LC-MS/MS

All analytical methods were in MRM mode where the parent ion was selected in Q1 of the mass spectrometer. The parent ion was fragmented and a characteristic fragment ion monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods were rapidly set up using Automaton^® (Applied Biosystems), where the compounds were listed with their name and mass in an Excel datasheet. Compounds were submitted in a 96-well plate to the HPLC autosampler and slowly injected without a column present. A narrow range centered on the indicated mass was scanned to detect the parent ion. The software then evaluated a few pre-selected parameters to determine conditions that maximized the signal for the parent ion. The molecule was then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass were determined. Three separate collision energies were evaluated to fragment the parent ion and the largest three ions were selected. Each of these three fragment ions was further optimized and the best fragment was chosen. The software then inserted the optimized masses and parameters into a template method and saved it with a unique name that indicated the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern were saved and reviewable later.

Solubility

The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Compounds were inverted for 24 hours in test tubes containing 1–2 mg of compound with 1 mL of PBS. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS/MS analysis was used.

Stability

Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at −80°C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed in a room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.

Determination of glutathione reactivity

One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 100 μM reduced glutathione. Final compound concentration was 10 μM unless solubility limited. The solution was allowed to incubate at 37°C for two hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized—a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of −129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans [20,21].

Primary uHTS assay to identify S1P4 receptor agonists (AID 1509)

Assay Overview: The purpose of this assay was to identify compounds that act as agonists of the S1P4 receptor. This assay uses Tango™ S1P4-BLA U2OS cells which contain the human Endothelial Differentiation Gene 6 (EDG6; S1P4) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P4 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P4 receptor agonists will activate S1P4 receptor and increase well FRET. Compounds were tested in singlicate at a final nominal concentration of 5 micromolar.

Protocol Summary: U2OS cells were cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of McCoy’s 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin-neomycin, 200 μg/mL zeocin, 50 μg/mL hygromycin, 100 μg/mL geneticin. Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO₂ and 95% RH. The next day, 25 nL of test compound (5 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to low control wells. S1P prepared in 2% fatty-acid free BSA was added to the high control wells to a final concentration of 5 μM. Plates were then incubated at 37 degrees C in 5% CO₂ for 4 hrs. After the incubation, 1 μL of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: compounds that exhibited greater than 10.98% agonism for S1P4 receptor were considered active.

Confirmation uHTS assay to identify S1P4 receptor agonists (AID 1523)

Assay Overview: The purpose of this assay was to confirm activity of compounds identified as active in the uHTS primary screen (AID 1509). This assay was run as described above (AID 1509). Compounds were tested in triplicate at a nominal concentration of 5 micromolar.

Protocol Summary: The assay was performed as described above (AID 1509). Assay Cutoff: compounds that exhibited greater than 10.98% agonism for S1P4 receptor were considered active.

Dose response uHTS assay to identify S1P4 receptor agonists (AID 1686)

Assay Overview: The purpose of this assay was to determine dose response for compounds identified as active in the uHTS primary screen (AID 1509) and that confirmed activity in the uHTS confirmation screen (AID 1523). This assay was run as described above (AID 1509). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.

Protocol Summary: The assay was performed as described above (AID 1509). Assay Cutoff: compounds with an EC₅₀ equal to or less than 10 micromolar were considered active.

Counterscreen uHTS assay to identify S1P1 receptor agonists (AID 1563)

Assay Overview: The purpose of this assay was to determine whether compounds identified as active in the primary uHTS assay (AID 1509), and that confirmed activity in the uHTS confirmation screen (PubChem AID 1523), were nonselective agonists due to activation of the S1P1 receptor. This assay uses Tango™ S1P1-bla U2OS cells which express S1P1 (EDG1) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P1 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P1 receptor agonists will activate S1P1 receptor and increase well FRET. Compounds were tested in triplicate at a final nominal concentration of 5 micromolar.

Protocol Summary: Cells were cultured as described above (AID 1509). Prior to the start of the assay, cells were suspended at a concentration of 625,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO₂ and 95% RH. The next day, 25 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone (low control), or S1P (high control; 5 μM final concentration) prepared in 2% fatty-acid free BSA, was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO₂ for 4 hours. After the incubation, 1 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: compounds that exhibited greater than 20.14% agonism for S1P1 receptor were considered active.

Counterscreen dose response uHTS assay to identify S1P1 receptor agonists (AID 1701)

Assay Overview: The purpose of this assay was to determine S1P1 agonist dose response for compounds identified as active in the uHTS S1P1 receptor counterscreen assay (AID 1563). The assay was performed as described above (AID 1563), except that compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.

Protocol Summary: The assay was performed as described above (AID 1563). Assay Cutoff: compounds with an EC₅₀ equal to or less than 10 micromolar were considered active.

Dose response assay to identify S1P4 receptor agonists with purchased compounds (AID 504460)

Assay Overview: The purpose of this assay was to determine S1P4 receptor agonist dose response with powder samples of purchased compounds. This assay was run as described above (AID 1509). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.

