Inneke Wynant was born on August 9, 1975, in Aalst, Belgium. She attended secondary school at the Dames Van Maria, Aalst, where she majored in Latin-Sciences as “First of her class”. In September 1993, she started her Biology studies at Universiteit Gent (UG). While she specialized in Animal Biology, she did a teaching practise on “Analyse van honing: gaschromatografie van suikers en bepalen van diastase-activiteit”, in the Lab. of Physiology, Entomology and Apiculture (Prof. Dr.
Jacobs). In 1997, she graduated with great honour and her licentiate’s thesis, entitled “Erwinia salicis, verwekker van de watermerkziekte bij wilg: karakterizering en ontwikkeling van een PCR- detectiemethode”, carried out at the Department of Biochemistry, Physiology and Microbiology, Lab.
of Microbiology, under supervision of Prof. Dr. Ir. J. Swings and under promotorship of Dr. L. Hauben, resulted in an interesting poster presentation as well as part of a research article.
With a passion for Science in general and for Research in particular, she started her professional career in 1997 as Scientific assistant at the University of Antwerp (UA), where she worked on the “Biochemical and physiological analysis of oxidative stress in tobacco BY-2 cell suspension cultures”, at the Department of Biology, Research Group of Plant Biochemistry and Biotechnology, under supervision of Prof. Dr. R. Caubergs. For two years, she was also involved in many teaching assignments (practical courses of Plant physiology and Microbiology to Biology and Bioengineering students and training of (doctoral) students) and organization of the International Scientific Congress on “Plasma Membrane Redox Systems and Their Role in Biological Stress and Disease” (5-8 April 1998). In order to learn new molecular techniques and RNA analyses, she got the opportunity to stay (31 May-14 June, 1998) at the University of Batch, School of Biology and Biochemistry (Prof. Dr. J. Beeching), England and to participate in multiple congresses, resulting in (co-)authorship on multiple posters and a research article.
However, her interest in human physiology and drug research kept on pursuing here. Around the millenium, she returned to her initial passion and started her doctoral research project on a renowned IWT project, entitled “Heterologous Expression Systems for the Production of Metabolites in Early Phase of Drug Development”. This project was a close collaboration between Janssen Pharmaceutica (under supervision of Dr. H. Bohets) and Institut Meurice (under supervison of Dr. J.- P. Simon), and under promotorship of Prof. Dr. L. Droogmans. During the first two and a half years at Institut Meurice, she obtained her Diplôme d’études approfondies en Sciences (2001) and a novel bioreactor technology was successfully optimized. The results obtained during her doctoral thesis were presented orally or by poster presentation at several national and international scientific congresses and resulted in publications in international peer-reviewed journals and several extended abstracts in (peer-reviewed) Conference Proceedings. As an extension of her own research, she is currently writing a review paper on drug metabolite synthesis in a prominent pharmaceutical journal.
In 2002, she applied for a permanent job as Study director/Scientist, which enabled her to personally validate the bioreactor technology at Janssen Pharmaceutica for many drug candidates. In 2006, she was promoted to Senior scientist, resulting in more active participation in drug development project teams. The last eight years, she was an important contact for mainly metabolism and drug-drug interaction studies in drug development, for which she attended several drug metabolism courses/conferences and advanced workshops on biotransformation for metabolite generation, resulting in several poster presentations and a Proceedings research article on DMPK- related topics. She is a member of the Cost (2001), the International Microencapsulation Society (2005), the Center of Expertise for Drug-Drug Interaction (2007) and a mentoring team (2008). She was nominated (Business Excellence award, 2006) as well as receiver (Vision Award, 2006; RED-EU award, 2006) of several awards. In view of GLP, she partcipated in audits of contract research organizations. In the margin, she also regularly invested in exploitation of her managerial and educational skills (organization of departmental scientific manifestations, “change agent”, promoter of two theses and teaching of university students (UA), consultant on immobilization for Karel de Grote Hogeschool).
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Faculté des Sciences
Institut de Recherches Microbiologiques
Heterologous expression systems for the production of metabolites
during early drug research
Project verricht met steun van het Vlaams Gewest – IWT
Thèse présentée en vue de l’obtention du grade légal de Docteur en Sciences
Promoteur de thèse: Pr. Dr. L. Droogmans
Co-promoteurs de thèse: Pr. Dr. J.-P. Simon & Dr. H. Bohets
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RESUME
La bio-transformation naturelle des médicaments peut produire des métabolites toxiques;
l’identification de ces métabolites est essentielle dans la stratégie de choix de molécules thérapeutiques. En appliquant les technologies de fermentation en bioréacteur des cellules hétérologues (souches d’E. coli recombinantes exprimant une iso-enzyme de cytochrome P450 humain avec la réductase humain), la bioconversion du substrat (principe actif) en ses métabolites de dégradation, a été réalisée à grande échelle (µg-g).
Notre choix s’est porté sur le complexe hCYP3A4/HR fonctionnel produit par un hôte E.
coli. Les cellules intactes ou les membranes cellulaires peuvent être exploitées comme biocatalyseur dans un système bioréacteur. Cependant, la faible solubilité des principes actifs dans des milieux de bioconversion aqueuse limitent le rendement. Un bioréacteur biphasique a été étudié. En solution, plusieurs combinaisons eau/solvants organiques conciliant la viabilité des cellules, la solubilité des principes actifs et produits de réaction et la catalyse des complexes enzymatiques ont conduit à l’établissement d’un mélange approprié. Cependant, ces combinaisons présentent toujours une inhibition importante du pouvoir catalytique des complexes enzymatiques. Pour minimiser un effet dénaturant possible des solvants sur le système enzymatique, ce dernier a été maintenu dans un environnement aqueux en immobilisant les cellules et/ou les membranes cellulaires dans une matrice hydrophile. L’alginate de calcium apparaît être une matrice d’immobilisation idéale pour les membranes assurant la fonctionnalité du complexe CYP/HR et permettant en outre un stockage à long terme des préparations. Par contre, l’immobilisation des cellules dans diverses matrices, si elle permet une viabilité et une conservation à long terme des souches recombinantes, ne permet aucune expression de l’activité enzymatique présente dans les cellules. La combinaison d’une localisation du complexe hCYP/HR fonctionnel dans la membrane interne et d’une perméabilité réduite des cellules d’E. coli (immobilisées) en est une explication possible mais non-démontrée. Entre-temps, cette technologie de bioréacteur homogène biphasique ou par immobilisation des membranes cellulaires a été utilisée plusieurs reprises pour produire des métabolites humains à partir de divers principes actifs. Ces métabolites ont été purifiés avec succès, démontrant que cette approche technologique est compétitive comparée aux procédures conventionnelles. Néanmoins, de nouvelles pistes de recherche seraient extrêmement intéressantes. La localisation des complexes enzymatiques recombinants en surface des
