High throughput reactive oxygen species-based cytochrome P450 inhibition assay

A method of determining an inhibitory potency of a candidate compound for a cytochrome P450, the method comprising:

• (a) providing a cytochrome P450 substrate and a candidate compound suspected of having an ability to inhibit a cytochrome P450;
• (b) mixing the candidate compound, the cytochrome P450 substrate, an indicator compound precursor, the cytochrome P450, and NADPH or an NADPH regenerating system, wherein a primary metabolic activity of the cytochrome P450 produces a chemical species in a side reaction;
• (c) producing the chemical species in the side reaction, whereby a fraction of the chemical species reacts with the indicator compound precursor to produce an indicator compound;
• (d) quantifying a rate at which the indicator compound is produced in the presence of the candidate compound;
• and
• (e) determining a concentration of the candidate compound that reduces the primary metabolic activity of the cytochrome P450 by a pre-set amount, whereby the inhibitory potency of the compound for a cytochrome P450 is determined.