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Engineered cytochrome P450 BM3 mutants as biocatalysts for metabolite production:Applications in biotechnology and toxicologyVenkataraman, H.

2014

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ENGINEERED CYTOCHROME P450 BM3 MUTANTS

AS BIOCATALYSTS FOR METABOLITE

PRODUCTION: APPLICATIONS IN

BIOTECHNOLOGY AND TOXICOLOGY

Harini Venkataraman

Engineered cytochrome P450 BM3 mutants as biocatalysts for metabolite production: applications in biotechnology and toxicology

Harini Venkataraman

Printed by Ipskamp Drukkers

Cover design by Dhwajal Chavan

Copyright Harini Venkataraman, Amsterdam 2014. All rights reserved. No part of this thesis may be reproduced in any form or by any means without permission from the author.

VRIJE UNIVERSITEIT

Engineered cytochrome P450 BM3 mutants as biocatalysts for metabolite production: Applications in biotechnology and toxicology

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan

de Vrije Universiteit Amsterdam, op gezag van de rector magnificus

prof.dr. F.A. van der Duyn Schouten, in het openbaar te verdedigen

ten overstaan van de promotiecommissie van de Faculteit der Exacte Wetenschappen op dinsdag 2 december 2014 om 9.45 uur

in de aula van de universiteit, De Boelelaan 1105

door

Harini Venkataraman

geboren te Chennai, India

promotor: prof.dr. N.P.E. Vermeulen copromotor: dr. J.N.M. Commandeur

There are only two days in the year that nothing can be done. One is called yesterday and the other is called tomorrow. Today is the right day to Love, Believe, Do and mostly Live.” Dalai Lama XIV

Reading committee: prof.dr. W.J.H.van Berkel prof.dr. L.Dijkhuizen prof.dr. P.D.J. Grootenhuis prof.dr.ir. R.V.A. Orru prof.dr. V.B.Urlacher

Part of the research described in this thesis was supported by the NWO-IBOS grant (# 053.63.013, entitled: Biocatalytic exploitation of Monooxygenases (BIOMOX) and was carried out in Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Section of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

Contents Chapter 1: General Introduction 9 Chapter 2: Regio- -ionone 47 enantiomers by engineered P450 BM3 mutants Chapter 3: Biosynthesis of a steroid metabolite by an engineered 73

Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme

Chapter 4: Stereoselective C-16 hydroxylation of testosterone by 91 P450 BM3 mutants Chapter 5: Application of engineered cytochrome P450 BM3 mutants as 113

biocatalysts for the synthesis of benzylic and aromatic hydroxylation products of fenamic acid NSAIDs

Chapter 6 : Application of engineered cytochrome P450 BM3 mutants to 135

study the effect of human GST P1-1 polymorphisms on the detoxification of reactive drug metabolites

Chapter 7: Cytochrome P450 mediated bioactivation of mefenamic acid 159 to benzoquinone imine intermediates and inactivation by glutathione S-transferases Chapter 8: Summary, conclusions and perspectives 189 Appendices: Nederlandse samenvatting List of publications Curriculum vitae Acknowledgements

Chapter 1

GENERAL INTRODUCTION

General Introduction

Outline The main focus of this thesis is about the use of engineered bacterial P450 mutants as biocatalytic tools for regio-and stereoselective hydroxylation of fine chemicals, steroids and drugs as well as a bioactivation system for the generation of reactive metabolites. The present chapter gives a general introduction to the various scientific aspects covered in this thesis as outlined below.

Regio-and stereoselective hydroxylation Oxidizing enzymes in biocatalysis Cytochrome P450 Monooxygenases (classification and catalytic cycle)

o Human cytochrome P450s o Microbial cytochrome P450s

Protein engineering methods Engineering substrate specificity and selectivity of P450 BM3 In vitro and in vivo product formation Microbial steroid oxidation Enzymatic drug metabolite synthesis Bioactivation of drugs and role in adverse drug reactions

o Generation and characterization of reactive metabolites Role of glutathione-S-transferases in the detoxification of reactive drug

metabolites Aim and scope of this thesis

10

CHAPTER 1

Regio-and stereoselective hydroxylation Selective oxidation of unactivated C-H bonds poses as one of the most challenging reactions in organic chemistry (1, 2). During the past two decades, considerable progress has been witnessed in the development of chemical strategies for

bonds involving the use of oxidizing agents, biomimetic supramolecular complexes or transitions metal catalysts. The combined use of directing groups and transition metal catalysts, for example, has provided a way to favor the oxidation of sp3 s located in proximity of a directing functionality (3-7). In spite of these advances, the control of oxidation selectivity by chemical means is still challenging. An overview of the use of redox biocatalysts in industrial applications (Figure 1) showed that enantioselective oxyfunctionalizations such as hydroxylation and epoxidation reactions comprise an important part of the enzymatic processes, underscoring the importance for the need of robust catalysts (8). The use of biocatalysts for regio- and/or stereoselective oxyfunctionalization has proved be an attractive alternative to chemical methods (9). Figure 1. Distribution of oxidation reactions amongst industrial biocatalytic oxidations. Adapted from (8).

11


Oxidizing enzymes as biocatalysts Over the years, the use of enzymes as biocatalysts for diverse applications has grown steadily. Biocatalysis in industrial synthetic applications has become indispensible particularly in specialty and bulk chemical processes (10, 11). Enzymes are able to catalyze a wide variety of reactions with high selectivity and specific activity under mild conditions of temperature and pressure. There is a high demand for the use of oxidative biocatalysts especially in view of certain unsolved problems of oxidation reactions such as lack of control and predictability of product structures and the high costs of oxidizing reagents in conventional chemical catalysis (12). On the other hand, oxidizing enzymes confer some key advantages as the oxidation reactions can be readily controlled with high degree of regio- and/or stereoselectivity (13, 14). Oxidizing enzymes are broadly classified into three categories which in turn have different subcategories based on the range of their different activities (Figure 2).

Figure 2.Classification of oxidizing enzymes. Adapted from (12).

12

CHAPTER 1

Cytochrome P450 monooxygenases Monooxygenases are a group of oxidizing enzymes which may be metal, haem or flavin dependent and catalyze the introduction of one atom of oxygen into a substrate molecule using cofactors like NADH or NADPH (15). Cytochrome P450s (P450s) constitute a superfamily of heme dependent monooxygenases (most of the 11000 CYPs are not all characterized) that catalyze oxyfunctionalization of a vast variety of chemical compounds (16). P450s have the ability to catalyze different reactions including some unusual reactions like nitration of tryptophan and the more recently reported cycloproponation and intramolecular C-H amination (17-22). Depending on the nature of the redox partners, P450s are classified as three-, two-, or single component systems (Figure 3) (5). The prokaryotic P450s are typically three component systems which are depending on a NAD(P)H-dependent FAD-containing reductase and a [2Fe-2S] ferredoxin or a FMN-dependent flavodoxin that shuttle electrons from the cofactor NAP(P)H to the monooxygenase protein (Figure 3a). Eukaryotic P450s such as drug-metabolizing human liver P450s and many plant and fungal P450s are two component systems in which electrons are delivered by a FAD

Figure 3. Different types of P450 systems: (a) bacterial, class I P450 system (e.g., P450cam); (b) eukaryotic, membrane-bound class II P450 systems (e.g., human liver P450s); (c) P450-CPR fusion system (e.g., P450BM3); (d) P450RhF-type fusion system. FeS = iron sulfur cluster, FMN = flavin mononucleotide, FAD = flavin adenine dinucleotide. Adapted from (5).

13


and FMN containing P450 reductase (CPR) to the membrane bound P450 (Figure 3b) (23, 24). Figure 3c-d illustrate the single component P450 systems that can operate as redox self sufficient enzymes in contrast to the other multi-component P450s. An excellent example of this single component system is P450 BM3 (CYP102A1) in which the heme and reductase domain are fused into a single polypeptide (Figure 3c) thereby making electron transfer highly efficient (25) . Genome analysis has led to the identification of other self sufficient P450s like CYP102A2 and CYP102A3 similar in architecture to that of P450 BM3. Yet another novel class of self sufficient P450s is P450 RhF from Rhodococcus sp. NCIMB 9784 in which electrons are supplied by a FMN- and Fe/S-containing reductase partner in a fused arrangement (Figure 3d) (26, 27). Cytochrome P450 catalytic cycle Most P450s are considered to function by a similar catalytic cycle. In the resting state of the P450 enzyme, the ferric iron atom is six-coordinated, and a water ligand occupies the axial position trans to the proximal thiolato ligand (1).

Figure 4: P450 cataltytic cycle. Adapted from (28).

14

CHAPTER 1

The catalytic cycle of P450 is initiated by binding of substrate to the enzyme (2) resulting in the displacement of water from heme distal face with simultaneous shift in the heme spin state from low spin to high spin (28). This spin transition can be monitored spectrophotometrically since the Soret band undergoes a blue or ‘‘type I’’ shift from ca. 418 nm to ca. 390 nm. A single electron supplied by a reduced pyridine nucleotide (NADH or NADPH) is shuttled to the haem iron via electron transfer partners, reducing it to the ferrous state (3). Oxygen binding leads to an oxy-complex (4). Subsequently, a second electron is transferred creating a ferric-peroxo intermediate (5a). This intermediate is protonated to form ferric hydroperoxy adduct (Compound 0) (5b). A second protonation triggers water loss via the heterolytic cleavage of the O–O bond to give ‘‘Compound I’’ (6), which has recently been characterized (29). The catalytic cycle ends with the oxygenation of the substrate to form a product complex (7) followed by release of product (Figure 4). During the P450 catalytic cycle, three major non-productive reactions collectively termed as “uncoupling” can occur. These are:

(i) autoxidation of the oxy-ferrous enzyme (4) resulting in the production of a superoxide anion and return of the enzyme to its resting state (2),

(ii) dissociation of hydrogen peroxide or hydroperoxide anion (5a,b) from the iron forming hydrogen peroxide

(iii) an oxidase uncoupling wherein the ferryl-oxo intermediate (6) is oxidized to water instead of oxygenation of the substrate (28, 30) .

Human P450 monooxygenases Human P450s are involved in the Phase I metabolism of approximately 75 % of marketed drugs, numerous dietery constituents as well as endogenous chemicals. The human genome contains 57 functional P450 genes arranged into 18 families and 42 subfamilies (31, 32). The general characteristics of different active human cytochrome P450 enzymes are listed in Table 1. Human P450s are known to be polymorphic in nature with clinically most important polymorphisms observed with CY2C9, CYP2C19 and CYP2D6 (33). A study of Shimada et al., 1994 reported that CYP1A2, CYP2A6, CYP2B6, CYP2C, CYP2D6, CYP2E1, and CYP3A accounted for approximately 70% of the total human liver P450 content; the remaining 30% consisted of then unknown enzymes (34) . In a recent study it was shown that the CYP4F family of enzymes also represents about 15 % of the overall human liver P450 pie (35) (Figure 5).

15


Table 1. General characteristics of human cytochrome P450 enzymes (33).

Enzyme Marker substrate reaction Substrate specificity Main tissue localization

CYP1A1 Ethoxyresorufin O-deethylation Pre-carcinogens, PAHs Extrahepatic CYP1A2 Phenacetin O-deethylation.

ethoxyresorufin O-deethylation Aromatic, amines, PAHs Liver

CYP1B1 Estradiol-4-hydroxylation DMBA, oestradiol Extrahepatic CYP2A6 Coumarin 7-hydroxylation Nicotine Liver CYP2A13 Coumarin 7-hydroxylation Olfactory mucosa

CYP2C8 Taxol hydroxylation Liver CYP2C9 Tolbutamide methylhydroxylation,

losartan hydroxylation, S-warfarin 7-hydroxylation

Drugs Liver

CYP2C18 Some drugs Extrahepatic CYP2C19 S-mephenytoin 4-hydroxylation,

omeprazole 5-hydroxylationDrugs Liver

CYP2D6 Dextromethorphan O-deethylation, bufuralol 1’-hydroxylation, debrisoquine 4-hydrohylation

Drugs Liver

CYP2E1 Chlorzoxazone 6-hydroxylation Solvents, drugs, pre-carcinogens

Liver

CYP2F1 Lung CYP2J2 Arachidonic acid hydroxylation Fatty acids Extrahepatic CYP2R1 Vitamin D25 hydroxylase Vitamin D Extrahepatic CYP2S1 Trans-retinol oxidation Small aromatic

hydrocarbons Extrahepatic

CYP3A4 -hydroxylation, midazolam 1’-hydroxylation, erythromycin N-demethylase

Drugs, pre-carcinogens, dietary components

Liver, intestine

CYP3A5 Same as CYP3A4 Similar as CYP3A4 Liver, intestine

Figure 5 . Revised human liver P450 pie. The inset figure depicts the percentage contributions of individual P450 enzymes based on the immunoquantification performed by Shimada et al. (1994). The larger figure shows the revised P450 pie with CYP4F contributing to 15% of the total hepatic P450s. Adapted from (35).

16

CHAPTER 1

Microbial P450 monoxygenases The P450 enzymes from microbes are enormously diverse and are involved in primary and secondary metabolism as well as act as potential drug targets (36, 37). Some examples of bacterial and fungal CYPs is shown in Table 2 with a selection of their respective activity/function. CYPs in bacteria are generally soluble proteins requiring ferredoxin and ferredoxin reductase for the two electrons needed in the catalytic cycle. CYPs in eukaryotic organisms are typically located in the endoplasmic reticulum with an associated NADPH-CPR providing the necessary reducing equivalents (38). Of the bacterial P450s, the most extensively studied are CYP101 (P450cam and CYP102A1 (P450 BM3) from Pseudomonas putida and Bacillus megaterium which have been structurally characterized extensively using X-ray crystallography and numerous other biophysical techniques (39). Table 2. Examples of bacterial and fungal CYPs with identified activity/function (36).

Microbe CYP activity/function Microbial eukaryotes

Saccharomyces cerevisiae CYP51 sterol biosynthesis (14-demethylation) Candida tropicalis CYP52A1 alkane and fatty acid catabolism Fusarium oxysporum CYP55A1 denitrification Saccharomyces cerevisiae CYP56A1 dityrosine formation for spore wall Nectria haematococca CYP57A1 phytoalexin detoxification Aspergillus nidulans CYP59A1, CYP60A2,

CYP60B1,CYP62A1 sterigmatocystin biosynthesis/mycotoxin

Rhizopus oryzae CYP509C1 -hydroxylase Chlamydomonas rheinhard

CYP710 (CYP61) sterol biosynthesis (22-desaturation)

Bacteria Pseudomas putida CYP101A1 catabolism of camphor Bacillus megaterium CYP102A1 fatty acid catabolism Streptomyces avermitils CYP105P1, CYP105D6 filipin biosynthesis/antifungal Saccharopolyspora erythrea CYP107A1 erythromycin biosynthesis/antibacterial Streptomyces lavendulae CYP107N1, CYP160A1 mitomycin c biosynthesis/antitumour Streptomyces hygroscopius CYP122A2 rapamycin

biosynthesis/immunosuppressant Mycobacterium tuberculosis CYP125A1 catabolism of cholesterol Jeotgalicoccussp. ATCC 8456 CYP152A3 decarboxylation of fatty acid Streptomyces fradiae CYP105L, CYP113B1 tylosin biosynthesis/veterinary

antibacterial Streptomyces noursei CYP105H1, CYP161A1 nystatin biosynthesis/antifungal Streptomyces spheroides CYP163A1 novobiocin biosynthesis/antibacterial Sorangium cellulosum CYP167A1 epothilone biosynthesis/antitumour Streptomyces avermitils CYP171A1 avermectin biosynthesis/anthelmintic

and insecticide

17


Protein engineering approaches to alter substrate selectivity Although P450s exhibit very high activity towards their natural substrates, their applicability towards unnatural substrates is generally limited. This can be overcome by various protein engineering strategies using both rational and evolutionary approaches (40, 41) (Figure 6). Rational approaches are characterized by the deliberate mutation of one or more amino acids based on mechanistic or structural information using molecular modeling and site directed mutagenesis. In the case of a semi-rational approach, mutations are introduced, for example, by simultaneous site-saturation mutagenesis at important active site residues. The resulting mutant P450s are subsequently screened for enhancement of desired property (42). Alternatively, directed evolution achieved by rounds of error prone PCR has also become a powerful tool in P450 engineering (43). Although most random mutations are either neutral or deleterious, a small percentage may be advantageous. The mutagenesis rounds are repeated in an iterative process until the desired catalytic activity or selectivity is achieved. Because large numbers of mutants need to be screened for the desired activity, the success of directed evolution is highly dependent on suitable high-throughput screening methodologies.

Figure 6. Protein engineering strategies. Adapted from (40).

18

CHAPTER 1

Table 3. Examples of P450 protein engineering (5, 42).

Substrate Protein engineering goal P450 Reference

Linear and cyclic terpenes Hydroxylation CYP102A1 (44)

Dopamine Improved binding CYP102A1 (45)

Amorphadiene Epoxidation CYP102A1 (46)

Propylbenzene, 3-methylpentane, fluorene

Increased coupling efficiency and activity CYP102A1 (47)

Protected cyclohexanol Selective hydroxylation CYP101A1 (48)

Vitamin D3

Increased activity for two sequential hydroxylations CYP105A1

(49, 50)

Progesterone Altered regioselectivity CYP106A2 (51)

Butane High rates of 1-butanol production CYP153A6

(52)

Deoxyaureothin Catalysis of second oxygenation-hetereocyclization step

CYP151A family

(53)

The appropriate method for protein engineering of enzymes should be decided case-by-case on the basis of the availability and quality of the structural and mechanistic information and the feasibility of a high-throughput screening (HTS) system for screening or selection (40, 54). Some examples illustrating protein engineering of microbial P450s is shown in Table 3. Engineered cytochrome P450 BM3 as biocatalysts Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium was identified as a medium to long chain fatty acid hydroxylase in the 1970s in the Fulco lab. Characterization of the 119 kDa protein revealed that the 55 kDa heme domain was fused to a 65 kDa reductase domain in a single polypeptide containing equimolar ratio of flavin groups FAD and FMN (55-57). P450 BM3 has the highest activity ever reported for a P450 of 17000 min-1 with arachidonic acid and exhibits a 100 to 1000 fold more efficient catalytic efficiency kcat/km when compared to that of the eukaryotic fatty acid hydroxylating CYP4 enzymes. In spite of this high native substrate activity, the wild type protein has a considerably narrow substrate scope. For potential use as biocatalysts for various applications, its substrate scope has been widened by the various protein engineering methods as discussed above.

19


Figure 7. Schematic representation of the active site of wild-type cytochrome P450 BM3 (PDB structure1BU7). Important amino acid residues are depicted in stick format, as well as the catalytically active moiety. Substrate free crystal structures revealed that P450BM3 features a long hydrophobic, largely non-aromatic, substrate channel connecting the protein surface to the heme pocket (58, 59). The structure of the palmitoleate-bound form of P450 BM3 provided clear insights into the binding mode for long-chain fatty acids and the key amino acids that are involved in substrate binding and recognition (60, 61). Important residues are shown in Figure 7. These residues have been targets for mutagenesis to modulate the substrate profile and regioselectivity of the enzyme. Computational techniques can be used to rationalize experimentally observed selectivity as well as to identify important substrate binding interactions by using molecular dynamics (MD) simulations based on crystal structure data (62-66). The most widely studied residue in P450 BM3 shown to affect selectivity and activity is F87. In the substrate free structure, F87 lies perpendicular to the porphyrin system, but in substrate bound form it is rotated by 90 degrees and occupies a position between iron centre and substrate tail (61). Modifications at F87, which is most proximal to the heme has led to an altered substrate selectivity as illustrated for a variety of substrates such as arenes, hetereoarenes, 2-aryl acetic acid esters, cycloalkanes, ionone, steroids (67-71). Apart from the F87 residue, other residues such as R47, A82, L437, S72 and A74 have also been shown to affect the substrate selectivity (72-74). Specifically, mutation at A82 position to a more bulky phenylalanine or tryptophan, increased the regioselectivity and coupling efficiency for hydroxylation of steroids and other drugs (75-77). S72 is the first residue of the B’ helix and modeling studies have revealed that fatty acids may tether to S72 when binding close to heme iron (78). Mutations at S72 have been shown to affect regio-and stereoselectivity of the substrate oxidation (79, 80).

20

CHAPTER 1

Combinatorial active site mutations have shown to have synergistic effects resulting in much higher selectivity and or activity. Mutations at positions 87 and 328 have greatly increased the selectivity of terpene oxidation (44), whereas mutations at 72 and 82 have been shown to affect stereoselectivity of steroid hydroxylation (80). Furthermore, 87 and 82 position mutations have been shown to affect stereoselectivity of styrene epoxidation (81). Thus, protein engineering approaches have led to the development of several mutants of cytochrome P450 BM3 capable of accepting non-natural substrates (Figure 8). In addition to conventional methods, efforts have also been made to control the substrate specificity of wildtype P450 BM3 without mutagenesis by using small decoy molecules (82). Recently, an activator-based strategy was applied to wild-type P450 BM3 in which the P450 BM3 catalytic cycle was triggered by long-chain perfluorocarboxylic acids (PFs), which mimic P450BM3 preferred substrates but contain non-oxidizable C F bonds in their side chains, resulting in the oxidation of various aliphatic substrates, such as cyclohexane, acyclic C6 and gaseous (C1 C4) alkanes (78, 83).