Protocol Summary: Cells were cultured as described above (AID 1509). Prior to the start of the assay, cells were suspended at a concentration of 1,000,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO₂ and 95% RH. The next day, 50 nL of test compound (50 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to low control wells. S1P prepared in 2% fatty-acid free BSA was also added to the high control wells to a final concentration of 6 μM. Plates were then incubated at 37 degrees C in 5% CO₂ for 4 hrs. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: compounds with an EC₅₀ equal to or less than 10 micromolar were considered active.

Dose response assay to identify S1P4 receptor agonists with synthesized compounds (AID 463107)

Assay Overview: The purpose of this assay was to determine S1P4 receptor agonist dose response with synthesized compounds. This assay was run as described above (AID 1509). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.

Protocol Summary: Cells were cultured as described above (AID 1509). Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO₂ and 95% RH. The next day, 25 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone (low control), or S1P (high control; 10 nM final nominal EC₈₀ concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO₂ for 4 hours. After the incubation, 1 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: compounds with an EC₅₀ equal to or less than 10 micromolar were considered active.

Counterscreen dose response assay to identify S1P1 receptor agonists (AID 463118)

Assay Overview: The purpose of this assay was to determine whether synthesized compounds identified as active in the previous assay (AID 463107) were nonselective agonists as assayed by activation of the S1P1 receptor. The assay was performed as described above (AID 1563), except that compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.

Protocol Summary: Cells were cultured as described above (AID 1509). Prior to the start of the assay, cells were suspended at a concentration of 275,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well of a 384-well plate (10,000 cells/well), followed by overnight incubation at 37 degrees C in 5% CO₂ and 95% RH. The next day, 50 nL of test compound in DMSO (0.5% final DMSO concentration), DMSO alone (low control), or S1P (high control; 40 nM final nominal EC₈₀ concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO₂ for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: compounds with an EC₅₀ equal to or less than 10 micromolar were considered active.

Counterscreen dose response assay to identify S1P2 receptor agonists (AID 463122)

Assay Overview: The purpose of this assay was to determine whether synthesized compounds identified as active in a previous assay (AID 463107) were nonselective agonists as assayed by activation of the S1P2 receptor. A Chinese Hamster Ovary (CHO) cell line stably transfected with the human S1P2 receptor and a cAMP Response Element-beta lactamase (CRE-BLA) reporter construct was used to measure S1P2 agonism. Under normal conditions, S1P2 has low basal activity and therefore cells express low BLA levels. Stimulation of the S1P2 receptor by agonist increases BLA gene transcription. This increase is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable fluorogenic cell-permeable BLA substrate. As designed, test compounds that act as S1P2 receptor agonists will activate S1P2 receptor and increase well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.

Protocol Summary: 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) supplemented with 10% v/v heat inactivated dialyzed fetal bovine serum, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, 5 mM L-glutamine, 2 mg/mL Geneticin and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin). Prior to assay, cells were suspended to a concentration of 1,250,000/mL in assay media, which consisted of phenol red-free Dulbecco’s Modified Eagle’s Media supplemented with 2% charcoal/dextran-treated fetal bovine serum, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, 5 mM L-glutamine and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin). The assay was initiated by dispensing 10 μL of cell suspension to each test well of a 384-well plate (6,000 cells/well) followed by incubation at 37 degrees C in 5% CO₂ for 16 hrs. To the appropriate wells were then added 50 nL of test compound in DMSO (final nominal concentration of 50 μM, final DMSO concentration of 0.5%) or DMSO only (low control). Next, 1 μL of S1P in 2% BSA (final concentration of 370 nM, i.e. a concentration that resulted in 80% activity) was added to the high control wells and plates were incubated again at 37 degrees C in 5% CO₂ for 2 hrs. The fluorogenic LiveBLAzer substrate mixture with 10 mM Probenicid was prepared according to the manufacturer’s protocol and 2.2 microliter of this mixture was then added to each well. After a further 2 hours of incubation at room temperature, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and fluorescence emission wavelengths of 535 nm & 460 nm. Assay Cutoff: compounds with an EC₅₀ equal to or less than 10 micromolar were considered active.

Counterscreen dose response assay to identify S1P3 receptor agonists (AID 463123)

Assay Overview: The purpose of this assay was to determine whether synthesized compounds identified as active in a previous assay (AID 463107) were nonselective agonists as assayed by activation of the S1P3 receptor. In this assay, a CHO cell line containing human S1P3 receptor and the beta-lactamase (BLA) reporter gene under control of the nuclear factor of activated T-cells (NFAT) promoter was used to measure S1P3 receptor agonism by test compound. Stimulation of S1P3 receptor by S1P 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 receptor agonist will increase NFAT-BLA transcription and increase well fluorescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.