Heterologous expression systems for the production of metabolites during early drug research
Thèse privée soutenue le 23 juin 2010, à Gosselies Thèse publique soutenue le 5 juillet 2010, à Gosselies
Composition du jury:
Président du jury: Pr. Dr. E. Pays, ULB Vice-président du jury: Pr. Dr. O. Leo, ULB Secrétaire du jury: Pr. Dr. A. M. Marini, ULB Promoteur de thèse: Pr. Dr. L. Droogmans, ULB Co-promoteurs de thèse: Dr. H. Bohets, J&J
Co-promoteurs de thèse: Pr. Dr. J.-P. Simon, UBT Rapporteur: Pr. Dr. E. Dubois, ULB Rapporteur: Pr. Dr. V. Kruys, ULB Expert extérieur: Dr. M. Vinken, VUB
Expert: Pr. Dr. P. Fickers, ULB
TABLE OF CONTENTS
THANKS TO…
LIST OF ABBREVIATIONS SUMMARY
INTRODUCTION ………..1
A. Facts on drug metabolism 1
A.1. Biotransformation of xenobiotics 1
A.2. Phase I enzymes and localization 1
A.3. CYP oxidative metabolism 2
A.4. CYP nomenclature 4
A.5. CYP variability 4
B. Pharmaceutical Research and Development 6
B.1. Drug metabolism studies 6
B.2. Reactive drug metabolites 7
B.3. Drug metabolite synthesis 8
B.3.1. Classical organic chemistry 9
B.3.2. Enzymatic synthesis 9
B.3.3 CYP mimicry 22
B.4. Purification 22
B.5. Conclusion 23
SCOPE OF THE THESIS ………24 RESULTS ………... 26
A. Recombinant hCYP/HR for metabolite production 26
A.1. Metabolite production on analytical scale 26
A.1.1. Recombinant E. coli co-expressing hCYP3A4/HR 26
A.1.2. hCYP3A4/HR plasmid construct 27
A.1.3. Expression for small culture volumes 27
A.1.4. Two biocatalytic systems: cells and cell membranes 27 A.1.5 Kinetic parameters of recombinant hCYP3A4 28 A.1.6. Stability of activity during conservation 28
A.2. Metabolite production on preparative scale 29
A.2.1. Application of minimal medium 29
A.2.2. Stability of plasmid construct and expression potential 30 A.2.3. Metabolite production in a controlled fermenter 31 A.2.1. Stability of activity during substrate turnover 32 A.3. Case study: validation of the E. coli bioreactor system 32
A.3.1. Setting the scene: human versus animal metabolism 32
A.3.2. CYP identification 34
A.3.3. Selective metabolite production 34
A.3.4. Preparative purification 35
B. Flexible multiphasic bioreactor system 35
B.1. Application of organic solvents 35
B.1.1. Biocompatibility 35
B.1.2. Drug partitioning 36
B.1.3. n-Hexadecane-diethyl ether mixture in a bioreactor 36
B.2. Application of immobilization 37
B.2.1. Immobilization in Ca-alginate microbeads 37 B.2.2. Initial proliferation and viability in alginate beads 37
B.2.3. hCYP/HR expression in alginate beads 38
B.2.4. Biomass evolution during culture/conservation in alginate beads 38 B.2.5. Metabolic potential of cells in alginate beads 38 B.2.6. Metabolic potential of cell membranes in alginate beads 41 B.2.7. Alternative cell immobilization techniques 41
DISCUSSION & PERSPECTIVES ………47
A.1 Lab-scale drug metabolite synthesis with recombinant hCYP/HR 47 A.2. Preparative metabolite synthesis with recombinant hCYP/HR 50 A.3. Validation of selective metabolite biosynthesis using bioreactor technology 53
B.1. Biphasic systems for improved drug solubility 54
B.2. Immobilized hCYP/HR for metabolite production 56
C.1. Conclusion and future perspectives 62
BIBLIOGRAPHY ………..
APPENDIX 1: Materials & methods ……….
A. Cooperation B. Molecular biology C. Microbiology D. Enzymology
E. Analytical techniques F. Fermentation technology
LIST OF ABBREVIATIONS
A660: absorbance at a wavelength of 660 nm δ-ALA: δ-aminolevulinic acid
AscA: ascorbic acid BM: Bacillus megaterium BSA: bovine serum albumin CMC: carboxymethyl cellulose CNS: central nervous system CHO: Chinese hamster ovary CFU: colony forming units
CDS: Chromatography Data System CYP: cytochrome P450
DO: dissolved oxygen DTT: dithiotreitol DMSO: dimethyl sulfoxide EC: electrochemical ER: endoplasmic reticulum E. coli: Escherichia coli
EDTA: ethylene diamine tetra-acetic acid EU: European union
FIH: First into Human
FDA: Food and Drug Administration GI: gastro-intestinal
G-6-P: glucose-6-phosphate
G-6-PD glucose-6-phosphate dehydrogenase GSH: glutathione
HPLC: high performance liquid chromatography hCYP: human cytochrome P450
HLM: human liver microsomes HR: human NADPH CYP reductase 6β-OHT: 6β-hydroxytestosterone ID: identification
ICH: International Conference on Harmonization IPTG: isopropyl-β-D-thiogalactopyranoside KP: Kansai Paint
LC: liquid chromatography LSC: liquid scintillation counting LLE: liquid-liquid extraction LB: Luria-Bertani
LBA: Luria-Bertani with ampicillin MS: mass spectrometry
MCB: Master cell bank MTB: modified terrific broth MWCO molecular weight cut-off MIST: metabolites in safety testing NCE: new chemical entity NMR: nuclear magnetic resonance PD: pharmacodynamic
PK: pharmacokinetic
PMSF: phenylmethylsulfonyl fluoride PhD: philosophical degree
PDA: photo diode array PGA: poly galacturonic acid PVA: polyvinyl alcohol PVDF: polyvinylidene fluoride POP: Proof of Principle ROI: return of investment rpm: rotations per minute SPE: solid phase extraction SD: standard deviation
SAR: structure-activity relationship THF: tetrahydrofurane
TSE: Tris-sucrose-EDTA TB: tuberculosis UV: ultraviolet US: United States WT: wild type WCB: Working cell bank
Summary
Biotransformation of drug can produce toxic metabolites; information on metabolites is essential. An "in-house" biological capacity to produce complex and in vivo relevant drug metabolites using one simple recombinant human cytochrome P450 isoenzyme (hCYP) co-expressed with human reductase (HR) in E.
coli cells was realized. Using fermentation and bioreactor technology for propagation of cells and substrate bioconversion, the scale-up of metabolite production was successful. Large amounts of functional hCYP3A4/HR complex were produced by intact cells, while whole cells as well as membranes were suitable as biocatalyst in a bioreactor system on a preparative scale for the fast and simple biosynthesis of milligram amounts of metabolite. However, the hydrophobic character of many molecules frequently resulted in low solubility in the aqueous bioconversion medium. A biphasic bioreactor was created: Application of several organic solvents resulted in a suitable and flexible organic solvent mixture. But, since even the best tolerated solvent system affected biocatalysis, the biocatalytic system was immobilized in a protective polymer network. Ca-alginate appeared an ideal immobilization matrix for the membranes, displaying hCYP/HR functionality as well as the ability to preserve enzyme activity during long time storage. An appropriate cell immobilization method, that protected the cells but that did not compromise their hCYP/HR functionality, was not found. The combination of localization of the functional hCYP/HR complex in the inner membrane with reduced permeability of (immobilized) cells is a logical explanation.Meanwhile, this innovative bioreactor technology is routinely used to produce “human”
metabolites for current drug candidates and their metabolites successfully purified, demonstrating that the technology is competitive with conventional procedures and will aid in conscientious decision-making. Still, recent advances in enzyme-, cell- and reaction-engineering are extremely exciting. Re-engineered activity and stability open perspectives. For membranes, elimination of expensive co-factors would be favorable. Seen the low permeability of cells, free as well as immobilized, surface expression of hCYP and HR might be considered for future bioreactor research. Additionally, solvent resistant cytochrome P450 activity might circumvent the need for immobilization.
