Probe Development Efforts for an Allosteric Agonist of the Sphingosine 1-phosphate Receptor 3 (S1P3)

LC-MS/MS

All analytical methods are in MRM mode where the parent ion is selected in Q1 of the mass spectrometer. The parent ion is fragmented and a characteristic fragment ion is 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 will be rapidly set up using Automaton^® (Applied Biosystems), where the compounds are listed with their name and mass in an Excel datasheet. Compounds are submitted in a 96-well plate to the HPLC autosampler and are slowly injected without a column present. A narrow range centered on the indicated mass is scanned to detect the parent ion. The software then evaluates a few pre-selected parameters to determine conditions that maximize the signal for the parent ion. The molecule is then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass are determined. Three separate collision energies are evaluated to fragment the parent ion and the largest three ions are selected. Each of these three fragment ions is further optimized and the best fragment is chosen. The software then inserts the optimized masses and parameters into a template method and saves it with a unique name that indicates the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern are saved and can be reviewed 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 at 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 is 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.

Primary, Confirmation, and Dose Response uHTS assays to identify S1P3 receptor agonists (AID 373 and AID 439)

Assay Overview: The purpose of these assays was to identify compounds that act as agonists of the S1P3 receptor. 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 Gα16 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). For the primary screen (AID 373), all compounds were tested once at a 4.5 micromolar final concentration, and for the confirmation screen (AID 373), all compounds were tested in triplicate at the same concentration. For the dose response screen (AID 439), compounds were tested in triplicate in a ten-point 1:3 dilution series starting at a nominal concentration of 45 μM.

Protocol Summary: 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 Gα16 pathway coupling protein was used. Cells were cultured in T-175 sq cm flasks at 37°C and 95% relative humidity (RH). The growth medium consisted of Dulbecco's Modified Eagle's Media containing 10% v/v heat inactivated bovine growth serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 2 mg/mL 5 mM L-Glutamine, 0.2 mg/mL Hygromycin B and 1× penicillin-streptomycin. Prior to the start of the assay, cells were suspended to a concentration of 1 × 10⁶/mL in phenol red free Dulbecco's Modified Eagle's Media containing 0.5% charcoal/dextran treated fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, and 5 mM L-Glutamine. The assay began by dispensing 5 mL 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, 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 μM. Plates were then incubated at 37°C in 5% CO₂ for 4 hours. After the incubation, 1 μL/well of the GeneBLAzer's fluorescent substrate mixture, prepared according to the manufacturer's protocol and containing 200 mM probenicid 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. Assay Cutoff: In the primary and confirmation screens, compounds that exhibited 4.22% agonism or greater for S1P3 receptor were considered active. In the dose response screen, compounds with EC50 > 10 μM were considered active.

Dose response assay to identify S1P3 receptor agonists with purchased analogues (AID 1192)

Assay Overview: The purpose of this assay was to determine dose response curves for purchased structural analogues of an S1P3 agonist (SID 7967985) identified as active in previous experiments. The assay was described above (AID 373). Compounds were tested in quadruplicate in 384-well plates in a 10-point 1:3 dilution series starting at a nominal test concentration of 10 micromolar.

Protocol Summary: Cells were cultured as described above (AID 373). Prior to the start of the assay, cells were suspended at a concentration of 1 × 10⁶/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, followed by overnight incubation at 37°C in 5% CO₂. The next day, 50 nL of test compound in DMSO, DMSO alone, or S1P (1 μM final nominal concentration) was added to the appropriate wells. After 4 hours of incubation, 2 μL/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. Assay Cutoff: Compounds with EC50 ≤ 10 μM were considered active.

Dose response assay to identify S1P3 receptor agonists with synthesized compounds (AID 540349)

Assay Overview: The purpose of this assay was to determine S1P3 receptor agonist dose response with synthesized compounds. This assay was run as described above (AID 373). Compounds were tested in triplicate using a 10-point 1:3 dilution series.

Protocol Summary: Cells were cultured as described above (AID 373). Prior to the start of the assay cells were suspended at a concentration of 1.25 × 10⁶/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 mL of cell suspension to each well of a 384 well plate (8 × 10³ cells/well), followed by overnight incubation at 37°C in 5% CO₂ and 95% RH. The next day, 50 nL of test compound (50 micromolar final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5% final concentration) was added to High Control wells. Next, S1P prepared in 2% BSA (0.7 micromolar final nominal concentration, corresponding to the EC80 of S1P) was added to the appropriate 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 EC50 ≤ 10 μM were considered active.

