Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites.
Recently, several mutants of cytochrome P450 BM3 (CYP102A1) with high activity toward drugs have been obtained by a combination of site-directed and random mutagenesis. In the present study, the applicability of these mutants as biocatalysts in the production of reactive metabolites from the drugs clozapine, diclofenac and acetaminophen was investigated. We showed that the four CYP102A1 mutants used in this study formed the same metabolites as human and rat liver microsomes, with an activity up to 70-fold higher compared to human enzymes. Using these CYP102A1 mutants, three novels GSH adducts of diclofenac were discovered which were also formed in incubations with human liver microsomes. This work shows that CYP102A1 mutants are very useful tools for the generation of high levels of reference metabolites and reactive intermediates of drugs. Producing high levels of those reactive metabolites, that might play a role in adverse drug reactions (ADRs) in humans, will facilitate their isolation, structural elucidation, and could be very useful for the toxicological characterization of novel drugs and/or drug candidates.
Damsten
Micaela C
MC
LACDR, Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
van Vugt-Lussenburg
Barbara M A
BM
Zeldenthuis
Tineke
T
de Vlieger
Jon S B
JS
Commandeur
Jan N M
JN
Vermeulen
Nico P E
NP
D000082:Acetaminophen / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D002384:Catalysis; D003024:Clozapine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004008:Diclofenac / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D001426:Bacterial Proteins; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D004008:Diclofenac; D000082:Acetaminophen; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D005978:Glutathione; D003024:Clozapine