Assessment of Bile Salt Export Pump (BSEP) Inhibition in Membrane Vesicles Using Radioactive and LC/MS‐Based Detection Methods

Lisa D. Marroquin1, Paul D. Bonin2, Julie Keefer1, Thomas Schroeter1

1 Hit Discovery and Optimization, Pfizer Inc, Groton, Connecticut, 2 Primary Pharmacology Group, Pfizer Inc, Groton, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.14
DOI:  10.1002/cptx.15
Online Posting Date:  February, 2017
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Abstract

The bile salt export pump (BSEP, ABCB11) belongs to the ATP‐binding‐cassette superfamily of transporters and is predominately found in the liver. BSEP is an efflux transporter that plays a critical role in the secretion of bile salts into the bile. Inhibition of BSEP function by drugs can result in the buildup of bile salts in the liver and eventually leads to cholestasis and drug‐induced liver injury (DILI). DILI is a major cause of withdrawal of drugs from the pharmaceutical market and accounts for >50% of acute liver failures. Therefore, early detection of BSEP inhibition by drugs can help to mitigate the possibility of BSEP‐associated liver injury. This unit describes two assays that investigate the relationship between drug interference with BSEP function and liver injury using membrane vesicles prepared from Hi5 insect cells transfected with human BSEP. Comprehensive protocols for assessing BSEP inhibition in a 384‐well format using radiolabeled and liquid chromatography/mass spectrometry (LC/MS)–based detection methods are described. © 2017 by John Wiley & Sons, Inc.

Keywords: bile salt export pump; BSEP; drug induced liver injury; DILI; cholestasis; ATP‐binding cassette transporter

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of BSEP Inhibition in Membrane Vesicles Using LC/MS Detection
  • Support Protocol 1: Establishing LC/MS Instrument Sensitivity, Signal Linearity, Assay Quality, and Assay Dynamic Range
  • Basic Protocol 2: Assessment of BSEP Inhibition in Vesicle Membranes Using Radiolabeled Substrate
  • Support Protocol 2: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assessment of BSEP Inhibition in Membrane Vesicles Using LC/MS Detection

  Materials
  • Test compounds
  • Dimethyl sulfoxide (DMSO; EMD, cat. no. MX‐1457‐7)
  • Glyburide (Sigma‐Aldrich, cat. no. G2539)
  • BSEP assay buffer (see recipe)
  • BSEP wash buffer I (see recipe), room temperature and ice cold
  • 2.04× taurocholic acid/ATP and AMP assay mixes (see recipe)
  • 5 mg/ml BSEP‐Hi5‐VT vesicles (0.5 ml/vial; Solvo Biotechnology, cat. no. SB‐BSEP‐Hi5‐VT)
  • 0.5 M EDTA (BioSolutions, cat. no. BIO260‐15)
  • 80% and 50% (v/v) methanol (Sigma‐Aldrich, cat. no. 494437; prepare using HPLC‐grade or better water)
  • 50 μg/ml internal standard (see recipe)
  • Mobile phase A: 0.1% formic acid in water (Fisher Chemicals, cat. no. LS118‐4)
  • Mobile phase B: 0.1% formic acid in acetonitrile (Fisher Chemicals, cat. no. LS120‐4)
  • 384‐well polypropylene plate (e.g., Costar cat. no. 3657)
  • Echo Liquid Handler (Labycyte) or equivalent (optional)
  • Recommended liquid‐dispensing equipment:
    • Electronic multichannel pipetter, 16‐channel, 2‐125 µl
    • Multidrop Combi Reagent Dispenser (Thermo Scientific) or equivalent
    • Apricot 384‐channel pipetters (Apricot Designs) or equivalent
  • Adhesive plate seals
  • Orbital microplate shaker
  • 10‐ml low‐residual‐volume disposable multichannel pipette reservoirs (Integra, cat. no. 4332)
  • 25 ml reservoirs
  • Refrigerated centrifuge (e.g., Allegra 6KR or R6R)
  • Swinging‐bucket rotor with plate holders (e.g., GH 3.8)
  • Multiscreen 384‐well FC filter plates (1.2 μm glass fiber; Millipore, cat. no. MZFCN0W10)
  • 384‐well receiver plate (any 384‐well plate)
  • 384‐well collection plates (Thermo Scientific, cat. no. 4325)
  • Sciex Triple Quad 6500 triple quadrupole mass spectrometer with electrospray IonDrive Turbo V Ion Source or equivalent
  • Apricot/Sound Analytics ADDA autosampler or equivalent, with appropriate software (ADDA software version 1.0.0.8760)
  • Agilent 1290 Infinity HPLC pump or equivalent
  • MAC‐MOD ACE 5 C18, 20 × 2.1–mm HPLC column or equivalent
  • AB SCIEX Analyst software (version 1.6.2)

Support Protocol 1: Establishing LC/MS Instrument Sensitivity, Signal Linearity, Assay Quality, and Assay Dynamic Range

  Materials
  • Test compounds
  • Dimethyl sulfoxide (DMSO; EMD, cat. no. MX‐1457‐7)
  • Glyburide (Sigma‐Aldrich, cat. no. G2539)
  • BSEP assay buffer (see recipe)
  • BSEP wash buffer II (see recipe), room temperature and ice cold
  • 2.2× taurocholic acid/ATP assay mix (see recipe)
  • 5 mg/ml BSEP‐Hi5‐VT vesicles (0.5 ml/vial; Solvo Biotechnology, cat. no. SB‐BSEP‐Hi5‐VT)
  • 0.5 M EDTA (BioSolutions, cat. no. BIO260‐15)
  • Ready Safe Scintillation Cocktail (Beckman Coulter, cat. no. 141349)
  • 384‐well polypropylene plate (Costar, cat. no. 3657)
  • Biomek FXP Liquid Handling Automation Workstation (Beckman Coulter) or equivalent
  • 10‐ml low‐residual‐volume disposable multichannel pipetter reservoirs (Integra, cat. no. 4332)
  • Electronic multichannel pipetter, 16‐channel, 2‐125 µl
  • TopSeal‐A plate seals (PerkinElmer, cat. no. 6005250)
  • Orbital microplate shaker
  • MultiScreenHTS 384‐well FC filter plates (Millipore, cat. no. MZFCNOW50)
  • PlateMate 2 × 2 Automated Liquid Handler with 384‐well/100‐µl dispensing head
  • MultiScreenHTS Vacuum Manifold (Millipore, cat. no. MSVMHTS00)
  • 1450 Microbeta TriLux Liquid Scintillation Counter (Wallac)

Basic Protocol 2: Assessment of BSEP Inhibition in Vesicle Membranes Using Radiolabeled Substrate

  Materials
  • Software capable of calculating IC 50 (e.g., IBDS E‐Workbook or GraphPad Prism)
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Figures

Videos

Literature Cited

Literature Cited
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