Production of metabolites of the anti-cancer drug noscapine using a P450BM3 mutant library
2019
Luke Richards | Adrian Lutz | David K. Chalmers | Ailsa Jarrold | Tim Bowser | Geoffrey W. Stevens | Sally L. Gras
Cytochrome P450 enzymes are a promising tool for the late-stage diversification of lead drug candidates and can provide an alternative route to structural modifications that are difficult to achieve with synthetic chemistry. In this study, a library of P450BM3 mutants was produced using site-directed mutagenesis and the enzymes screened for metabolism of the opium poppy alkaloid noscapine, a drug with anticancer activity. Of the 18 enzyme mutants screened, 12 showed an ability to metabolise noscapine that was not present in the wild-type enzyme. Five noscapine metabolites were detected by LC-MS/MS, with the major metabolite for all mutants being N-demethylated noscapine. The highest observed regioselectivity for N-demethylation was 88%. Two hydroxylated metabolites, a catechol and two C-C cleavage products were also detected. P450-mediated production of hydroxylated and N-demethylated noscapine structures may be useful for the development of noscapine analogues with improved biological activity. The variation in substrate turnover, coupling efficiency and product distribution between the active mutants was considered alongside in silico docking experiments to gain insight into structural and functional effects of the introduced mutations. Selected mutants were identified as targets for further mutagenesis to improve activity and when coupled with an optimised process may provide a route for the preparative-scale production of noscapine metabolites. Keywords: Cytochrome P450, N-demethylation, Hydroxylation, Site-directed mutagenesis, Biocatalysis
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