INTRODUCTION
The main aim of this thesis was the development and optimization of a bioreactor for the biological synthesis of milligram amounts of oxidative drug metabolites using the combination of
1. enzymatic bioconversion with recombinant human cytochrome P450(hCYP)/human reductase (HR) enzymes expressed in Escherichia coli (E. coli) 2. fermentation (and immobilization) technology and
3. purification techniques
The bioreactor was designed for application in a drug development setting in the pharmaceutical industry.
Therefore, this introduction will particularly elaborate on the following:
- Relevant aspects of cytochrome P450 (CYP) related to the optimization of the bioreactor technology will be described. In addition, the importance of CYP- mediated drug metabolism will be demonstrated. Where appropriate, the link between characteristic features of CYP enzymes and the bioreactor technology will be highlighted (A).
- The importance of preparative synthesis and purification of oxidative drug metabolites will be illustrated, resulting in an extensive collection of available techniques to synthesize metabolites, enzymatically as well as non-enzymatically (B).
A. Facts on drug metabolism
A.1. Biotransformation of xenobiotics
Xenobiotics are chemical compounds that are not part of the normal composition of the human body. The principal route of elimination of xenobiotics from the body is through biotransformation. Biotransformation of xenobiotics is controlled by metabolic reactions that can be divided in Phase I and Phase II (conjugations) reactions. Based on their evolutionary history, many of the enzymes involved in the metabolism of xenobiotics are principally involved in the metabolism of endogenous compounds such as steroids, bile and eicosanoids (Parkinson, 2001; Danielson, 2002).
A.2. Phase I enzymes and localization
Phase I reactions are functionalization reactions which can be broadly classified into 4 categories: oxidation (epoxidation, peroxidation, heteroatom oxidation and oxidation of aromatic rings), reduction, hydroxylation and oxidative N-, O- or S-dealkylation
Figure 1: In eukaryotes CYPs are membrane bound enzymes (Bernhardt, 2006) mainly localized in the smooth ER (Werck-Reichhart and Feyereisen, 2000; Danielson, 2002; Neve and Ingelman-Sundberg, 2010).
CYP
(Danielson, 2002; Baader and Meyer, 2004; Gillam, 2005; Wisniewska and Mazerska, 2009). This divers set of reactions is mainly catalyzed by CYP enzymes, the major xenobiotic metabolizing enzymes (Parkinson, 1996a). As a result, although also non-CYP- mediated oxidations can play an important role in the metabolism of xenobiotics (Strolin et al., 2006), Phase I biotransformation of drugs is also mainly catalysed by CYP enzymes (Guengerich, 1993b; Maurel, 1996; Danielson, 2002; Kumar, 2010), with aliphatic and aromatic hydroxylation (at C-centres), N- and O-dealkylation, heteroatom oxidation (at N- and S-centres) and epoxide formation from alkenes and aromatic rings and oxidation of aldehydes to acids being the most commonly found in human drug metabolism (Guengerich, 2001; Gillam, 2005; Isin and Guengerich, 2007; Gillam, 2008). This makes hCYP enzymes extremely valuable for generation of in vivo relevant oxidative metabolites by the use of a bioreactor technology.
Since the majority of CYPs is localized in mainly liver cells (about 2.5% of total hepatic microsomal protein), the primary organ of drug metabolism is the liver (Karlgren et al., 2004; Wilkinson, 2005). The liver thus acts as the gatekeeper of the body, the more since after absorption in the gut, all orally administered drugs are transported through the portal vein directly to the liver. However, some CYPs also occur extra-hepatically (Parkinson, 1996a; Danielson, 2002; Ding and Kaminsky, 2003; Karlgren et al., 2004;
Wilkinson, 2005; Daly, 2006; McLean et al., 2007).
Subcellularly, in eukaryotes, CYP are membrane bound enzymes (Bernhardt, 2006) mainly localized in the smooth ER (Werck-Reichhart and Feyereisen, 2000; Danielson, 2002; Neve and Ingelman-Sundberg, 2010; Preissner et al., 2010) (Figure 1). Axelrod (1955) and Brodie et al. (1955) were first to report on CYP enzymes. Both found that certain xenobiotics were oxidized by an enzyme system present in the endoplasmic reticulum (ER) of rat liver microsomes.
A.3. CYP oxidative metabolism
In general, the CYP monoxygenase system constitutes a large superfamily of heme- containing enzymes, which catalyze mainly oxidative conversion reactions of a wide range of compounds (Porter and Coon, 1991), endogenous and exogenous (Guengerich, 1987; Guengerich and Shimada, 1991; Gonzalez, 1992; Danielson, 2002). In a few instances, however, CYPs also carry out reductions (under anaerobic conditions) or peroxidations (Blake and Coon, 1980; Estabrook et al., 1984; Marnett and Kennedy, 1995).
Figure 2: Proposed CYP-mediated oxidative catalytic reaction cycle involving as well HR, cofactors and oxygen.
Although CYP was initially thought of as a single enzyme, the functional complex consists of
(1) an electron-transporting protein that contains a heme-prosthetic group, a hemoprotein, where heme is an iron-containing protoporphyrin IX and the protein an apoprotein with a molecular weight of 45 000 – 55 000 Da (i.e. CYP, the substrate- and O-binding site).
(2) a flavoprotein (NADPH cytochrome P450 reductase or NADH-cytochrome b5
reductase, the e- shuttle) (Smith et al., 1994; Bernhardt, 2006). Since CYPs are not self- sufficient and E. coli do not possess endogenous reductase activity, coexpression of CYPs and their redox partners in heterologous expression systems was introduced in order to allow shuttling of electrons (Gillam et al., 1993; Blake et al., 1996; Pritchard, 1997;
Bernhardt, 2006). The latter E. coli cells were also used to optimize the bioreactor.
While the details of the catalytic cycle are still subject of ongoing research, the hydroxylation mechanism is well understood (Rabe et al., 2008) and the overall stochiometry of the mixed function oxidase function of CYP is as follows:
substrate (RH) +O2 +NADPH +H+ → product (ROH) + H2O + NADP+
(Ortiz de Montellano, 1989; Sono et al., 1996, Danielson, 2002 ; Gillam, 2005; Bernhardt, 2006)
Essentially, one oxygen atom of dioxygen is incorporated into the substrate (Parkinson, 1996a), while the other oxygen atom is reduced by two electrons to give water.
Therefore, molecular oxygen is an essential requirement for CYP dependent oxidation of drugs, as is the cofactor NADPH, who usually provides the two electrons needed to catalyze activation of molecular oxygen (Johansson et al., 2007). Based on the importance of oxygen for CYP-mediated oxidative drug bioconversion, oxygen is often the rate limiting factor when applying the bioreactor technology. The oxidative catalytic cycle is demonstrated in Figure 2.
Additionally, if the catalytic cycle is interrupted and, therefore, not completed, uncoupling reactions can occur resulting in the formation of reactive oxygen species such as superoxide anion radicals and/or hydrogen peroxide (Parkinson, 1996a;
Schenkman and Jansson, 2003; Bernhardt, 2006; Myasoedova, 2008).
