Primary HTS and Confirmation Assays for S1P1 Agonists and Agonism Potentiators
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: 315
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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 S1P1:
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 endothlial barriers and therefore potentially for the treatment of multiple sclerosis, transplant rejection and adult respiratory distress syndrome .
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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).
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Keywords: Sphingosine Receptor, S1P1, 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 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.
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.
For the confirmation assay, all compounds were tested in triplicate in identical conditions to the primary HTS assay. If there was insufficient quantity of a particular compound, it was not tested.
The results of counterscreen and potency assays will be published in future submissions.
For all assays, a Chinese Hamster Ovary (CHO) cell line was used, stably transfected with human S1P1 receptor (pcDNA3.1-S1P1) and a cyclic AMP response element (CRE) reporter construct.
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), 2 mg/ml Geneticin (Invitrogen part#10131-027), and 1x penicillin-streptomycin (Invitrogen part#15140-122).
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.
Data Table (Concise)