A High‐Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC‐MS/MS

Xiaofeng Li1, Tom Suhar1, Lateca Glass1, Ganesh Rajaraman1

1 AbbVie, Drug Metabolism, North Chicago, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 9.18
DOI:  10.1002/0471141755.ph0918s64
Online Posting Date:  March, 2014
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Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug‐drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high‐throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro–in vivo extrapolation. Described in this unit is a high‐throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Curr. Protoc. Pharmacol. 64:9.18.1‐9.18.10. © 2014 by John Wiley & Sons, Inc.

Keywords: enzyme reaction phenotyping; recombinant human CYP; cytochrome P450

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • 10 mM carbutamide (Sigma‐Aldrich) prepared in DMSO (10.0 mg carbutamide in 3.69 ml DMSO; store at room temperature)
  • Acetonitrile (J.T. Baker or Sigma‐Aldrich)
  • 0.5 M potassium phosphate buffer, pH 7.4 (purchase from BD Gentest or see recipe in appendix 2A)
  • Test compounds, 5 mM in DMSO
  • 10 mM NADPH in 50 mM potassium phosphate buffer (see recipe)
  • Human rCYPs expressed from human cDNA using a baculovirus expression system:
    • Human CYP1A2 (BD Gentest, cat. no. 456203)
    • Human CYP2C8 (BD Gentest, cat. no. 456252)
    • Human CYP2C9*1 (BD Gentest, cat. no. 456258)
    • Human CYP2C19 (BD Gentest, cat. no. 456259)
    • Human CYP2D6*1 (BD Gentest, cat. no. 456217)
    • Human CYP3A4 (BD Gentest, cat. no. 456202)
  • 384‐well, 120‐µl well volume polypropylene plates (Greiner Bio‐One)
  • Echo liquid handler (Labcyte)
  • 384‐deep‐well, 200‐µl well bottom polypropylene plates (Greiner Bio‐One)
  • Multidrop Combi and Multidrop Dispensing Cassette (Thermo Scientific)
  • Biomek FXP Laboratory Automation Workstation (Beckman Coulter)
  • PlateLoc Thermal Plate Sealer (Agilent Technologies)
  • Centrifuge (Eppendorf AG) with plate bucket
  • ADDA High‐Speed Dual Arm Autosampling System (Apricot Designs)
  • LC/MS instrument: API‐5500 Triple Quad (AB Sciex)
  • LC/MS pump: 20AD single‐channel pump (Shimadzu)
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Literature Cited

Literature Cited
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