Abstract
In the present study, a site-saturation mutagenesis library of drug metabolising CYP102A1 M11H with all 20 amino acids at position 87 was applied as biocatalyst for the production of stable and reactive metabolites of clozapine. Clozapine is an atypical antipsychotic drug where 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 non-enzymatic and glutathione S-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 amino acid at position 87 of CYP102A1 M11H strongly determines both 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 both 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 presence of recombinant human glutathione S-transferase P1-1. Five human-relevant GSH adducts were produced at high levels enabling structural characterization by 1H-NMR. This work shows that drug metabolizing CYP102A1-mutants, in combination with GST's, are very useful tools for the generation of GSH-conjugates of reactive metabolites of drugs in order to enable their isolation and structural elucidation.
- bioactivation
- cytochrome P450 catalyzed oxidations
- glutathione conjugates
- reactive metabolites
- site-directed mutagenesis
- Received June 7, 2011.
- Accepted September 2, 2011.
- The American Society for Pharmacology and Experimental Therapeutics