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

Paper ID Volume ID Publish Year Pages File Format Full-Text
31770 44837 2011 12 PDF Available
Title
Facile production of minor metabolites for drug development using a CYP3A shuffled library ☆☆☆
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.

Keywords
COSY, correlation spectroscopy; hNPR, human NADPH cytochrome P450 reductase; HMBC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; P450, cytochrome P450. heme-thiolate protein P450; LC-MS, liquid chromatography mass
First Page Preview
Facile production of minor metabolites for drug development using a CYP3A shuffled library ☆☆☆
Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 13, Issue 6, November 2011, Pages 682–693
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering