Biochemical Pharmacology xxx (2017) xxx–xxx

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Biochemical Pharmacology

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Efficient substrate screening and inhibitor testing of human CYP4Z1 using permeabilized recombinant fission yeast ⇑ Qi Yan a, David Machalz b, Andy Zöllner c, Erik J. Sorensen a,d, Gerhard Wolber b, Matthias Bureik a, a School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin 30072, China b Pharmaceutical and Medicinal Chemistry (Computer-Aided Drug Design), Institute of Pharmacy, Free University Berlin, Germany c Lab Logistics Group GmbH, D-53340 Meckenheim, Germany d Department of Chemistry, Princeton University, Princeton, NJ 08544, USA article info abstract

Article history: We have established a protocol for the preparation of permeabilized fission yeast cells (enzyme bags) that Received 18 July 2017 recombinantly express human cytochrome P450 enzymes (CYPs). A direct comparison of CYP3A4 activity Accepted 21 September 2017 gave an eightfold higher space-time yield for enzyme bag-catalyzed biotransformation as compared to Available online xxxx whole-cell biotransformation, even though the total number of cells employed was lower by a factor of 150. Biotransformation of the luminogenic substrate Luciferin-H using CYP2C9-containing enzyme Chemical compounds studied in this article: bags proceeded efficiently and stably for 24 h. CYP4Z1 is of interest because it is strongly overexpressed Aminobenzotriazole (PubChem CID: 1367) both in breast cancer cells and in breast cancer metastases; however, current knowledge about its Arachidonic acid (PubChem CID: 444899) catalytic properties is very limited. Screening of CYP4Z1-containing enzyme bags with 15 luminogenic 1-Benzylimidazole (PubChem CID: 77918) Econazole (PubChem CID: 3198) substrates enabled us to identify two new hydroxylations and eleven ether cleavage reactions that are Eicosadienoic acid (PubChem CID: 6439848) catalyzed by CYP4Z1. By far the best substrate found in this study was Luciferin benzyl ether Lauric acid (PubChem CID: 3893) (Luciferin-BE). On the basis of the recently published crystal structure of CYP4B1 we created a new Miconazole (PubChem CID: 4189) homology model of CYP4Z1 and performed molecular docking experiments, which indicate that all active Myristic acid (PubChem CID: 11005) substrates show a highly similar binding geometry compared to the endogenous substrates. The model Tolazoline (PubChem CID: 5504) predicts that Ser113, Ser222, Asn381, and Ser383 are key hydrogen bonding residues. We also identified five new inhibitors of CYP4Z1: miconazole, econazole, aminobenzotriazole, tolazoline, and Keywords: Human cytochrome P450 1-benzylimidazole respectively, with the last compound being the most potent giving an IC50 value of Biotransformation 180 nM in our test system. Fission yeast Ó 2017 Elsevier Inc. All rights reserved. Hydroxylation Ether cleavage

1. Introduction include a broad range of active pharmaceutical ingredients (APIs), environmental toxins, carcinogens, and food-derived chemicals [1]. Cytochrome P450 enzymes (CYPs) are encoded by a large gene On the other hand, many human CYPs are involved in the biosyn- family present in all biological kingdoms. In humans, there are 57 thesis, metabolism and degradation of a plethora of endogenous structural CYP genes encoding enzymes that oxidize both endoge- compounds, such as steroids, seco-steroids, fatty acids and many nous compounds and xenobiotics. The importance of CYPs is illus- more [2]. Since CYPs often have a huge influence on drug clearance, trated by the fact that they are responsible for the majority of their activity is a critical focus of drug development and the deter- Phase I reactions in human drug metabolism, and their substrates mination of the CYP reactivity profile for a small molecule is important for predicting its metabolic fate in vivo. However, the diversity and overlap of CYP reaction profiles and also of their Abbreviations: ADMET, absorption, distribution, metabolism, excretion, and expression patterns pose a significant challenge for dissecting the toxicity; API, active pharmaceutical ingredient; CPR, NADPH-cytochrome P450 roles of individual CYPs in the metabolization of drugs and other oxidoreductase; CYP, cytochrome P450; EMM, Edinburgh Minimal Medium; HLMs, molecules. Moreover, some prodrugs are activated by CYPs in their human liver microsomes; RLU, relative luminescence unit; UGDH, UDP-glucose 6- target tissues. Finally, several CYPs are drug targets, and some CYP dehydrogenase. ⇑ Corresponding author. inhibitors are already on the market, while others are being tested E-mail address: [email protected] (M. Bureik). in clinical trials [3,4]. Thus, CYP-dependent activity assays are in https://doi.org/10.1016/j.bcp.2017.09.011 0006-2952/Ó 2017 Elsevier Inc. All rights reserved.

