Biotechnological synthesis of drug metabolites using human cytochrome P450 2D6 heterologously expressed in fission yeast exemplified for the designer drug metabolite 4′-hydroxymethyl-α-pyrrolidinobutyrophenone☆
Introduction
Reference standards of drug metabolites are needed for their structural confirmation and pharmacologic and toxicologic characterization, including studies on their pharmacodynamic and pharmacokinetic properties, on enzyme kinetics of their formation, and on phase II metabolism. However, such metabolite standards are often not commercially available, particularly in the case of new therapeutic drugs or drugs of abuse. The classical chemical synthesis of drug metabolites can be cumbersome and stereochemically demanding and hence go beyond the possibilities of most biochemistry or pharmacology/toxicology oriented laboratories. Custom-made metabolite standards are a possible but usually time-consuming and very expensive solution.
Biotechnological synthesis of drug metabolites using cytochrome P450 (CYP) enzymes could be a versatile alternative to classical chemical synthesis and possibly have important advantages over the latter. Firstly, they would yield the metabolites of interest as products, at least if the CYP isozymes responsible for the in vivo formation of the respective metabolites were used. Secondly, one would expect only one (major) product when using specific isozymes, because they usually catalyze one metabolic step with high preference or even exclusively. Moreover, the metabolic reactions are generally highly stereoselective. Finally, they can be carried out under mild conditions in comparison to classical chemical reactions that often require high temperatures, high pressure or aggressive and/or toxic chemicals.
The aim of the presented study was evaluating the feasibility of biotechnological synthesis of drug metabolites using human CYP2D6 heterologously expressed in the fission yeast Schizosaccharomyces pombe as model enzyme and 1-(4-methylphenyl)-2-pyrrolidin-1-ylbutan-1-one (4′-methyl-α-pyrrolidinobutyrophenone, MPBP) as model substrate. CYP2D6 was chosen as model enzyme, because it is involved in the metabolism of many therapeutic and illicit drugs in humans [1], [2], [3]. In addition, CYP2D6 and metabolic steps catalyzed by this isozyme are of particular interest in pharmacology and toxicology because CYP2D6 is polymorphically expressed [3]. In CYP2D6 poor metabolizers, the metabolic clearance of drugs predominantly metabolized by CYP2D6 can be dramatically reduced increasing the risk of side effects and drug toxicity. Fission yeast, which was chosen for heterologous expression of human CYP2D6, was first isolated by Lindner from East African millet beer in 1893 [4]. The species name is derived from the Swahili word for beer “pombe”. It is an ancient species diverged from bakers yeast 400 million years ago. This organism was chosen for expression of human CYP2D6 for various reasons. It is a eukaryote with structures and processes that resemble the ones encountered in higher eukaryotes [5], [6], [7], [8], [9]. Its genome has been fully sequenced [10] and genetic techniques for working with this species are well established [11]. Moreover, fission yeast has only two putative endogenous CYPs by homology [10] decreasing the risk for unwanted side reactions. Finally, it has already been successfully used for expression of human CYPs [12], [13], [14], [15], [16]. The model drug MPBP is a new designer drug. Together with α–pyrrolidinopropiophenone (PPP) [17], [18], 4′-methyl-α-pyrrolidinopropiophenone (MPPP) [17], [18], [19], 4′-methyl-α-pyrrolidinohexanophenone (MPHP) [20], 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP) [21], 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP) [17], [22], and α–pyrrolidinovalerophenone (PVP) it belongs to the class of pyrrolidinophenone-type designer drugs, some of which are scheduled as controlled substances in Germany. Their chemical structures are given in Fig. 1. MPBP was seized as powder by the German police. It is assumed to be taken orally as the other pyrrolidinophenones, which are distributed among drug abusers as tablets, capsules, or powders [17]. Statements on the frequency of occurrence of the pyrrolidinophenones cannot be made, because they cannot be detected with usual routine analysis procedures [18], [19], [20], [21], [22], [23] and might, therefore, have been overlooked. So far, little information about the dosage of as well as the pharmacologic and toxicologic effects of the pyrrolidinophenones is available. However, they may be expected to be very similar to those of pyrovalerone (4′-methyl-α-pyrrolidinovalerophenone), a potent psychostimulant, because of their close structural relation to this drug as shown in Fig. 1. Concerning its mechanism of action, it has been reported that pyrovalerone releases dopamine and norepinephrine from respective nerve terminals [24], [25] and inhibits the reuptake of these neurotransmitters [26]. In comparison to amphetamine, it has been reported to have similar psychostimulant effects but less influence on motor function in animals and humans [27], [28], [29]. Pyrovalerone had been studied as a therapeutic drug [30], [31], [32], but was withdrawn from the market and scheduled as a controlled substance after a report of its intravenous abuse [33]. A similar pharmacological profile of the pyrrolidinophenones would clearly be in line with their abuse as stimulant designer drugs. The qualitative metabolism of MPBP has been studied in rats and 4′-methyl hydroxylation followed by oxidation to the respective carboxylic acid was found to be the main metabolic pathway [23]. This is in line with the metabolism of other 4′-methyl pyrrolidinophenones in animals [18], [20], [23], [34], [35], [36], [37] and humans [34]. An initial screening study in the authors’ laboratory using insect cell microsomes with cDNA expressed human CYPs showed that CYP2D6, CYP2C19, and CYP1A2 were involved in the 4′-methyl hydroxylation of MPBP. For further studies on the contribution of each of these isozymes the reference standard of the resulting metabolite 4′-hydroxymethyl-α-pyrrolidinobutyrophenone (HO-MPBP) was needed. This is why biotechnological semi-preparative synthesis of HO-MPBP from MPBP was chosen as the model reaction in the present paper.
Section snippets
Chemicals and reagents
MPBP-HNO3 was provided from Hessisches Landeskriminalamt (Wiesbaden, Germany) for research purposes. The purity and identity had been proven by mass spectrometry, infrared and 1H-NMR spectroscopy [38]. N-Methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) was obtained from Fluka (Steinheim, Germany). Hydrobromide of dextromethorphan (DXM) and tartrate of dextrorphan (DXOH) were from MP Biomedicals. All other chemicals and biochemicals used were obtained from Merck (Darmstadt, Germany) and were
Construction of S. pombe strain CAD58
Growth and phenotype of CAD58 cells were similar to those of the parental strain. Functional expression of human CYP2D6 in CAD58 was tested by incubation experiments with DXM. In subsequent GC-MS analysis, DXOH was detected in the extracts of supernatants from CAD58 incubations but not in those of the wild-type strain NCYC2036.
Biotechnological synthesis and isolation of HO-MPBP
CAD58 was used for biotechnological synthesis of the MPBP metabolite HO-MPBP. HPLC analysis of the incubation supernatants showed that approximately 250 μmol (75 mg) of
Discussion
The aim of the present study was evaluating the feasibility of biotechnological synthesis of drug metabolites using human CYP2D6 heterologously expressed in the fission yeast Schizosaccharomyces pombe as model enzyme and MPBP as model substrate. After transformation, fission yeast strain CAD58 constructed for this purpose had acquired the capability to catalyze the CYP2D6 specific DXM O-demethylation absent in the wild-type strain. This proved expression of functional CYP2D6 enzymes in CAD58. A
Acknowledgments
Part of the presented study was supported by NanoBioNet e.V. The authors thank Dr. Giselher Fritschi, Anette Wohnsland, Armin A. Weber, and Andrea Schwaninger for their support.
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Part of these results were reported at the 44th International Meeting of the International Association of Forensic Toxicologists (TIAFT), Ljubljana, Aug. 26 to Sept. 1, 2006.