control was added. One microliter of a mixture of forskolin and SEW2871 was dispensed to all compound & control wells at a final concentration of 4 micromolar (forskolin#s EC80) and 2 nanomolar (SEW2871#s EC20), respectively. Additionally, 48 nL of SEW2871 was also added to designated positive control wells to a final concentration of 1 micromolar (i.e. its EC100). Plates were then incubated at 37 C in 5% CO2 for 4 hrs. After the incubation, 1 microliter/well of the GeneBLAzer 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.
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