Counterscreen dose response assay to identify S1P1 receptor agonists (AID 540368)

Assay Overview: The purpose of this assay was to determine whether powder samples of compounds identified as active for S1P3 agonist were nonselective agonists as assayed by activation of S1P1. This assay uses Tango S1P1-bla U2OS cells which express the human Endothelial Differentiation Gene 1 (EDG1; S1P1) 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 agonists will activate S1P1 and increase well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series.

Protocol Summary: U2OS cells were cultured in T-175 sq cm flasks at 37°C and 95% 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 micrograms/mL Zeocin, 50 micrograms/mL Hygromycin, and 100 micrograms/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 2.75 × 10⁵/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 (1 × 10⁴ cells/well), followed by overnight incubation at 37°C in 5% CO₂ and 95% RH. The next day, 50 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone, or S1P (40 nM final nominal EC80 concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37°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 EC50 ≤ 10 μM were considered active.

Counterscreen dose response assay to identify S1P2 receptor agonists (AID 540367)

Assay Overview: The purpose of this assay was to determine whether powder samples of compounds identified as active for S1P3 agonist were nonselective agonists as assayed by activation of S1P2. A 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 S1P1 agonists will activate S1P1 and increase well FRET. Compounds were tested in quadruplicate at 50 micromolar.

Protocol Summary: Cells were cultured as described above (AID 373). Prior to assay, cells were suspended to a concentration of 1.25 × 10⁶/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 × 10³ cells/well) followed by incubation at 37°C in 5% CO₂ for 16 hours. To the appropriate wells were then added 50 nL of test compound in DMSO (final nominal concentration of 50 micromolar, final DMSO concentration of 0.5%) or DMSO only (for high control wells) followed directly afterwards by 1 μL of S1P in 2% BSA (final concentration of 370 nanomolar, i.e. a concentration that resulted in 80% activity). The high control (EC80 challenge) and low control (100% antagonism) were added to the appropriate control wells and plates were incubated again at 37°C in 5% CO₂ for 2 hours. The fluorogenic LiveBLAzer substrate mixture with 10 mM Probenicid was prepared according to the manufacturer's protocol and 2.2 μL 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: Where treatment with 50 μM compound did not result in greater than 50% activation, the EC50 was determined manually as greater than 50 μM. Compounds with an EC50 ≤ 50 μM were considered active.

Counterscreen dose response assay to identify S1P4 receptor agonists (AID 540366)

Assay Overview: The purpose of this assay was to determine whether powder samples of compounds identified as active for S1P3 agonist were nonselective agonists as assayed by activation of S1P4. 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 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 agonists will activate S1P1 and increase well FRET. Compounds were tested in triplicate at 50 micromolar.

Protocol Summary: U2OS cell were cultured as described above (AID 540368). Prior to the start of the assay, cells were suspended at a concentration of 2.5 × 10⁵/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°C in 5% CO₂ and 95% RH. The next day, 25 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone, or S1P (10 nM final nominal EC80 concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37°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: Where treatment with 50 μM compound did not result in greater than 50% activation, the EC50 was determined manually as greater than 50 μM. Compounds with an EC50 ≤ 50 μM were considered active.

Counterscreen dose response assay to identify S1P5 receptor agonists (AID 540369)

Assay Overview: The purpose of this assay was to determine whether powder samples of compounds identified as active for S1P3 agonist were nonselective agonists as assayed by activation of S1P4. 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 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 sextuplet using a 10-point, 1:3 dilution series starting at a nominal concentration of 25 micromolar.

Protocol Summary: U2OS cell were cultured as described above (AID 540368). Prior to the start of the assay, cells were suspended at a concentration of 2.75 × 10⁵/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 (1 × 10⁴ cells/well), followed by overnight incubation at 37°C in 5% CO₂ and 95% RH. The next day, 50 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone, or S1P (1.5 nM final nominal EC80 concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37°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 EC50 ≤ 10 μM were considered active.

