Abstract
Drug metabolites generated by UDP glycosyltransferases (UGTs) are needed for drug development and toxicity studies, especially in the context of safety testing of metabolites during drug development. Because chemical metabolite synthesis can be arduous, various biological approaches have been developed; however, no whole-cell biotransformation with recombinant microbes that express human UGTs was yet achieved. In this study we expressed human UDP glucose-6-dehydrogenase together with several human or rat UGT isoforms in the fission yeast Schizosaccharomyces pombe and generated strains that catalyze the whole-cell glucuronidation of standard substrates. Moreover, we established two methods to obtain stable isotope-labeled glucuronide metabolites: the first uses a labeled aglycon, whereas the second uses 13C6-glucose as a metabolic precursor of isotope-labeled UDP-glucuronic acid and yields a 6-fold labeled glucuronide. The system described here should lead to a significant facilitation in the production of both labeled and unlabeled drug glucuronides for industry and academia.
Footnotes
This work is part of the following patent application: Dragan C-A, Bureik M, and Buchheit D (2008) Drug metabolism. European patent application EP 08 164 826.3.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.030965.
-
ABBREVIATIONS:
- P450
- cytochrome P450
- UGT
- UDP glycosyltransferase
- UDP-GA
- UDP glucuronic acid
- 4MU
- 4-methylumbelliferone
- T
- testosterone
- TG
- testosterone glucuronide
- 4MUG
- 4-methylumbelliferone-β-D-glucuronide
- HPLC
- high-performance liquid chromatography
- UGDH
- UDP glucose-6-dehydrogenase
- EMM
- Edinburgh minimal medium
- LC/MS
- liquid chromatography/mass spectrometry
- FTMS
- Fourier transform mass spectrometry
- EIC
- extracted ion currents.
- Received October 29, 2009.
- Accepted December 11, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|