Furthermore, phospholipids, important components of biological membranes, are also necessary for the optimal interaction of CYP and its electron transport partner reductase
Figure 3: Schematic drawing of the membrane topology of E. coli-expressed CYP and HR, demonstrating that CYP and HR are both situated in the inner cell membrane, probably with their catalytic site facing the cytosol (based on unpublished LINK data).
cytochrome P450 HR e-, ...
cell membrane out
in
substrate metabolite O2
(Kumar, 2010). Thus, since E. coli do not possess an ER, but functionality depends on a lipid environment, hCYP/HR systems are generally directed to the hosts cell membrane (Figure 3). Additionally, it is imperative that hCYP and HR are close to each other in the membranar system allowing efficient channeling of the electron transport in the membrane from HR to hCYP.
A.4. CYP nomenclature
The nomenclature system for these CYP isoforms, named after the typical absorption maximum of the reduced CO-bound complex at 450 nm (Werch-Reichhart and Feyereisen, 2000; Danielson, 2002; Bernhardt, 2006), was developed by Nelson et al. in 1996. Based on primary AA sequence homology, the CYP superfamily is devided in gene families (indicated with an arabic number e.g. CYP3) and gene subfamilies (indicated by a capital letter e.g. CYP3A). While numerous exceptions exist, within the same family the CYP proteins share more than 40% protein sequence homology (Danielson, 2002; Rabe et al., 2008). CYP proteins with a sequence homology greater than 55% belong to the same subfamily and those that are more than 97% identical are considered to represent alleles unless there is evidence to the contrary (Danielson, 2002). Further, molecular techniques have allowed cloning, expression and improved characterization (generation of better antibodies, development of molecular probes) of individual members of CYP subfamilies. Each is indicated with an arabic number (e.g. CYP3A4, CYP3A5, CYP3A7, CYP3A43, Li et al., 1995; Daly, 2006) and is numbered sequentially as it is characterized.
Each member represents a single gene and a single protein.
A.5. CYP variability
CYP are ubiquitous enzymes found from bacteria to humans (Werck-Reichhart and Feyereisen, 2000; Danielson, 2002; Urlacher et al., 2004, Gillam, 2005). Over 450 different CYP isoforms have been found in plants, bacteria, insects yeast and mammals (Johansson et al., 2007). Although in bacteria some very interesting CYP activities are found lately (Gillam, 2008; Julsing et al., 2008; Rabe et al., 2008), E. coli lack cytochrome P450 enzymes (Wolf, 1999). However, this is one of the traits that make E. coli extremely attractive for this bioreactor technology, resulting in low background activity as well as clear metabolic profiles.
The CYPs display a tissue- as well as species-specific expression (Ding and Kaminsky, 2003). Because of these interspecies differences it is not often advisable to predict human oxidative metabolism of a drug based on animal models alone (Wrighton et al., 1993; Nedelcheva and Gut, 1994; Ding and Kaminsky, 2003). Additionally, large
Figure 4: CYP3A4 is the most important CYP involved in drug metabolism. Left: In human liver the CYP3A4 isoform is most predominantly present. Right: Most drugs are mainly metabolized by CYP3A4.
CYP2B6 (0,2%)
CYP2D6 (2%) CYP2A6 (4%)
CYP2E1 (7%) CYP1A2 (13%)
CYP2C (18%) unknown (27%)
CYP3A (29%)
CYP2E1 (1%)
CYP1A2 (5%)
CYP2C (20%)
CYP2D6 (25%) CYP3A (55%)
intraspecies differences exist in the expression and catalytic capabilities of the CYP enzymes.
In human 57 CYP isoenzymes (Meyer, 1996; Danielson, 2002; Miller et al., 2009;
Wisniewska and Mazersk, 2009; Kumar, 2010), divided over 18 families and 43 subfamilies (Danielson, 2002; Preissner et al., 2010), occur. The most important human CYP isoenzymes are CYP1A2, 2A6, 2B6, 2C, 2D6, 2E1 and 3A (Shimada et al., 1994). The percentage of different CYP isoforms present in the human liver is displayed in Figure 4.
Three human CYP gene families CYP1, CYP2 an CYP3 (Ding and Kaminsky, 2003; Karlgren et al., 2004; Wilkinson, 2005), including especially human CYPs CYP1A2, 2C9, 2C19, 2D6 and 3A4, are involved in ~95% of the CYP-mediated metabolism of drugs (Wisniewska and Mazersk, 2009) (about ~ 75% of drug metabolism (Lamb et al., 2007)). The percentage of CYP isoforms that metabolize existing drugs is given in Figure 4. This clearly illustrates the importance of CYP3A in drug metabolism. Especially CYP3A4 is qualitatively and quantitatively the most important CYP involved in drug metabolism (Wrighton et al., 1996; Anzenbacher and Anzenbacherova, 2001; Danielson, 2002; Daly, 2006; Sawayama et al., 2009), followed by CYP2D6 and CYP2C9 (Wisniewska and Mazersk, 2009). CYP3A4 and CYP2D6 are responsible for 80% of all CYP-related drug metabolism (Guengerich, 2003; Gillam, 2008). Therefore, development of the current bioreactor technology focused mainly on CYP3A4 activity.
CYPs are characterized by unique, but broad and frequently overlapping substrate specificity and regio- and stereoselectivity (Crespi et al., 1993; Danielson, 2002;
Wilkinson, 2005), reflecting the wide variety of compounds they have to cope with, thus underscoring their catalytic versatility (Danielson, 2002; Gillam, 2005). Therefore, recombinant (human) CYP isoenzymes are particularly valuable for selective generation of (potentially human-)specific oxidative metabolites of drugs in development. The details of the mechanism underlying the unusual substrate diversity of these CYPs are currently not fully understood (Urlacher et al., 2004).
Finally, extensive inter- (Danielson, 2002) and intra-individual differences exist. These differences can be due to environmental factors such as sex, age (e.g. CYP3A5 and CYP3A7 in human fetal liver, Wilkinson, 1996), diet (caffeine), hormonal influences, disease state (cirrhosis, hepatitis), drug consumption (induction and inhibition), chemicals (smoke, alcohol), but also genetic factors (genetic polymorphism: CYP2C9, CYP2C19 and 2D6, London et al., 1996; Ingelman-Sundberg; 2005 Wilkinson, 2005) play an important role (Wrighton et al., 1996; Danielson, 2002; Wilkinson, 2005). For example, there are no individuals lacking CYP3A, although high inter-individual variation
Figure 5: The whole process of drug development takes more than a decade and costs hundreds of millions. During this process the efficacy of a NCE as well as its safety are rigorously investigated.
(>10-fold in vivo) is not exceptional (Danielson, 2002; Wilkinson, 2005). Therefore, the bioreactor technology was also investigated for CYP2C9 and CYP2D6 activity. Another previously unappreciated source of genetic variation in drug metabolism are variants in CYP oxidoreductase (Hart et al., 2008; Miller et al., 2009).
B. Pharmaceutical Research and Development
- Publication: “Metabolite synthesis in drug development: a partnership between biological and chemical methodologies”, Wynant I., Cuyckens F., Vliegen M., Scheers E., Bohets H. (for Current Drug Metabolism) (Accepted abstract in Appendix 2).
