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| | In Vitro M4 &M2 Functional Assay |
| Description: | The functional activity of compounds at the M4 and M2 receptors was determined by measuring changes in the level of intracellular calcium ions caused by signalling cascades mediated by the receptor. Intracellular Calcium levels were measured using a calcium sensitive fluorescent dye, calcium 5 (Molecular Devices) The changes in fluorescence were monitored by a fluorescent imager, FLiPR Tetra (Molecular devices). Increases in intracellular calcium were readily detected upon activation of both receptors by the muscarinic receptor agonist Acetylcholine.CHOK1 cells stably expressing human M4 or M2 receptor and co-expressing the accessory g-protein Gα16 were routinely grown as monolayers in Hams-F12 medium (Invitrogen) supplemented with 10% foetal bovine serum (FBS) (Hyclone), 500 ug/mL Geneticin and 250 ug/mL zeocin (both invitrogen) in 5% CO2 at 37° C. Once confluent is cells cryopreserved by freezing at −186° C. in freezing solution (90% FBS 10% DMSO) (Sigma-Aldrich Co.). Twenty-four hours prior to testing cells resuscitated and freezing media removed via centrifugation, cells then seeded in a black walled clear bottom 384 well plates (Corning) at a density of 15,000 cells/well in Hams F12 media supplemented with 10% FBS. On the day of assay, growth media was removed and replaced with 63 μl of Calcium 5 dye solution (Molecular Devices) in assay buffer (HBSS, 20 mM HEPES, 0.1% BSA, 1 mM Probenecid pH7.4 (Sigma-Aldrich Co.)) per well (each vial of Calcium 5 resuspended in 27 mL of assay buffer). Cells were then incubated for 45 minutes at 37° C., 5% CO2. Compound was serially diluted in DMSO (log/half log) before being diluted 1:20 with assay buffer. 7 μl of compound diluted in assay buffer was then added to cells on FLiPR tetra and fluorescence intensity measured for 5 minutes. EC50 values for compounds were determined from ten point half log scale dose-response studies and represent the concentration of compound required to prevent 50% inhibition of its own maximal response. Curves were generated using the average of duplicate wells for each data point and analyzed using non-linear regression of four parameter dose response. Percentage Relative efficacy (RE) to an EC100 concentration of acetylcholine was reported for all compounds. The results are set out in Table 3 below in which the term No Response means that there was no significant response of calcium flux in the assay indicative of agonism. |
| Affinity data for this assay | |
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