Please cite this article in press as: Q. Yan et al., Efficient substrate screening and inhibitor testing of human CYP4Z1 using permeabilized recombinant fis- sion yeast, Biochem. Pharmacol. (2017), https://doi.org/10.1016/j.bcp.2017.09.011 2 Q. Yan et al. / Biochemical Pharmacology xxx (2017) xxx–xxx constant demand for many reasons and the availability of conve- inducers, and inhibitors being available for some enzymes (like nient and robust CYP test systems is a conditio sine qua non for CYP3A4 or CYP2D6) while for others (such as CYP3A43 or modern pharmaceutical research. CYP20A1) hardly anything is known. Recombinant CYP enzymes Different types of CYP assays exist and have been shown to be facilitate CYP reaction profiling by unambiguously assigning a useful to varying degrees for different applications. In comparison reaction to the single CYP present in the reaction mixture. How- to other variables that characterize such assays, the source of the ever, all recombinant expression systems have their own issues: enzyme(s) and the choice of the test substrate are of special impor- Since CYPs are membrane-bound enzymes, bacterial expression tance. As a matter of course, these aspects in turn depend on the systems typically produce truncated mutants that render the properties of the enzymes. Human CYPs are membrane bound enzymes soluble [14,15]; but they have the advantage that enzymes that are typically located either on the cytoplasmic side unwanted P450 side-reactions are absent due to the lack of native of the endoplasmic reticulum or on the matrix side of the inner P450s in the expression host E. coli. Recombinant yeasts are typi- mitochondrial membrane; in general, they are the terminal oxi- cally used for whole-cell biotransformations due to their genetic dases of short electron transfer chains that consist of the CYP accessibility, cost-effectiveness and rapid growth, with the added enzyme proper together with one or two electron transfer proteins benefit that the expensive cofactor NADPH is produced within the that are colocalized with it; most human CYPs depend on NADPH- cells, which is especially advantageous for preparative production cytochrome P450 oxidoreductase (CPR) [5]. Most commonly, the [16,17]; they do have some endogenous CYPs – two in fission CYPs oxidize hydrophobic compounds, rendering them more water yeast Schizosaccharomyces pombe and three in baker’s yeast soluble and amenable to direct elimination or to additional modi- Saccharomyces cerevisiae – but these have very low homology to fications by conjugating enzymes such as UDP glucuronidases [6]. human drug metabolizing CYPs and typically don’t give much The substrate selectivity profile of individual human CYP enzymes background [18]. However, in the yeast system substrates and varies considerably, from rather narrow (consider CYP11B potential inhibitors need to cross multiple biological barriers to enzymes involved in steroid hormone synthesis) [7] to very broad reach the target enzymes and if product detection and/or purifica- (CYP3A4 in drug metabolism) [8]. CYP activity and inhibition tion are done from the supernatant, the product molecules also assays are designed around probe substrates that CYPs convert to need to pass the membranes. This drawback is especially pro- readily detected products. A huge variety of such substrates is nounced for charged molecules as they are much less likely to available today and methods of product detection include mass permeate membranes easily; in some cases the problem can be spectrometry, absorbance, fluorescence, radiometry, and biolumi- overcome by pH variation [19]. Microsomal CYP preparations nescence. As a matter of course, the choice of the substrate are also available from recombinant insect and mammalian cells, employed in a certain investigation depends on many factors, respectively, where CYPs are expressed in a much more native including aim of the study, analytical equipment, convenience, environment than in microbes and no biological barriers need to robustness, and others. For example, choosing a physiological sub- be overcome [20]. However, in these cell types background reac- strate is important when comparing the activities of different poly- tions by other CYPs present in the cells get again more important; morphic variants (or other mutants) of an individual CYP as and economically, they