A quantitative high-throughput 96-well plate fluorescence assay for mechanism-based inactivators of cytochromes P450 exemplified using CYP2B6

Nat Protoc. 2010 Sep;5(10):1652-8. doi: 10.1038/nprot.2010.125. Epub 2010 Sep 23.


Mechanism-based inactivators such as bergamottin are useful chemical tools for identifying the functions of specific active-site amino acid residues in the reactions catalyzed by cytochromes P450 (CYPs or P450s), which are responsible for the metabolism of a wide variety of drugs and endogenous substrates. In clinical settings, mechanism-based inactivation of P450s involved in xenobiotic metabolism has the potential to lead to adverse drug-drug interactions, and assays to identify and characterize drug candidates as P450 inactivators are important in drug discovery and development. Here we present a quantitative high-throughput protocol for investigating cytochrome P450 mechanism-based inactivators; we use the example of CYP2B6 and bergamottin to illustrate the finer points of this protocol. This protocol details the adaptation of a 7-ethoxytrifluoromethyl coumarin O-deethylation fluorescence activity assay to a 96-well microtiter plate format and uses a plate reader to detect the fluorescence of the product. Compared with previous methods, this protocol requires less P450 and takes significantly less time while greatly increasing throughput. The protocol as written takes ∼2 h to complete. The principles and procedures outlined in this protocol can be easily adapted to other inactivators, P450 isoforms, substrates and plate readers.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids / chemistry
  • Aryl Hydrocarbon Hydroxylases / antagonists & inhibitors*
  • Aryl Hydrocarbon Hydroxylases / metabolism
  • Coumarins / chemistry
  • Cytochrome P-450 CYP2B6
  • Cytochrome P-450 Enzyme Inhibitors*
  • Cytochrome P-450 Enzyme System / metabolism
  • Enzyme Inhibitors / chemistry
  • Fluorescent Dyes
  • Fluorometry / methods
  • Furocoumarins / chemistry
  • High-Throughput Screening Assays / methods*
  • Humans
  • Oxidoreductases, N-Demethylating / antagonists & inhibitors*
  • Oxidoreductases, N-Demethylating / metabolism
  • Time Factors


  • Amino Acids
  • Coumarins
  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Furocoumarins
  • 7-ethoxy-4-trifluoromethylcoumarin
  • Cytochrome P-450 Enzyme System
  • Aryl Hydrocarbon Hydroxylases
  • CYP2B6 protein, human
  • Cytochrome P-450 CYP2B6
  • Oxidoreductases, N-Demethylating
  • bergamottin