Figure 8 . Spectrum of compounds that have been used as substrates for engineered P450 BM3 variants.

Apart from improving the substrate scope and selectivity, efforts have been made to improve the robustness of P450 BM3 as a biocatalyst for biotechnological applications (24, 42, 84). Extensive work has been done to improve the thermostability and solvent tolerance of BM3 variants (85-87). Improving total turnover numbers was shown to positively affect coupling efficiency (42, 88). Novel methods of incorporation of non-

Engineered P450 BM3

Flavor, fragnance

compounds

Pharmaceuticals

Aliphatic hydrocarbons

Steroids and other

compounds

Aromatics

Indole

Testosterone

n-alkanes

Naphthalene

Anthracene

Alpha-ionone

Alpha-pinene

Propanolol

Dextromethorphan

21


canonical amino acids have proven to be effective to increase total turnover numbers to more than 30000 for non-natural substrates like ibuprofen methyl ester and nootkatone (89). In vitro and in vivo product formation For non-cellular in vitro P450 catalysis, enzymatic regeneration systems are usually employed for regeneration of the cofactor NADPH (15, 57). The most commonly used systems with their advantages and disadvantages are presented in Table 4. By mutations at residue W1046, P450 BM3 has been engineered to accept NADH as co-factor in place of the more expensive NADPH (90). Alternative methods which circumvent the use of NAD(P)H as electron donor include light driven catalysis, peroxide driven catalysis and the use of zinc and cobalt sepulchrate system (91-93). Table 4. Examples of NAD(P)+-dependent (de)hydrogenases that can be applied for coenzyme regeneration (15).

Enzyme Organism Advantages/Disadvantages Alcohol dehydrogenase

Thermoanaerobium brockii + Thermostable + Commercially available

Only selective for NADP+ Unfavorable thermodynamic

equilibrium High [substrate] required

Formate dehydrogenase

Candida boidinii + + Favorable thermodynamic equilibrium + Thermostable + Selective for NAD(P)+

Low activity Glucose dehydrogenase

Bacillus cereus + Commercially available

+ Thermostable + Selective for NAD(P)+

Product may complicate workup procedure

Glucose-6-phosphate dehydrogenase

Leuconostoc mesenteroides + Commercially available

+ Selective for NAD(P)+

substrate Product may complicate

workup procedure Phosphite dehydrogenase

Pseudomonas stutzeri + Favourable thermodynamic equilibrium + Thermostable + Selective for NAD(P)+

Low activity

22

CHAPTER 1

P450 catalysis using whole cellular systems has gained considerable importance as a more cost effect way of product formation (16). Several different host organisms like E. coli (94) , B. megaterium (95), R. erythropolis (96), Y. lipolytica (97), S. cervisiae (98), S. pombe(99), P. pastoris (100) have been employed for P450 whole cell biocatalysis. Although the effective intrinsic NADPH concentration can sometimes be rate limiting even in whole cell systems, this has been easily addressed by co-expression of NADPH regenerating enzymes (101, 102). One of the most often encountered problems with whole cell biocatalysis is the inefficient cellular uptake of substrates (103). Many P450 substrates are highly hydrophobic and do not readily cross cell membranes (104). In such cases, the use of another expression host can be beneficial. For example, it was demonstrated that the triterpene acid 11-keto- -boswellic acid can efficiently cross the membrane of B. megaterium, while no activity was observed with this substrate when using recombinant Escherichia coli cells (95). Microbial steroid hydroxylation Microbial steroid transformation is a powerful tool for generation of novel steroidal drugs, as well as for efficient production of key pharmaceutical intermediates (105, 106). Biotransformation of steroids can be carried out by microbial P450s with the desired regio- and stereoselectivity which is hard to achieve by chemical means. Several reactions can be completed in one biotechnological step making the process much easier. Bacterial cytochrome P450s play an important role in the steroid conversion (107). Different bacterial CYPs are capable of highly selective ring oxidation of testosterone as shown in Figure 9.

Figure 9. Hydroxylation of testosterone by bacterial P450s (108).

23


Table 5. Selected commercial and industry relevant CYP processes for steroids. Adapted from (16).

Steroid pharmaceuticals have a high market demand next to antibiotics. Therefore mirobial steroid hydroxylation plays an important role in the industrial scale applications. For example, the microbial hydroxylation of progesterone into 11 -hydroxyprogesterone catalyzed by Rhizopus arrhizus significantly increased the efficiency of the multi-step chemical synthesis of corticosteroid hormones as well as led to a reduction in the cortisone manufacturing costs (105, 113). Some of the commercialized microbial steroid biotransformations are shown in Table 5. Drug metabolite production

Drug discovery and development is a costly and time-consuming process involving many steps starting from target discovery up to clinical trials. Because drug metabolites sometimes have high pharmacological or toxicological properties, they

Steroid product Microorganism and/or CYPs involved

Reference/company

Cortisiol

Biotransformtion with Curvuaria sp. Denovo synthesis in S. cerevisiae; Mammalian CYP11A1, CYP17A1, CYP21, CYP11A1, 3 -HSD

(109) / Schering (1982), now Bayer (110) / Sanofi

Cortisone

Biotransformation with Rhizopus sp.

(111) / Upjohn(1952), now Pharmacia and Upjohn company, Pfizer

Pregnenolone

De novo synthesis in S.cerevisiae; mammalian CYP11A1

(112) / Sanofi

24

CHAPTER 1

sometimes play an important role in drug action and adverse drug reactions. It is therefore important to thoroughly study the metabolism of potential drugs as well as to characterize the pharmacological and toxicological properties of drug metabolites (114). Drug metabolism studies play an important role in the lead optimization stage of drug discovery in order to avoid undesirable adverse effects. In accordance with the MIST guidelines, identification, quantification and characterization of major metabolites are considered to be important for drug safety assessment (115, 116). Such studies can require large quantities of the pure metabolites which may be difficult to synthesize by conventional chemical methods. Therefore, effective strategies are required to obtain sufficient amount of drug metabolites to facilitate their structural elucidation as well as for toxicological evaluation (117). Although human P450s are involved in Phase I metabolism of many drugs (118), they are often inefficient for the production of large quantities of drug metabolites because of their membrane bound nature and lower activity (39). Engineered bacterial P450s with high activity and selectivity have therefore better perspectives for large scale drug metabolite synthesis (119). Figure 10 illustrates the construction of a CYP expression library for bacterial P450 monooxygenases for screening and optimization of the most efficient biocatalyst (117). Of the bacterial P450s, cytochrome P450 BM3 has been engineered for drug metbaolite production harnessing the inherent advantages of easily scalability and high selectivity (120, 121). Engineered P450 BM3 variants were able to catalyzed the formation of several drug metabolites with high turnover numbers, as summarized in Table 6.

Figure 10. Generalized strategy for the use of bacterial cytochrome P450 for drug metabolite production and the use of a bacterial CYP reaction array. Adapted from (117).

25


Table 6. Summary of drug metabolite production by engineered P450 BM3 variants. Adapted from (108).

Drug Metabolite produced by Engineered P450 BM3 variants

Main human P450 involved

Reference

Acetaminophen N-acetyl-p-benzoquinone imine 2E1 (122)

Amitriptyline Nortriptyline 2C19, 3A4, 1A2 (123)

Amodiaquine N-desethylamodiaquine 2C8 (124)

Astemizole

6-hydroxyastemizole 6-hydroxydesmethylastemizole Desmethylastemizole

2D6 2D6 2D6

(125)

Buspirone (R)-6-hydroxybuspirone 3A4 (126)

Clozapine

N-demthylclozapine Clozapine N-oxide

1A2 1A2

(127)

Dextromethorpha n 3-methoxymorphinan 2D6, 3A4 (124)

Diclofenac 4’-hydroxy diclofenac 5-hydroxy diclofenac

2C9 3A4

(128, 129)

Diltiazem

Demethyldiltiazem Di-demethyldiltiazem

3A4 3A4

(123)

Ibuprofen 2-hydroxy-ibuprofen 2C9 (130) Irbesartan

Hydroxyirbesartan Di-hydroxyirbesartan

2C9 2C9

(123)

Lovastatin 6b-hydroxylovastatin 6'-exomethylenelovastatin

3A4/5 3A4/5

(131)

Meclofenamic acid 3’-hydroxy-methyl-meclofenamic acid 4’-hydroxy-meclofenamic acid 5-hydroxy-meclofenamic acid

2C19 2C9 1A2

(132)

Naproxen Desmethylnaproxen 2C9, 1A2 (130)

Omeprazole 5-hydroxy-omeprazole 2C19 (75)

Ondansetron Hydroxylondansetron Dih d d

3A4, 2D6, 1A2 3A4 2D6 1A2

(123) Phenacetin Acetaminophen 1A2 (133)

Propafenone

Hydroxylated metabolites N-despropylpropafenone

2D6 3A4/1A2

(123)

Propranolol 4'-hydroxypropranolol 5'-hydroxypropranolol N-despropylpropranolol

2D6 2D6 1A2

(134)

Simvastatin 6b-hydroxysimvastatin 6'-exomethylenesimvastatin

3A4 3A4

(131)

Tolbutamide 4-hydroxytolbutamide 2C (128)

Tolfenamic acid 4’-hydroxy-meclofenamic acid 5-hydroxy-meclofenamic acid

1A2 1A2

(132)

Verapamil

Norverapamil 3A4 (125)

26

CHAPTER 1

Role of bioactivation in adverse drug reactions Serious adverse drug reactions (ADRs) are major causes of patient morbidity and mortality as well as for drug recalls and black box warnings (135, 136). Four different types of ADRs are known of which the Type B idiosyncratic ADRs (IADRs) pose significant problem due to their unpredictable nature, non-dependence on dose-response relationship and lack of animal models (137, 138). Although the exact mechanism is as yet unknown, drug metabolism and reactive metabolite formation seem to play an important role in ADRs and IADRs (139-141). Under normal circumstances, lipophopilic drugs are converted into hydrophilic more soluble metabolites that can be easily excreted from body. In some cases, however, metabolism can lead to bioactivation of drugs and to the formation of reactive metabolites (142, 143). Both phase I and phase II metabolic enzymes can be involved in the generation of reactive species of which cytochrome P450 and glucuronyl transferases are commonly involved. Figure 11 depicts the generally proposed scheme for the role of reactive drug metabolites in drug toxicity. Figure 11. Proposed role of bioactivation in adverse drug reaction (140).

27


Table 7 lists examples of drugs that are known to be bioactivated to reactive metabolites and the adverse effects associated with them. Identification of the common functional group capable of undergoing bioactivation during the drug design stage can help mitigate bioactivation risk (141, 144). The main approaches used to minimize bioactivation risk are (a) eliminating the suspect functional group, (b) blocking the potential for metabolism and (c) incorporating metabolic soft spots to direct metabolism away from the suspect group (144).

Table 7. Examples of drugs and reactive metabolites possibly involved in ADRs (143, 145).

Drugs Reactive intermediate Toxicity Acetaminophen Benzoquinoneimine Hepatotoxicity Carbamazepine 2-Hydroxy, Benzoquinoneimine Agranulocytosis, Aplastic

anemia

Clarithromycin Nitroalkane Hypersensitivity Clozapine Nitrenium ion Agranulocytosis Dapsone Hydroxulamine, Nitroso Hemolysis, hypersensitivity Diclofenac Acylglucuronide, Benzoquinone imine Hepatotoxicity Halothane Trifluoroacetyl Hepatitis Indomethacin Iminoquinone Hypersensitiv, ,

Hepatotoxicity

Isonizaid Isonicotinic acid, acetylating species Hypersensitivity

Mefenamic acid Benzoquinoneimine Hepatoxicity, Nephrotoxicity Nefazadone Benzoquinoneimine Hepatotoxicity Nevaripine Arene oxide, quinine methide Hepatotoxicity, Skin allergy Phenacetin p-Nitrosophenetole Hepatotoxicity Tacrine 7-OH-tacrine Hepatotoxicity Tamoxifen N-oxide, N-oxide-epoxide Carcinogenesis

Ticlodipine Keto, S-oxide Agranulocytosis, aplastic anemia

Tielinic acid Thiopene S-oxide Hypersensitivity, hepatitis

Troglitazone Conjugates, Benzoquinone, Quinone epoxide

Hepatotoxicity

Valproic acid Acyl glucuronides Hypersensitivity, Hepatotoxicity

28

CHAPTER 1

Generation and characterization of reactive intermediates Three key components are involved in general trapping experiments namely the formation of the reactive metabolites, the trapping of electrophiles and the subsequent analytical procedures. In vitro generation of electrophilic reactive drug metabolites is often done with human or rat liver microsomes in the presence of the cytochrome P450 co-factor NADPH (146, 147). Pooled human liver microsomes are commonly used as bioactivation system (148). The contribution of the individual human CYPs in metabolic activation pathway can be studied using commercially available human recombinant CYP supersomes (149, 150). An alternative for the use of human enzyme systems are engineered P450 BM3 mutants. Several P450 BM3 mutants obtained by a combination of random- and site-directed mutagenesis were shown to bioactivate drugs like clozapine, diclofenac, mefenamic acid to the same human relevant reactive intermediates (127, 151). Most of the reactive metabolites with a possible exception of acyl glucuronides are highly reactive and short lived, thus making their detection difficult (152). The most common way to screen for reactive metabolites is to perform trapping experiments (139, 152), with model nucleophiles such as glutathione (GSH) followed by analysis of the formed adducts. Although intermediates such as quinones, quinoneimines, iminoquinone methides, epoxides, arene oxides and nitrenium ions can be trapped by GSH, it is a soft nucleophile and does not always trap all the reactive intermediates. For this reason, the hard nucleophile cyanide is also used as a nucleophilic trapping agent (153). Drug candidates are routinely subjected to in vitro evaluation for reactive metabolite formation through the GSH trapping experiments. In view of this, rapid, sensitive and selective methods for detecting and characterizing reactive metabolites are highly desired in the drug discovery process (154, 155). The conjugates thus formed are analyzed using LC-MS/MS (156). The most common MS/MS mode for screening of GSH conjugates is the neutral loss scan of 129 which is based on the loss from [M + H]+ ion of the elements of pyroglutamic acid in combination with positive ionization (157). However, this method is not always successful in detection of some conjugates like aliphatic and benzylic thioether conjugates (158). Alternatively, precursor ion scanning (m/z 272; loss of H2S) in the negative ionization mode can also be used (159). Although mass spectrometry based analyses is the most often used approach for detection of GSH conjugates (160), in most cases the unequivocal structures cannot be determined by using LC-MS/MS techniques only. In such cases, the additional use of NMR is required for structural elucidation which can provide insights into the

29


mechanism of bioactivation (146, 161). However NMR typically requires high quantity of the sample for recording a good quality spectrum.

Figure 12. Graphical representation of the generation and characterization of reactive intermediates in trapping experiments. Adapted from (162). Large scale production and isolation of GSH conjugates can be facilitated by the use of bacterial CYPs as biocatalysts. Unequivocal structural elucidation of GSH conjugates of drugs such as clozapine, mefenamic acid has been achieved by this method (163, 164). A generalized scheme for generation and characterization of reactive metabolites is depicted in Figure 12. Role of glutathione-S-transferases in detoxification of reactive drug metabolites The conjugation of GSH to a reactive intermediate can be spontaneous non-enzymatic or catalyzed by glutathione-S-transferases (GSTs) which is dependent on the nature of the electrophile and the concentration of GSH (165). For example, N-acetyl-p-benzoquinone imine (NAPQI) may react spontaneously with GSH at physiological concentrations but requires GSTs at low concentrations of GSH indicating the importance of GSTs in detoxification (166).

30

CHAPTER 1

GSTs are a superfamily of dimeric phase II enzymes, which play an important role in the cellular detoxification of electrophiles and oxidants (167, 168). The human GSTs exhibit broad catalytic diversity due to the existence of eight cytosolic classes namely alpha, mu, pi, sigma, theta, omega, zeta and kappa, classified according to their amino acid sequence similarity, substrate specificity and primary and tertiary structures (169, 170). The expression pattern of GSTs is distinct in different organs (171, 172). For example in the liver, GST A1 and A2 contribute to about 78% of the human hepatic cytosolic GSTs (172, 173) (Figure 13). Although the expression of GST P1 is relatively low in liver, it has high extrahepatic tissue expression (174, 175). Figure 13. Distribution of hepatic cytosolic human GSTs. Percentages were calculated based on references (172, 173). As previously described, many drugs are bioactivated by cytochrome P450s to short-lived reactive metabolites that may lead to ADRs. The balance between the rate of formation of these highly reactive metabolites and the rate of inactivation by GSH conjugation, which may be catalyzed by GSTs, will determine the level of protein adduct formation and consequently may determine the risk for ADRs (165, 176). It has been shown that GSTs are involved in activation of reactive intermediates of several drugs (177). For example, reactive metabolites of valproic acid and acetaminophen were efficiently inactivated by addition of rat GSTs (166, 178). Human liver GSTs were found to catalyze GSH conjugation of reactive metabolites of clozapine and troglitazone (179, 180). Moreover, human GSTs also showed marked differences in the catalysis of 2-phenylpropenal and 2-acetylbenzothiophene-S-oxide, the reactive intermediates of felbamate and zileuton (181, 182), respectively. An overview of the GSH conjugation to reactive intermediates catalyzed by GSTs is presented in Table 8.

52%26%

18%

3 % 0.7% 0.3%

GST A1-1GST A2-2GST M1-1GST P1-1GST T1-1GST M3-3

31


Table 8. Effects of human glutathione S-transferases on the conjugation of drug reactive intermediates.

Drug Reactive electrophile GST activity ** Ref.

Acetaminophen *

N-acetyl-p-benzoquinoneimine

GST P1> GST M1> GST A1

(166)

Diclofenac

1’,4’-diclofenac-quinoneimine

2,5-diclofenac-quinoneimine

GST P1~ GST M1> GST A1 GST P1>> GST M1> GST A1

(183)

Clozapine clozapine nitrenium ion

GST P1> GST M1> GST A1

(179)

Mefenamic acid

2,5-mefenamic acid-

quinoneimine

GST P1> GST A2> GST M1> GST M3

(164)

Felbumate

2-phenyl-propenal

GST M1> GST P1> GST A1

(181)

Zileuton

2-acetylbenzothiophene-S-oxide

GST M1> GST P1>

(182)

* Study was performed using rat liver GSTs; similar results were also observed when human GSTs were used ** Total GSH conjugation is taken into account for comparing the activity of GSTs

32

CHAPTER 1

Several human GSTs are known to be genetically polymorphic due to point mutations, gene deletion and gene duplication (168, 184, 185). In this regard, interindividual variations in the detoxification of reactive drug metabolites due to GST genetic polymorphism can increase susceptibility to cancers and ADRs (185-187). Individuals with GST null genotypes might be at higher risk for toxicity due to the combined effect of higher exposure to the reactive metabolites and lower detoxification capacity. Gene deletions have been observed for GST M1 and GST T1 with a frequency of the null allele at 50% and 18% respectively in Caucasian population (188, 189). For drugs such as diclofenac, tacrine, troglitazone, carbamazepine, GST M1 and T1 null alleles have been associated with enhanced risk of hepatotoxicity (190-193). A number of single nucleotide polymorphisms (SNPs) have also been identified for GSTP1 (194, 195).The variants that result from polymorphisms at position 105 and 114, GST P1*A (Ile105/Ala114), GST P1*B (Val105/Ala114), GST P1*C (Val105/Val114) and GST P1*D (Ile105/Val114) are the most sudied so far. In view of the importance of GST P1 in detoxfication process, a large number of in vitro studies have been performed to investigate the effect of the mutations at position 105 and 114 using various drugs and model substrates (196-200). Aim and scope of this work The main goal of this thesis was to study the catalytic potential of cytochrome P450 BM3 enzymes for applications in biotechnology and toxicology. The first section of this thesis was performed as a part of a NWO funded, Integrated Biosynthesis and Organic Synthesis (IBOS) project titled “Biocatalytic exploitation of monooxygenases” (BIOMOX) with a common goal to develop new biocatalytic platforms (isolated enzymes and whole cell systems) capable of enantioselective hydroxylation reactions. Four academic and three industrial partners were actively involved in this project. Chapters 2-4 of this thesis cover the use of P450 BM3 mutants for selective oxidation of organic compounds to tackle the challenging task of regio-and stereoselective hydroxylation. A collection of novel P450 BM3 mutants were engineered and the catalytically active and selective variants were characterized specific for each of the applications. In the last three chapters 5-7, the potential use of P450 BM3 mutants in pharmaceutical industry for drug discovery and metabolite safety testing is covered. Engineered cytochrome P450 BM3 variants were applied for both stable and reactive drug metabolite production. Additionally, they have been used to facilitate the study of the role of human GSTs in the inactivation of P450 mediated reactive metabolites.

