Automated screening with confirmation of mechanism-based inactivation of CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2 in pooled human liver microsomes

Drug Metab Dispos. 2005 Aug;33(8):1211-9. doi: 10.1124/dmd.104.003475. Epub 2005 Apr 28.


A strategy is proposed to profile compounds for mechanism-based inactivation of CYP3A4, CYP2C19, CYP2C9, CYP2D6, and CYP1A2 based on an apparent partition ratio screen. Potent positives from the screen are confirmed by time- and concentration-dependent inactivation assays. Quasi-irreversible inhibitions are then differentiated from irreversible inactivations by oxidation with potassium ferricyanide and/or dialysis. The three-step screening procedure has been validated with acceptable accuracy and precision for detection and confirmation of mechanism-based inactivators in drug discovery. We report here the apparent partition ratios for 19 mechanism-based inactivators and four quasi-irreversible inhibitors obtained under the same experimental conditions. The apparent partition ratio screen was automated to provide throughput for determining structure-mechanism-based inactivation relationships. Information about reversibility can be used to assess potential toxicity mediated by covalent adducts, as well as the potential for pharmacokinetic drug-drug interactions. Direct comparison of known mechanism-based inactivators and quasi-irreversible inhibitors, based on our screening of apparent partition ratios, has identified ritonavir, mibefradil, and azamulin as highly effective mechanism-based inactivators; e.g., 1 mol of CYP3A4 was inactivated on turnover of about 2 mol of compound. Other mechanism-based inactivators we identified include bergamottin (CYP1A2 besides previously reported CYP3A4), troglitazone (CYP3A4), rosiglitazone (CYP3A4), and pioglitazone (CYP3A4). Comparison of the apparent partition ratios and inactivation clearance data for the three glitazones suggests that the chromane moiety on troglitazone contributes to its greater potency for mechanism-based inactivation.

Publication types

  • Comparative Study

MeSH terms

  • Aryl Hydrocarbon Hydroxylases / antagonists & inhibitors
  • Aryl Hydrocarbon Hydroxylases / metabolism*
  • Automation
  • Chromans / pharmacology
  • Chromatography, Liquid
  • Cytochrome P-450 CYP1A2 / metabolism*
  • Cytochrome P-450 CYP1A2 Inhibitors
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2C9
  • Cytochrome P-450 CYP2D6 / metabolism*
  • Cytochrome P-450 CYP2D6 Inhibitors
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Drug Evaluation, Preclinical / methods
  • Enzyme Inhibitors / administration & dosage
  • Enzyme Inhibitors / pharmacology*
  • Enzyme Reactivators / pharmacology
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Mass Spectrometry
  • Microsomes, Liver / enzymology*
  • Mixed Function Oxygenases / antagonists & inhibitors
  • Mixed Function Oxygenases / metabolism*
  • Pharmaceutical Preparations / metabolism*
  • Pioglitazone
  • Reproducibility of Results
  • Rosiglitazone
  • Thiazolidinediones / pharmacology
  • Troglitazone


  • Chromans
  • Cytochrome P-450 CYP1A2 Inhibitors
  • Cytochrome P-450 CYP2D6 Inhibitors
  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Enzyme Reactivators
  • Pharmaceutical Preparations
  • Thiazolidinediones
  • Rosiglitazone
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • CYP2C9 protein, human
  • Cytochrome P-450 CYP2C9
  • Aryl Hydrocarbon Hydroxylases
  • CYP2C19 protein, human
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human
  • Troglitazone
  • Pioglitazone