Fluorometric high-throughput screening for inhibitors of cytochrome P450

Ann N Y Acad Sci. 2000;919:26-32. doi: 10.1111/j.1749-6632.2000.tb06864.x.


Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug-drug interactions and associated toxicities. We have developed microtiter plate-based, direct, fluorometric assays for the activities of the principal human drug-metabolizing enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as well as for CYP2A6, which is an important enzyme in environmental toxicology. These assays are rapid and compatible with existing high-throughput assay instrumentation. For CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, the potency of enzyme inhibition (IC50) is consistent regardless of the probe substrate or assay method employed. In contrast, CYP3A4 inhibition for an individual inhibitor shows significant differences in potency (>300-fold) depending on the probe substrate being used. We have investigated these differences through the use of several structurally distinct fluorescent substrates for CYP3A4 and several classical substrate probes (e.g., testosterone, nifedipine, and midazolam), with a panel of known, clinically significant, CYP3A4 inhibitors. The use of multiple probe substrates appears to be needed to characterize the inhibition potential of xenobiotics for CYP3A4.

MeSH terms

  • Coumarins / metabolism
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / metabolism
  • Drug Evaluation, Preclinical / methods*
  • Enzyme Inhibitors / analysis
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / toxicity*
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • Fluorometry / methods*
  • Humans
  • Inhibitory Concentration 50
  • Isoenzymes / analysis
  • Isoenzymes / antagonists & inhibitors
  • Kinetics
  • Mixed Function Oxygenases / antagonists & inhibitors
  • Mixed Function Oxygenases / metabolism
  • Xenobiotics / analysis
  • Xenobiotics / metabolism
  • Xenobiotics / toxicity


  • Coumarins
  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Isoenzymes
  • Xenobiotics
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human