33


In chapter 2, the regio- and stereoselective hydroxylation of the small isoprenoids -ionone by engineered P450 BM3 variants is discussed. P450 BM3 mutants which displayed high selectivity and high total turnover numbers were characterized. The origin of the selectivity was rationalized using docking studies. In Chapter 3 the potential of Rhodococcus erythropolis as a new host for P450 BM3 mediated whole cell biocatalysis was evaluated. A model steroid substrate (norandrostenedione) was used to study the conversion. The combination of mutant Rhodococcus strain and a highly active engineered P450 BM3 variant is a promising option for whole cell biocatalysis of steroid hydroxylation. In chapter 4, a semi-rational approach was used to identify the key active site residues of P450 BM3 and create a minimal library for the oxidation of a larger substrate like testosterone. In-silico modelling studies provided insights into active site interactions responsible for this change in selectivity. This study showed that small differences in the key substrate interaction residues in P450 BM3 can have a profound effect on the stereoselectivity of hydroxylation. Chapter 5 describes the applicability of P450 BM3 mutants for the generation of human relevant drug metabolites of fenamic acid NSAIDs. A library of P450 BM3 mutants was screened to identify catalytically active and selective mutants which were then used for large scale metabolite production to enable structural elucidation of metabolites by NMR. These enzymatically synthesized metabolites were used for further toxicological studies in chapter 7. In Chapter 6, P450 BM3 mutants were used as a bioactivation system in addition to human liver microsomes to investigate the effect of four allelic variants of hGSTP1-1 in their ability to inactivate the reactive drug metabolites of paracetamol, clozapine, and diclofenac. Chapter 7 describes the use of cytochrome P450 BM3 as a tool for bioactivation of mefenamic acid and for the structural elucidation of the GSH conjugates. The role of human cytchrome P450s involved in the bioactivation pathway as well as the effects of a panel of seven major recombinant human GSTs on the formation of GSH conjugates of mefenamic acid were also studied. Finally, Chapter 8 provides an overall summary of the work presented in this thesis as well as perspectives for future work.

34

CHAPTER 1

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44

45

46

Chapter 2

REGIO- AND STEREOSELECTIVE HYDROXYLATION -IONONE ENANTIOMERS BY ENGINEERED CYTOCHROME P450 BM3 MUTANTS

Adapted from Harini Venkataraman, Stephanie B.A. de Beer, Daan P. Geerke, Nico P. E. Vermeulen and Jan N. M. Commandeur (2012) Advanced Synthesis and Catalysis, 354, 2172–2184.

Regio- and stereoselective hydroxylation of -ionone

Abstract

Selective hydroxylation of unactivated C-H bond is a crucial step in the synthesis of fine chemicals such as hydroxylated terpenoids. In the present study, the ability of 40 cytochrome P450 BM3 mutants to perform regio- and stereoselective hydroxylation of

-ionone has been investigated. Based on their activity and selectivity to produce 3-OH- -ionone from racemic -ionone, 6 BM3 mutants were selected. Out of these, 3 mutants (M01 A82W, M11 A82W and M11 V87I) showed high selectivity for trans-3-OH- -ionone formation while 3 other mutants (M11 L437N, M11 L437S and M11 L437T) formed almost equal amounts of both cis-3-OH- and trans-3-OH- -ionone. Incubation with individual enantiomers showed that M11 L437N, M11 L437S and M11 L437T exhibited opposite stereoselectivity producing (3S,6S)-OH- -iononewith (6S)-enantiomer and (3S,6R)-OH- -ionone with (6R)-enantiomer. Thus for the first time, BM3 mutants that can selectively produce diastereomers of 3-hydroxy- -ionone (>90 % de), with high turnover numbers and minimal secondary metabolism, have been identified. Docking studies have been performed to rationalize the basis of the experimentally observed selectivity. In conclusion, engineered P450 BM3s are promising biocatalysts for regio- -ionones for industrial applications.

CHAPTER 2

Introduction

Ionones are a class of norisoprenoids which are widely used in flavor and fragrance industry (1) and as building blocks for many chemicals (2). -Ionone is one of the norisoprenoids which possesses characteristic organoleptic properties. Interestingly, the individual enantiomers of -ionone possess different odors (3), thereby making them important for fragrance industry. The hydroxylated variants of -ionone are also of commercial interest. For example, derivatives of 3-hydroxy- -ionone are used as an intermediate for the total synthesis of lutein and its stereoisomers (4), Also, 3-hydroxy- -ionone has been shown to be a potent attractant for B.latifrons during pollination (5). The synthesis of hydroxylated variants of natural products such as terpenes and terpenoids still poses a challenge due to the formation of a variety of side products and the lack of selectivity of conventional synthetical methods (2, 6). In particular, the stereoselective hydroxylation of unactivated sp3 C-H bonds is a challenging aspect in modern day chemistry (7, 8). Because enzymes can catalyze reactions with remarkable regio- and stereoselectivity under mild conditions, their use as biocatalyst for selective production of fine chemicals has gained increased interest over the years (9, 10). A variety of mono-oxygenases has been used for selective oxyfunctionalization of organic substrates (11). An important class of mono-oxygenases are the cytochrome P450s, which are heme-dependent enzymes with the ability to perform regio- and stereoselective hydroxylations of a wide range of chemicals (12). Efforts over the last decade have been focussed on engineering P450s to obtain robust and useful biocatalysts for industrial applications (13, 14). Protein engineering of P450s has proved to be an excellent means to broaden their substrate scope and specificity as well as to improve activity (15-18). For example, engineered variants of cytochrome P450cam have been shown to exhibit increased regio- and stereoselectivity towards terpenes like pinene and limonene (19, 20). Cytochrome P450 CYP102A1 from Bacillus megaterium, commonly referred to as P450 BM3, has good perspectives for the use as biocatalyst for hydroxylation reactions of industrial significance because it is the most active P450 so far identified (21). The fact that the heme and reductase domain of P450 BM3 are fused into a single polypeptide makes the electron transfer very efficient (22). The active site of P450 BM3 has been re-engineered to accept a broader variety of substrates including terpenoids such as ionones (23). Although wild type P450 BM3 exhibits low activity

-ionone hydroxylation, some cytochrome P450 BM3 mutants engineered by rational design showed an increased activity. A triple mutant containing A74G F87V


-ionone to 3-hydroxy- -ionone

(49%) in addition to a number of other products (24). Also, BM3 mutant F87V produced a mixture of 4 oxidized products with -ionone without any selectivity (25).

-ionone at the C-3 position can result in the formation of four different diastereomers of 3-hydroxy- -ionone. Individual enantiomers (6R)- or (6S)- -ionone can be hydroxylated to (3R,6R) and (3S,6R) or (3R,6S) and (3S,6S), respectively, depending on the selectivity of the reaction. Structures of the 4 possible hydroxylated diastereomers are shown in Figure 1.

Figure 1. -ionone enantiomers and their 3´ hydroxylated products. A: (6R)- -ionone B: (6S)-

-ionone C: (3S,6R)-OH- -ionone D: (3R,6S)-OH- -ionone E: (3R,6R)-OH- -ionone F: (3S,6S)-OH- -ionone. C &D are cis isomers and E & F are trans isomers.

-ionone using different Streptomyces strains (26), fungi such as Aspergillus Niger (27), immobilized cells of Nicotium tabacum (28), and cultured cells of Caragana chamlagu (29) has been reported. Moreover, different bacterial P450s from Bacillus Subtillus, Sorangium cellulosum, Novaspinghobium

-ionone (30-34). -ionone that have been reported

so far, trans-3-OH- -ionone was found to be the main product (24, 30, 32). Other studies reported the formation of 2 diastereomers of 3-hydroxy- -ionone from

-ionone hydroxylation (24, 30, 32). However, from these studies it is not clear whether these enzymes are able to catalyze both cis- and trans hydroxylation on

CHAPTER 2

each individual enantiomer, or whether they hydroxylate each enantiomer with different diastereoselectivity. In the present study a library of P450 BM3 mutants was evaluated for their ability to selectively produce the individual diastereomers of 3-hydroxy- -ionone. This library was generated by a combination of site-directed mutagenesis of active site residues and random mutagenesis by error-prone PCR as described previously (35-37). Thirty one mutants of the library were selected based on their proven catalytic diversity towards a variety of drugs and steroids (35, 37-39). Nine additional mutants were prepared by site-directed mutagenesis of active site residues A82, V87 and L437 using M01 or M11 as template, see Table S1. Because some of our P450 BM3 mutants showed both cis- and trans- -ionone as substrate, it was decided to investigate the hydroxylation of each enantiomer individually. In this chapter, it is shown for the first time that BM3 mutants can hydroxylate individual

-ionone in a highly regio- and stereoselective manner and with high turnover numbers for 3 out of the 4 possible diastereomers. Thus the introduction of a hydroxyl group in a single step by engineered BM3 in a highly regio- and stereoselective manner gives them good perspectives for industrial applications. Materials and methods Materials

-ionone, enantiomers (R)-and (S)- -ionone and the hydroxy diastereomers (3S,6R)-OH- -ionone and (3S,6S)-OH- -ionone were obtained from DSM Innovative synthesis B.V. , Geleen, The Netherlands. All other chemicals were of analytical grade and purchased from Sigma unless otherwise mentioned. Restriction endonucleases were obtained from Westburg, Pfu polymerase and isopropyl -D-thiogalactopyranoside (IPTG) were obtained from Fermentas. Enzymes and plasmids

31 P450 BM3 mutants used in this work were constructed as described previously (35, 37, 40). Detailed information about the mutations present is tabulated in Table S2. 9 other mutants were made by site directed mutagenesis using appropriate oligonucleotides). In general, the mutations were introduced in the corresponding templates in pBluescript II KS(+) vector by the QuikChange mutagenesis protocol


using the forward primers mentioned in. The reverse primers were exactly complementary to the forward primers. After mutagenesis, the presence of right mutations was verified by DNA sequencing (Service XS, Leiden, The Netherlands). The whole gene was subcloned to pET28a+ vector using BamHI and EcoRI sites and later transformed to BL21 (DE3) cells for expression. Expression and purification of P450 BM3 mutants

-ionone hydroxylation, cytosolic fractions of the enzymes were used. Wild-type P450 BM3, mutants M01, M11, M01 A82W and M11 L437N were grown on a large scale and purified as follows: 600 mL terrific broth medium with 30 μg/mL kanamycin was inoculated with 15 mL of overnight culture. The cells were grown at 37 °C and 175 rpm until the OD600 reached 0.6. The protein expression was then induced by the addition of 0.6 mM IPTG. The temperature was lowered to 20 °C and 0.5 mM of heme precursor delta aminolevulinic acid was added. Expression was allowed to proceed for 18h. Cells were harvested by centrifugation (4600 g, 4 °C, 20 min) and the cell pellet was resuspended in 20 mL KPi-glycerol buffer (100 mM potassium phosphate [KPi] pH 7.4, 10% glycerol, 0.5 mM EDTA, and 0.25 mM DTT). Cells were disrupted using a French press (1000 psi, 3 repeats), and the cytosolic fraction was separated from the membrane fraction by ultracentrifugation of the lysate (120,000 g, 4 °C, 60 min). Then the enzymes were purified using Ni-NTA agarose (Sigma). To prevent aspecific binding, 1 mM histidine was added to the cytosolic fraction. 3 mL of Ni-NTA slurry was added to 20 mL of cytosol and the mixture was equilibrated at 4 °C for 2 hours. The Ni-NTA agarose was retained in a polypropylene tube with porous disc (Pierce,Rockford,USA) and was washed 4 times with 4 mL Kpi-glycerol buffer containing 2 mM histidine. The P450 was eluted in 10 mL Kpi-glycerol containing 200 mM histidine. The histidine was subsequently removed by repeated washing with Kpi-glycerol buffer in Vivaspin 20 filtration tube (10,000 MWCO PES, Sartorius) at 4000 g until the histidine concentration was below 250 nM. Incubations of P450 BM3 mutants with -ionone and analysis by GC-MS

For the biotransformation reaction, cytosolic fraction containing 200 nM of P450 BM3 mutants was incubated with 500 μM of racemic -ionone in a final volume of 250 μL. The reaction was initiated by the addition of 25 μL NADPH regeneration system containing 5 mM NADPH, 50 mM glucose-6-phosphate and 20 units of glucose-6-phosphate dehydrogenase. The reaction was allowed to proceed for 1 hour and then

CHAPTER 2

stopped by placing on ice. After addition of 10 μL of 5 mM carbazole as internal standard, samples were extracted by vortexing with 2 mL of ethyl acetate for 30 sec. Subsequently samples were centrifuged at 4000 g for 5 minutes to separate phases. About 1 mL of the organic layer was then transferred to GC vials for analysis. The extracts were analyzed using a Hewlett-Packard 6890 series with ATAS Programmable Injector 2.1. Samples were separated on a DB-5 GC-column (30 m x 0.25 mm). Ionization was done by electron impact (EI) at 70 eV with the mass

temperature program for the GC was as follow-1

enantiomers, same conditions were used as described above. Dissociation constant determination by optical spectroscopy

The binding of (R)- or (S)- -ionone to wild-type P450 BM3 and mutants M01, M11, M01 A82W and M11 L437N was analyzed by optical titrations using a Shimadzu UV-2501PC spectrophotometer (Shimadzu Duisburg, Germany). The spectra for the substrate- e recorded at 24 °C in 1 mL of 100 mM potassium

-ionone enantiomers. Increasing amounts of (R)- or (S -ionone dissolved in methanol were added from appropriate stocks up to a final volume of not more than 2% of the total volume of the solution. Spectra were recorded after each addition of substrate, and a difference spectrum was computed by subtraction of the starting (substrate-free) spectrum from those generated at each point in the titration. The maximum apparent absorption change

390-422) was determined by subtraction of the minimum absorption value at the trough in each difference spectrum from the maximum value at the peak and these values were plotted versus corresponding concentrations of (R)- or (S)- -ionone after correction for dilution. Data were analyzed by nonlinear regression using GraphPad Prism 4 (GraphPad Sotware, San Diego, CA, USA). The spectral dissociation constants (KD) were determined by fitting the titration binding curves to the following equation

-to- max is the maximum absorbance difference, [S] is the substrate concentration and KD is the dissociation constant of the enzyme–substrate complex. For substrate showing tight binding (Kd <

(41)

SKSAA

D

max


-to- max is the maximum absorbance difference, [S] is the substrate concentration and [E] is the enzyme concentration. Determination of coupling efficiency

NADPH oxidation rates were measured at 25°C using Libra S12 Biochrom UV-VIS spectrophotometer and 1 cm path length cuvettes. To measure the NADPH consumption rate, P450 BM3 (final concentration 200 nM) was mixed with 100 mM KPi buffer pH 7.4 containing 2 -ionone enantiomers(R or S) in methanol (2% final) in a volume of 1 mL. The reaction was initiated by the addition of NADPH (end

seconds. Rates were calculated based on extinction co- -

1cm-1 (42). To measure the coupling efficiency, the product formation was quantified under the same conditions. The percentage of coupling was determined as the ratio of the amount of product formed to the amount of NADPH consumed. Determination of total turnover numbers of M01 A82W and M11 L437N

200 nM of purified enzymes (M01 A82W and M11 L437N) was incubated with 1 mM of (R)- or (S)-enantiomers (2% methanol) in a total volume of 500 μL. The reaction was initiated by the addition of 50 μL NADPH regenerating system as mentioned in section 2.4. The reaction was performed in duplicate in 1.5 mL eppendorf tubes at 24 for 12 hours with shaking. The samples were then extracted and analyzed by GC-MS as described above. Total turnover numbers were calculated as nmol of product formed per nmol enzyme by using the calibration curve of (3S,6S)-OH- -ionone (R2 Preparative scale synthesis

Large scale incubation was performed in a 3L baffled flask with 250 nM of purified mutant M01 A82W, 48 mg of (S)- -ionone (final concentration 1 mM) in a volume of 250 mL in 100 mM potassium phosphate buffer (pH 7.4) at 24 . The reaction was initiated by addition of 25 mL NADPH regenerating system. The reaction was allowed to proceed for 6 hours and then the products were extracted by using ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and analyzed by GC-MS to determine the substrate conversion and product formation.

][2*42

max ESEKSEKSE

AA dd

CHAPTER 2

Computational methods

Mutants M01 A82W and M11 L437N were selected for molecular docking studies as they showed distinct stereoselectivity in C-3 hydroxylation. Molecular structures of the mutants were modeled based on chain A of the substrate-free crystal structure (PDB code 1BU7),(43) using the molecular modeling software MOE (Molecular Operating Environment).(44) Side-chains of the mutated amino acids were manually replaced using the builder tool, and polar and aromatic hydrogen atoms were added. The mutant structures were energy minimized in vacuo using the GROMOS05 biomolecular simulation package (45, 46) and the GROMOS force field parameter set 45A4.(47) Subsequently, aliphatic hydrogen atoms were added using MOE. Molecular structures of the substrate for use in docking were manually built using the MOE program and energetically optimized using the MMFF94x force field, as implemented in MOE. Subsequently, (R)- and (S)- -ionone were docked into the M01 A82W and M11 L437N active sites using GOLD (Genetic Optimization for Ligand Docking),(48) version 4.0, and the Chemscore scoring function.(49) The center point of the docking sphere was placed in the middle of the protein binding cavity, in between the side chain of residue 437 and the heme iron atom, from which the radius was set to 1.5 nm to define the active site of the protein. For each enantiomer, four independent docking simulations were performed. Per docking simulation, maximally 50 docked poses were retained. A maximum of 50000 operations in the GOLD genetic algorithm were performed, using a population of 100 genes. Generated binding poses for which all of the substrate’s carbon atoms were positioned more than 6 Å away from the heme iron center, (which is the hypothetical cut-off for catalytic activity,(50) and which is comparable to the distance of 5.6 Å between the heme iron and the C- -ionone in the crystal structure with PDB code 3OFU(34)) were discarded for further analysis. The remaining binding poses were visually inspected using MOE. Results and Discussion Screening of the P450 BM3 library with racemic -ionone

The goal of the initial screening was to identify P450 BM3 mutants that can produce 3-OH- -ionone with high selectivity. 40 cytochrome P450 BM3 mutants that were tested

-ionone to trans-3-OH- and cis-3-OH- -ionone with different selectivities and activities. As shown in Figure 2, three distinct product profiles were

-ionone predominantly to trans-3-OH- -ionone (Figure 2A), mutants which produced almost equal amounts of both cis- and trans-3-hydroxy- -ionone (Figure 2B) and mutants with produced significant


Figure 2. -ionone by (A) M01 A82W (B) M11 L437N (C) M11 A74E. Identification of the peaks: (1) cis-3-OH- R 9.84 (2) trans-3-OH-ionone tR 9.92 (3) 3-oxo- R 10.02 (4) unknown product tR 10.84. amounts of over-oxidation products (Figure 2C). The retention times (tR) of cis-3-OH-

-ionone and trans-3-OH- -ionone were 9.84 and 9.92 minutes respectively based on the tR of the available reference compounds. The product profile of all the mutants

-ionone is shown in Figure 3. In general, most of the mutants were capable of highly regio-selective C-3 hydroxylation of rac -ionone. C-3 is energetically the most favorable site for hydroxylation because it is an allylic methylene group. P450 BM3 mutants exhibiting high selectivity are highlighted in

-ionone mainly to trans-3-OH- -ionone (>85% de), whereas mutants M11 L437N, M11 L437S and M11 L437T produced almost equal amounts of trans-3-OH- -ionone and cis-3-OH- -ionone. Consistent with previous study [17], wild-type P450 BM3 showed only very low activity, with preference for trans-hydroxylation.

