Facile production of minor metabolites for drug development using a CYP3A shuffled library
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 hypo...
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| Veröffentlicht in: | Metabolic engineering 2011-11, Vol.13 (6), p.682-693 |
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| creator | 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. |
| description | 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. |
| doi_str_mv | 10.1016/j.ymben.2011.09.001 |
| format | Article |
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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
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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.</description><subject>Cytochrome P-450 CYP3A - genetics</subject><subject>Cytochrome P-450 CYP3A - metabolism</subject><subject>Cytochrome P450</subject><subject>DNA Shuffling</subject><subject>Drug development</subject><subject>Drug Discovery - methods</subject><subject>Drug metabolites</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Gene Library</subject><subject>Hydroxytestosterones - metabolism</subject><subject>Laboratory-scale bioreactors</subject><subject>Substrate Specificity</subject><subject>Testosterone - metabolism</subject><issn>1096-7176</issn><issn>1096-7184</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkM9rFDEYhoMotlb_AkFz87RjvpnMJDl4KItVoaCgPegl5MeXNcvMZE1mCv3vzbq1R_GUBJ73Je9DyEtgDTAY3u6bu8ni3LQMoGGqYQwekXNgatgIkPzxw10MZ-RZKfsKQK_gKTlrQQ0dl_05-XFlXByRHnLyq1timmkKdIpzynTCxdg0xgULDfXt87qjHm9xTIcJ54WuJc47auj2-5fukpafawgjejpGm02-e06eBDMWfHF_XpCbq_ffth83158_fNpeXm8cB75sAAN2succrECJYFWQPWKwxirJlTFCDQ45-uBYy62wXilh_MD7YCSwrrsgb069dcOvFcuip1gcjqOZMa1FKyZA8br2P8hWQiu6I9mdSJdTKRmDPuQ41VEamD7a13v9x74-2tdM6Sq3pl7d9692Qv-Q-au7Aq9PQDBJm12ORd98rQ1DTYuBybYS704EVmO3EbMuLuLs0MeMbtE-xX9-4TcuX6Dl</recordid><startdate>20111101</startdate><enddate>20111101</enddate><creator>Hunter, D.J.B.</creator><creator>Behrendorff, J.B.Y.H.</creator><creator>Johnston, W.A.</creator><creator>Hayes, P.Y.</creator><creator>Huang, W.</creator><creator>Bonn, B.</creator><creator>Hayes, M.A.</creator><creator>De Voss, J.J.</creator><creator>Gillam, E.M.J.</creator><general>Elsevier Inc</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20111101</creationdate><title>Facile production of minor metabolites for drug development using a CYP3A shuffled library</title><author>Hunter, D.J.B. ; 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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
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Cytochrome P-450 CYP3A - genetics Cytochrome P-450 CYP3A - metabolism Cytochrome P450 DNA Shuffling Drug development Drug Discovery - methods Drug metabolites Escherichia coli Escherichia coli - enzymology Escherichia coli - genetics Escherichia coli Proteins - genetics Gene Library Hydroxytestosterones - metabolism Laboratory-scale bioreactors Substrate Specificity Testosterone - metabolism |
| title | Facile production of minor metabolites for drug development using a CYP3A shuffled library |
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