title: Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs.
abstract: Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3'-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4'-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000-6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4' position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.
authors.affiliation: AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Harini
authors.initials: H
authors.lastname: Venkataraman
authors.affiliation: AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Marlies C A
authors.initials: MC
authors.lastname: Verkade-Vreeker
authors.affiliation: AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Luigi
authors.initials: L
authors.lastname: Capoferri
authors.affiliation: AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Daan P
authors.initials: DP
authors.lastname: Geerke
authors.affiliation: AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.affiliation: AIMMS Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. Electronic address: j.n.m.commandeur@vu.nl.
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000378:metabolism*; D001410:Bacillus megaterium / Q000201:enzymology / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D062367:ortho-Aminobenzoates / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D001426:Bacterial Proteins; D062367:ortho-Aminobenzoates; D003577:Cytochrome P-450 Enzyme System; C001209:fenamic acid; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases
keywords: Biocatalysis; Drug metabolites; Engineered enzymes; Fenamic acid NSAIDs; Regioselectivity; Total turnover numbers