Analysis of cytotoxicity (AID 540344)

Assay Overview: The purpose of this assay was to determine cytotoxicity of the S1P3 agonist compound CID 17253208, ML249. 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 ug/ml (20 μL, 4 × 10³ cells/well) into the wells of a 384-well plate. Eight 1:3 serial dilutions of compound (100 μM in growth media) were made. 5μL of diluted compound or media were added to wells, giving final compound concentrations of 0–20 μM. The plate was incubated at 37°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).

Competition binding assay with physiological ligand S1P (AID 588327)

Assay Overview: The purpose of this assay was to determine whether ML249 competes competitively or noncompetitively for binding of [³³P]S1P to cells that contain the S1P3 receptor. In this assay, S1P3 Jump-In CHO cells containing human S1P3 are incubated with a fixed concentration of [³³P]S1P and increasing concentrations of S1P3 agonist. If the S1P3 agonist compound competes with the [³³P]S1P for binding to the S1P3 receptor, the radioactivity bound to the cells will decrease as the concentration of cold S1P3 increases. If the S1P3 agonist compound does not compete with the [³³P]S1P for binding to the S1P3 receptor, the radioactivity bound to the cells will remain unchanged as the concentration of S1P3 agonist increases. S1P3 agonist compound was tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 micromolar (Experiment 1). An experiment to demonstrate that cold S1P competes competitively for binding of [³³P]S1P to cells that contain the S1P3 receptor serves as a control (Experiment 2). In this assay, S1P3 Jump-In Chinese Hamster Ovary (CHO) cells containing human S1P3 are incubated with a fixed concentration of [³³P]S1P and increasing concentrations of cold S1P. If the cold S1P competes with the [³³P]S1P for binding to the S1P3 receptor, the radioactivity bound to the cells will decrease as the concentration of cold S1P3 increases. Cold S1P was tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 micromolar.

Protocol Summary: S1P3 Jump-In CHO cells were cultured in T-175 sq cm flasks at 37°C and 95% RH. The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) (with GlutaMAX) containing 10% v/v heat inactivated fetal bovine serum (dialyzed), 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, and 1 × penicillin-streptomycin-neomycin. On the day before the assay, cells were suspended at a concentration of 0.2 × 10⁶/mL in the growth media and plated at 0.1 × 10⁶/well in a 24-well plate. On the day of the assay, the growth medium was replaced with serum-starvation medium consisted of Dulbecco's Modified Eagle's Media (DMEM) (with GlutaMAX) containing 0.1 mM NEAA, 1 mM sodium pyruvate, and no antibiotics. The cells were incubated at 37°C for 4 hours. After 4 hours of serum starvation, the medium was removed and the cells were rinsed with 200 μL of ice-cold binding buffer. The binding buffer consisted of 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 15 mM NaF, 0.5 mM EDTA, 1 mM Na₃VO₄, 0.5% fatty acid-free BSA, and 1× protease inhibitor cocktail. Experiment 1: For the S1P3 agonist competition assay, 30 μL of increasing concentrations of ML249 (final ML249 concentrations of 0.001 nM to 10 μM) and 270 μL ice-cold binding buffer containing [³³P]S1P (final concentration of 0.1 nM) were added to the cells in each well. Experiment 2: For the control assay, 30 μL of increasing concentrations of cold S1P (final S1P concentrations of 0.001 nM to 10 μM) and 270 μL ice-cold binding buffer containing [³³P]S1P (final concentration of 0.1 nM) were added to the cells in each well. For both experiments, the cells were incubated at 4 °C for 30 minutes. The cells were then washed three times with 500 μL ice-cold binding buffer. The cells were lysed with 300 μL 0.5% SDS and transferred to scintillation vials. 5 mL of scintillation cocktail was dispensed into each vial and the vial vortexed. The ³³P radioactivity (cpm) was counted for 5 minutes/vial in a Beckman LS 6000SC counter.

Counterscreen panel assay for S1P3 agonists: Ricerca HitProfilingScreen + CYP450 (AID 540351)

Assay Overview: The purpose of this panel of binding assays performed by Ricerca Biosciences, LLC, was to identify a subset of potential receptors, transporters, ion channels, etc. for which the S1P3 agonist compound CID 17253208, ML249, 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.