can hardly compete with microbial sys- structurally unrelated probe substrates may yield different results tems. In short, due to the nature of CYPs there is no production [9]. However, in the many cases where the effects of transcrip- system which is free of disadvantages. Rather, for any given study tional inducers or small molecule inhibitors on the activity of a an enzyme source needs to be chosen whose drawbacks matter as wild-type CYP are studied, there is no fundamental reason not to little as possible to the question at hand. To address this lacuna, it use unphysiological probe substrates. In this investigation biolumi- was the first aim of this investigation to expand the list of nescent CYP assays [10] were employed for the main part of the available CYP preparations by combining the advantages of study, as the reaction products from all the different substrates recombinant expression in yeast with a microsomal-like biotrans- have the unique property of producing photons with luciferase, formation procedure. When cells are treated with suitable surfac- which is very convenient when testing multiple substrates in tants (e.g. detergents or solvents) at the right concentration, the parallel. cell envelope becomes permeable to low molecular weight com- Another important consideration for designing CYP activity pounds while large molecules (including enzymes) stay within assays regards the source of the enzymes. A fundamental divide the cells. After washing away small molecules, these enzyme bags exists between recombinant and non-recombinant systems. Clas- can be used for enzymatic catalysis under defined conditions, and sic biosynthesis is done in vitro using (more or less) purified through a careful choice of substrate the number of reactions that enzyme preparations or in vivo with whole living cells. Typical proceed effectively are limited [21]. In a previous study aimed at non-recombinant sources of human hepatic CYP systems are liver transferring this concept to CYP-dependent biotransformations in cells or human liver microsomes (HLMs). HLMs contain a wide fission yeast we could only obtain rather moderate increases in variety of drug metabolizing enzymes and are commonly used activity [22]. However, recently we succeeded in significantly for in vitro absorption, distribution, metabolism, excretion, and optimizing the reaction conditions, as is illustrated by the efficient toxicity (ADMET) studies such as the identification and character- biotechnological production of UDP glucuronic acid from UDP glu- ization of CYP inhibitors, while cell-based assays are done to mon- cose (5 mM substrate concentration, 3 h reaction time, 100% yield itor CYP induction [11]. However, a fundamental challenge in the and selectivity) using permeabilized recombinant fission yeast use of native samples is the presence of multiple potentially [23]. In the present study we sought to apply the newly found cross-reactive CYPs. To overcome this problem, CYP-selective inhi- reaction conditions for the permeabilization of fission yeast to bitors and substrates are used to pinpoint CYP-selective effects. CYP-dependent biotransformations and to elucidate their For instance, a particular drug transformation is assigned to a usefulness for the identification of new CYP substrates and for given CYP when a selective inhibitor of that CYP blocks the trans- the evaluation of CYP inhibitors. For this purpose we employed formation. Conversely, inhibitors and inducers of specific CYPs are fission yeast strains that recombinantly express human CYP2C9, identified with selective probe substrates: activity with the probe CYP3A4, or CYP4Z1, respectively. We then identified a number is respectively increased or decreased by an inducer or an inhibi- of new CYP4Z1 substrates and inhibitors and, moreover, devel- tor [12,13]. However, our knowledge about the different human oped a new homology model of this enzyme that explains our CYPs is highly variable, with huge datasets on substrates, experimental findings.

Please cite this article in press as: Q. Yan et al., Efficient substrate screening and inhibitor testing of human CYP4Z1 using permeabilized recombinant fis- sion yeast, Biochem. Pharmacol. (2017), https://doi.org/10.1016/j.bcp.2017.09.011 Download English Version: https://daneshyari.com/en/article/8524506

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