Temporal Changes In Ic50 Values

Some CYP inhibitors covalently modify and inactivate the enzymes. Such mechanism-based inhibitors may not be potent competitive inhibitors and thus may not be detected in brief assays for competitive inhibition. Different experimental approaches (typically time- and NADPH-dependent inhibition) are needed to identify this class of inhibitors. Two aspects of the experimental design facilitate the detection of mechanism-based inhibitors. First, the incubation times are relatively long, therefore there is an opportunity for time- and NADPH-dependent inactivation to occur. Second, most of the assays are direct, therefore the plates can be scanned at multiple time points and temporal changes in IC 50 analyzed. With this approach, a 1000-fold decrease in IC50 values was observed for tienilic acid [24] inhibition of CYP2C9 (Fig. 2). Such a substantial time-dependent decrease in IC50 values implies that mechanism-based inhibition is occurring. Similar time-dependent decreases were observed for CYP1A2 and furafylline, another

Figure 2 Time-dependent inhibition of CYP2C9 by tienilic acid. The assay was conducted as described in the materials and methods section. Tienilic acid was dissolved in water, and the highest concentration was 1 mM. At the indicated times the plate was scanned and the IC50 value calculated. The values above the individual bars are the calculated IC50 values.

mechanism-based inhibitor [24]. This type of observation indicates that more detailed studies may be appropriate.

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