| Assay Method Information | |
| | In Vitro Cellular Assay |
| Description: | Following agonist data acquisition, cells were then incubated for a further 30 min at room temperature before either an EC20 concentration (to measure positive allosteric modulator activity) or an EC50 concentration (to measure antagonism) of carbachol was added using a fluorometric imaging plate reader. Positive allosteric modulator pEC50 values and antagonist pIC50 values for each compound were then determined. PAM Emax values were generated following normalisation between the EC20 base line fluorescence (0%) and maximal carbachol effect (100%). Antagonist Emax values were generated following normalisation between the EC80 fluorescence (0%) and baseline EC0 (DMSO) response (100%). Data analysis was carried out using a 4 parameter logistic nonlinear regression model with the XLFIT (IDBS) excel add-in10. 1-10 Examples 76-80 and 82-89 were tested in a slightly modified procedure. The points of modification and the alternate reagents, concentrations or equipment employed in these examples were: 1200 μg/ml hygromycin B; 2G418 not added; 3CELLBANKER 1; 410E6; 5 40 μl; 6 1 in 99; 7 20 μl; 8FDSS6000 (Hamamatsu Photonics); 9(480Ex, 540Em); 10Spotfire (TIBCO).As measured by the above in vitro assay, compound Examples 1 to 105 are positive allosteric modulators of mAChR M1 displaying the pEC50 values for positive allosteric modulation given in Table 2. |
| Affinity data for this assay | |
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