Production of metabolites of the anti-cancer drug noscapine using a P450BM3 mutant library

Luke Richards; Adrian Lutz; David K Chalmers; Ailsa Jarrold; Tim Bowser; Geoffrey W Stevens; Sally L Gras

doi:10.1016/j.btre.2019.e00372

Production of metabolites of the anti-cancer drug noscapine using a P450_BM3 mutant library

Biotechnol Rep (Amst). 2019 Aug 24:24:e00372. doi: 10.1016/j.btre.2019.e00372. eCollection 2019 Dec.

Authors

Luke Richards^{1

2}, Adrian Lutz³, David K Chalmers⁴, Ailsa Jarrold⁵, Tim Bowser⁶, Geoffrey W Stevens¹, Sally L Gras^{1

2}

Affiliations

¹ Department of Chemical Engineering, The University of Melbourne, Parkville, VIC, 3010, Australia.
² The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Rd, Parkville, VIC, 3010, Australia.
³ Metabolomics Australia, School of Botany, The University of Melbourne, Parkville, VIC, 3010, Australia.
⁴ Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Pde, Parkville, VIC, 3010, Australia.
⁵ Sun Pharmaceutical Industries Ltd., Princes Highway, Port Fairy, VIC, 3281, Australia.
⁶ Impact Science Consulting, Unit 2/52 Swanston St, Heidelberg Heights, VIC, 3081, Australia.

Abstract

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 P450_BM3 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: Biocatalysis; Cytochrome P450; DMSO, dimethyl sulfoxide; Hydroxylation; IPTG, isopropyl β-D-1-thiogalactopyranoside; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MOPS, 3-(N-morpholino)propanesulfonic acid; N-demethylation; NADPH, nicotinamide adenine dinucleotide phosphate; P450, cytochrome P450; P450BM3, CYP102A1 from Bacillus megaterium; QTOF, quadrupole time-of-flight; Site-directed mutagenesis; WT, wild-type.