Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug Metabolizing Bm3 M11: Application for Structural Characterization of Clozapine Gsh Conjugates

In the present study, a site-saturation mutagenesis library of drug metabolizing BM3 M11 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 BM3 M11 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 P11. Five human relevant GSH adducts were produced at high levels enabling structural characterization by 1 H-NMR. This work shows that drug metabolizing BM3-mutants, in combination with GSTs, are very useful tools for the generation of GSH conjugates of reactive metabolites of drugs in order to enable their isolation and structural elucidation. Chapter 3 Generation of reactive intermediates