Facile production of minor metabolites for drug development using a CYP3A shuffled library

Hunter, D.J.B., Behrendorff, J.B.Y.H., Johnston, W.A., Hayes, P.Y., Huang, W., Bonn, B., Hayes, M.A., De Voss, J.J. and Gillam, E.M.J (2011) Facile production of minor metabolites for drug development using a CYP3A shuffled library. Metabolic Engineering, 13 6: 682-693. doi:10.1016/j.ymben.2011.09.001

Author Hunter, D.J.B.
Behrendorff, J.B.Y.H.
Johnston, W.A.
Hayes, P.Y.
Huang, W.
Bonn, B.
Hayes, M.A.
De Voss, J.J.
Gillam, E.M.J
Title Facile production of minor metabolites for drug development using a CYP3A shuffled library
Journal name Metabolic Engineering   Check publisher's open access policy
ISSN 1096-7176
Publication date 2011-11
Sub-type Article (original research)
DOI 10.1016/j.ymben.2011.09.001
Volume 13
Issue 6
Start page 682
End page 693
Total pages 12
Place of publication United States
Publisher Academic Press
Collection year 2012
Language eng
Abstract Metabolic profiling of new drugs is limited by the difficulty in obtaining sufficient quantities of minor metabolites for definitive structural identification. Biocatalytic methods offer the potential to produce metabolites that are difficult to synthesize by traditional medicinal chemistry. We hypothesized that the regioselectivity of the drug metabolizing cytochrome P450s could be altered by directed evolution to produce minor metabolites of drugs in development. A biocatalyst library was constructed by DNA shuffling of four CYP3A forms. The library contained 11±4 (mean±SD) recombinations and 1±1 spontaneous mutations per mutant. On expression in Escherichia coli, 96% of mutants showed detectable activity to at least one probe substrate. Using testosterone as a model drug-like substrate, mutants were found that preferentially formed metabolites produced in only trace amounts by parental forms. A single 1.6. L batch culture of one such mutant enabled the facile isolation of 0.3. mg of the minor metabolite 1Β-hydroxytestosterone and its ab initio structural determination by 1D- and 2D-NMR spectroscopy.
Keyword Cytochrome P450
Drug development
Drug metabolites
Escherichia coli
Laboratory-scale bioreactors
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2012 Collection
School of Chemistry and Molecular Biosciences
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Created: Tue, 18 Oct 2011, 10:50:15 EST by Dr James De Voss on behalf of School of Chemistry & Molecular Biosciences