Rapidly Distinguishing Reversible and Time-Dependent CYP450 Inhibition Using Human Liver Microsomes, Co-incubation, and Continuous Fluorometric Kineti

In this chapter we have provided a step-by-step protocol of a 384-well plate fluorescence-based assay used for rapid identification of reversible and time-dependent CYP450 inhibition. This was accomplished by comparing the time-dependence pattern of IC₅₀ values of potential test inhibitors using a co-incubation approach with continuous fluorometric kinetic measurements. Briefly, test compounds were mixed with NADPH and were serially diluted to eight different concentrations. The enzymatic reaction was initiated by adding a single recombinant CYP pre-mixed with its corresponding fluorescent substrate and a mixture of NADP⁺ , G6P and MgCl₂ . The enzyme activity was measured every 2 min by fluorescence intensity (CYP product) over typically a 30-min time period. Inhibition percentages were calculated relative to controls that contained no inhibitors at each time point and IC₅₀ values of inhibitors were calculated at different incubation time intervals. Plotting IC₅₀ values vs. incubation time revealed three different patterns for test inhibitors that could be used to distinguish reversible and time-dependent inhibitors. IC₅₀ values of reversible inhibitors either maintained within a narrow range, or increased with incubation time because of losing inhibitor as a result of metabolism or non-specific binding to the matrix. In contrast, IC₅₀ values decreased with incubation time for time-dependent inhibitors because of irreversible reactions caused by progressive enzyme inactivation by reactive metabolite species generated during the incubation or other inactivation mechanisms. Results clearly suggest that this co-incubation in vitro continuous fluorometric kinetic assay using recombinant CYPs and fluorometric generating substrates is a valuable high-throughput assay for distinguishing reversible and time-dependent inhibitors for large compound collections.