| Assay Method Information | |
| | S1P Assay |
| Description: | Selected Compounds of the Examples were evaluated at Millipore Corporation, USA, using S1P1 receptor; [35S]-GTPgamaS binding assay. A [35S]-GTPgamaS binding assay at Millipore was conducted by GPCR Profiler Custom Service Laboratory, Temecula, Calif., Millipore, Inc. to monitor dose-dependent agonist selectivity for selected Examples against the S1P1 receptors. The assay was completed with sample compounds subjected to an eight-point, four-fold dose response curve with starting concentration of 10 μM. Selectivity was determined upon initial addition of compounds followed by a 30 minute incubation at 30° C. Following compound incubation, bounded [35S]-GTPgamaS was determined by filtration and scintillation counting. Percentage activation and inhibition values were determined relative to the reference agonist at S1P1 and are shown in Table 10. Independently, selected compounds were evaluated for S1P1 and S1P3 agonistic activity. The S1P1 assay system was GTPgama-S35 binding in membranes from CHO K1 cells, expressing S1P1 human receptor. The S1P3 assay system was calcium mobilization in CHO K1 cells expressing S1P3 human receptor. There was no significant background response to S1P in the CHO K1 cells with either assay. Compounds were tested initially at a concentration of 10 μM. Those compounds with significant efficacy (Emax>0.15 relative to S1P) at either receptor type were used to generate concentration-effect (dose response) curves at that receptor. |
| Affinity data for this assay | |
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