Optimization and Characterization of a Second Antagonist for G-protein Coupled Receptor 7 (GPR7)

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 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 50 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 (22–23).

Primary uHTS assay to identify GPR7 antagonists (AID 1861, AID 1952, and AID 2251)

Assay Overview: The purpose of this assay was to identify compounds that inhibit GPR7 activity. Although GPR7 is naturally coupled to Gαi, which decreases cAMP levels upon activation, this assay employs a chimeric cell line that forces the receptor to use Gqi3, and therefore the assay readout is calcium release. In this assay, HEK cells stably co-transfected with the human GPR7 receptor and Gqi3 (hGPR7 HEK293T/Gqi3 cell line) were treated with test compounds, followed by measurement of intracellular calcium as monitored by the FLUO-8 fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as GPR7 antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye, and thus decreased well fluorescence. Test compounds were assayed in singlicate at a final nominal concentration of 4.4 μM (AID 1861), in triplicate at a final nominal concentration of 4.4 μM (AID 1952), or in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 44 μM (AID 2251).

Protocol Summary: The hGPR7 HEK293T/Gqi3 cell line was routinely cultured in T-175 sq cm flasks at 37°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 qualified fetal bovine serum, 25 mM HEPES, 200 ug/mL Hygromycin-B, 200 ug/mL Geneticin, 0.625 ug/mL Puromycin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay 1.5 × 10³ cells in 3 μL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37°C, 5% CO₂, and 95 % RH for 23 hours. Next, 2 μL of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer’s protocol) was added to each well. After incubation for 1 hour at 37°C, 5% CO₂, and 95 % RH, 22 nL of test compound in DMSO, or DMSO alone, were dispensed to the appropriate wells. The assay was started after a 30-minute incubation at room temperature by performing a basal read of plate fluorescence (470–495 nm excitation and 515–575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of GPR7 agonist NPW (2 nM final concentration) in DMSO, or DMSO alone, were dispensed to the appropriate wells. Then a real-time fluorescence measurement was immediately performed for the remaining 180 seconds of the assay. Assay Cutoff: 1) Plate-based cutoff (AID 1861); 2) Compounds that inhibited GPR7 greater than 23.76% were considered active (AID 1952); 3) Compounds with an IC50 < 10 μM were considered active.

Counterscreen uHTS assay to identify MCH1 antagonists (AID 2148 and AID 2257)

Assay Overview: The purpose of this assay was to identify compounds that inhibit the melanin-concentrating hormone receptor 1 (MCH1) (GPR24), the receptor for MCH, a cyclic hypothalamic neuropeptide that promotes food intake (17), increases leptin and insulin release (18), and modulates energy metabolism (19). This assay also serves as a counterscreen for compounds identified as active in a previous set of experiments entitled, “Fluorescence-based primary cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7)” (AID 1861), and that confirmed activity in a previous set of experiments entitled, “Fluorescence-based confirmation cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7)” (AID 1952). This assay employs HEK cells stably co-transfected with human MCH1 and a chimeric Gαqi3. Cells are treated with test compounds followed by measurement of intracellular calcium as monitored by the FLUO-8 fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as human MCH1 antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye, and thus decreased well fluorescence. Test compounds were assayed in triplicate at a final nominal concentration of 4.4 μM (AID 2148) or in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 44 μM (AID 2257).

Protocol Summary: The hMCHR1 HEK293T/Gqi3 cell line was routinely cultured in T-175 sq cm flasks at 37°C and 95% RH. The growth media consisted of DMEM supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, 200 ug/mL Hygromycin-B, 100 ug/mL Zeocin, 200 ug/mL Geneticin and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay, 1.5 × 10³ cells in 3 μL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37°C, 5% CO₂, and 95 % RH for 23 hours. Next, 2 μL of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer’s protocol) was added to each well. After incubation for 1 hour at 37°C, 5% CO₂, and 95 % RH, 22 nL of test compound in DMSO, or DMSO alone, were dispensed to the appropriate wells. The assay was started after an additional 30-minute incubation at room temperature by performing a basal read of plate fluorescence (470–495 nm excitation and 515–575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of MCH peptide agonist (0.2 nM final concentration) in DMSO, or DMSO alone, were dispensed to the appropriate wells. Then a real-time fluorescence measurement was immediately performed for the remaining 180 seconds of the assay. Assay Cutoff: 1) Compounds that inhibited MCH1 greater than 21.36% were considered active (AID 2148); 2) Compounds with an IC50 ≤ 10 μM were considered active.