CHAPTER 2

Figure 3. -ionone by wild-type P450 BM3 and engineered variants. Conversions in % were measured from substrate consumption after 1 hour incubation of 500 μM of -ione with 200 nM of P450 BM3. Values represent averages of 2 independent experiments with less than 10% error. Product selectivity in % was calculated from gas chromatograms by integrating the product peaks. Representative P450 BM3 variants showing high selectivity towards formation of trans-3- -ionone (M11 V87I, M11 A82W, M01 A82W), variants that were not selective (M11 L437N, M11 L437S, M11 L437T, M11 L437N V87F) and a variant showing high over-oxidation product formation (M11 A74E) are highlighted. Wild-type P450 BM3 and mutants M01 and M11 are also highlighted for comparison. Of the 40 mutants tested, only one variant showed <0.5% conversion (M11 V87C) and was therefore not included in Figure 3. Conversion to 3-oxo- -ionone

-ionone to 3-oxo- -ionone and an unidentified over-oxidation product (m/z 206), in addition to trans-3-OH- and cis-3-OH- -ionone. A representative gas chromatogram illustrating this product profile is shown in Figure 2C. Incubation of purified P450 BM3 mutants with

-ionone also resulted in the formation of 3-oxo- -ionone indicating that the secondary oxidation of alcohol to ketone is also mediated by P450 BM3. 3-Oxo- -ionone has been recently shown to be a phagostimulant and also has commercial value because of its fragrance (51). The exact mechanism of formation of 3-oxo- -ionone is still to be investigated, but the formation of ketones by engineered P450 has been reported before for octane hydroxylation (52, 53). A possible mechanism as


mentioned in these studies is theformation of a geminal diol that readily dehydrates to form the corresponding ketone group. Also, in the case of 7-hydroxy-delta8-tetrahydrocannabinol (THC) hydroxylation by hepatic microsomes from Japanese monkeys, the formation of 7-oxo-delta8-THC by a similar mechanism has been reported (54).

-ionone

As seen in Figure 3 -ionone, mutants M11 L437N, M11 L437S and M11 L437T initially appeared to be not stereoselective by producing almost equal amounts of trans- and cis-3-OH- -ionone.

-ionone enantiomer, these three mutants were also incubated with the individual enantiomers (6R)- and (6S)- -ionone. All three mutants turned out to be highly stereoselective with individual enantiomers as substrate, producing (3S,6S)-OH- -ionone with (6S)-enantiomer and (3S,6R)-OH- -ionone with (6R)-enantiomer. .

Figure 4. Representative gas chromatograms showing opposite stereoselectivity for (A) hydroxylation of (6R)- -ionone by M11 L437N and (B) hydroxylation of (6S)- -ionone by M11 L437N and similar stereoselectivity for (C) hydroxylation of (6R)- -ionone by M01 A82W and (D) hydroxylation of (6S)- -ionone by M01 A82W. In all cases, the substrate (6R)- or (6S)- -ionone was completely oxidized to respective products.

CHAPTER 2

Figure 4 (A, B) shows the results obtained with mutant M11 L437N. Incubation of M11 L437T with (6R)- -ionone at a concentration of 500 μM resulted in the formation of about 80% of (3S,6R)-OH- -ionone, 12% of (3R,6R)-OH- -ionone and 8% of 3-oxo-

-ionone -ionone was completely converted to 3-oxo- -ionone In contrast, 12 hour incubations of M11

-ionone enantiomer showed only small amounts of 3-oxo-ionone. This shows that BM3 mutant M11 L437N is capable of highly selective hydroxylation with minimal formation of secondary oxidation products whereas mutant M11 L437T can result in the formation of significant amount of 3-oxo- -iononeThe fact that incubations with mutant M11 showed also significant amounts (30%) of (3R,6R)-OH-

-ionone with the (6R)-enantiomer as substrate, Table 1, shows that substitution L437N in mutant M11 has improved stereoselectivity of (6R)- -ionone hydroxylation. Interestingly, L437 has been recently reported to be a ‘hotspot’ affecting the selectivity of terpene hydroxylation (55). To test whether the L437N substitution also influences stereoselectivity in other M11-related mutants, the enantiomers of -ionone were also incubated with the available combinations M11 V87 and M11 V87F L437N, and M11 A82W and M11 A82W L437N. As shown in Figure 5A, substitution of L437N also increased relative amount of (3S,6R)-OH- -ionone when applied to M11 V87F and M11 A82W although to a much lower extent when compared to that when applied to M11. When incubated with (6S)-

-ionone, the L437N substitution led to a slight increase in formation of (3R,6S)-OH- -ionone when applied to M11 V87F, whereas the substitution had no effect when applied to M11 A82W, Figure 5B. These results confirm that the effect of single amino acid substitution is strongly dependent on additional nearby mutations (55, 56). As shown in Figure 3, M01 A82W metabolized racemic -ionone with high activity, producing mainly trans-3-OH- -ionone, implicating that both enantiomers of -ionone were stereoselectively hydroxylated. This was confirmed by incubation of M01 A82W with the individual enantiomers. As shown in Figure 4 (C, D), (6R)- -ionone was specifically metabolized to (3R,6R)-OH- -ionone, whereas (6S)- -ionone was specifically metabolized to (3S,6S)-OH- -ionone. In both cases only minimal amounts of secondary oxidation products were observed. When (6R)- -ionone was incubated with purified M01, significant amounts of both diastereomers were found, Table 1, indicating that mutation A82W has strongly increased stereoselectivity of hydroxylation of -ionone.


Figure 5. Effect of L437N mutation on stereoselectivity of C-3 hydroxylation of -ionone enantiomers. Panel A and Panel B represent hydroxylation of (6R) and (6S)- -ionone respectively. Wild-type P450 BM3, which showed only very low activity when incubated with

-ionone, showed very low diastereoselectivity, when incubated with the -ionone, Table 1. When incubated with (S)- -ionone, almost equal

amounts of (3S,6S)- and (3R,6S)-diastereomers were found, next to significant amounts of unidentified oxidation products. When incubated with (R)- -ionone, the diastereomeric excess for the (3R,6R)-diastereomer was found to be 51%. Total turnover numbers for stereoselective hydroxylation

An optimal biocatalyst should ideally be able to catalyze the formation of the desired product in highly selective manner (regio-, enantio- or stereo-) in combination with high catalytic turnovers and with minimal amount of secondary products (57).

CHAPTER 2 �

Among M11 L437N, M11 L437S and M11 L437T, M11 L437N, which showed the highest diastereoselectivity (>90%), see Figure 4, was selected for determination of

-ionone by P450s, mutant M11 L437N is the first engineered P450 BM3 specifically producing one of the cis-3-OH products, i.e. (3S,6R), with high stereoselectivity. Additionally, among mutants that produced predominantly trans-3-OH- -ionone, M01 A82W was selected for incubation with individual enantiomers because it exhibited the highest diastereoselectivity as well as activity. M01 A82W catalyzed the formation of (3S,6S)-OH- -ionone and (3R,6R)- -ionone with (6S)- and (6R)- enantiomers respectively, with remarkable selectivity. The fact that the P450 BM3 mutants M01 A82W and M11 L437N exhibit different stereoselectivities towards hydroxylation of (R)-and (S)- enantiomers, makes them highly useful for industrial biotechnological applications. M01 A82W catalyzes the formation of (3S,6S)-OH- -ionone and (3R,6R)-OH- -ionone with turnover numbers in the range of 3500-4000, Table 1. Similarly M11 L437N can also catalyze the formation of (3S,6R)-OH- -ionone with high turnover numbers of about 3000 nmol product/nmol enzyme. These high turnover numbers indicate that these enzymes might be useful for preparative scale biosynthesis (58). For both M01 A82W and M11 L437N these reactions are carried out with high selectivity to the desired product with minimal formation of side products. Purified M01 A82W was used as biocatalyst with (S)- -ionone (48 mg) as substrate in a volume of 250 mL to illustrate the preparative use of this engineered P450 BM3. The reaction resulted in > 90% substrate conversion to produce (3S,6S)-OH- -ionone (95% de) under the experimental conditions mentioned. Therefore, apparently the biotransformation reaction can be scaled up without any loss of diastereoselectivity

-ionone enantiomers

-ionone enantiomers with the stereoselective mutants M01 A82W and M11 L437N and their respective templates M01 and M11 were studied by spectral binding experiments. Titration of (R)- or (S)- -ionone to solutions of purified P450 BM3 mutants in all cases resulted in typical type I difference spectra, with a peak at 390 nm and a trough at 422 nm, indicative of the conversion of heme iron from low spin to high spin. The dissociation constants obtained from these titration studies are shown in Table 2. The introduction of the A82W mutation in M01 increased binding affinity towards both enantiomers: the dissociation constant (Kd) for M01 A82W was decreased approximately 3.5 fold in the case of R-enantiomer and approximately 22 fold in the case of S-enantiomer when compared to M01.

Regi

o- a

nd st

ereo

sele

ctiv

e hy

drox

ylat

ion

of

-iono

ne

Tabl

e 1.

Ste

reos

elec

tive

hydr

oxyl

atio

n of

(R)-

-iono

ne a

nd (S

)--io

none

by

wild

-typ

e P4

50 B

M3

engi

neer

ed P

450

BM3

mut

ants

.

[a] V

alue

s (in

%) w

ere

calc

ulat

ed fr

om 2

inde

pend

ent e

xper

imen

ts w

ith n

ot m

ore

than

10%

err

or. F

or w

ild-ty

pe P

450

BM3

the

activ

ity to

war

ds b

oth

enan

tiom

ers w

as

low

to a

ccur

atel

y de

term

ine

the

prod

uct d

istr

ibut

ion

unde

r th

e co

nditi

ons

used

for

othe

r P4

50 B

M3

mut

ants

. The

refo

re, t

he in

cuba

tions

wer

e pe

rfor

med

at a

hig

her

-oxi

datio

n re

fers

to p

eak

with

m/z

208

as d

etec

ted

on G

C-M

S.

[b] T

otal

tur

nove

r nu

mbe

rs a

re r

epor

ted

as n

mol

pro

duct

per

nm

ol e

nzym

e. T

he v

alue

s ar

e ca

lcul

ated

from

2 in

depe

nden

t ex

peri

men

ts w

ith ±

10%

err

or. T

otal

tu

rnov

er n

umbe

rs w

ere

mea

sure

d on

ly fo

r the

enz

ymes

whi

ch sh

owed

hig

h di

aste

reos

elec

tivity

tow

ards

pro

duct

form

atio

n

Mut

ant

Subs

trat

e Pr

oduc

t Dis

trib

utio

n (%

)[a]

D

iast

ereo

mer

ic

exce

ss (d

e %

)

Tota

l tu

rnov

er

num

ber[b

]

Wild

-typ

e P4

50 B

M3

(R)-

-iono

ne

3R,6

R (5

3%),

3S,6

R (1

7%),

othe

r-ox

idat

ion* (

30%

) 51

(3R,

6R)

-

(S)-

-iono

ne

3S,6

S (4

6%),

3R,

6S (3

6%),

oth

er-o

xida

tion* (

18%

) 12

(3S,

6S)

-

M01

(R

)--io

none

3R

,6R

(59%

), 3S

,6R

(30%

), 3

-oxo

--io

none

(11

%)

32 (3

R,6R

) -

(S)-

-iono

ne

3S,6

S (8

4%),

3R,

6S (1

4%),

3-o

xo-

-iono

ne (2

%)

71 (3

S,6S

) -

M11

(R

)--io

none

3R

,6R

(35%

), 3S

,6R

(55%

), 3

-oxo

--io

none

(10%

) 22

(3S,

6R)

-

(S)-

-iono

ne

3S,6

S (8

3%),

3R,

6S (7

%),

3-

oxo-

-iono

ne (

10%

) 84

(3S,

6S)

-

M01

A82

W

(R)-

-iono

ne

3R,6

R (9

1%),

3S,6

R (5

%),

3-

oxo-

-iono

ne (

4%)

91 (3

R,6R

) 35

00

(S)-

-iono

ne

3S,6

S (9

5%),

3R,

6S (3

%),

3-

oxo-

-iono

ne (

2%)

95 (3

S,6S

) 40

00

M11

L43

7N

(R)-

-iono

ne

3R,6

R (4

%),

3S,

6R (9

0%),

3-o

xo-

-iono

ne (

6%)

90 (3

S,6R

) 30

00

(S)-

-iono

ne

3S,6

S (9

1%),

3R,

6S (5

%),

3-

oxo-

-iono

ne (

4%)

91 (3

S,6S

) 38

00

CHAPTER 2

Improved affinity of hydrophobic substrates by A82W mutation in P450 BM3 has been reported previously with other substrates like fatty acids and testosterone (38, 59). Mutation L437N in M11 also apparently increases affinity towards both

-ionone; dissociation constants were lowered 2.4-3.6 fold in comparison to its precursor M11, which exhibited Kd values of 17 μM and 27 μM for R-enantiomer and S-enantiomer respectively. Wild-type P450 BM3 also showed a typical Type I difference spectrum. Dissociation constants obtained were comparable to those obtained with the much more active P450 BM3 mutants, being 17.5 and 10.3 μM, respectively, for R- -ionone and S- -ionone. NADPH consumption and product formation

To study the coupling efficiency of the mutants and wild-type P450 BM3, the rates of NADPH oxidation and product formation were determined at 200 μM of substrate, Table 2. In case of wild-type BM3, relatively low (4-6%) coupling efficiencies were observed for both enantiomers: the NADPH oxidation rates upon addition of R- or S- -ionone were found to be approximately 30 min-1 whereas the product formation was only about 1.5 min-1. Wild-type P450 BM3 produced a mixture of 2 diastereomers of C-3 hydroxylation and an additional peak with m/z of 208 indicating hydroxylation at another carbon with both the enantiomers. Interestingly, no over-oxidation peaks were observed with both enantiomers (Table 1). In case of the P450 BM3 mutants significantly higher coupling efficiencies, up to 45%, were observed when compared to wild-type P450 BM3. Introduction of mutation A82W in M01 did not significantly affect the product formation with R- -ionone as substrate, but decreased NADPH-oxidation 3-fold. The increased affinity towards R- -ionone due to the A82W mutation in combination with a more productive binding mode therefore may explain the increased coupling efficiency For M11 the introduction of L437N did not increase coupling efficiency of C-3 hydroxylation of both enantiomers, Table 2. However, the rate of both NAPDH oxidation and product formation was increased almost 3 to 4 fold. The higher affinity to M11 L437N variant and the higher stereoselectivity when compared to M11 are indicative to a different, more productive, binding mode due to the L437N mutation. For S- -ionone, the A82W mutation in M01 led to approximately 1.7 fold increase in rate of NADPH oxidation and 2.7 fold increase in the rate of product formation, resulting in an increased coupling efficiency of 44%. For S-enantiomer all four variants formed predominantly trans-OH- -ionone with M01 A82W exhibiting the highest diastereoselectivity (95%).


The mutant producing the highest amounts of (3R,6S)-3-OH- -ionone was found to be M11 L437N V87F, which also showed an aselective product profile with racemic- -ionone. This mutant produced (3R,6S)-OH- -ionone with 40% selectivity Interestingly, wild-type P450 BM3 also produced about 36% (3R,6S)-OH- -ionone but with a much lower activity compared to the BM3 mutant M11 L437N V87F. Apparently, the presence of phenylalanine at position 87 seems to increase the amount of (3R,6S)-OH- -ionone produced. Although this mutant still shows only moderate selectivity for the formation of this product, it could be a good starting point to improve the stereoselectivity by directed evolution. Docking studies

Molecular docking studies were performed in order to rationalize the basis for the experimentally observed regio- and stereoselectivity of hydroxylation of -ionone enantiomers. Figure 6 shows representative binding poses as observed in our docking studies on the binding of (R)- and (S)- -ionone to M01 A82W and M11 L437N. In case of mutant M01 A82W, the majority of obtained binding poses for both (R)- and (S)- -ionone was found in a position suitable for C-3 hydroxylation, i.e. C-3 was found within 6 Å from the heme iron catalytic center, Figure 6A and 6B (50) in line with the experimental observation that C-3 is the dominant site-of-metabolism The introduction of 82W substantially reduces the size of the binding cavity, which results in directing the substrate closer to the heme group and in an increase in activity, in line with previous suggestions based on a study on the hydroxylation of palmitate by a A82W mutant of BM3 (59). Also among the docking poses obtained for M11 L437N in which the substrate was positioned within the catalytic cut-off, C-3 was found to be in close proximity of the heme iron atom (Figure 6C and 6D). Based on the poses in which C-3 was found to be the carbon atom closest to the heme iron, the experimentally observed regioselective hydroxylation at C-3 can be explained by the formation of a hydrogen bond between the carbonyl oxygen and active site residue Ser72, which seems to anchor the ligand such that C-3 points toward the heme catalytic center, see Figure 6. Interestingly, substitution of alanine by tryptophan has been shown to affect the regioselectivity (38) and along with hydrophobic substitutions at position 72 to affect stereoselectivity of hydroxylation of larger substrates like steroids (60) -ionone by Ser72 is

-ionone by His94 upon binding to CYP109D1 as observed in a recent docking study of Khatri et al., (32) and by Thr285 as observed in a recent docking study by Ly et al. (33) -ionone binding to CYP264B1.

CHAP

TER

2

Tabl

e 2.

-io

none

ena

ntio

mer

s by

wild

-typ

e P4

50

BM3,

mut

ants

M01

A82

W, M

11 L

437N

and

thei

r res

pect

ive

prec

urso

rs M

01 a

nd M

11

[a] R

ates

of N

ADPH

oxi

datio

n w

ere

mea

sure

d ov

er 3

0 s a

t 340

nm

as n

mol

NAD

PH co

nsum

ed/m

in/n

mol

enz

yme.

The

reac

tion

cont

aine

d pu

rifie

d P4

50 B

M3

(200

nM

), N

ADPH

(20

0 μM

) an

d R-

or

S--io

none

(20

0 μM

) in

met

hano

l (2%

) an

d po

tass

ium

pho

spha

te b

uffe

r (1

00 m

M, p

H 7

.4).V

alue

s ar

e ca

lcul

ated

from

3 in

depe

nden

t ex

peri

men

ts a

nd e

xpre

ssed

as m

ean±

S.D.

[b

] Rat

es o

f pro

duct

form

atio

n w

ere

mea

sure

d ov

er 3

0 s a

s nm

ol to

tal p

rodu

ct fo

rmed

/min

/nm

ol e

nzym

e un

der s

ame

cond

ition

s as a

bove

. [c

] Cou

plin

g ef

ficie

ncy

is th

e ra

tio o

f tot

al a

mou

nt o

f pro

duct

s to

the

amou

nt o

f NAD

PH co

nsum

ed e

xpre

ssed

in p

erce

ntag

e [d

] Dis

soci

atio

n co

nsta

nts (

μM) w

ere

dete

rmin

ed b

y UV

-VIS

spec

tros

copy

usi

ng 1

μM

enz

yme

in 1

00 m

M p

otas

sium

pho

spha

te b

uffe

r, pH

7.4

, titr

ated

with

a so

lutio

n of

ei

ther

(R)-

or (

S)-

-iono

ne d

isso

lved

in m

etha

nol.

All s

ubst

rate

s pro

duce

d a

type

I bi

ndin

g sp

ectr

um w

ith a

pea

k at

390

nm

and

trou

gh a

t 420

-422

nm

.

R-

-iono

ne

S--io

none

Wild

ty

pe

BM3

M01

M

01

A82W

M

11

M11

L4

37N

Wild

type

BM

3 M

01

M01

A8

2W

M11

M

11

L437

N

NAD

PH o

xida

tion

[a]

31±5

.6

260.

9±7.

7 83

±4.9

12

8.6±

5.5

393±

21.0

29

.1±2

.7

124±

16.0

22

1±6.

4 51

.7±3

.1

210±

12

Prod

uct

form

atio

n[b]

1.4±

0.6

38.4

±3.4

35

.2±2

.7

58.5

±5.1

15

2±8.

3 1.

9±0.

9 36

.8±2

.0

97.8

±8.2

20

.1±1

.7

85.5

±9.3

Co

uplin

g ef

ficie

ncy[c

] 4.

1±0.

5 14

.8±1

.7

42.3

±1.6

45

.6±3

.0

38.6

±4.3

6.

5±0.

1 30

.1±4

.3

44.2

±3.6

38

.7±4

.2

40.7

±2.6

Di

ssoc

iatio

n co

nsta

nt[d

] 17

.5±2

.5

13.5

±2.1

3.

8±0.