B.1. Drug metabolism studies
The history of the pharmaceutical industry shows that many important drugs have been discovered by a combination of fortuity and luck. Today, however, new chemical entities (NCEs) are designed to achieve an improved efficacy (pharmacological activity) in combination with limited side effects (toxicology). A large number of potential NCEs are generated via combinatorial chemistry and high-throughput screening techniques (Guengerich and MacDonald, 2007). The process of drug development takes more than a decade and costs hundreds of millions and besides the efficacy of a NCE, its safety is rigorously investigated (Figure 5). Since lots of drug failures result from lack of efficacy, toxicity (Guengerich and MacDonald, 2007; Guengerich, 2008) and/or inadequate pharmacokinetics (PK), the toxicological and PK properties are examined to have a better understanding of the human drug safety profile. Therefore, it is important to develop in vitro drug metabolism models which allow rapid and flexible screening of unfavorable properties from a series of drug candidates (Figure 6). Reactive or toxic metabolites can be one of the potential cases of failure. Gathering information on metabolites in an early stage of development helps in judgment of potential safety and efficacy outcome.
One of the selection criteria in early phase of development (discovery) is the metabolic stability of a NCE. Therefore, the metabolism is investigated in vitro in the different toxicological and/or pharmacological animal species and man, supported by in vivo metabolism studies in animals. Resulting data primarily give information on the relevance of the toxicological and/or pharmacological species and on the (semi- )quantitative occurrence of potentially (re)active and/or human-specific metabolites.
Next to the metabolism, data on the enzymes involved in the metabolism are gathered.
Therefore, investigation of the metabolism in human liver microsomes in the presence of specific CYP inhibitors (Halpert et al., 1994; Bourrie et al., 1996; Guengerich, 1996)
Figure 6: Early elimination of potential drug candidates is important in order to facilitate lead optimization and ultimately attempt reduction in the overall development cost of new drug substances. Bringing new or better drugs to the market is the main objective of the pharmaceutical industry, although elimination of drug candidates at an early stage that would eventually not make it to the clinic is just as important.
and in heterologous expression systems expressing a single CYP are conducted (Ono et al., 1996; Guengerich, 1996; Van ‘t Klooster and Lavrijsen, 1997; McGinnity et al., 2000) to identify CYPs involved. Metabolism data allow assessing the impact of potential toxicological and pharmacological characteristics of the NCE and can drive initiation of specific metabolite synthesis. The scope of the current research is the development of a bioreactor to produce metabolite. As mentioned before, these metabolites are mainly needed to assess the pharmacological and toxicological properties of metabolites formed. The basis of our bioreactor system is E. coli expressing recombinant hCYP.
B.2. Reactive drug metabolites
The ultimate goal of xenobiotic metabolism is the ''elimination'' of xenobiotics (Porter and Coon, 1991; Ioannides, 1996) by altering their physical properties from those favouring absorption (lipophylicity) to those favouring excretion in urine or faeces (hydrophilicity) (Taavitsainen, 2001; Gillam, 2005). Therefore, the drug metabolism reaction cascade generally tends to increase hydrophilicity, which can influence the pharmaco-toxicological drug properties. Therefore, biotransformation does not always stand for bioinactivation or detoxification, especially among oxidative reactions (Blaauboer, 1996). Numerous examples are known of compounds which are biotransformed into pharmacologically or toxicologically active metabolites (bioactivation) with desirable (prodrugs) or adverse effects, including hepatotoxicity, cellular necrosis, hypersensitivity, carcinogenicity, teratogenicity and mutagenicity (Wrighton and Stevens, 1992; Guengerich, 1993a; Park et al., 1995; Blaauboer, 1996;
Parkinson, 1996a; Friedberg and Wolf, 1996; Parkinson, 2001; Purnapatre et al., 2008) due to the production of electrophylic species that can react with cellular nucleophiles, such as glutathione, lipids, DNA and proteins (Gillam, 1998; van Bladeren, 2000). Over the last few years, several high profile late stage failures and post-approval drug recalls caused by toxicity or negative drug-drug interactions have led to an increased focus on metabolite studies, including metabolite synthesis.
Regulatory documents in this perspective include the Food and Drug Administration (FDA) guidance for industry on safety testing of drug metabolites, published in February 2008. The guidance mainly focuses on Phase I metabolites. The guidance provides recommendations to industry on how to deal with safety of drug metabolites (MIST or metabolites in safety testing) and especially deals with human unique metabolites, i.e.
metabolites which are not present in the toxicology species used to support the clinical development program. Disproportionately higher levels in humans than in any of the animal species used during standard nonclinical toxicology testing are discussed as well.
Based on the FDA guidance, nonclinical characterization of a human metabolite is only warranted when that metabolite is observed at exposures greater than 10% of the total
parent drug at steady state concentration seen in the toxicity studies. This guidance triggered a lot of discussions as referred to in the following review articles: Smith and Obach, 2005; Vishwanathan et al., 2009; Smith et al., 2009; Leclercq et al., 2009;
Frederick and Obach, 2010. One of the major concerns by the different scientists authoring the reviews and by industry was the “10% of drug-related material exposure rule”. Further discussions between industry and regulators resulted in an updated International Conference on Harmonization (ICH) M3 (R3) guidance “Non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals.” The guidance is intended to be applicable in the EU, Japan and the US and is less conservative compared the MIST guidance of FDA. In the M3 guidance nonclinical characterization of a human metabolite is only warranted when that metabolite is observed at exposures greater than 10% of total drug-related exposure and at significantly greater levels in humans than the maximum exposure seen in the toxicity studies.
Pharmacological activity and/or toxicity of metabolites can be put into different categories as described by Park et al. (1998). The different categories and how to put this in the context of MIST was further discussed in the review of Frederick and Obach (2010). Some examples of drugs with metabolites that can potentially result in toxicity are acetaminophen, clozapine, carbamazepine, troglitazone…. (Park et al., 2009; Smith and Obach, 2006). Although clinical relevance of toxic metabolites will depend on specific clinical situations. Acetaminophen is seen as a safe drug in clinical use but can result in severe hepatotoxicity at overdose. Reactive metabolites which are not trapped by glutathione is one of the causes of this type of toxicity. Troglitazone was withdrawn from the market due to safety issues. Examples of drugs with active metabolites, such as atrovastatin, amitryptyline (active metabolite nortriptiline) codeine (active metabolite morphine), propranolol, risperidone, tolbutamide, loratidine, zolmitriptan, tramadol,…
are described in reviews of Gad (2003), Fura (2006) and Smith and Obach (2005, 2009).
Both the occurrence of reactive or active metabolites can significantly affect the drug development process, both in terms of developmental activities and cost. As mentioned earlier, it is therefore important to understand the specific features of a metabolite in an early phase of development. The CYP bioreactor technology can play a major role in this process.
B.3. Drug metabolite synthesis
These documents state that already in the early phase of development of a NCE, information about the identity and pharmacological or toxicological activity of drug metabolites can be useful in the further development process. Therefore, sufficient amounts (in the order of milligrams) of drug metabolites are desired (Kumar, 2010):
1) to identify metabolite structure via Nuclear Magnetic Resonance (NMR), 2) to screen the metabolites for their pharmacological activity,
3) as reference material for the optimization of bioanalytical methods, 4) for screening in receptor binding studies,
5) for PK tests (such as drug-drug interaction studies at the level of CYP) and 6) for in vitro investigation of potential (cyto)toxicity
7) data on drug metabolites will be used for selection of the toxicological species and
8) to support regulatory filings.
