salicylates and 1-naphthol

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.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Chromatography, High Pressure Liquid; Drug Discovery; Drug Evaluation, Preclinical; Drug Interactions; Enzyme Inhibitors; Fluorescence; Glucuronides; Glucuronosyltransferase; High-Throughput Screening Assays; Humans; Molecular Targeted Therapy; Naphthols; Pharmaceutical Preparations; Pharmacokinetics; Salicylates

The ability of Pseudomonas fluorescens 26K strain to utilize naphthalene at concentrations up to 600 mg/liter as the sole source of carbon and energy in mineral liquid media was shown. Using HPLC, TLC, and mass-spectrometry, the intermediates of naphthalene transformation by this strain were identified as naphthalene cis-1,2-dihydrodiol, salicylaldehyde, salicylate, catechol, 2-hydroxymuconic semialdehyde, and 1-naphthol. Catechol 2,3-dioxygenase (a homotetramer with native molecular mass 125 kDa) and NAD+-dependent homohexameric naphthalene cis-1,2-dihydrodiol dehydrogenase with native molecular mass 160 kDa were purified from crude extract of the strain and characterized. NAD+-dependent homodimeric salicylaldehyde dehydrogenase with molecular mass 110 kDa was purified and characterized for the first time. Based on the data, a pathway of naphthalene degradation by P. fluorescens 26K is suggested.

Topics: Aldehyde Oxidoreductases; Aldehydes; Catechol 2,3-Dioxygenase; Catechols; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Fatty Acids, Unsaturated; Mass Spectrometry; Naphthalenes; Naphthols; Oxidoreductases Acting on CH-CH Group Donors; Pseudomonas fluorescens; Salicylates