| Assay Method Information | |
| | Evaluation of In Vitro Activity |
| Description: | a. Cloning, Sequencing, Transfection and Selection of Positive Clones Expressing Human TRPM8A functional cell-based assay for the identification of TRPM8 receptor antagonists, optimised to allow high throughput screening at FLIPRTETRA, was developed in HEK293 cells by stable pure clone selection and functional characterization with a fluorescent calcium sensitive dye.TRPM8 was cloned into the multiple cloning site of pcDNA3 mammalian expression vector; the obtained construct pcDNA3/hTRPM8 was fully sequence verified and used for the transfection of HEK293 cell line. HEK293 cells stably transfected with TRPM8 gene were maintained in Minimum essential medium. The cells were transfected with the pcDNA3/hTRPM8 vector by electroporation and then selected with medium containing 0.8 mg/ml G418 for 10-15 days.The following commercial compounds were used as TRPM8 channel reference compound to test HEK293/hTRPM8 cell line for both agonist and antagonist activity:Activators: Menthol (SIGMA cat. #M2772) WS-3, (N-Ethyl-5-methyl-2-(1-methylethyl) cyclohexanecarboxamide) (SIGMA cat. #W345501)Blocker: Capsazepine (SIGMA cat. #C191)The experimental activities were performed using FLIPR instruments.The functional clones were selected at FLIPR384 on the basis of 1 mM menthol response. Two best responder clones were selected, diluted at a cell density of 1 cell/well and analysed at FLIPR384 with 1 mM menthol.The TRPM8 receptor was analysed for the response to reference agonist, menthol, using a calcium-dependent fluorescence signal.Patch clamp recordings were also obtained in voltage-clamp configuration on HEK/TRPM8 clones in order to verify the receptor pharmacology and to determine the agonist dose-response curve and EC50 value. HEK293 cells were maintained at room temperature on an fire-polished borosilicate glass pipettes having 1.5-2.5 MΩ resistance were used to record currents following drug application. Menthol application induced a dose-dependent inward current in a selected HEK/hTRPM8 clone (calculated EC50 value=58 μM). No menthol-induced currents were recorded in not transfected HEK293 cells.In order to determine the capsazepine antagonist activity on menthol agonist response and to verify the antagonist response stability throughout different days of experiments, the selected clone of TRPM8 was analysed after 24 h at FLIPR384 in presence of variable concentrations of antagonist (from 100 nM to 316 μM). The selected clone showed very good stability and reproducibility of the antagonist activity (calculated IC50 value=20 μM).Summarizing, the best clone was characterized for: 1 pharmacology: agonist EC50 and antagonist IC50 determination over different experiments;2 optimal cell density and seeding time;3 DMSO sensitivity;4 ligand stability;5 patch clamp analysis.b. Screening Set Up for the Identification of TRPM8 AntagonistsThe following commercial compounds were used as ligands:Activator: Cooling Agent 10 (Takasago CAS N. 87061-04-9)Blocker: Capsazepine (SIGMA cat #D_5879)The experimental activities were performed using FLIPRTETRA instruments.HEK293 cells stably transfected with TRPM8 gene were maintained in Minimum essential medium.The TRPM8 cell line was analysed for the response to a library of compounds using a Ca2+ mobilization-dependent fluorescence signal in 384 wells microtiter plate format. The analysis was performed using the FLIPRTETRA (MDC) with the ICCD Camera.The execution of the assay involved the use of three microtiter plates:1. Assay plate, containing cells loaded with dye and prepared as follows:Cells were seeded at 15000 c/well in Poly-D-Lysine coated 384 wells Microtiter Plates in complete medium (25 μl/well).24 h after seeding, the cell plates were washed with Tyrode assay buffer by the Microplate Washer and 10 μL of Tyrode assay buffer was left in each well.Cells were then loaded with 10 μL/well of the Fluo-4 NW dye solution by CyBio-Well pipettor. |
| Affinity data for this assay | |
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