Early identification (ID) of potential therapeutic problems related to drug metabolites is vital to drive a more profound evaluation of metabolite safety and reduce the development costs of NCEs.
B.3.1. Classical organic chemistry
Hitherto drug metabolites are still mainly produced by traditional chemical synthesis because this approach generally allows large quantities to be synthesized. However, depending on the nature of the metabolite, this process is often time consuming, frequently in the order of weeks or even months and expensive, depending on the raw materials. The technique is extra complicated when dealing with a chiral mixture and/or when several steps are involved, what requires development of specific purification methods (Griffin et al., 1998). Additionally, the exact structure of the metabolite to be synthesized is not always known from previous in vitro and in vivo experiments reducing chemical synthesis often to a play of trial and error. Sometimes it is simply impossible to produce metabolites by chemical synthesis. Complementary to chemical synthesis, stress-induced degradation of product to generate drug metabolites is currently gaining more interest, since some metabolites are sometimes also formed during natural degradation, although generally in smaller amounts. Further reading is supplied in Appendix 2.
B.3.2. Enzymatic synthesis
Isolation and purification of metabolites from animal biological fluids (excreta such as urine, faeces and bile and blood or plasma) and tissues (e.g. liver, brain), following in vivo studies, have been common practice for decades. Some of the possible limitations are difficult purification from complex matrices resulting in considerable losses, need for human-specific metabolites and insufficient yield of one particular metabolite due to multiple metabolic pathways. Therefore, alternative in vitro methods are used to evaluate human drug metabolism. Well-established methods using biological matrices are liver-derived models and pure enzymes:
Table 1: General advantages and limitation of in vitro liver preparations, adopted from Guillouzo, 1998.
Liver-derived models
Since the liver is the key organ involved in drug biotransformation (Lohmann and Karst, 2008), human liver tissues (from liver subcellular fractions, cultured hepatocytes, liver slices to perfused liver, Table) are valuable in vitro models for drug metabolism studies (Donato et al., 2008). However, from a scientific point of view, in vitro phenotypic instability of hepatocytes, high functional variability in the population according to the background of human donors, low levels of single drug-metabolizing enzymes (Fujita and Kamataki, 2002; Elaut et al., 2006) and technical complexity hinder the routine use of human-derived tissue for drug metabolite synthesis (Donato et al., 2008), as will also be illustrated in this thesis. Another reason that restricts the application of human liver specimens is limited availability of suitable human tissue (McGinnity et al., 1999; Fujita and Kamataki, 2002; Donato et al., 2008).
Meanwhile, different human-derived cell line models have been proposed to overcome these limitations given their higher availability, unlimited life span, stable phenotype and their ease to handle. Examples of metabolically competent immortalized hepatocytes are transformation of human hepatocytes with plasmids that encode immortalizing genes, hepatocyte-like cells derived from stem cells, cell lines generated from transgenic animals, and hepatocyte/hepatoma hydrid cells. Currently, while upregulation of the expression of drug-metabolize enzymes in cell lines of human origin is intensely explored (Donato et al., 2008), human cell lines remain principally used as toxicology models (Moshage and Yap, 1992) and are not in use for metabolite production at larger scale.
Although prediction of human drug metabolism based on animal test systems alone is dangerous, partially due to pronounced species-species differences in the catalytic properties of enzymes involved in drug metabolism (Wrighton and Stevens, 1992), animal liver tissue can be used for metabolite biosynthesis (Lohmann and Karst, 2008).
However, in order to synthesize µg up to mg amounts of drug metabolites, especially human-specific metabolites, this approach might be challenging and excessively large amounts of hepatocytes may be needed.
Pure enzymes
Alternatively, isolated oxidative heme enzyme systems, such as simple peroxidases and more sophisticated CYPs, can be used to produce metabolites. Prokaryotic CYPs are easier to handle as they are often soluble, more stable and far more active than their eukaryotic counterparts (Urlacher and Schmid, 2006). However, they lack the typical regio- and stereospecificity of eukaryotic CYPs, required for authentic metabolite
synthesis. The application of eukaryotic CYPs on an industrial scale has been limited by substrate specificity, low activity, poor stability (Rabe et al., 2008) and the need for cofactor (regeneration) and membrane association (Urlacher and Schmid, 2002; Chefson and Auclair, 2006). However, pure enzymatic production of rapontigenin (metabolite of the glycosylated parent compound, rhaponticin) for pharmaceutical studies, an active anti-cancer compound, was described by Roupe et al. (2005). To overcome stability issues, CYP enzyme immobilization was introduced to synthesize metabolites (Lamb et al., 1998). According to Wiseman (2003) immobilization can mimic natural cell compartmentalization. Immobilized enzyme reactors based on human recombinant CYP were used by Nicoli et al. (2008) and have the potential to be used for bulk synthesis of metabolites in the future (Wollenberg et al., 2008; Kumar, 2010).
However, today none of the above described in vitro models have the capacity to produce milligram amounts of drug metabolites. Therefore, for the past twenty years organic chemists have asked the biotechnology community for alternative enzymatic tools to generate regio- and stereospecifc hydroxylated compounds (Julsing et al., 2008;
Kumar, 2010). As a result, novel biological systems for the preparative synthesis of complex metabolites were exploited and developed, based on the use of endogenous CYP in microbial cells and recombinant hCYP in heterologous expression systems, respectively. Hence, the latter is also the main topic addressed in this thesis. We had access to E. coli expressing different hCYP forms in combination with HR. Starting from this enzymatic system, bioreactor conditions were initially optimized in terms of production of biomass and catalytically active hCYP/HR. Secondly, a system with a physical barrier in terms of immobilization between the test compound (drug) and the bacteria was investigated. The concept of immobilization was designed to allow enzyme protection under high solvent conditions and to ease preparative purification of the metabolites. Finally, the concept was implemented in an industrial setting.
Endogenous CYP in microbial cells
In order to avoid the costly addition of cofactors and to avoid problems associated with isolation, immobilization, low activity and instability of pure enzymes, whole cell systems are often used as a bioreactor (Gillam, 2005, Urlacher and Schmid, 2006;
Straathof et al., 2002; Julsing et al., 2008; Rabe et al., 2008). Microorganism such as fungi and bacteria are known to contain a number of endogenous enzymes able to catalyze a tremendous variety of chemical transformations (Azerad, 1999; Abourashed et al., 1999), including the stereo- and regioselective conversion pathways of mammalian metabolism (Venisetty and Ciddi, 2003). Therefore, transformation of drugs by microorganisms can be used to produce specific metabolites which are difficult to
obtain synthetically (Venisetty and Ciddi, 2003). Traditionally, a large collection of bacterial and fungal strains (sometimes more than 200 microorganisms) are screened for their ability to mimic a specific mammalian CYP oxidizing activity (Gillam, 2005;
Bernhardt, 2006). Preparative generation of metabolites of drug candidates (mg-g amounts) with a selected microbial strain appeared competitive with the use of cloned CYP (Rushmore et al., 2000; Vail et al., 2005) in terms of yield, cost and speed.
Nevertheless, the potential of microbial CYP in biotechnology in general (Bernhardt, 2006) and for drug metabolite production in particular has so far been scarcely used (Rabe et al., 2008).
