Assay Report Card

ID: ALA3707545

Type: Binding

Description: Inhibition Assay: To gain insight into the selectivity of the synthetic compounds for inhibition of other CYPs, we examined the major CYPs present in human liver. Prior to these studies we showed that the nicotine analogs were quite metabolically stable in the presence of mouse or rat or human liver microsomes (i.e., T1/2>60 mins). That the nicotine analogs showed low or no inhibitory activity against other CYPs suggests that the inhibitors examined selectively inhibited CYP2A6. A typical incubation mixture (final volume 0.25 mL) contained 50 mM Tris or KPhos buffer (pH 7.5), 0.5 mM NADP+, 2.0 mM G6P, 1 U of G6P dehydrogenase and 0.6 mg DETAPAC and the inhibitor was added last to minimize interaction with the protein. After mixing on ice, the reaction was initiated by the addition of substrate and incubated at 37° C. with shaking in air. Organic extracts were analyzed by fluorescence (for fluorometric substrates) or injected onto a Hitachi L-7100 system equipped with a Hitachi L-7400 UV detector.

Tissue: Liver