AID	Name	Type	Comment
891	qHTS Assay for Inhibitors and Substrates of Cytochrome P450 2D6	confirmatory
883	qHTS Assay for Inhibitors and Substrates of Cytochrome P450 2C9	confirmatory
884	qHTS Assay for Inhibitors and Substrates of Cytochrome P450 3A4	confirmatory
899	qHTS Assay for Inhibitors and Substrates of Cytochrome P450 2C19	confirmatory
885	qHTS Assay for Activators of Cytochrome P450 3A4	confirmatory

NCGC Assay Overview:

The P450 gene superfamily is involved in metabolism and the clearance of xenobiotics. This assay used human CYP1A2 to measure the demethylation of luciferin 6' methyl ether (Luciferin-ME; Promega-Glo) to luciferin. The luciferin is then measured by luminescence after the addition of a luciferase detection regeant. Luciferin-ME concentration in the assay was equal to its Km for CYP1A2.

NCGC Assay Protocol Summary:

Two uL of enzyme and substrate mix (final concentrations: 20nM CYP1A2, 100uM Luciferin-ME, 100mM KPO4) were dispensed into a white, solid bottom 1536-well microtiter plate. 23nL of compound was transferred by a pin tool. The plate was incubated at room temperature for 10 min. Two uL of an NADPH regeneration solution (Promega supplied reagent) was dispensed and the reaction was allowed to continue for 60 min at room temperature. Then the reaction was stopped by reconstituted detection reagent. After another 20 min of incubation the plate was read on the ViewLux (Perkin Elmer) at 60 sec exposure and 2x2 binning. This protocol was carried out with Promega-Glo reagents and further information can be found at http://www.promega.com/tbs/tb325/tb325.pdf. Data were normalized to the to AC100 inhibition (57 uM furafylline).