title: Fluorescence-based high-throughput screening assay for drug interactions with UGT1A6.
abstract: The increasing awareness and the rising importance of UDP-glucuronosyltransferases (UGTs) in the pharmacokinetics of drugs have evoked a need to develop more powerful tools for studying the role of UGTs in the metabolism of drug candidates. To this end, we have developed a fluorescent high-throughput screening assay for screening potential inhibitors and/or substrates for recombinant human UGTs-here, for the UGT1A6. The assay is based on the increase in fluorescence intensity when 1-naphthol is glucuronidated. The formation of the highly fluorescent product, 1-naphthylglucuronide, is followed at excitation wavelengths of 295 and 300 nm with fixed emission (335 nm) in real time directly from the reaction mixture. A probe concentration of 5 μM with 2.5 μg of total protein in phosphate buffer at pH 7.4 with 5% dimethyl sulfoxide resulted in optimal linearity and acceptable signal separation (signal-to-base, 3.0) for the probe reaction. The interactions of test compounds with the enzyme are detected as lower rate of 1-naphthylglucuronide formation and thus lower rate of fluorescence increase. The success of the assay was first demonstrated with the known UGT1A6 substrates 4-hydroxyindole and scopoletin (Z' factor ≥0.5) and later with nonsteroidal anti-inflammatory drugs and salicylate derivatives. Diclofenac, 5-methylsalicylic acid, 5-bromosalicylic acid, 5-chlorosalicylic acid, and 5-fluorosalicylic acid decreased the probe glucuronidation rate at 500 μM by >50%. Further, the results gained with the high-throughput screening assay correlated well with the results obtained, in parallel, with the reference high-performance liquid chromatography method.
authors: Division of Pharmaceutical Technology, University of Helsinki,Viikinkaari 5E, Helsinki, Finland. anne.soikkeli@helsinki.fi
authors: Anne
authors: A
authors: Soikkeli
authors: Mika
authors: M
authors: Kurkela
authors: Jouni
authors: J
authors: Hirvonen
authors: Marjo
authors: M
authors: Yliperttula
authors: Moshe
authors: M
authors: Finel
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000032:analysis / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000032:analysis / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D005453:Fluorescence; D020719:Glucuronides / Q000737:chemistry / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D058990:Molecular Targeted Therapy; D009284:Naphthols / Q000737:chemistry / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010599:Pharmacokinetics; D012459:Salicylates / Q000032:analysis / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D004791:Enzyme Inhibitors; D020719:Glucuronides; D009284:Naphthols; D004364:Pharmaceutical Preparations; D012459:Salicylates; C029350:1-naphthol; C110655:UDP-glucuronosyltransferase, UGT1A6; D014453:Glucuronosyltransferase