Abstract
In the present study, the validity of using a cocktail screening method in combination with a chemometrical data mining approach to evaluate metabolic activity and diversity of drug-metabolizing bacterial Cytochrome P450 (CYP) BM3 mutants was investigated. In addition, the concept of utilizing an in-house-developed library of CYP BM3 mutants as a unique biocatalytic synthetic tool to support medicinal chemistry was evaluated. Metabolic efficiency of the mutant library towards a selection of CYP model substrates, being amitriptyline (AMI), buspirone (BUS), coumarine (COU), dextromethorphan (DEX), diclofenac (DIC) and norethisterone (NET), was investigated. First, metabolic activity of a selection of CYP BM3 mutants was screened against AMI and BUS. Subsequently, for a single CYP BM3 mutant, the effect of co-administration of multiple drugs on the metabolic activity and diversity towards AMI and BUS was investigated. Finally, a cocktail of AMI, BUS, COU, DEX, DIC and NET was screened against the whole in-house CYP BM3 library. Different validated quantitative and qualitative (U)HPLC-MS/MS-based analytical methods were applied to screen for substrate depletion and targeted product formation, followed by a more in-depth screen for metabolic diversity. A chemometrical approach was used to mine all data to search for unique metabolic properties of the mutants and allow classification of the mutants. The latter would open the possibility of obtaining a more in-depth mechanistic understanding of the metabolites. The presented method is the first MS-based method to screen CYP BM3 mutant libraries for diversity in combination with a chemometrical approach to interpret results and visualize differences between the tested mutants.Graphical abstractGeneral worklfow in screening mutant enzyme libraries for catalytic efficiency and diversityElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-9241-x) contains supplementary material, which is available to authorized users.
Highlights
Cytochrome P450 monooxygenases (CYPs) play an essential role in metabolism of a wide range of xenobiotics and are involved in biosynthesis of prostaglandins and steroid hormones
The aim of the present study is to investigate if a cocktail approach can be used to screen CYP BM3 libraries for metabolic activity and diversity
It was decided to employ a CYP BM3 library consisting of mutants with as much structural diversity as possible by selecting mutants with mutations that preferably were introduced throughout the whole protein structure
Summary
Cytochrome P450 monooxygenases (CYPs) play an essential role in metabolism of a wide range of xenobiotics and are involved in biosynthesis of prostaglandins and steroid hormones. CYPs catalyze hydroxylation, epoxidation, reduction and other oxidative reactions on substrates that range from alkanes to complex endogenous molecules such as drugs, steroids and fatty acids [1]. The many reactions that CYPs can catalyze make these enzymes highly versatile and very interesting as tools to generate rather diverse and unique medicinal compound libraries not always accessible via classical synthetic routes. Metabolites produced by CYPs are well known to display important pharmacological activities or may be responsible for the toxicity or other unwanted side effects of xenobiotics [2, 3]. For drug discovery purposes metabolites are very interesting during lead optimization as they may display improved properties, such as target selectivity or even solubility without significant loss of the pharmacological properties compared to the parent drug itself [4]
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