Fluorescence-based high-throughput screening assay for drug interactions with UGT1A6.
Assay Drug Dev Technol
; 9(5): 496-502, 2011 Oct.
Article
em En
| MEDLINE
| ID: mdl-21438674
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Preparações Farmacêuticas
/
Glucuronosiltransferase
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Inibidores Enzimáticos
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Descoberta de Drogas
/
Ensaios de Triagem em Larga Escala
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article