EA432

ExpressPlus UGT Inhibition Using Recombinant Enzymes and LC-MS/MS

T.his assay is used to assess the potential of a test compound to inhibit individual human UDP-glucuronosyltransferase (UGT) isoforms

Required from Customer

  • Either a minimum of 600 µL of test compound at 10 mM in DMSO, or 10 mg of powder
  • Exact molecular mass of test compound and its salt form
  • MSDS or handling and storage information (e.g., light sensitive, store at -20°C, etc.)
  • Stability of test compound in the presence of individual human UGTs
  • Solubility of test compound in assay buffer

Deliverables

  • Table of % inhibition of each UGT isoform by a single concentration of test compound
  • Table of % inhibition of each UGT isoform by a positive control inhibitor

Substrate

  • Test compound at a single concentration (typically between 10 and 100 µM, depending on solubility and customer input) dissolved in aqueous buffer, DMSO, acetonitrile, or methanol (final concentration of organic solvent ≤1%)

Assay System

  • Individual human recombinant UGT isoforms, with 1 mM UDP-glucuronic acid (UDPGA) and alamethicin added
  • Individual substrates in separate incubations
  • LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to the glucuronide conjugate of each probe substrate divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

  • The standard protein concentration is 0.25 mg/mL
  • Run the assay in duplicate (N=2 separate incubations)
  • Include a maximum activity control, a single concentration of test compound, and a single concentration of a positive control inhibitor
  • Initiate reaction by adding 1 mM UDPGA
  • Sample reaction mixture at a singe time point (typically between 30 and 60 minutes)

Assay QC

  • Significant inhibition by each positive control run in parallel

Notes

  1. The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
  2. Percent inhibition is calculated from the amount of metabolite formed in the presence of test compound relative to that formed in the absence of test compound, based on the peak area response ratio (PARR): % Inhibition = [(PARR without test compound) – (PARR with test compound) / (PARR without test compound)] x 100%.
  3. Pricing is based on screening for inhibition of at least five UGT isoforms per test compound.
UGT Isoforms, Probe Substrates and Metabolites
UGT Probe Substrate Conc. (µM) Metabolite
UGT1A1, UGT1A3, UGT1A6, UGT1A9, UGT2B7 7-HFC 100 7-HFCG
UGT1A4 TFP 100 TFPG

 

Positive Control Inhibitors for Standard (hepatic) UGT Isoforms
UGT Positive Control Inhibitor
UGT1A1 Bilirubin
UGT1A3 Buprenophine
UGT1A4 Hecogenin
UGT1A6 1-Naphthol
UGT1A9 Niflumic acid
UGT2B7 Diclofenac

7-HFC = 7-hydroxy-4-trifluoromethylcoumarin 7-HFCG = 7-HFC glucuronide TFP = trifluoperazine TFPG = TFP glucuronide

Options

  1. The customer must specify: • the concentration of test compound • which UGT isoforms to assay

 

****JAKES NOTE****

This Chart and bottom information had no structure in the current sites “Assay Data Sheet” option.

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