Recombinant hCYP in heterologous expression systems
Heterologous expression of mammalian CYPs resulted initially from the need to address problems with sourcing (Gillam, 2007). Resulting purified recombinant CYPs were used to study protein structure and the mechanism of catalysis and isolated microsomal forms were used for CYP phenotyping, metabolic stability screening and inhibitory potential evaluation (Tang et al., 2005). However, the scientific community rapidly recognized that the stereo- and regiospecificity of recombinant hCYP made the systems extremely useful to produce human-specific drug metabolites. Several heterologous expression systems were developed in the past (virus, bacteria, yeast, insect cells, mammalian cells…) (Coon et al., 1992; Doehmer et al., 1994; Waterman et al., 1995;
Friedberg and Wolf, 1996; Guengerich et al., 1997; Friedberg et al., 1999; Sakaki and Inouye, 2000; Fujita and Kamataki, 2002; Bernhardt, 2006; Gillam, 2007). Yun et al., (2007) and Purnpatre et al. (2008) both provided very detailed reviews on all methods and aspects involved in producing recombinant hCYP enzymes in various heterologous expression systems, demonstrating that depending on the endpoint each system has its advantages and limitations.
Mammalian cells (COS 1 African green monkey kidney fibroblasts, V79 Chinese hamster ovary (CHO) cells, AHH-1 TK± human lymphoblastoid cell line) have been used to express CYP heterologously as described by Aoyama et al. (1990), Crespi et al. (1993), Doehmer et al. (1994), Doehmer et al. (1995), Ding et al. (1997), Krebsfaenger et al., 2003. Mammalian cell lines are very attractive since they most closely resemble the in vivo environment of the native enzyme (i.e. ER) and as such are potentially the most likely to produce the protein in its functional state by correct configuration (post-translational modification), usually without requiring modifications of the cDNA in its non-coding or coding regions. Additionally, the correct complement of CYP reductase, as required electron transport partner, is also present. Disadvantages, however, are potential interference by endogenous
CYP enzymes, relatively low expression levels compared to other systems and poor heme incorporation. Moreover, cell culture facilities are required and scale up is costly and technically difficult (Guengerich, 1996). Therefore, expression of CYP in mammalian cells is most appropriate for toxicological purposes (Friedberg et al., 1999), such as genotoxicity and cytotoxicity (Tang et al., 2005; Doehmer, 2009).
Baculovirus expression system is a highly effective system for the large-scale production of recombinant protein in insect cells, which also provides a eukaryotic environment, resulting in the fact that again extensive modification of the mammalian cDNA is not required. Although, as in mammalian cells, this expression system also suffers from low incorporation of heme, it is difficult to achieve stable expression and the virus frequently kills the cells by down regulating housekeeping genes. Additionally, production of transgenic e.g. drosophila is a complicated and expensive procedure requiring specialist experience and equipment (Lee et al., 1995; Lee et al., 1996).
Thus, while mammalian cells and baculovirus systems offer some clear advantages, they are clearly not amenable for industrial application (Gillam, 2007) such as large-scale metabolite production (Loft et al., 1996).
Yeast (e.g. Saccharomyces cerevisiae or bakers yeast) is characterized by higher expression levels of functional CYP/HR than mammalian cells (Gillam, 2007). Since yeast cells are eukaryotes, they have many features of subcellular organization found in mammalian cells, such as smooth endoplasmic reticulum and mechanisms underlying membrane targeting resulting in preserved membrane topology and post-translational modification enzymes (Gillam, 2007). Additionally, they are readily adapted for large-scale work (Bligh et al., 1992; Renaud et al., 1993; Pompon et al., 1996; Urban et al., 1996; Lavrijsen et al., 1996; Masimirembwa et al., 1999).
However, uptake of compounds may prove difficult because yeast cells possess a rigid cell wall. The cell wall also makes yeast cells difficult to break without the use of hydrolytic enzymes (Guengerich, 1996). Interference by endogenous CYP enzymes (Engler et al., 2000) is another disadvantage. Finally, yeast express endogenous reductase which can be coupled with the recombinant CYP, but the interaction between both enzymes is not always optimal (Gillam, 2007). Despite some disadvantages, expression of various microbial and human CYP involved in drug biotransformation in yeast (Saccharomyces cervisiae, Pichia pastoris and Yarrowia lipolytica, Schizosaccharomyces pombe) is still exploited to study drug metabolism (Cheng et al., 2006) and produce metabolites necessary for
characterization of new potential drugs (Fickers et al., 2005; Dragan et al., 2005;
Bernhardt, 2006). In 2009, Peters et al. (2009a, b) reported the successful drug metabolite synthesis in fission yeast strains heterologously expressing human CYP2D6.
Alternatively, bacterial expression is technically straightforward. The genetic constructs are generally stable and the level of expression can be very high. A frequently used bacterial system to express foreign proteins such as drug metabolizing enzymes are E. coli cells (Fujita and Kamataki, 2002; Gillam, 2007).
Functional CYP protein could first be expressed by the modification of the N- terminal amino acid sequence in E. coli cells in 1991 by Barnes et al. (Fujita and Kamataki, 2002). Since then many forms of hCYP have been successfully expressed in E. coli cells (Fujita and Kamataki, 2002). The E. coli cell system is well understood genetically and can, therefore, be manipulated to suit the requirements, particularly since a wide variety of strain variants and vectors with powerful promoters are available. Moreover, E. coli cells are easy to grow, easy to manipulate, have low cost of culture (Doehmer et al., 1994; Waterman et al., 1995; Friedberg and Wolf, 1996;
Blake et al., 1996; Gillam, 2007). Additionally, endogenous CYP is absent, circumventing interference by basal transcriptional background activity, which -in combination with expression of one single hCYP isoenzyme- resulted in more interpretable metabolic profiles and easier metabolite purification. However, most importantly E. coli cells display higher CYP expression levels (Friedberg et al., 1999), which explains why the present project also focused on E. coli for drug metabolite synthesis. On the other hand, major drawbacks are that E. coli show little similarity to mammalian (human) cells as they have no ER, no endogenous reductase (Blattner et al., 1997; Kelly et al., 2001) and possess little capability for posttranslational modification (Guengerich, 1996; Guengerich et al., 1997).
However, since the CYP enzymes require the presence of (phospho)lipids and the reductase to be functional, human CYP is expressed (Barnes et al., 1991; Gillam, 2007; Kumar, 2010) together with human HR (Gillam et al.,1993; Blake et al., 1996;
Pritchard, 1997; Bernhardt, 2006) in the cell membrane. Detailed membrane topology of co-expressed CYP and HR in E. coli is not yet fully understood. It might be expected that the two proteins are located close to each other and in the bacterial inner membrane, with at least the active site of HR oriented towards the cytoplasm, where NADPH is located, since CYP and HR are able to couple and work efficiently in intact cells in the absence of endogenous NADPH. This was confirmed by protease protection experiments since CYP3A4 was protected from digestion by trypsin in intact spheroplasts (where the protease only has access to the
periplasmic side of the inner membrane). On the other hand, digestion was almost complete in detergent-solubilised spheroplasts (where the protease also has access to the cytoplasmic side of the inner membrane). This was however, not yet performed on reductase (unpublished LINK data). This internal localization of the recombinant hCYP/HR system is frequently a complicating factor when using whole microorganisms as complex biocatalysts because of reduced reactivity due to the diffusion barrier of their cell membrane (Kaderbhai et al., 2001; Kaderbhai et al., 2006 Yim et al. (2006), especially in case of gram negative cells, which will also be illustrated here (Wynant et al., 2009).