5 17

.6±3

.0

7.4±

0.4

10.3

±3.1

11

.6±0

.9

0.5±

0.06

27

.5±2

.0

7.5±

1.1


When docking either (R)- or (S)- -ionone into M01 A82W, binding poses were predominantly obtained in which the C-3-bound trans-hydrogen is oriented toward the heme iron atom (Figure 6A and 6B, respectively). Such binding poses will be referred to as “trans-orientated” binding poses. Experimentally, more than 90% of the products were found to be hydroxylated at the trans position. These findings suggest that the observed binding orientation (Figure 6A and 6B) directs the stereoselectivity of the hydroxylation process, in which the substrate is oxidized by the activated iron-oxo ferryl species (61). When docking (S)- -ionone into M11 L437N, predominantly trans-orientated binding poses were obtained as well (Figure 6D), but interestingly, only for (R)- -ionone, a number of docking poses were found in which the cis-hydrogen was found closest to the heme iron atom (Figure 6C). This offers a possible explanation for the experimental finding that hydroxylation at C-3’s cis-position was observed for this substrate by M11 L437N. For M01 A82W, the (R)-enantiomer is oriented with C-3’s trans-hydrogen closest to the heme iron atom, due to a steric conflict of the two methyl groups bound to C1 and the bulky tryptophan side chain at the 82 position (Figure 6A), whereas M11 L437N (lacking this bulky side chain) leaves more space in the active site, making it possible for the double methyl groups to point towards the residue-82 side of the cavity as seen in Figure 6C. For the (S)-enantiomer, no such direction of the binding orientation to prevent sterical hindrance is observed, as seen in Figure 6B and 6D. The role of A82W mutation was experimentally further confirmed by incubating P450 BM3 mutant M11 L437N A82W with R- -ionone. In line with the above discussion, the addition of A82W to M11 L437N was found to substantially decrease the stereoselectivity and cis-hydroxylation at C-3 position of R- -ionone, see Figure 5. In conclusion, these docking studies support the observations that residue 82W plays

-ionone enantiomers by M01 A82W and M11 L437N. The presented docking studies used a wild-type BM3 crystal structure (PDB code 1BU7) as structural template for the mutants and did not explicitly account for the role of possible differences in active-site conformations and water molecule orientations, which may affect docking outcomes (62-65). As a first exploration of the substrate binding orientation. Currently, the structural analysis of the binding of (R)-and (S)- -ionone to BM3 mutants such as M01 A82W and M11 L437N is extended towards a more rigorous study using molecular dynamics simulations, thereby allowing inclusion of the effects of protein plasticity (64, 66) and water- mediated protein-substrate interactions (67), to make possible a more detailed

-ionone binding process.

CHAPTER 2

Figure 6. Binding poses of (R)- -ionone (depicted in green in A and C) and of (S)- -ionone (depicted in magenta in B and D) in mutant M01 A82W (A and B) and M11 L437N (C and D), as obtained from molecular docking studies. Selected amino acid residues within the binding cavity of the mutants, and the catalytic active heme moiety are depicted as well. The substrate’s main site-of-metabolism (C-3) is marked by a sphere. Hydrogen atoms are omitted, except for the C-3 bound hydrogens, which are shown in small spheres. Cis-hydrogens are depicted in grey, and trans-hydrogens are depicted in orange. In addition, the hydrogen atom at the stereo-center (C-6) are explicitly shown, in small grey spheres, to highlight which stereoisomer is depicted. The blue thin line represents the hydrogen bond formed between the side-chain

-ionone; the dashed line represents distances (in Å) of C-3 toward the heme iron atom. Conclusion

The present s -ionone can be hydroxylated by P450 BM3 mutants with high regio- and stereoselectivity along with high turnover numbers, which makes them an attractive option for the green production of diastereomers of 3-hydroxy- -ionone. This is the first time that such stereoselectivity

-ionone hydroxylation by P450 BM3 mutants has been reported. Moreover, the formation of (3S,6R)-OH- -ionone by M11 L437N (90% de) exhibits the ability of engineered P450 BM3 to produce cis-3-OH- -ionone. While initial docking results yield some insights into the regio- -ionone


hydroxylation and the role played by the A82W mutation in M01 A82W, extensive free energy calculations and binding affinities of the individual enantiomers to M01 A82W and M11 L437N are needed to further understand the basis of stereoselectivity. Acknowledgements This work was financially supported by the Netherlands Organization for Scientific Research (NWO) within the IBOS (Integration of Biosynthesis and Organic Synthesis) program

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Supplementary Information: Table S1. Forward primers used for site directed mutagenesis

Mutation Oligonucleotide sequences (5´-3´) Templates

A82Y GT CAA GCG CTT AAA TTT GTT CGC GAT ATT TAC GGA GAC GGG

M11 L437N M11 L437S

GT CAA GCG AAT AAA TTT GTT CGC GAT TTT TAC GGA GAC GGG M01 L75N A82W GT CAA GCG CTT AAA TTT GTT CGC GAT ATT TGG GGA GAC GGG M11 L437N

V87F GCA GGA GAC GGG TTA TTC ACT AGT TGG ACG CAT M11 L437N M11 L437S

V87I GCA GGA GAC GGG TTA ATC ACT AGT TGG ACG CAT M11 L437N M11 L437S

L437E

C TAC GAG CTC GAT ATT AAA GAG ACT GAG ACG TTA AAA CCT GAA GGC TTT GTG G M01

Chapter 3

BIO-SYNTHESIS OF STEROID METABOLITE BY AN ENGINEERED RHODOCOCCUS ERYTHROPOLIS

STRAIN EXPRESSING A MUTANT CYTOCHROME P450 BM3 ENZYME

Adapted from Harini Venkataraman*, Evelien M. te Poele* , Kamila

, Robert van der Geize, and ( )

Synthesis of keto-steroid metabolite in Rhodococcus

Abstract

Rhodococcus erythropolis

biotransformation of a 17-

to form 16- - R. erythropolis

-

formation is P450 R. erythropolis is

CHAPTER 3

Introduction

(1).

(2-4). -- and

or stereo (5, 6).

(7). -

(8). Bacillus megaterium,

(9, 10). Protein -

for different regio- and stereoalkenes - (10).

(11-13) Engineered P450

(14). -

(15). Escherichia coli (16-21), also

-(22-27).

Rhodococcus

Rhodococcus

(28, 29). Rhodococcus strains are -kn

(30, 31). (32, 33).

Rhodococcus e


of Rhodococcus

(31).

Rhodococcus erythropolis strain SQ1 3-ketosteroid 1-

- -degradation (34-37). R. erythropolis SQ1, -androstene-3,17-

kstD, kstD2 and kshA1 steroid ring opening (36). of R. erythropolis, n

- (38, 39). erythropolis

Experimental Section Materials

All mentioned. Bacterial strains and growth conditions Rhodococcus erythropolis (40)

kstD kstD2 kshA1 van der Geize, et al. . Pre- Rhodococcus

(41)Rhodococcus

CHAPTER 3

Expression and purification of BM3 mutant M02

E. coli -

(38). Determination of dissociation constant and coupling efficiency

-

(42).

Product isolation and NMR analysis

Large-norandrostenedione

- -

- semi-

preparative mm i.d.) from

norandrostenedione and metabolites. Metabolites

H

-d4. 1H- and 1H-1H-

operating at 500.13MHz.. S -

-of-


.

Cloning and heterologous expression of P450 BM3 mutant M02 in R. erythropolis strain RG9

-amplified - - Nco -

XhoNcoI/XhoI and liga NcoI/XhoI sites of pTip- (43)

- R. erythropolis (37).

In vivo biotransformation of norandrostenedione

In vivo

600 of 16-(43)

norandrostenedione. Representative -MS

Results and discussion In vitro biotransformation of norandrostenedione by P450 BM3 M02 using purified P450 BM3

m/z -

CHAPTER 3

1H NMR and Figure

2 -

minal to H-16 (44). Figure 1. in vitro biotransformation of

B -15 proton single H-16. -16.

- -16 (Figure 1

–(39)assigned as 16- - (Figure 2).

4.0 3.5 3.0 2.5 2.0 1.5 1.0F2 Chemical Shift (ppm)

1.0

1.5

2.0

2.5

3.0

3.5

4.0

F1

Ch

em

ica

l Sh

ift

(pp

m)

15/16

15/16


Figure 2. In vitro .

-VIS

eme Figure 3).

Figure 3. -

OCH3

O

OCH3

O

OHP450 BM3 MO2

Norandrostenedione 16- -OH-norandrostenedione

CHAPTER 3

d

Table 1

l -(45).

Table 1. Invitro

[a]

7.4) [b] over 6

/

-

- Invivo biotransformation of NAND in Rhodococcus erythropolis wildtype and RG9 cells

Figure 4 R. erythropolis strain

d strain. -MS of a representative sample a mono-

e R. erythropolis 3-keto-steroid - and 3-keto-steroid

(31) adati s are be -OH- and 1,4-estradiene-3,17-dione ( ) . After

-OH-

in vitro R. erythropolis P450-mediated steroid biotransformation in vivo ontrol

- Figure 5A

Enzyme NADPH oxidation[a]

Product formation[b]

Coupling efficiency Dissociation constant (Kd ) [c]

355.4 .4 .1


Figure 6).

R. erythropolis -

Figure 4. norandrostenedione R. erythropolis.

dotted lines

- R. erythropolis -

i.e. P450 protein expression in E. coli

Figures 5B & 5Cin vivo same mono- - -

in vitro -

16- -

CHAPTER 3

Figure 5. Representative H (A) Profile of Rhodococcus

on. 16- -norandrostenedione (16- -OH-P450-


Figure 6. norandrostenedione R. erythropolis ) and R. erythropolis -

35 g/L of 16- - not observed after (Figure 6).

ivated. formation 16- -OH

- s. R.

Erythropolis -OH-

degraded (46) -ketosteroid 1- - -

(47, 48). - - - -NAN .

CHAPTER 3

Figure 7. or Rhodococcus strain and

Rhodococcus total of

s. E. coli is as a it P450s

for E. coliof steroids is not (14). for Rhodococcus

(30). T , in Rhodococcus

organism.

-keto-steroid, norandrostenedione as

O

OCH3 O

OCH3

O

OCH3

OH

4-estrene-3,17-dione (NAND)

1,4-estradiene-3,17-dione (NANDD)

9 -hydroxy-4-estrene-3,17-dione (9OH-NAND)

KSTD2

KSTD1

metabolic block in strain RG9

O

OCH3

OH

Heterologously expressed P450 BM3 M02 in RG9

16 -hydroxy-4-estrene-3,17-dione (16OH-NAND)

Wildtype strain

KSHm/z 273

m/z 271

m/z 289

m/z 289


R. erythropolis. ered Rhodococcus

degradation seems to be a promising formed.

Acknowledgements

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(43) Nakashima, N. and Tamura, T. rolling- -plasmid from Rhodococcus erythropolis - -protein expression. Appl Environ Microbiol 70, 5557-

(44) Kirk, D. N., Toms, H. C., Douglas, C., White, K. A., Smith, K. E., Latif, S. and Hubbard, R. W. P. -Field H-1- -

- Journal of the Chemical Society-Perkin Transactions 2, 1567-

(45) Li, Q. S., Ogawa, J., Schmid, R. D. and Shimizu, S. -3 for Appl Environ Microbiol 67, 5735-

(46) es. Dissertation, University of Groningen.

(47) Knol, J., Bodewits, K., Hessels, G. I., Dijkhuizen, L. and van der Geize, R. -Keto-- -

Biochem J 410 -346. van der Geize, R., Hessels, G. I., Nienhuis-Kuiper, M. and Dijkhuizen, L.

- --

Appl Environ Microbiol 74 -

Chapter 4

CYTOCHROME P450 BM3 MEDIATED STEREOSELECTIVE HYDROXYLATION OF

TESTOSTERONE Adapted from Harini Venkataraman, Stephanie B.A. de Beer, Laura A.H van Bergen,

Nick van Essen, Daan P. Geerke, Nico P. E. Vermeulen, and Jan N. M. Commandeur (2012) Chembiochem, 13, 520-523.

Stereoselective C-16 hydroxylation of testosterone

Abstract

Screening of a minimal mutant library revealed a cytochrome P450 BM3 variant M01 A82W S72I capable of producing -OH-testosterone. Remarkably, a single active site mutation S72I in M01 A82W reversed the stereoselectivity of hydroxylation from

-OH selective variant M11 V87I also resulted in similar inversion of stereoselectivity. Thus engineered BM3 are

-face of testosterone D-ring with >90 %(e.e).

CHAPTER 4

Introduction

CYP102A1, commonly known as cytochrome P450 BM3 is a well known fatty acid hydroxylase from Bacillus Megaterium with one of the highest hydroxylation activities ever reported for a P450 (1). Although the natural substrates of P450 BM3 are long chained fatty acids, its substrate specificity has been broadened by site directed and random mutagenesis to a wide variety of other substrates (2-4). Moreover, engineered P450 BM3 mutants have gained importance over the years for their potential usage as biocatalysts for production of several pharmaceutically relevant human metabolites (5-7). Among such metabolites are hydroxylated steroids which are used for various therapeutic purposes. Chemical synthesis of specific hydroxylated variants of steroids are difficult as it requires multistep synthetic pathways and may yield a lot of side products (8). Steroid hydroxylation by microbial biotransformation has gained importance through the years. Several microbial strains and isolated P450s have been reported to hydroxylate steroids at different positions (9-12). Engineered P450 BM3 variants have also been described as biocatalysts for steroid hydroxylation with different regioselectivities on A or D ring. Since there are multiple positions at which steroids can be hydroxylated, they are ideal probes to study the effects of active site mutations on regio- and stereoselectivity. The first BM3 variant to show testosterone (henceforth abbreviated as TES) metabolism was a triple mutant (R47L, L188Q F87V), constructed by rational design and produced 3 mono-hydroxylated metabolites (13) - - -OH TES. Recently, it was shown that the residue at position 87 of a drug metabolizing enzyme M11 has a strong effect on the regioselectivity of TES hydroxylation (14). BM3 variant M11 V87I selectively hy (84%) whereas M11 V87A was

More recently, a library of BM3 mutants evolved by iterative saturation mutagenesis

positions (15). Other studies show that BM3 mutants can hydroxylate steroids by combinatorial alanine substitutions in the active site (16). We have also shown that restriction of active site of BM3 mutant M01 by replacing an alanine at 82 position with tryptophan resulted in selective hydroxylation of TES on D- (17). However, D-ring hydroxylation of TES by P450 BM3 mutants has been reported

-face until now. The regio- or stereoselectivity of the P450 mediated oxidation reactions depend upon the orientation of the substrate relative to the reactive iron-oxo species which is determined by the active site configuration of the P450 enzyme (18). Small variations in the active site of P450s could alter or improve


the substrate scope, regio- or stereoselectivity, activity and coupling efficiency (19). It is therefore of great interest to modify the active site of cytochrome P450 enzymes in order to create novel hydroxy steroids. Materials and methods

Materials Testosterone was purchased from Sigma. Rat liver microsome cytochrome CYP2C11 was purchased from BD Biosciences, The Netherlands. All other chemicals were of analytical grade and purchased from Sigma unless otherwise mentioned. Restriction endonucleases were obtained from Westburg. Pfu polymerase and isopropyl -D-thiogalactopyranoside (IPTG) were obtained from Fermentas. Sited directed mutagenesis

Mutations at positions 72, 82, 87 and 437 were made by site directed mutagenesis using M01 and M11 as template. In general, the mutations were introduced in the corresponding templates in pBluescript II KS(+) vector by the QuickChange mutagenesis protocol using the forward primers mentioned in Table 1. The reverse primers were exactly complementary to the forward primers. After mutagenesis, the presence of right mutations were verified by DNA sequencing (Service XS, Leiden, The Netherlands).The whole gene was subcloned to pET28a+ vector using BamHI and EcoRI sites and later transformed to BL21 (DE3) cells for expression. Table 1. Oligonucleotide sequences

Target sites Oligonucleotide sequences

72 5’- CGCTTTGATAAAAACTTAXXXCAAGCGCTTAAATTT -3’ a

82 5’-GTCAAGCGCTTAAATTTGTTCGCGATTTTTGGGGAGACGGG-3’ (W)

87 5’-GCAGGAGACGGGTTAXXXACTAGTTGGACGCAT-3’ b

a XXX represents codon for hydrophobic and charged substitutions ( Ile ATT, Val GTT, Asp GAT, Glu GAA)

b XXX represents codon for hydrophobic substitutions (Ala GCC, Ile ATC, Val GTG, Phe TTC).Mutations at position 437 were constructed using primers described in (20)

Expression and purification of BM3 mutants

BM3 mutants were grown on a large scale and purified as follows: 600 mL terrific broth medium with 30 μg/mL kanamycin was inoculated with 15 mL of overnight culture. The cells were grown at 37 °C and 175 rpm until the OD600 reached 0.6.The

CHAPTER 4

protein expression was then induced by the addition of 0.6 mM IPTG. The temperature was lowered to 20°C and 0.5 mM of the heme precursor delta aminolevulinic acid was added. Expression was allowed to proceed for 18h. Cells were harvested by centrifugation (4600 g. 4°C, 20 min) and the pellet was resuspended in 20 mL KPi-glycerol buffer (100 mM potassium phosphate [KPi] pH 7.4, 10% glycerol, 0.5 mM EDTA, and 0.25 mM DTT). Cells were disrupted using a French press (1000 psi, 3 repeats), and the cytosolic fraction was separated from the membrane fraction by ultracentrifugation of the lysate (120,000 g, 4°C, 60 min). Then the enzymes were purified using Ni-NTA agarose (Sigma). To prevent aspecific binding, 1 mM histidine was added to the cytosolic fraction. 3 ml of Ni-NTA slurry was added and the cytosolic fraction was equilibrated at 4°C for 2 hours. The Ni-NTA agarose was retained in a polypropylene tube with porous disc (Pierce,Rockford,USA) and was washed 4 times with 4 ml Kpi-glycerol buffer containing 2 mM histidine. The P450 was eluted in 10 mL Kpi-glycerol containing 200 mM histidine. The histidine was subsequently removed by repeated washing with Kpi-glycerol buffer in Vivaspin 20 filtration tube (10,000 MWCO PES, Sartorius) at 4000 g until the histidine concentration was below 250 nM. Testosterone metabolism by P450 BM3 mutants and CYP2C11

For the UPLC based library screening, 200nM of cytosolic fraction of BM3 variants was incubated with 0.5 mM testosterone in a total volume of 250 μL in 100 mM potassium phosphate buffer (pH 7.4). The reaction was initiated by the addition of 25 μL NADPH regeneration system containing 2 mM NADPH, 3 mM glucose 6-phosphate and 4 U/mL of glucose 6-phosphate dehydrogenase. The reaction was allowed to proceed for 1 hour at 24°C and then stopped with 250 μL of ice-cold methanol. Based on the library screening, for selected mutants (M01 A82W ,M01 A82W S72I, M01 A82W S72V, M11 87I and M11 87I S72I) incubations were performed with purified enzymes at a concentration of 500 nM containing 0.5 mM testosterone in a total volume of 250 μL in 100 mM potassium phosphate buffer (pH 7.4). The reaction was initiated by the addition of 25 μL NADPH regeneration system as described above. Incubation of testosterone with CYP2C11 was performed at an enzyme concentration of 10 pmol with 0.5 mM testosterone in a total volume of 250 μL buffer. The reaction was allowed to proceed for 30 minutes at 37°C and then stopped with 250 μL of ice-cold methanol. Samples were centrifuged for 10 min at 14,000 rpm, after which supernatants were transferred to a 96 well plate. Testosterone and metabolites were analyzed by high-resolution UPLC(21) using an Agilent 1200 system using a Zorbax Eclipse XDB-C18 column (1.8 μm, 50 mm x 4.6 mm; Agilent, USA). Metabolites and the substrate were eluted at a flow rate of 1 mL/ min using an isocratic flow of methanol/water


(60:40).The column temperature was maintained at 40°C. UV detection was done at 254 nm. LC-MS and GC-MS analysis of new metabolite

Representative samples from incubation of testosterone with M01A82W S72I were analyzed using LC-MS/MS. The metabolites of testosterone were seperated by reversed-phase liquid chromatography. A Luna 5 m C18 column (150 mm × 4.6 mm i.d.) from Phenomenex was used as the stationary phase, protected by a 4.0 mm × 3.0 mm i.d. security guard (5 m) C18 guard column (Phenomenex, Torrance, CA.). An isocratic flow of 60:40 MeOH/water (0.1% formic acid) was used as mobile phase. For the identification of metabolites, an Agilent 1200 Series Rapid resolution LC system was connected to a hybrid quadrupole-timeof- flight (Q-TOF) Agilent 6520 mass spectrometer (Agilent Technologies,Waldbronn, Germany), equipped with an electrospray ionization (ESI) source and operating in the positive mode. The MS ion source parameters were set with a capillary voltage at 3500V; nitrogen was used as the desolvation and nebulizing gas at a constant gas temperature of 350°C; drying gas, 8 L/min; and nebulizer, 40 psig. Nitrogen was used as a collision gas with collision energy of 25 V. MS spectra were acquired in full scan analysis over an m/z range of 50-1000 using a scan rate of 1.003 spectra/s. The MassHunter Workstation Software (version B.02.00) was used for system operation and data collection. For GC-MS analysis, samples from incubation of M01 A82W S72I with testosterone were extracted with ethyl acetate and the organic layer was analyzed using a Hewlett-Packard 6890 series with ATAS Programmable Injector 2.1. Samples were separated on a DB-5 GC-column (30 m x 0.25 cm). Ionization was done by electron impact (EI) with the mass selective detector 5973. The injector temperature was maintained at

ramp at 20 -1 Large scale incubation and NMR analysis

Large-scale incubations were performed with 1 μM of mutant M01 A82W S72I, 500 μM of testosterone in a volume of 25 mL in 100 mM potassium phosphate buffer (pH 7.4). The reaction was initiated by addition of 2.5 mL NADPH regenerating system containing 2 mM NADPH, 3 mM glucose 6-phosphate and 4 U/mL glucose 6-phosphate dehydrogenase. The reaction was allowed to proceed for 6 hours and then the products were extracted by using 2 x 50mL dichloromethane. The organic layers were collected in a round-bottom flask and evaporated using a rotary evaporator. The dried

