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

ABSTRACT:In the present study, a site-saturation mutagenesis library of drugmetabolizing 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.

Characterization of Human Cytochrome P450s Involved in the Bioactivation of Clozapine

“…CYP1A1, CYP1A2, CYP1B1, CYP2J2, and CYP3A5 showed less than 20% of the activity of CYP3A4 activity at these two substrate concentrations. The ratios of the three major GSH conjugates formed in the presence of hGST P1-1 did not significantly change between the different enzymes, suggesting that all GSH conjugates are formed from the same reactive intermediate, as proposed previously (Rea et al, 2011).…”

Glutathione trapping of reactive drug metabolites produced by biomimetic metalloporphyrin catalysts

“…loss of C 5 H 7 NO 3 = 129 u), confirmed the presence of the glutathione moiety, but gave no indication of the position of the conjugation. Based on earlier reports, glutathione was most likely attached to the same carbon from which the chlorine had been lost . RM2 was detected only in S9 incubations, but not in any of the BMO incubations, which suggests the lack of a reaction mechanism leading to this particular reactive intermediate from the latter.…”

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

“…The 40 other previously described mutants have been based upon these four templates [28][29][30][31][32][33][34]. The 44 mutants have been shown to metabolize a wide range of substrates, including marketed drugs [28,30,35,36], steroids [29,32,34], ionones [33], kinase inhibitors [47], alkoxyresorufins [29,31], and endocrine disrupting chemicals [31,48], with varying catalytic activities while also displaying significant differences in the metabolic profiles generated. The 22 mutants that have not been described before were all created inhouse by site-directed mutagenesis using the appropriate mutant templates (see the Supporting Information, Table S2).…”

“…Also within our own lab, isolated CYP BM3 mutants have previously been successfully applied for biosynthesis of drug metabolites on a preparative scale [32][33][34][35][36]39,47,48]. During our in vitro experiment, bioconversion was monitored in time (see Fig.…”

“…Over the years, a diverse library of CYP BM3 mutants has been constructed within our group by a combination of random and site-directed mutagenesis [27][28][29][30][31][32][33][34]. It has been shown that these mutants metabolize a wide range of substrates, including marketed drugs [28,30,35,36] and steroids [29,32,34], with varying catalytic activities and regio-and stereoselectivities.…”