Industrial paradigm
Already in the 90s, biotechnological application of recombinant hCYP as bioreactor for synthesis of authentic molecules and metabolites was claimed competitive with chemical procedures in terms of manipulation, cost, yield and selectivity (Holland, 1992;
Pritchard et al., 2006; Waterman et al., 1995; Donato et al., 2008). Additionally, it was accepted that any heterologous expression system that can be scaled up can potentially be adapted to the large-scale production of metabolites (Gillam, 1998). Still, the use of native microbial CYP enzymes dominates the industrial fermentation processes. A well- known technical example is the CYP-mediated microbial oxidation involving 11β- hydroxylation of Reichstein S (cortexolone), a progesterone derivative, which leads to corticosteroids with anti-inflammatory activity (Urlacher et al., 2004). This process is performed by fungi of the genus Curvularia (Suzuki et al., 1993) at a scale of about 100 ton per year (Urlacher and Eiben, 2006) and demonstrated that bioconversion can result in much faster synthesis of metabolites and be a valuable tool to help the organic chemist (Urlacher et al., 2004), resulting in a gain in resources. Additionally, the fungal strain Beauveria bassian Lu 700 was identified to selectively hydroxylate 2- phenoxypropionic acid to 2-(4’-hydroxy-phenoxy)propionic acid which is used for the synthesis of a number of agrochemicals. This strain has also proved a suitable catalyst for the selective monohydroxylation of other aromatic carboxylic acids (Oliveira et al., 2003); With regards to industrial degradation of environmental contaminants, the white-rot fungus Pleurotus streatus was found to be capable of metabolizing phenanthrene to phenenthrene-trans-9,10-dihydrodiol and 2,2-diphenic acid as well as mineralizing it to CO2 as part of the fermentation process (Urlacher et al., 2004). Even for authentic drug metabolite production, a lot of companies still use large microbial databases to screen for production of specific drug metabolites.
While some initial reports on large scale production of recombinant CYP in E. coli emerged in 2003 (Kanamori et al., 2003), there is scarce reporting on preparative metabolite synthesis of CYP probe substrates using an in vitro bioreactor system based
on recombinant hCYP (Rushmore et al., 2000; Vail et al., 2005; Uno et al. 2008). Up to date, no data on the industrial application of recombinant CYP for metabolite production of drugs in development have been published so far (Urlacher and Eiben, 2006; Eiben et al., 2006). Hence, this development/optimization of a recombinant E.
coli-based bioreactor for preparative CYP-mediated drug metabolite synthesis was a scope of this project.
However, although recombinant CYPs looked very promising, these systems -as other in vitro systems- also suffered from high substrate specificity, relatively low activity, poor operational and storage stability and need for cofactor (regeneration) and lipid environment. Nevertheless, although substantial challenges have to be overcome, these systems were the basis for further research (Yun et al., 2006), as described below.
Current innovations
In general, new evolutions were attempts to overcome problems encountered with natural as well as recombinant CYPs via engineering at several fronts.
Firstly, achieving successful heterologous expression in bacteria was the aim of lots of engineering studies (Gillam, 2008) based on the discovery that some modification of the CYP nucleotide sequence, and often also amino acid sequence, was required to facilitate expression of significant amounts of recombinant hemoprotein in bacteria (Gillam, 2008). As described above, the first critical success was already obtained by Barnes et al.
(1991), who expressed bovine CYP17A1 with altered codons to minimize secondary structure formation and match E. coli codon preferences. Still today alike strategies are applied (Wu et al., 2006). In 2009, Wu et al. still demonstrated that codon optimization of the N-terminus of the entire gene alone or combined with co-expression of molecular chaperone can increase heterologous expression levels of mammalian CYP in E. coli.
Another important progress was fusion of bacterial leader sequences such as ompA signal sequence, to the 5’-end of mammalian CYP genes (Pritchard et al., 1997) to direct expression. A similar strain was used in this research project. Other modification approaches are discussed in detail by Gillam et al., but it can be concluded that after all these years of successful bacterial expression it is still not possible to predict if a sequences modification will give the intended effect (Gillam, 2008). Additionally, to circumvent the need to supplement expensive heme precursors, such d-ALA (Kaderbhai et al., 2006), coexpression of the hemA gene, which encodes a glutamyl-t RNMA reductase catalyzing the rate-limiting step in heme biosynthesis, was co-expressed by Harnastai et al. (2006). Other ways to improve expression are culture medium and culturing at lower temperature (Yun et al., 2006; Gillam, 2008).
However, since bacterial CYPs generally exhibit higher catalytic rates, are easier to handle due to their soluble nature and have higher expression levels in heterologous hosts than their mammalian counterparts, they are ideal targets for protein engineering (Bernhardt, 2006). Thus, in view of industrial application mainly microbial CYPs were genetically engineered to change selectivity (Bernhardt, 2006; Julsing et al., 2008), which can enhance activity (Julsing et al., 2008). In case there is some understanding of the structure-activity relationship (SAR) of the enzyme concerned, re-engineered enzymes with altered selectivity can be produced by rational protein design through site-directed mutagenesis (Gillam, 2005), introducing a limited and defined number of mutations (Rabe et al., 2008). However, probably due to lacking knowledge on SAR (Gillam, 2008), there was only scarce reporting on improved activity by engineering (Parikh et al., 1999; Urlacher et al., 2004; Bernhardt, 2006) up to 2002. Now, when only limited information is available on the mechanism of the enzyme (Gillam, 2005), methods of directed evolution or random mutagenesis (Gillam, 2007; Rabe et al., 2008) such as error-prone PCR and saturation mutagenesis (Urlacher and Schmid, 2002;
Urlacher et al., 2004; Gillam, 2007) are more successful. Based on these approaches libraries of mutants (of mutants) are generated (Rabe et al., 2008) followed by screening and selection of the desired characteristic (Kumar, 2010). A third approach is sequence independent chimeragenic or recombinatorial approaches (e.g. via common gene shuffling or molecular breeding) which might even result in new activities (Urlacher and Eiben, 2006; Lamb et al., 2007). For example, Bernhardt (2006) reported on engineered solubility via chimeragenesis. Improving solubility of mammalian CYPs is important mainly for crystallization and structure determination (Cosme and Johnson, 2000;
Gillam, 2008; Li et al., 2009). Hence, the structure of CYP3A4 was recently published (Williams et al., 2004). However, divers CYPs and reductases expressed as active recombinant cytosolic forms in E. coli have yielded preparations with increased drug transformation activities. All three enzyme engineering approaches posses their specific advantages and disadvantages (Rabe et al., 2008). Other key approaches used for directed evolution of enzymes and numerous examples of improved activity through engineering, including xenobiotic metabolizing CYPs, are discussed by Gillam (2005;
2008).
Although for industrial application also evolutions towards better stability are vital, improved stability remained unreported for a long time (Parikh et al., 1999; Urlacher et al., 2004; Bernhardt, 2006). One way is forcing better coupling of CYP with its redox partner via enzyme engineering (Gillam, 2007) leading not only to better activity but also improved operational and storage stability (Urlacher and Eiben, 2006; Gillam, 2008;
Julsing et al., 2008).