CHAPTER 4

product was dissolved in 1 mL methanol and the sample was applied by manual

mm x 100 mm i.d.) from Phenomenex . An isocratic flow rate of 2 mL/min of eluent containing 60% methanol/ 40% water was used to separate testosterone and metabolites. Metabolites were detected using UV detection (254 nm) and collected manually. Collected fractions were first analyzed for purity by UPLC method as described above. The samples were evaporated to dryness under nitrogen stream and dissolved in 500 μL of methyl alcohol-d4.Structural identification of the new metabolite was performed by a combination of 1D-1H, 1H-1H-COSY and 1H-1H-NOESY NMR experiments. NMR spectra were recorded at room temperature using on Bruker Avance 500 (Fallanden, Switzerland), equipped with cryoprobe at 500.13MHz. Computational Methods MD simulation and docking settings

A combined MD/docking study as previously described for mutants M01 and M01 A82W in reference 8 was performed on the orientation of testosterone to the heme domain of BM3 mutant M01 A82W S72I solvated in water. The starting structure for MD simulations of mutant M01 A82W S72I was modeled based on the M01 A82W coordinates taken from reference 8, using the molecular modeling software MOE (Molecular Operating Environment)(22). The side chain of 72I was manually introduced using MOE’s builder tool such that it had minimal overlap with surrounding residue atoms. After introduction of the mutation S72I, polar and aromatic hydrogen atoms were added and the mutant’s structure was energy minimized in vacuo. Energy minimizations were carried out using the GROMOS05 biomolecular simulation package(23, 24) and the GROMOS force field parameter set 45A4(25). Subsequently, the protein was solvated in a periodic rectangular box, containing 18,176 SPC water molecules(26), with a periodic box volume of 8.6 x 8.6 x 8.6 nm, using a minimum solute to wall distance of 0.8 nm, and 15 Na+ ions to neutralize the system’s charge. The force-field parameters that were used to describe the BM3 protein and sodium ions were taken from the GROMOS parameter set 45A4(25). In a first set of (equilibration) MD simulations, the system was gradually heated up to 298 K, in seven separate simulation steps of 20 ps each (at 50, 100, 150, 200, 250 and 298 K, respectively), in which the volume of the system was kept constant. In the first five simulations, position restraints were applied on the protein atoms using a gradually decreasing force constant (of 25000, 2500, 250, 25 and 2.5 kJ


mol-1 nm-2, respectively) for the harmonic restraints. After the equilibration process, a production MD simulation at 298 K with a total length of 3 ns of the systems followed. In the production MD simulations, no position restraints were applied, and the pressure was kept constant to a target value of 1 atm, using a Berendsen barostat with a coupling time of 0.5 ps. The isothermal compressibility was set to 4.575 x 10-4 (kJ mol-1 nm-3)-1. In all simulations, a time step of 2 fs was used, and the temperature was kept constant at its target value by using a Berendsen thermostat with a coupling time of 0.1 ps. Bonds were kept constrained using the SHAKE algorithm(27), with a geometric tolerance of 10-4. At every time step, nonbonded interactions were calculated up to a distance of 0.8 nm, by means of a pair list, generated every five time steps. At every fifth time step, intermediate-range interactions at distances up to 1.4 nm were calculated and kept constant. A reaction-field contribution was added to the energies and forces, to account for the electrostatic interactions outside the cut-off radius of 1.4 nm, with a dielectric permittivity of 61. During the production run, energies were stored every 250 time steps (0.5 ps) for analysis. After the 3 ns MD run of the proteins, 1500 coordinate frames were generated by writing out protein coordinate files every 1000 time steps (2 ps), which were used as protein template structures in further docking studies (after adding aliphatic hydrogens using the corresponding tool in MOE). Molecular structures of the substrate testosterone were used for individual docking runs as described previously (28). Note that two distinct molecular conformations can be distinguished for testosterone: a ‘flat’ conformation and a ‘bent’ conformation, characterized by a trans- or gauche-orientation of the dihedral angle defined by atoms C2, C1, C10 and C9, respectively. In both conformations, the B and C ring are in a chair conformation. Combining the 2 x 4 different testosterone orientations and the 1500 protein structures obtained from the MD simulation of M01 A82W S72I, testosterone was docked into the protein active-sites using the GOLD (Genetic Optimization for Ligand Docking)(29) software, version 4.0, and the Chemscore scoring function(30) optimized for heme groups (31). The center point for docking was defined by the mirror image of the sulfur atom of Cys400 on the opposite site of the heme iron, from which the docking-active radius was set to 1.5 nm to define the active-site of the protein. A maximum of 50000 operations in the GOLD genetic algorithm were performed, using a population of 100 genes. After every docking simulation, (maximally) 50 of the highest-ranked obtained poses were retained for further analysis. In total, circa 50,000 protein-TES complexes were thus obtained and were used as input for post-processing analysis. Obtained binding poses were collected for which C16 was the carbon atom in closest proximity

CHAPTER 4

to the catalytic heme iron, after discarding the poses for which C16 was positioned more than 0.7 nm away from the heme iron center (which is slightly larger than the hypothetical cut-off of 0.6 nm for catalytic activity(32)) or closer than 0.3 nm to the heme iron (considering that accommodation of a reactive oxygen molecule should be possible). Binding constant determination by optical spectroscopy

The binding of testosterone to P450 BM3 mutants M01 A82W and M01 A82W S72I was analyzed by optical titrations using a Shimadzu UV-2501PC spectrophotometer (Shimadzu Duisburg, Germany). The spectra for the substrate- M) were recorded at 24 °C in 1 mL of 100 mM potassium phosphate buffer (pH 7.4) prior to addition of testosterone. Increasing amounts of testosterone dissolved in methanol were added (2 - than 2% of the total volume of the solution. Spectra were recorded after each addition of substrate, and a difference spectrum was computed by subtraction of the starting (substrate-free) spectrum from those generated at each point in the titration. The maximum apparent absorption ch 392-

427 412-432) for M01 A82W S72I was determined by subtraction of the minimum absorption value at the trough in each difference spectrum from the maximum value at the peak and these values were plotted versus corresponding testosterone concentrations. Data were analyzed by nonlinear regression using GraphPad Prism 4 (GraphPad Sotware, San Diego, CA, USA). Results and Discussion Minimal library creation

In our previous work, we provided structural rationalization for the observed differences in regioselectivity of TES hydroxylation by BM3 mutants M01 and M01 A82W (28). During this in silico rationalization study, key active site residues (72, 82, 87, and 437) were identified which may have an effect on the orientation of TES in the active site. In the present study, a minimal library consisting of 25 combinatorial single and double mutants was constructed with substitutions at these key active site residues based on M01 and M11 (33) as templates. Since BM3 mutants capable of hydroxylation at the A or D ring on the beta face with high selectivities have been reported before (15, 34), the goal of this screening was to identify mutants with

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CHAPTER 4

altered regio- and stereoselectivities towards TES hydroxylation. All the BM3 variants were successfully expressed and showed a P450 peak as determined from CO difference spectrum. UPLC based screening of the mutant library revealed that most of

- - and -OH testosterone in different product ratios. The screening results depicting the

differences in selectivities are shown in supplementary material Figure 1. Interestingly two mutants from the library produced significant amount of a new metabolite not reported before for P450 BM3 mediated testosterone hydroxylation (35). Identification of the new metabolite

The new metaboli - and 16 -OH testosterone under the conditions described in Methods section. LC –MS analysis of the metabolite indicated that it was mono-hydroxylated metabolite with an [M+H]+ ion at m/z 305. Moreover, the characteristic ions m/z 97, m/z 123 as observed from MS/MS spectrum were indicative of D-ring hydroxylation. Rat liver microsomes P450 2C11 is known to produc - -OH metabolites with testosterone (36). As an additional confirmation, UPLC analysis showed that the newly isolated metabolite and 16- -OH-TES produced by rat liver microsomes co-eluted as shown in Figure 2. The pure metabolite collected by preparative HPLC was also analyzed by GC-MS which gave a highly reproducible fragmentation pattern comparable to previous report (37). Based on these observations, the new metabolite was tentatively assigned to be 16- -OH-testosterone. In order to confirm the structure unequivocally, large scale incubations were performed to isolate sufficient amount of metabolite for NMR analysis. The identity of the metabolite was confirmed by 1 0.81 (18-CH3) 1.23 (19-CH3 3.39 (doublet 17- with a characteristic J value 5.8) and 4.03 (16-

as shown in Figure 3. The 1H NMR chemical shifts were similar to previously reported values for 16- -OH-testosterone (38, 39). The COSY spectrum also helped to understand the site of hydroxylation better. The mutual correlation observed between

4.03 ppm confirmed that hydroxylation was at C-16 as reported previosuly (9). The combination of LC-MS, GC-MS fragmentation pattern combined with in depth NMR analysis helped to confirm that the new metabolite formed by the P450 BM3 mutant is 16- -OH-testosterone.


Figure 2. Overlay of UPLC chromatograms depicting co- -OH-T (tR 1.30 min) isolated from incubation of testosterone with BM3 mutant M01 A82W S72I and incubation of testosterone with rat liver cytochrome 2C11(36). Effect of the mutation S72I

Analysis of the mutation position revealed that two of the mutants which produced -OH-TES were based on M01 A82W and had hydrophobic substitutions at position

72. M01 A82W is a selecti produce even trace -OH-TES. The introduction of hydrophobic residues at position 72 in

M01 A82W had a profound effect on the stereoselectivity. The S72V mutation in M01 -OH-TES, 15- -OH-TES and

2- -OH-TES -OH-TES still being the major metabolite (40%). Interestingly, the introduction of isoleucine at position 72 in M01 A82W resulted in a

hydroxy TES was formed (Figure 1

(Table 2). The overall specific hydroxylation activity was lowered 2-3 fold upon the introduction of hydrophobic residues at position 72 in M01 A82W.

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CHAPTER 4

Analysis of the dissociation constants revealed that the new mutant M01 A82W S72I binds about 2.5 fold less tightly (Kd =102.4 μM) as compared to M01 A82W (Kd =42.5 μM). Titrations of testosterone with M01 A82W produced a typical type I spectrum with a peak at 392 nm and trough at 427 nm indicative of the conversion of the heme iron from low spin to high spin. On the other hand, M01 A82W S72I produced an altered type I spectra with a red shift in the peak maxima from 392 nm to 412 nm as observed in the difference spectrum (Figure 4). Previously, the binding of lanosterol to Mycobacterium tuberculosis 14- -demethylase containing an active site mutation F89H exhibited a similar shift in peak maxima from 390 to 412 nm (40). Binding of piperonylic acid to a plant P450 CYP73A1 also resulted in Soret peak maxima shift to 412 nm. Although the mechanism for such a shift is not completely elucidated yet, it may possibly indicate a ligand induced alteration in the heme protein structure (41).

[a]Testosterone hydroxylation activities were measured with 500 nM of purified P450 BM3 mutants and 0.5 mM testosterone. The values represent average calculated from 2 independent experiments with ± 5% error. Hydroxylation activity is reported as nmol product/nmol enzyme/hour. It was interesting to observe that the complete reversal in stereoselectivity was seen only in the combination of A82W and S72I mutations. Single mutants with hydrophobic substitutions at position 72 in M01 did not have a big effect on the product distribution compared to M01 itself. Such synergistic effects of P450 BM3 double mutants have been shown to affect regio- and stereoselectivity. For example, hydrophobic substitutions at positions 87 and 328 improved selectivity of terpene hydroxylation (42) and combined substitutions at 82 and 438 residues have been shown to control stereoselectivity of styrene epoxidation (43).

Table 2. Testosterone C-16 hydroxylation activity by M01 A82W and the constructed 72 position mutants.

Mutant C-16 hydroxylation activity [a] -

OH -OH - -OH

M01 A82W - 397 0 M01 A82W S72V 63 127 0.49 M01 A82W S72I 174 7 24.8


Figure 4. Spectral shifts induced by the binding of testosterone to A. M01 A82W and B. M01 A82W S72I by addition of increasing concentrations of testosterone (2 - 250 μM) dissolved in methanol. The inset shows the absorption changes plotted against the concentrations of testosterone used in titrations. For calculating Kd values, data points were fitted using Graphpad Prism software as explained in Materials and methods. Binding constants were found to be A. 42.5 μM and B. 102.4 μM

CHAPTER 4

-OH-TES selective mutant could also

produce similar change in stereoselectivity, we introduced the S72I mutation in BM3 variant M11 87I which has been shown previously to hydroxylate testosterone

(17). Interestingly, the combinatorial V87I S72I double mutation also inverted -OH-TES with >90% e.e. As also observed for M01A82W S72I, the overall hydroxylation activity was reduced approximately 3 fold in comparison with M11 87I itself. In future work, the activity of the selectiv be improved by random mutagenesis. Computational studies

Position 72 is the first residue in the B’-helix (44) and it can play an important role in substrate interactions. To our knowledge, only a few studies involving the effect of mutations at position 72 on BM3 hydroxylation activity and selectivity have been reported (45, 46). From our results, the substitution of hydrophobic amino acids in place of serine at position 72 had a significant effect on the selectivity and activity of TES hydroxylation. Prior computational studies in our laboratory suggested that active site residue serine 72 may have a potential anchoring effect on ligand binding orientation in the BM3 binding pocket by facilitating a hydrogen bond possibility with the carbonyl group of TES A-ring in BM3 mutant M01 A82W. By removing this hydrogen bonding possibility (by replacing serine by a hydrophobic residue), the preferred binding orientation of TES in BM3 mutant M01 A82W might be affected. To rationalize the observed enantioselectivity for TES hydroxylation by BM3 mutant M01 A82W S72I, in silico site-of-metabolism (SOM) prediction was performed using a combined Molecular Dynamics (MD) and ligand docking approach (as described previously) to account for the flexibility and plasticity of the protein and to identify possible orientations of TES binding in the M01 A82W S72I active site. Detailed computational studies investigating the favorable binding orientation of substrates in the P450 active site to rationalize and predict the substrate’s preferred SOM have been reported before (47). In accordance with those studies we found that in the

was observed, C16 and H16 were within a distance of 2-6 Å from the heme iron atom

and the heme iron atom (Fe-H-C angle) was in the range of 120-180°. For the obtained poses in which C16 was found to be the carbon atom closest to the heme iron atom


It

was observed that for the poses with H16 closer to Fe, a higher density for which the hydrogen is within close proximity (2-4 Å) is observed than for H16 , in line with the

Figure 5 depicts typically observed poses coconformations showing the two predominant rotamers of the I72 side chain. Poses for

-OH (in green) are found to form a steady hydrogen bond to the backbone moiety of Ala330, which is not observed for binding poses -OH (in black). Moreover, different rotamers of I72 show sterical hindrance of its side chain

and sterical hindrance with I72 offer a structural explanation of the observed

The present study shows that small differences in key substrate interaction residues in P450 BM3 can have profound effect on the stereoselectivity of TES hydroxylation. To our knowledge, M01 A82W S72I and M11 V87I S72I are the first BM3 variants

the stereoselectivity also makes it an ideal candidate for further mechanistic studies. In conclusion, these results lead to a better understanding of substrate orientation in the BM3 active site, opening up possibilities to design mutants with novel selectivities as well as scope to improve existing variants towards steroid hydroxylation. Acknowledgements

This research was financially supported by the Integrated Biosynthesis Organic Synthesis (IBOS) program of The Netherlands Organization for Scientific Research (NWO).The authors would also like to thank Frans de Kanter for NMR experiments.

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Stereoseelctive C-16 hydroxylation of testosterone

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(39) Numazawa, M. and Osawa, Y. (1978) Improved synthesis of 16alpha-hydroxylated androgens: intermediates of estriol formation in pregnancy. Steroids 32, 519-527.

Stereoseelctive C-16 hydroxylation of testosterone

(40) Bellamine, A., Lepesheva, G. I. and Waterman, M. R. (2004) Fluconazole binding and sterol

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Chapter 5

APPLICATION OF ENGINEERED CYTOCHROME P450 BM3 MUTANTS AS BIOCATALYSTS FOR THE

SYNTHESIS OF BENZYLIC AND AROMATIC HYDROXYLATION PRODUCTS OF FENAMIC ACID

NSAIDs Adapted from Harini Venkataraman, Marlies C.A. Verkade-Vreeker, Luigi Capoferri, Daan P. Geerke, Nico P. E. Vermeulen and Jan N. M. Commandeur (2014) Bioorganic and Medicinal chemistry, 22, 5613-5620.

Generation of fenamic acid NSAIDs metabolites

Abstract

Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3’-hydroxy-methyl- (1a), 5-hydroxy-meclofenamic acid (1b) and 4'-hydroxy- (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of MCFA respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000- 6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4’ position of all three drugs tested with high regioselectivity. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.

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Introduction

Meclofenamic acid (1), mefenamic acid (2) and tolfenamic acid (3) belong to the group of fenamic acid containing NSAIDs (fenamates) which are used for the treatment of pain and inflammation (1). They inhibit the production of prostaglandins formed by cyclo-oxygenase and thereby exhibit anti-inflammatory effect (2). This group of drugs is structurally similar to diclofenac, a widely studied NSAID for its idiosyncratic hepatotoxic effect. Unlike diclofenac which can undergo only aromatic hydroxylation, these compounds can undergo both P450 mediated benzylic and aromatic hydroxylation (3, 4). For example, hydroxylation on the benzylic group (3’-hydroxy-methyl) is reported to be the major P450 mediated metabolite for 2 in addition to aromatic ring hydroxylated metabolites (5). Benzylic alcohols can be bioactivated to toxic reactive intermediate catalyzed by human sulfotransferases which can react with DNA leading to genotoxicity (6). Additionally, the aromatic hydroxylated metabolites of these compounds can lead to the formation of quinone imines which are reactive towards sulfhydryl groups of proteins, and therefore can be responsible for the adverse reactions caused by these drugs (7, 8). In the case of structurally similar drug diclofenac, quinone imines have been reported to be more cytotoxic compared to the quantitatively more abundant acyl glucuronides (9). The recent regulatory guidance of Food and Drug Administration (FDA) has made it increasingly important to perform toxicological evaluation of drugs metabolites which are formed more than 10% of the parent drug systemic exposure (10, 11). Therefore, the generation of both benzylic and aromatic hydroxylated metabolites of these drugs to facilitate pharmacological and toxicological evaluation is of significant importance. Selective oxyfunctionalization of many compounds including drugs by chemical means still poses a challenge (12). Enzymes which are capable of introducing an oxygen atom in a highly regioselective manner are of high demand in organic synthesis (13). The use of engineered bacterial cytochrome P450s for the production of drug metabolites has gained importance over the years (14, 15). Among the bacterial P450s, cytochrome CYP102A1 (Cytochrome P450 BM3) from Bacillus megaterium is a well known fatty acid hydroxylase. In the last two decades, P450 BM3 was widely studied in order to expand its substrate specificity for biotechnological purposes (16). Directed evolution combined with rational and semi-rational techniques have resulted in a wide array of catalytically active and selective mutants that can be employed for hydroxylation of various compounds (17). Of particular interest is the exploitation of these monooxgenases for the production of drug metabolites (18).

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Recent studies have reported the ability of cytochrome P450 BM3 mutants to produce human relevant drug metabolites of several drugs including NSAIDs (19-21). Engineered P450 BM3 mediated hydroxylation of NSAIDs like diclofenac, ibuprofen and naproxen have been reported (22-24). In the present study, fenamic acid NSAIDs (Figure 1) have been used as examples to demonstrate the ability of P450 BM3 mutants to produce human relevant metabolites. The aim of this study is to identify P450 BM3 variants capable of selective benzylic and aromatic hydroxylation with minimal secondary oxidation of fenamic acid NSAIDs that can be used as biocatalysts for large-scale drug metabolite production. A library of catalytically diverse P450 BM3 mutants were screened with 1. Large scale incubations were performed with the selective BM3 variants and the structures of the metabolites were elucidated by NMR analysis. Application of these selective mutants towards structurally related 2 and 3 has also been studied. Docking studies were performed to get preliminary insights into the experimentally observed ring selectivity and to identify the key interactions in the active site.

Figure 1.Structure of fenamic acid NSAIDS used in this study.