Late-stage assay to identify GPR7 antagonists (AID 504868 and AID 540365)

Assay Overview: The purpose of this assay was to determine dose response curves for synthesized compounds in a 384-well plate format for antagonism of GPR7. The assay was as described above for the uHTS assay (AID 1861, AID 1952, and AID 2251). Test compounds were assayed in triplicate in an 8-point 1:3 dilution series starting at a nominal test concentration of 20 μM.

Protocol Summary: The hGPR7 HEK293T/Gqi3 cell line was routinely cultured in T-75 sq cm flasks at 37°C and 95% RH. The growth media consisted of DMEM supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, 200 ug/mL Hygromycin-B, 200 ug/mL Geneticin, 0.625 ug/mL Puromycin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay 1 × 10⁴ cells in 25 μL of growth media were seeded into each well of 384 well microtiter plates and allowed to incubate at 37°C, 5% CO₂, and 95 % RH for 23 hours. Next, 25 μL of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer’s protocol) was added to each well. After incubation for 50 minutes at 37°C, 5% CO₂, and 95 % RH, 100 nL of test compound in DMSO, or DMSO alone, were dispensed to the appropriate wells. The assay was started after an additional 15-minute incubation at room temperature by performing a basal read of plate fluorescence (470–495 nm excitation and 515–575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 5.5 nL of GPR7 agonist (20 nM final concentration) in FLIPR buffer (HBSS/20 mM Hepes/0.1% BSA) was dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 180 seconds of the assay. Assay Cutoff: Compounds with an IC50 ≤ 10 μM were considered active.

Late-stage counterscreen assay to identify MCH1 antagonists (AID 504889 and AID 540371)

Assay Overview: The purpose of this assay was to determine dose response for a set of synthesized compounds in a 384-well format counterscreen assay for antagonism of MCH1. The assay was as described above for the uHTS assay (AID 2148 and AID 2257). Test compounds were assayed in triplicate in an 8-point 1:3 dilution series starting at a nominal test concentration of 20 μM.

Protocol Summary: The hMCHR1 HEK293T/Gqi3 cell line was routinely cultured in T-75 sq cm flasks at 37°C and 95% RH. The growth media consisted of DMEM supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, 200 ug/mL Hygromycin-B, 100 ug/mL Zeocin, 200 ug/mL Geneticin and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay, 1 × 10⁴ cells in 3 μL of growth media were seeded into each well of 384 well microtiter plates and allowed to incubate at 37°C, 5% CO₂, and 95 % RH for 23 hours. Next, 25 μL of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer’s protocol) was added to each well. After incubation for 50 minutes at 37°C, 5% CO₂, and 95 % RH, 100 nL of test compound in DMSO, or DMSO alone, were dispensed to the appropriate wells. The assay was started after an additional 15-minute incubation at room temperature by performing a basal read of plate fluorescence (470–495 nm excitation and 515–575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 5.5 nL of MCH peptide agonist (30 nM final concentration) in FLIPR buffer (HBSS/20 mM Hepes/0.1% BSA) were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 180 seconds of the assay. Assay Cutoff: Compounds with an IC50 ≤ 10 μM were considered active.

Cytotoxicity assay (AID 504886 and AID 588326)

Assay Overview: The purpose of this assay was to determine cytotoxicity of a powder sample of a compound identified as active in the late-stage assay to identify GPR7 antagonists (AID 463251). In this assay, U2OS cells were 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 sextuplet in a 7-point 1:3 dilution series starting at a nominal test concentration of 20 micromolar.

Protocol Summary: U2OS cells were grown in DMEM supplemented with 10% v/v fetal bovine serum, 2 mM L-Glutamine, and 100U/mL penicillin and streptomycin. Prior to the start of the assay, 2 × 10⁵ U2OS cells in 20 μL HBSS were dispensed into wells of a 384-well tissue culture-treated microtiter plate. Test compound was diluted 1:100 in growth medium (100 μM final concentration) and then serially diluted 1:3 in growth medium. The assay was started immediately by dispensing 5 μL of test compound, media alone, or rotenone as a positive control (150 μM final concentration) to the appropriate wells. The plates were then incubated for 2 hours at 37°C. The plate was then equilibrated at room temperature for 30 minutes. The assay was stopped by dispensing 25 μL of CellTiter-Glo reagent to each well followed by incubation in the dark at room temperature for 10 minutes. Well luminescence was measured on the ViewLux plate reader. Activity Cutoff: Compounds with a CC50 value of ≤ 10 μM were considered active (cytotoxic).

Ricerca HitProfilingScreen + CYP450 panel assay (AID 540345)

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 GPR7 antagonist compound CID 46172919 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 ≥ 50% inhibition was considered active.

Table 2 gives an overview of the PubChem assays associated with the GPR7 antagonist project and indicates which are associated with the current probe development effort for ML250.

Table 2

Overview of PubChem assays for GPR7 antagonist project.