| Assay Method Information | |
| | GTP γ35S Binding Assay |
| Description: | The S1P1 activity of the compounds according to the invention, were tested using the GTP γ35S binding assay. The compounds were assessed for their ability to activate or block activation of the human S1P1 receptor in cells stably expressing the S1P1 receptor. Some of the results are presented in Table 3.GTP γ35S binding was measured in the medium containing (mM) HEPES 25, pH 7.4, MgCl2 10, NaCl 100, dithiothreitol 0.5, digitonin 0.003%, 0.2 nM GTP γ35S, and 5 μg membrane protein in a volume of 150 μl. Test compounds were included in the concentration range from 0.08 to 5,000 nM unless indicated otherwise. Membranes were incubated with 100 μM 5′-adenylylimidodiphosphate for 30 min, and subsequently with 10 μM GDP for 10 min on ice. Drug solutions and membrane were mixed, and then reactions were initiated by adding GTP γ35S and continued for 30 min at 25° C. Reaction mixtures were filtered over Whatman GF/B filters under vacuum, and washed three times with 3 mL of ice-cold buffer (HEPES 25, pH7.4, MgCl2 10 and NaCl 100). Filters were dried and mixed with scintillant, and counted for 35S activity using a 8-counter. |
| Affinity data for this assay | |
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