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Materials and Methods Materials

Mefenamic acid, meclofenamic acid sodium salt and tolfenamic acid were purchased from Sigma (Schnelldorf, Germany). Human liver microsomes (HLM) pooled from different individual donors were obtained from BD Gentest TM (San Jose, USA) and contained 20 mg/mL protein (Cat. No. 452161). All other chemicals were of analytical grade and purchased from standard suppliers unless otherwise mentioned. Expression and purification of P450 BM3 mutants

Expression of the CYP102A1 mutants was performed by transforming competent E.Coli BL21 cells with the corresponding pET28+vectors. P450 BM3 mutant M11, M11 V87I , M11 L437N V87I and M11 V87F were grown on a large scale and purified using nickel affinity column as described previously (25). CYP concentrations were determined using carbon monoxide (CO) difference spectrum assay as described by Omura et al., (26). Metabolism of fenamic acid containing NSAIDS by engineered P450 BM3 mutants

For the biotransformation reaction, cytosolic fraction containing 150 nM of 63 different P450 BM3 mutants and wildtype BM3 was incubated with 500 μM of 1 (from DMSO stock solution), in a final volume of 250 μL in 100 mM potassium phosphate buffer. The reactions were initiated by addition of an NADPH regenerating system (final concentrations of 0.5 mM NADPH, 10 mM glucose 6-phosphate, and 0.4 unit/mL glucose-6-phosphate dehydrogenase). The reaction was allowed to proceed for 1 hour at 24 -cold methanol. The protein was removed by centrifugation for 15 min at 13000 rpm. The supernatant was analysed by HPLC method as described below. Selected mutants were incubated with 2 and 3 to evaluate the applicability of these mutants towards these fenamic acid NSAIDS using 150 nM enzyme and 500 μM drug. Human liver microsomes (HLM) incubations were also performed for 2 mg/mL HLM. The reactions were initiated as above and allowed to proceed for 1 hour at 37 C. Analytical methods

The analyses of metabolites were performed by reversed-phase liquid chromatography on a Shimadzu HPLC equipped with two LC-20AD pumps, a SIL20AC

117


mm) as stationary phase. For separation of parent compound and metabolites, a gradient method was used with two mobile phases: solvent A (0.8 % acetonitrile, 99% water, 0.2% formic acid) and solvent B (0.8% water, 99% acetonitrile, 0.2% formic acid). For 2, the first 5 min was isocratic at 40% solvent B. From 5 till 30 min, the concentration of solvent B linearly increased to 100%, followed by linear decrease back to 40% until 30.5 min. Isocratic re-equilibration at 40% solvent B was maintained until 45 min. For separation of metabolites of 1 and 3, a similar gradient was used starting with 5% solvent B instead of 40%. The flow rate was 0.5 mL/min and the detection was at 254 nm. A standard curve of respective metabolites of 2 was used to determine the concentrations of the formed metabolites of 2. For 1 and 3, the concentration of the products formed was calculated based on a calibration curve of the parent drug by taking into account a correction factor for the extinction coefficients.Representative samples were analyzed by using an Agilent 1200 Series Rapid resolution LC system connected to a time-of-flight (TOF) Agilent 6230 mass spectrometer equipped with electrospray ionization (ESI) source and operating in the positive mode. The MS ion source parameters were set with a capillary voltage at 3500 V. Nitrogen was used as drying gas (10 L/min) and nebulizing gas (pressure 50 psig) at a constant gas temperature of 350 °C. Data analysis was performed using Agilent MassHunter Qualitative analysis software. Dissociation constant determination by optical spectroscopy

The binding of fenamic acid NSAIDs to BM3 mutants was studied by optical titration using a Shimadzu UV-2501PC spectrophotometer (Shimadzu Duisburg, Germany) as described previously (27). Isolation of metabolites for NMR analysis

To obtain the oxidative metabolites of 1, 2 and 3, incubations were performed in a 5 mL reaction volume containing 250 nM purified P450 BM3 mutant, 500 μM respective fenamic acids and a NADPH regenerating system in 100 mM potassium phosphate buffer pH 7.4. The reactions were allowed to continue for 4 h at 25°C. The reactions were terminated by adding an equal volume of ice cold methanol containing 2 % (v/v) of aqueous 50 mM ascorbic acid and centrifuged for 15 min at 14000 rpm. After precipitation of the proteins, a series of 1 mL injections of the sample was applied on a semi-preparative HPLC column Luna 5 μm C18(2) (250 mm x 10 mm i.d.) from Phenomenex (Torrance, CA, USA) which was previously equilibrated with 100% eluent A. A flow rate of 1.5 mL/min and a gradient using the eluent A (0.8% acetonitrile, 99% water, and 0.2% formic acid) and B (99% acetonitrile, 0.8% water, and 0.2% formic acid) was applied for separation of formed metabolites. The first 10

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min was isocratic at 20% eluent B: from 10 to 40 min, the percentage of eluent B increased linearly to 100% followed by linear decrease back to 20% until 45.5 min. Isocratic re-equilibration at 20% solvent B was maintained until 60 min. Metabolites were detected using UV detection (254 nm) and collected by automated fraction collector. Collected fractions were first analyzed for purity and identity by the analytical HPLC and LC-MS methods as described above. The samples were evaporated to dryness under a nitrogen stream and redissolved in 500 μL of methyl alcohol-d4. 1H-NMR-analysis was performed on Bruker Avance 500 (Fallanden, Switzerland), equipped with cryoprobe. 1H-NMR measurements were carried out at 500.23 MHz. Determination of kinetic parameters

For the determination of kinetic parameters, the enzyme concentration and incubation time of the reaction were chosen in such a way that product formation was linear with time (data not shown). Incubations were performed in 100 mM potassium phosphate buffer (pH 7.4), with 150 nM purified P450 BM3 mutants. The final volume

te concentrations ranging from 3.125-(final concentrations of 0.5 mM NADPH, 10 mM glucose 6-phosphate, and 0.4 unit/mL glucose-6-phosphate dehydrogenase). All incubations were performed in duplicates. The reaction was allowed to proceed for 5 min at 24 °C and then stopped by addition

-cold methanol. The protein was removed by centrifugation for 15 min at 13000 rpm. The supernatant was analyzed by HPLC method as described above. The data were fitted with the Hill’s equation, using the Graphpad Prism5 software. Determination of total turnover numbers (TTN)

To determine total turnover numbers, 150 nM of purified enzymes was incubated with 1 mM of fenamic acid NSAIDs (2% DMSO) in a total volume of 250 μL. The reaction was initiated by the addition of 50 μL NADPH regenerating system as mentioned above. The reaction was performed in duplicate in 1.5 mL eppendorf tubes

were analyzed by HPLC as described in section 4.4. Total turnover numbers were calculated as nmol of product formed per nmol enzyme.

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Preparative scale synthesis

Large scale incubation was performed in a baffled flask with 150 nM of purified mutant M11 V87F, 12 mg of 2 (final concentration 1 mM) in a volume of 50 mL in 100 mM potassium phosphate buffer (pH 7.4addition of 5 mL NADPH regenerating system. The reaction was allowed to proceed for 6 hours and then the stopped with ice-cold methanol. The metabolites were separated by semi-preparative HPLC method as described in section 4.6. The pure fractions containing 2b were evaporated to dryness using a vacuum centrifuge to result in an isolated yield of 6.8 mg 2b. Results and Discussion Selection of P450 BM3 variants

In the present study, 78 mutants of P450 BM3 were selected for screening with 1. These variants were pre-selected based on their catalytic diversity towards a variety of drugs and steroids. Mutants M01, M02, M05, and M11 were previously constructed from a triple mutant by subsequent random-mutagenesis. Mutants M01 and M11 were used as templates for additional site-directed mutagenesis of active site residues, guided by computational modelling of the active site of P450 BM3. Positions which were selected for mutagenesis were residues 72, 74, 82, 87 and 437 which appear to be key active site residues and have profound influence on regio- and stereoselectivity of P450 BM3 mutants (20, 22, 28, 29). Oxidation of meclofenamic acid by engineered P450 BM3 mutants and structural identification of products

Wildtype P450 BM3 was tested for its ability to convert 1. Consistent with previous studies reporting about conversion of NSAIDs like diclofenac and naproxen, only very low substrate conversion (<0.5% of the parent drug concentration) was observed with wildtype BM3 and 1. On the other hand, engineered P450 BM3 mutants showed much higher substrate conversions, with distinct product profiles. For example, BM3 variant M11 was able to convert 1 to three mono-hydroxylated products 1a, 1b and 1c (m/z 312) with no particular selectivity. (Figure 2A) In order to identify the metabolites, large scale incubations of 1 with BM3 mutant M11 were carried out to generate enough material for NMR analysis. Figure 2B-D shows the aromatic region of 1H NMR of the 3 metabolites. As a characteristic feature, dichloro-ring hydroxylated

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Figure 2. Metabolism of meclofenamic acid (1) by P450 BM3 mutant M11 and structural elucidation of metabolites A. Representative HPLC chromatogram obtained after incubation of 1

(150 nM). Aromatic region of 1H NMR spectrum of the metabolites B. 1a C. 1c and D. 1b metabolites of 1 show a signal coming from H-doublet pattern with an ortho and meta coupling (Figure 2B,C). In the case of C-5 hydroxylation on the carboxy phenyl ring, the signal corresponding to H-6 shifted

Additionally, in the case of benzylic hydroxylation at the 3’ position, a characteristic singlet corresponding to –CH2 appears arThe structure of metabolite 1awas assigned based on NMR analysis to be the benzylic

121


hydroxylated product of 1 at its 3’ position. Metabolite 1a was found to co-elute with the major metabolite 3-hydroxy-methyl-meclofenamic acid (30), formed after incubation of 1 with human liver microsomes (HLM). Metabolites 1c and 1b were identified as products of aromatic hydroxylation at position C-4’of the dichloro phenyl ring and at position C-5 of carboxy phenyl ring, respectively (see Supplementary Information). These aromatic hydroxylated metabolites were also formed in HLM incubations and therefore engineered P450 BM3 can produce human relevant metabolites of 1. Selective benzylic vs aromatic hydroxylation of 1 by engineered P450 BM3 mutants

Figure 3 shows the diversity of P450 BM3 mutants towards either benzylic or aromatic hydroxylation of 1. Most of the mutants were able to convert 1 with different activities and hydroxylation selectivities.

Figure 3. Product profile and relative activity of P450 BM3 engineered variants towards meclofenamic acid (1) hydroxylation. Activity in % were measured from substrate consumption after 1 hour incubation of 500 μM of 1 with 150 nM of P450 BM3 relative to the most active mutant M11 L437N V87F. Values represent averages of 2 independent experiments with less than 10% error. Product selectivity in % was calculated from HPLC chromatograms by integrating the product peaks and represented by different shades in each bar. Representative P450 BM3 variants showing high selectivity towards benzylic hydroxylation (M11 V87I, M11 L437N V87I and variants showing high selectivity towards aromatic hydroxylation (M11 V87F, M11 L437N V87F) are highlighted.

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Wild-type P450 BM3 showed less than 0.5 % conversion of 1. Of all the tested variants, mutant M11 L437N V87F showed the highest activity with 72% selectivity for hydroxylation at the 4’position. Interestingly, mutants of M11 with mutations (either isoleucine or phenylalanine) at position 87 showed a remarkable increase in selectivity of product formation. Representative chromatograms obtained after incubation of 1 with P450 BM3 mutants M11 L437N V87I and M11 V87F are shown in Figure 4. M11 L437N V87I showed a high selectivity (83%) towards formation of 1a, whereas M11 V87F showed a high selectivity (89%) for formation of 1c. Because of the higher aromatic selectivity, mutant M11 V87F was selected for further studies. Residue 87 is located in close proximity to the propionate group of the haem and therefore affects the accessibility of the substrate to the reactive centre. Previously, it was shown that the residue at position 87 can play a critical role in substrate binding and in the change of the spin state of the haem iron, thereby possibly affecting trends in regioselectivity by P450 BM3 mutants (31).

Figure 4. Representative HPLC chromatograms showing high selectivity of meclofenamic acid hydroxylation on 3’-methyl group by BM3 mutant M11 L437N V87I (solid line) and high selectivity of hydroxylation at 4’position on aromatic ring by P450 BM3 variant M11 V87F (dotted line)

123


In terms of aromatic or benzylic hydroxylation of compounds by engineered P450 BM3, hydroxylation of xylenes have been studied and it has been previously shown that the active site architecture and the residue at position 87 can play a role in determining the preference for hydroxylation in line with our experimental observations (31, 32). Moreover, kinetic isotope effects have also been used to delineate the importance of residue at position 87 for aromatic or benzylic hydroxylation (33). Application to other structurally related NSAIDS mefenamic acid and tolfenamic acid

In order to evaluate the applicability of selective P450 BM3 mutants M11, M11 V87I, M11 L437N V87I, M11 V87F towards the metabolism of structurally similar drugs, conversion of 2 and 3 were also studied. The product selectivities are shown in Table 1. Mutant M11 converted 2 to three mono-hydroxylated metabolites (m/z 258). These three metabolites were identified by NMR to be 3’- hydroxymethyl-mefenamic acid (2a), 4’-hydroxy-mefenamic acid (2b) and 5-hydroxy-mefenamic acid (2c) which are corresponding to the regioselectivity of the metabolites formed with 1. Introduction of phenyl alanine at 87 position was found to increase the regioselectivtity towards the formation of 2b to 76%. In case of V87I mutation, the selectivity shifted towards the hydroxylation of benzylic methyl group which was observed as the major site of metabolism. For 3, the P450 BM3 variants M11, M11 V87I and M11 V87F formed two mono-hydroxylated metabolites (m/z 278) which were identified as aromatic ring hydroxylation products: 4’-hydroxy-tolfenamic acid (3a) and 5-hydroxy-tolfenamic acid (3b) (Supplementary information). As observed for 1 and 2 as well, the introduction of the V87F mutation promoted aromatic hydroxylation specifically at 4’ position. This observation is consistent with previous studies reporting aromatic hydroxylation of substrates like mono-substituted benzenes and o-xylene by P450 BM3 variants containing phenylalanine at position 87 (32, 34). Interestingly, no benzylic hydroxylation was observed for 3. Aromatic ring preference for hydroxylation by P450 BM3 mutants

In general, among the aromatic hydroxylation products of fenamic acid NSAIDs, the P450 BM3 variants showed a preference towards hydroxylation at the dichloro phenyl ring (1) or dimethyl phenyl ring (2) compared to the carboxy phenyl ring (Table 1). Previously, it has been shown that for the arylacetic acid containing NSAID diclofenac, hydroxylation at the 4’ position was the major product produced by engineered P450

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BM3 mutants (22, 24). In order to rationalize the observed ring preference for hydroxylation, in silico docking studies were performed with BM3 mutant M11 and 2 as a model, using the GOLD (version 5.2) software (35) and the docking settings as previously described (27). Docking of 2 into M11 predominantly shows the occurrence of binding poses in which 2 is docked with its dimethyl phenyl ring toward the heme group, with either 3’-methyl or C-4’ in closest proximity of the heme iron center (see Figure 5, panels A and B, respectively). These binding orientations are stabilized by formation of a hydrogen bond between the 2 carboxyl group and the side-chain hydroxyl group of Ser72. In only a single binding pose obtained from docking, the carboxy phenyl ring was found in close proximityof the porphyrin ring (Figure 5, panel C). In this pose, the carbon atoms of the phenyl ring are situated further than 6 Å from the heme iron, which is typically taken as a cut-off distance for possible catalytic activity. Importantly, no stabilizing hydrogen bonding interactions (or salt-bridge formation) of 2’s carboxyl group with the protein was observed with the C-5 ring in the closest proximity of Fe (Figure 5, panel C), which can explain the observed preference for hydroxylation of the other phenyl ring of the fenamic acids.

Figure 5. Binding poses of mefenamic acid (depicted in ball-and-stick and with green carbon atoms) in M11 (A,B,C) as observed in docking studies. Selected amino acid residues within the binding cavity of the mutants and the heme group are explicitly depicted in stick representation while aromatic and aliphatic hydrogens are omitted. Atoms prone to 3’-methyl aliphatic hydrogen abstraction (A) or to aromatic oxidative addition at C-4’position (B) are marked with a red circle, and the dashed red lines represent their distances (in Å) from the heme iron atom. In (C), the C-5 position is marked with a red circle. Dashed blue lines represent hydrogen bonds.

125

CHAP

TER

5

Tabl

e 1.

Cha

ract

eriz

atio

n of

P45

0 BM

3 va

rian

ts to

war

ds h

ydro

xyla

tion

of fe

nam

ic a

cid

NSA

IDs.

[a] T

otal

turn

over

num

bers

are

rep

orte

d as

nm

ol p

rodu

ct p

er n

mol

enz

yme.

[b] D

isso

ciat

ion

cons

tant

s (μ

M)

wer

e de

term

ined

by

UV-V

IS s

pect

rosc

opy

usin

g 1

μM

enzy

me

in 1

00 m

M p

otas

sium

pho

spha

te b

uffe

r, pH

7.4

, titr

ated

with

a so

lutio

n of

resp

ectiv

e dr

ugs d

isso

lved

in e

than

ol. [

c] C

atal

ytic

effi

cien

cy V

max

/Km

is e

xpre

ssed

t d

eter

min

ed.

Mef

enam

ic A

cid

Pr

oduc

t Dis

trib

utio

n (%

)

TTN

(4C)

a TT

N (2

5C)

a K D

b V m

ax/K

mc

3'

-OH

4'

-OH

5-

OH

3'

-OH

4’

-OH

5-

OH

M11

40

55

5

31

0 46

5 59

M11

V87

F 21

76

3

10

0 37

6 50

M11

V87

I 75

16

9

40

6 92

35

M11

V87

I L43

7N

68

25

7

0

.08

ND

ND

ND

Mec

lofe

nam

ic A

cid

Prod

uct D

istr

ibut

ion

(%)

TT

N (4

C)

TTN

(25

C)

K D

V max

/Km

3'

-OH

4’

-OH

5-

OH

3'

-OH

4'

-OH

5-

OH

M11

36

42

22

306

495

201

M11

V87

F 9

89

2

198

556

114

M11

V87

I 63

20

17

ND

ND

ND

M11

V87

I L43

7N

85

13

2

729

120

191

Tolfe

nam

ic A

cid

Pr

oduc

t Dis

trib

utio

n (%

)

TTN

(4C)

TT

N (2

5C)

K D

V m

ax/K

m

4'-O

H

5-OH

4'-O

H

5-OH

M

11

71

29

727

343

M11

V87

F

87

13

10

28

188

M11

V87

I

57

43

75

0 55

0

M11

V87

I L43

7N

58

42

ND

ND

126


This docking study showed that upon hydrogen bonding to Ser72, 2 can adopt binding conformations that are consistent with either benzylic oxidation at 3’-methyl or aromatic hydroxylation at C-4’ (Figure 5, panels A and B). In addition, 3’-methyl and/or C-4’ were consistently found to be in closer proximity of Fe than 2’-methyl, which offers a structural rationalization of the fact that no hydroxylation of the 2’-methyl group has been experimentally observed, although a similar reactivity can be expected for the two methyl groups of 2. It also explains why no benzylic hydroxylation is found for 3, in which the methyl group in position 3’ is substituted by a chlorine atom. The observed benzylic and aromatic hydroxylation selectivity in different mutants will be further rationalized in near future in extensive molecular dynamics (MD) simulation based studies. Characterization of fenamic acid metabolism by P450 BM3 variants

Enzyme kinetic parameters were determined for the three fenamic acid NSAIDs and BM3 variants which showed high selectivity for benzylic and aromatic hydroxylation. For 1 and 2, introduction of V87F mutation in M11 led to a 1.6 and 3 fold decrease respectively in the catalytic efficiency (Vmax/Km) towards formation of benzylic hydroxylation at 3’position, while the catalytic efficiency towards aromatic hydroxylation (4’-OH) was not affected (Table 1). This resulted in the higher selectivity for aromatic hydroxylation. For 3, V87F mutation led to a 1.4 fold increase in Vmax/Km resulting in a higher catalytic efficiency for 4’ hydroxylation of all the three drugs tested. On the other hand, the introduction of V87I mutation in M11 led to a 5 fold decrease in the catalytic efficiency (Vmax/Km) towards formation of 2b, while the catalytic efficiency towards benzylic hydroxylation was increased by approximately 1.2 fold. The marked decrease in aromatic hydroxylation efficiency by V87I mutation can be attributed to increased selectivity towards benzylic hydroxylation. Similar results were also observed for 1. For 3, the introduction of V87I mutation increased the C-5 hydroxylation catalytic efficiency by 1.5 fold. In comparison to 2 and 1, BM3 variants showed higher C-5 hydroxylation efficiency for 3 which can be attributed to the lack of benzylic hydroxylation. In all cases, the Hill coefficients were not significantly different from one which indicates that co-operativity does not take place. In order to evaluate the effect of mutations on the binding of the fenamic acid drugs in the active site of P450 BM3, spectroscopic titrations with the respective drug were carried out. Binding of fenamic acid NSAIDs to the tested BM3 variants resulted in a typical type I spectra (Figure 6) with a peak around 390 nm and trough at 420 nm

127

CHAPTER 5

Figure 6. UV-VIS difference spectra of (A) meclofenamic acid binding to M11 L437N V87I and (B) mefenamic acid binding to M11 V87I.

which is indicative of the conversion of the haem spin state from low to high. For mutant M11 with 1 and 3, a reverse type I spectra was observed. Further studies are currently being done to elucidate the binding mode of these two drugs in mutant M11. Table 1 shows the dissociation constants (KD) as obtained for the binding of fenamates to the BM3 mutants. The mutations showed varied effect with respect to dissociation constant values depending on the substrate. For example, in the case of 2, the introduction of V87I mutation in M11 led to a 3 fold increase in KD value while the

128


V87F mutation led to a 12.6 fold increase. The binding parameters could not be determined for wildtype P450 BM3 owing to lack of spin state shift. Previously, it was reported that the structurally similar compound diclofenac did not show any spin state shift with wild type P450 BM3 (24). Total turnover numbers and scalability of the reaction

To determine the preparative scale use of the BM3 variants for the synthesis of metabolites of fenamic acid NSAIDs, total turnover numbers (TTNs) for selective mutants were calculated. As shown in Table 1, the TTNs ranged from 2000- 6000 depending on the mutant and drug combination. Higher TTNs were observed for 2 hydroxylation. These values are in the order of magnitude required for preparative scale synthesis (Table 2). Additionally, TTNs were also calculated at 4 °C. No remarkable difference in TTNs was observed in comparison to those at 25 °C. Interestingly, at lower temperatures, an increase (upto 10 %) in aromatic hydroxylation at C-5 position was observed. Previously, it has been reported that the P450 BM3 mediated regioselectivity of butane hydroxylation can be altered by changing the reaction temperatures (36). Table 2.Total turnover numbers towards metabolism of drugs by engineered P450 BM3 variants.