Protocol Summary: Cells were cultured in T-175 sq cm flasks at 37 degrees C and 95% RH. The growth media consisted of 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-neomycin. Prior to the start of the assay, cells were suspended at a concentration of 1,250,000/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 μL of cell suspension to each well of a 384-well plate (8,000 cells/well), followed by overnight incubation at 37 degrees C in 5% CO₂ and 95% RH. The next day, 50 nL of test compound (50 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to low control wells. Next, S1P prepared in 2% BSA (0.7 μM final nominal concentration, corresponding to the EC₈₀ of S1P) was added to the high control wells. After 4 hours of incubation, 2.2 μL/well of the GeneBLAzer fluorescent substrate mixture, prepared according to the manufacturer’s protocol and containing 10 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 535 nm and 460 nm. Assay Cutoff: compounds with an EC₅₀ equal to or less than 10 micromolar were considered active.

Counterscreen dose response assay to identify S1P5 receptor agonists (AID 463129)

Assay Overview: The purpose of this assay was to determine whether synthesized compounds identified as active in a previous assay (AID 463107) were nonselective agonists as assayed by activation of the S1P5 receptor. This assay uses Tango™ S1P5-BLA U2OS cells which contain the human Endothelial Differentiation Gene 8 (EDG8; S1P5) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P5 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P5 receptor agonists will stimulate migration of the fusion protein, thus increasing proteolysis of GAL4-VP16 and BLA transcription, leading to an increase in well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.

Protocol Summary: Cells were cultured as described above (AID 1509). Prior to the start of the assay, cells were suspended at a concentration of 275,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well of a 384-well plate (10,000 cells/well), followed by overnight incubation at 37 degrees C in 5% CO₂ and 95% RH. The next day, 50 nL of test compound in DMSO (0.5% final DMSO concentration), DMSO alone (low control), or S1P (high control; 1.5 nM final nominal EC₈₀ concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO₂ for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: compounds with an EC₅₀ equal to or less than 10 micromolar were considered active.

Analysis of cytotoxicity (AID 463119)

Assay Overview: The purpose of this assay was to determine cytotoxicity of a synthesized compound identified as active in a previous assay (AID 463107). In this assay, U2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 7-point 1:3 dilution series starting at a nominal test concentration of 20 micromolar.

Protocol Summary: This assay was started by dispensing U2OS cells in McCoy’s 5A medium plus 10% FBS, penicillin 100 U/ml and streptomycin 100 μg/mL (20 μL; 4,000 cells/well) into the wells of a 384-well plate. Eight 1:3 serial dilutions of compound (100 μM in growth media) were made. 5 μL of diluted compound or media were added to wells, giving final compound concentrations of 0–20 μM. The plate was incubated at 37 degrees C in a humidified incubator for 24 hours, then equilibrated to room temperature for 30 minutes. 25 μL CellTitre-Glo reagent was added to each well, followed by incubation of the plate in the dark for 10 minutes. Well luminescence was measured on the Envision plate reader. Assay Cutoff: compounds with a CC₅₀ equal to or less than 10 micromolar were considered active (cytotoxic).

Inhibition by S1P4-selective antagonist (AID 463225)

Assay Overview: The purpose of this assay is to determine whether a test compound with S1P4 agonist activity is inhibited by an S1P4-selective antagonist. This assay uses Tango™ S1P4-BLA U2OS cells which contain the human Endothelial Differentiation Gene 6 (EDG6; S1P4) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P4 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. The presence of an S1P4-selective antagonist compound in the assay inhibits S1P4 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. In the presence of a test agonist compound, the S1P4 receptor will be stimulated, leading to an increase in well FRET. Compound was tested in triplicate using a 6-point, 1:3 dilution series starting at a nominal concentration of 10 micromolar.

Protocol Summary: Cells were cultured as described above (AID 1509). Prior to the start of the assay, cells were suspended at a concentration of 1,000,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO₂ and 95% RH. The next day, 50 nL of test compound in DMSO was added to sample wells, and DMSO alone (0.5% final concentration) was added to control wells. Next, 370 nM S1P4-selective antagonist SR2-304 was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO₂ for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. Assay Cutoff: Compounds with an IC₅₀ equal to or less than 10 micromolar were considered active.

Counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450 (AID 504400)

Assay Overview: The purpose of this panel of binding assays performed by Ricerca Biosciences, LLC, is to identify a subset of potential receptors, transporters, ion channels, etc. for which the S1P4 agonist compound CID 44620892 displays affinity.

Protocol Summary: Assays for CYP450, 1A2; CYP450, 2C19; CYP450, 2C9; CYP450, 2D6; and CYP450, 3A4 were enzyme assays using human recombinant insect Sf9 cells with 5 μM 3-cyano-7-ethoxycoumarin as substrate (except for CYP450, 3A4, which used 50 μM 7-benzyloxy-4-(trifluoromethyl)-coumarin as substrate). Detection was based on spectrofluorimetric quantitation of the enzymatic product produced. Assays for the other targets were radioligand binding assays. Assay Cutoff: A response of at least 50% inhibition or stimulation was considered “active”. Negative inhibition represents a stimulation of binding.