A fluorescence polarization-based functional assay for cyclic AMP (cAMP) production in cells has been proven effective for the detection of agonist-stimulated cAMP production in a HEK 293 recombinant cell line expressing the corticotropin-releasing factor subtype 2alpha (CRF2alpha) receptor. Assays were completed in a single well of a 384-well microplate with no transfer, separation, or wash steps incurred. The assay performance is excellent for adaptation to the high throughput screening environment in terms of speed of analysis, magnitude of displaced signal, precision, and detection limits for cAMP quantitation. Relative potencies of agonists and antagonists are maintained with respect to radiometric assays. The assay withstands up to 5% DMSO and up to 10 microM concentrations of highly colored compound. These attributes suggest that accurate assessment of drug binding can be measured using this assay.

A fluorescence polarization-based functional assay for cyclic AMP (cAMP) production in cells has been proven effective for the detection of agonist-stimulated cAMP production in a HEK 293 recombinant cell line expressing the corticotropin-releasing factor subtype 2cr (CRF2a) receptor. Assays were completed in a single well of a 384-well microplate with no transfer, separation, or wash steps incurred. The assay performance is excellent for adaptation to the high throughput screening environment in terms of speed of analysis, magnitude of displaced signal, precision, and detection limits for cAMP quantitation. Relative potencies of agonists and antagonists are maintained with respect to radiometric assays. The assay withstands up to 5% DMSO and up to 10 μM concentrations of highly colored compound. These attributes suggest that accurate assessment of drug binding can be measured using this assay.

Inositol-specific phospholipase Cs(PLCs) are a group of enzymes involved in the signal transduction pathway of many plasma membrane receptor mediated events. We developed a modified solid surface to capture [(3)H] PIP(2) onto the Basic FlashPlate(R) in order to monitor PLC activity. Our results clearly demonstrate the utility of [(3)H] PIP(2)-Coated Phospholipid FlashPlate(R) microtiter plates for assessing PLC activity for HTS of receptor-coupled functional assays. The results show that PLC activity can be measured easily from a variety of sources including purified recombinant enzyme preparations, crude HL60 cell lysates and permeabilized A431 human carcinoma cells. Moreover, this format provides a surface comparable to that used for classical solution based radiolabeled mixed phospholipid micelle studies and illustrates the feasibility of this assay for measuring PLC activation in a variety of different drug screening assays.

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