| Solution Information | help | |
| Enzyme: | Sphingosine 1-phosphate receptor 2 | |
| inhibitor: | BDBM41597 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: A cell line expressing both the beta-lactamase (BLA) reporter-gene under control of the cAMP Response Element (CRE) promoter and the human S1P2 receptor was used to identify compounds that inhibit S1P2 activity. Under normal conditions, S1P2 has low basal activity and the cells express low BLA levels. Treatment of the cells with an S1P agonist stimulates the S1P2 receptor and activates BLA gene transcription. An antagonist is identified as a compound that prevents activation by S1P, and as a result decreases transcription of the BLA gene. 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 selection criteria from a previous set of experiments entitled, "Primary Cell-Based High-Throughput Screening to Iden | |
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