| Solution Information | help | |
| Enzyme: | Sphingosine 1-phosphate receptor 2 | |
| inhibitor: | BDBM41616 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: A cell line expressing the beta-lactamase (BLA) reporter-gene under control of the cAMP Response Element (CRE) promoter was used to measure nonspecific antagonism of the cAMP signaling pathway. Under normal conditions, the CRE promoter has low basal activity and therefore cells express low BLA levels. In this assay, cells have been stimulated with forskolin, which stimulates adenylate cyclase, hence increasing BLA gene transcription. An antagonist is identified as a compound that prevents activation by exogenously added forskolin. Therefore, in the presence of a potential cyclic AMP pathway antagonist, transcription of the BLA gene will decrease. This decrease is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable fluorogenic cell permeable BLA substrate. Protocol Summary: The purpose of this assay is to determine dose response curves for 61 available of 64 compounds that had greater than 50 percent inhibition and passed hit selecti | |
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