title: Role of residue 87 in the activity and regioselectivity of clozapine metabolism by drug-metabolizing CYP102A1 M11H: application for structural characterization of clozapine GSH conjugates.
abstract: In the present study, a site-saturation mutagenesis library of drug-metabolizing CYP102A1 M11H with all 20 amino acids at position 87 was applied as a biocatalyst for the production of stable and reactive metabolites of clozapine. Clozapine is an atypical antipsychotic drug in which formation of reactive metabolites is considered to be responsible for several adverse drug reactions. Reactive intermediates of clozapine can be inactivated by GSH to multiple GSH conjugates by nonenzymatic and glutathione transferase (GST)-mediated conjugation reactions. The structures of several GST-dependent metabolites have not yet been elucidated unequivocally. The present study shows that the nature of the amino acid at position 87 of CYP102A1 M11H strongly determines the activity and regioselectivity of clozapine metabolism. Some mutants showed preference for N-demethylation and N-oxidation, whereas others showed high selectivity for bioactivation to reactive intermediates. The mutant containing Phe87 showed high activity and high selectivity for the bioactivation pathway and was used for the large-scale production of GST-dependent GSH conjugates by incubation in the presence of recombinant human GST P1-1. Five human-relevant GSH adducts were produced at high levels, enabling structural characterization by (1)H NMR. This work shows that drug-metabolizing CYP102A1 mutants, in combination with GSTs, are very useful tools for the generation of GSH conjugates of reactive metabolites of drugs to enable their isolation and structural elucidation.
authors: Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors: Vanina
authors: V
authors: Rea
authors: Sanja
authors: S
authors: Dragovic
authors: Jan Simon
authors: JS
authors: Boerma
authors: Frans J J
authors: FJ
authors: de Kanter
authors: Nico P E
authors: NP
authors: Vermeulen
authors: Jan N M
authors: JN
authors: Commandeur
mesh_terms: D014150:Antipsychotic Agents / Q000378:metabolism* / Q000493:pharmacokinetics; D001483:Base Sequence; D001711:Biotransformation; D002853:Chromatography, Liquid; D003024:Clozapine / Q000378:metabolism* / Q000493:pharmacokinetics; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D017931:DNA Primers; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry
chemical_list: D014150:Antipsychotic Agents; D017931:DNA Primers; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione; D003024:Clozapine