Drug Therapeutic Type TTNs Reference

Artemisinin Antimalarial agent 434 Zhang et al., 2010 (37) Buspirone Antianxiety drug 3800 Landwehr et al., 2006 (38) Cyclopentanecarboxylic acid Precursor to carbovir 9200 Munzer et al., 2005 (39) Ibuprofen NSAID 650 Rentmeister et al., 2011(23) Mefenamic acid NSAID 6000 This study Naproxen NSAID 1010 Rentmeister et al.,2011 (23) Parthenolide Antileukemic agent 4980 Kolev et al.,2013 (40) Phenacetin Analgesic 930 Kim et al., 2010 (41) Verapamil Antiarrhythmic agent 1500 Sawayama et al.,2009 (21)

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To illustrate the scalabitity of the reaction, 2 was chosen as an example. Incubations in a volume of 50 mL containing 12 mg of 2 (1 mM) and 150 nM of mutant M11 V87F in the presence of a glucose-6-phosphate dehydrogenase catalyzed NADPH regenerating system resulted in 90 % substrate conversion with 75 % regioselectivity for formation of 2b. This illustrates that the reaction can be scaled up without any loss of regioselectivity. Additionally, very minimal secondary oxidation to quinoneimines was observed indicating that this P450 BM3 mutant catalyzes the first step very efficiently. After isolation of the metabolite, 6.8 mg 2b was obtained. 2b is an important ring hydroxylated metabolite that can be bioactivated to reactive quinoneimines. Therefore, this enzymatically synthesized metabolite can be used as a standard in cellular studies as well as a substrate for further toxicological evaluation. Conclusions

Tailoring cytochrome P450 BM3 mutants for selective drug metabolite production has gained considerable importance over the past years. In the current work, we studied the ability of P450 BM3 variants to produce metabolites of three drugs belonging to the fenamate class of NSAIDs and associated with adverse reactions. Our study has identified engineered P450 BM3 variants that are capable of synthesis of benzylic or aromatic ring hydroxylated metabolites of the fenamic acid NSAIDs as an alternative to their human P450 counterparts. As a result, these mutants with high total turnover numbers can be employed as biocatalytic tools for large-scale production of the respective drug metabolite to facilitate toxicological evaluation. In general, amino-acid substitution at the active site residue 87 of P450 BM3 from valine to isoleucine or phenylalanine led to more selective hydroxylation at either a benzylic or aromatic position respectively. Future studies will focus to understand the structural determinants of the selective hydroxylation which in turn will help to design mutants with novel regioselectivites. References (1) Jiang, H. N., Zeng, B., Chen, G. L., Bot, D., Eastmond, S., Elsenussi, S. E., Atkin, S. L., Boa, A. N.

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132


Supplementary Information Characterization of the hydroxylated metabolites of 1, 2 and 3 by 1H NMR spectroscopy 3’ -OH-methyl-meclofenamic acid (1a) 1H NMR (500 MHz, METHANOL-d4 J=8.5 Hz, 1 H) 6.69 - 6.76 (m, 1 H) 7.14 - 7.22 (m, 1 H) 7.47 (d, J=8.5 Hz, 1 H) 7.51 (d, J=8.5 Hz, 1 H) 8.02 (dd, 1 H) 4’ -OH-meclofenamic acid (1c) 1H NMR (500 MHz, METHANOL-d4 J=8.2 Hz, 1 H) 6.63 - 6.67 (m, 1 H) 6.92 (s, 1 H) 7.14 (td, J=7.8, 1.7 Hz, 1 H) 7.99 (dd, J=7.4, 1.7 Hz, 1 H) 5 -OH-meclofenamic acid (1c)

1H NMR (500 MHz, METHANOL-d4) ppm 2.41( s, 3 H) 6.15 (d, J=8.2 Hz, 1 H) 6.75 (dd, J=8.4, 1.1 Hz, 1 H) 7.16 (d, J=8.5 Hz, 1 H) 7.35 (d, J=7.6 Hz, 1 H) 7.43 (d, 1H) 3’ -OH-methyl-mefenamic acid (2a) 1H NMR (500 MHz, METHANOL-d4 - 6.70 (m, 1 H) 6.72 - 6.76 (m, 1 H) 7.20 - 7.28 (m, 4 H) 7.97 (dd, J=8.2, 1.6 Hz, 1 H) 4’ -OH-mefenamic acid (2b) 1H NMR (500 MHz, METHANOL-d4 J=8.2 Hz, 1 H) 6.55 (t, J=7.6 Hz, 1 H) 6.66 (d, J=8.5 Hz, 1 H) 6.89 (d, J=8.5 Hz, 1 H) 7.11 (ddd, J=8.5, 7.6, 1.6 Hz, 1 H) 7.91 (dd, J=7.9, 1.6 Hz, 1 H) 5 -OH-mefenamic acid (2c) 1H NMR (500 MHz, METHANOL-d4) J=8.8 Hz, 1 H) 6.83 (dd, J=8.8, 2.8 Hz, 1 H) 6.91 (d, J=7.3 Hz, 1 H) 7.03 (t, J=7.6 Hz, 1 H) 7.09 (d, J=7.9 Hz, 1 H) 7.42 (d, J=2.8 Hz, 1 H) 4’ -OH-tolfenamic acid (3a) 1H NMR (500 MHz, METHANOL-d4) ppm 2.25 (s, 3 H) 6.46 (d, J=8.5 Hz, 1 H) 6.61 - 6.66 (m, 1 H) 6.83 (d, J=8.5 Hz, 1 H) 7.04 (d, J=8.5 Hz, 1 H) 7.21 (td, J=6.9, 1.7 Hz, 1 H) 7.95 (dd, J=8.0, 1.7 Hz, 1 H) 5 -OH-tolfenamic acid (3b) 1H NMR (500 MHz, METHANOL-d4 (m, J=6.6 Hz, 1 H) 6.93-6.96 (m, 1 H) 7.00 (d, J=7.3, 1 H) 7.06 (t, J=7.9,1 H) 7.20 (d, J=7.6, 1 H) 7.45 (d, 1 H)

133

134

Chapter 6

APPLICATION OF ENGINEERED CYTOCHROME P450 BM3 MUTANTS TO STUDY THE EFFECT OF

HUMAN GLUTATHIONE S-TRANSFERASE GSTP1-1 POLYMORPHISMS ON THE DETOXIFICATION OF

REACTIVE DRUG METABOLITES

Adapted from Harini Venkataraman*, Sanja Dragovic*, Selina Begheijn, Nico P.E. Vermeulen and Jan N.M. Commandeur, Toxicology Letters (2014) 224, 272-281 * Equal contribution

Role of hGSTP1-1 polymorphisms

Abstract

Recent association studies suggest that genetically determined deficiencies in GSTs might be a risk factor for idiosyncratic adverse drug reactions resulting from the formation of reactive drug metabolites. hGSTP1-1 is polymorphic in the human population with a number of single nucleotide polymorphisms that yield an amino acid change in the encoded protein. Three allelic variants of hGSTP1-1 containing an Ile105Val or Ala114Val substitution, or a combination of both, have been the most widely studied and showed different activity when compared to wild-type hGSTP1-1*A (Ile105/Ala114). In the present study, we studied the ability of these allelic variants to catalyze the GSH conjugation of reactive metabolites of acetaminophen, clozapine, and diclofenac formed by bioactivation in in vitro incubations by human liver microsomes and drug metabolizing P450 BM3 mutants. The results show that effects of change of amino acid at residue 105 and 114 on conjugation reactions was substrate dependent. A single substitution at residue 105 affects the ability to catalyze GSH conjugation, while when both residue 105 and 114 were substituted the effect was additionally enhanced. Single mutation at position 114 did not show a significant effect. The different hGSTP1-1 mutants showed slightly altered regioselectivities in formation of individual GSH conjugates of clozapine which suggests that the binding orientation of the reactive nitrenium ion of clozapine is affected by the mutations. For diclofenac, a significant decrease in activity in GSH-conjugation of diclofenac 1',4'-quinone imine was observed for variants hGSTP1-1*B (Val105/Ala114) and hGSTP1-1*C (Val105/Val114). However, since the differences in total GSH conjugation activity catalysed by these allelic variants were not higher than 30%, interindividual differences in inactivation of reactive intermediates by hGSTP1-1 are not likely to be a major factor in determining interindividual difference in susceptibility to adverse drug reactions induced by the drugs studied.

CHAPTER 6

Introduction

Glutathione S-transferases (GSTs) are a superfamily of dimeric phase II enzymes, which play an important role in the cellular defense system (1, 2). The human GSTs exhibi(alpha mu pi sigma theta kappa omega) (zeta) and microsomal GSTs (3). The major role of these GSTs is to catalyze the conjugation of reduced glutathione (GSH) to electrophilic compounds and the reduction of organic hydroperoxides, thereby preventing cytotoxicity or mutagenicity. Furthermore, upregulation of GSTs in tumor tissues can contribute to resistence against alkylating antitumor agents (2). It is well established that many drugs are bioactivated by cytochrome P450s to short-lived reactive metabolites that may be responsible for adverse drug reactions (ADRs) (4). The balance between the rate of formation of these highly reactive metabolites and their inactivation by GSH conjugation, which may be catalyzed by GSTs, will determine the level of protein adduct formation and may determine the risk for ADRs (4, 5). In humans, several GSTs are known to be genetically polymorphic due to point mutations, gene deletion and gene duplication (1, 6). Therefore, interindividual variations in the GSTs due to genetic polymorphism have been proposed as potentially important factors determining susceptibility to cancer and ADRs (7-10). One of the polymorphic GSTs, human GSTP1-1 (hGSTP1-1). is highly expressed in most extrahepetic tissues (11,12) and overexpressed in many tumor forms (1,2,9). Because hGSTP1-1 has high activity in the inactivation of many environmental carcinogens and antitumor agents, its genetic polymorphisms have been subject of many studies to test whether they might affect susceptibility of individuals to different types of cancer and the efficacy of chemotherapy (6, 9). For hGSTP1-1, a number of single nucleotide polymorphisms (SNPs) have been identified that yield an amino acid change in the encoded protein, i.e. Ile105Val (13), Ala114Val (14), Asp147Tyr (15), Phe151Leu (16), and Gly169Asp (17). Four allelic forms of hGSTP1-1, resulting from combinations of the first two SNPs, have been subject to most studies so far. The variants that result from polymorphisms at position 105 and 114 are hGSTP1*A (Ile105/Ala114), hGSTP1*B (Val105/Ala114), hGSTP1*C (Val105/Val114) and hGSTP1*D (Ile105/Val114). The allele frequencies of the genotypes in Caucasian poulations are: hGSTP1*A, 0.685; hGSTP1*B, 0.262, and hGSTP1*C, 0.0687 (18). Although frequently tested in in vitro studies the occurrence of allele hGSTP1*D remains to be established. The effect of the mutations at position 105 and 114 of hGSTP1 has been studied in vitro using approximately thirty different electrophilic drugs and model substrates,


including 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid, chlorambucil, thiopeta, acrolein and diol epoxides of several polycyclic aromatic hydrocarbons (PAHs), see supplementary Table S1. However, the activity of hGSTP1-1 variants in inactivation of very short-lived reactive drug metabolites, which can only be tested in presence of a bioactivating system, has not yet been evaluated. In the present study, the effect of GSTP1-1 allelic variants on the GSH-dependent inactivation of the highly reactive metabolites of acetaminophen (APAP), clozapine (CLZ) and diclofenac (DF) was studied because in vitro studies have shown that hGSTP1 showed high activity in inactivation of the reactive metabolites of these drugs formed by cytochrome P450s (19-21). APAP is bioactivated by cytochrome P450 to N-acetyl-p-benzoquinone imine (NAPQI), a highly reactive intermediate which covalently binds to proteins, thereby causing hepatotoxicity (4, 22). Among the GSTs investigated, GSTP1-1 was the most active catalyst in GSH-conjugation of synthetical NAPQI in both rats and humans (19). Although GSTP1-1 is expressed in only low amounts in human liver (12), a recent clinical study by Buchard et al. suggests that homozygous carriers of the hGSTP1*C variant have a decreased risk for APAP hepatotoxicity (23). However, it has not been tested whether GSTP1-variants differ in the activity in inactivation of NAPQI. Figure 1. Reactive intermediates of acetaminophen (a), clozapine (b) and diclofenac (c,d) and identified structures of GSH-conjugates formed by wild-type hGSTP1-1. NAPQI, N-acetyl-p-benzoquinoneimine; DF-1',4'-QI, diclofenac 1',4'-quinoneimine; DF-2,5-QI, diclofenac 2,5-quinoneimine.

CHAPTER 6

The use of the antipsychotic drug CLZ is restricted because of the risk of life-threatening agranulocytosis and hepatotoxicity, both of which are considered to the result of bioactivation to a reactive nitrenium ion by myeloperoxidase and cytochrome P450s, respectively (24-26). The nitrenium ion of CLZ is efficiently inactivated by GSH, forming multiple regioisomeric GSH conjugates as summarized in Table 1. Dragovic et al. showed that hGSTP1-1 has high activity in the inactivation of reactive nitrenium ion by catalyzing both chlorine-substitution and GSH-addition reactions, Figure 1 (20). GSTP1-1 is highly expressed in white blood cells (27) and hence polymorphism of GSTP1-1 might be a risk factor for occurrence of agranulocytosis in case mutations strongly affect the inactivation of the nitrenium ion. Treatment with the nonsteroidal anti-inflammatory drug DF is associated with serious gastrointestinal injury and rare cases of idiosyncratic hepatotoxicity (28,29). Although the exact mechanisms of these ADRs remain to be established, formation of reactive metabolites by cytochrome P450s is considered to play an important role in the onset of these toxic events (29,30). Based on the structure of GSH-conjugates identified in microsomal incubations at least four different reactive intermediates have been proposed for DF. Quantitatively the major ones are quinoneimines resulting from dehydrogenation of 4’-hydroxy diclofenac and 5-hydroxy diclofenac, Figure 1 (31). Minor GSH-conjugates result from a quinonemethide formed via oxidative decarboxylation (32), and an as yet unidentied reactive intermediate resulting in chlorine substitution of DF (33). We recently showed that hGSTP1-1 has high activity in catalysing GSH-conjugation of all reactive intermediates. Because hGSTP1-1 is abundantly expressed in the gastrointestinal tract, polymorphisms of hGSTP1-1 might be risk factor for the occurrence of gastrointestinal injury in case the amino acid substitutions influence GSH-conjugation of the different reactive intermediates formed by P450s. The aim of the present study was to investigate whether the allelic variants of hGSTP1-1 have different activity in the GSH conjugation of reactive metabolites generated by P450s in incubations of APAP, CLZ, and DF. Because the reactive intermediates of CLZ and DF can be conjugated to GSH at different positions, Figure 1, also the effect of the mutations on regioselectivity of GSH-conjugation will be studied, which might be indicative for different binding orientations. Reactive intermediates were generated using human liver microsomes (HLM), and purified drug metabolizing mutants of bacterial CYP102A1 as bioactivation systems. The CYP102A1 mutants were selected because of their ability to produce human relevant GSH conjugates at high activity and to circumvent interference of microsomal GSTs (5, 20).


Materials and methods

Enzymes, plasmids and chemicals

The bacterial CYP102A1 mutants CYP102A1 M11H, CYP102A1 M11H Phe87, and CYP102A1 M11 L437N were prepared and purified as described previously (20, 35, 36). Human liver microsomes (HLM), pooled from 50 donors, were obtained from Xenotech (Lenexa, United States) and contained 20 mg/mL protein. Escherichia coli XL-1 Blue cells containing the expression plasmid for human GSTP1-1, was kind gift from Prof. Mannervik (Department of Biochemistry and Organic chemistry, Uppsala University, Sweden). All other chemicals and reagents were of analytical grade and obtained from standard suppliers. Site directed mutagenesis of hGSTP1-1 isoforms

Three variants of wild type hGSTP1-1 (hGSTP1*A), namely hGSTP1*B (Val105, Ala114), hGSTP1*C (Val105, Val114) and hGSTP1*D (Ile105, Val114), were constructed by site directed mutagenesis. The single mutants hGSTP1*B and hGSTP1*D were constructed using the expression vector pKKD-hGST P1-1 as the template. The double mutant hGSTP1*C was constructed by introducing Ile104Val mutation in pKKD-hGSTP1*D (Ile105, Val114). The mutations were introduced by the Quikchange mutagenesis protocol using the following forward primers: Ile104Val : CGC TGC AAA TAC GTC TCC CTC ATC TAC Ala113Val : C ACC AAC TAT GAG GTG GGC AAG GAT GAC The mutated amino acid position is indicated in bold. The reverse primers were exactly complementary to the forward primers. After mutagenesis, the presence of right mutations was verified by DNA sequencing (Service XS, Leiden, The Netherlands). Expression and purification of hGSTP1-1 enzymes

Human GSTP1*A and its variants hGSTP1*B, hGSTP1*C and hGSTP1*D were expressed in E. coli XL-1 Blue cells. pKKD expression plasmids containing the different hGSTP1-1 genes, were transformed into E.coli XL-1 Blue cells. Expression and purification were essentially conducted as previously described (20). Purity of the enzymes was checked by SDS-polyacrylamide gel electrophoresis on a 12% gel with Coomassie staining as well as by reversed-phase liquid chromatography using a method adapted from Jenkins et al. (37).

CHAPTER 6

Protein concentrations were determined according to the method of Bradford (38) with reagent obtained from Bio-Rad. The specific activity of the purified GSTs was assayed according to Habig et al. (39). The specific activities of the purified recombinant human GSTP1*A and its polymorphic variants using CDNB as a substrate

17.3

Bioactivation of the drugs and GSH conjugates formation in the presence of hGSTP1-1 variants

Incubations were performed using purified CYP102A1 M11H as bioactivation system for APAP (34). Mutant CYP102A1 M11H Phe87 was used for CLZ (35) and CYP102A1 M11 L437N for DF (21) as we have shown that these enzymes have high activity and selectivity for bioactivation to reactive intermediates. CLZ and APAP incubations were performed at a final enzyme concentration of 250 nM while for DF incubations were performed at a final BM3 concentration of 500 nM. All incubations were performed in

from the stock solutions was not more than 2% in the incubations. The allelic variants of hGSTP1-1

the linearity of product formation with protein concentration and to compare their -1 was used in

the incubations to investigate enzymatic GSH conjugation of the other substrates. Incubations with hGSTP1- GSH. Reactions with CLZ were started by adding 0.5 mM NADPH (final concentration) and performed for 30 min at 24°C, as described previously (20). For the other substrates, an NAPDH regenerating system (final concentrations: 0.5 mM NADPH, 20 mM glucose-6-phosphate and 2 units/mL of glucose-6-phosphate dehydrogenase) was used to start the reaction and incubations were performed for 60 min at 24 °C. Reactions

-cold HClO4. In case of D -cold methanol with 2% 50 mM ascorbic acid in water was added to stop incubations. All samples were centrifuged for 15 min at 14000 rpm. The supernatants were analyzed by HPLC and LC-MS, as described below. Incubations were also performed usinincubated in 100 mM Kpi buffer, pH 7.4, for 30 minutes at 37°C with 1 mg/mL

-1. Reactions were started with 0.5 mM NADPH (final concentration). For DF and APAP,


-1. Reactions were initiated by the addition of NADPH regenerating system (final concentrations: 0.5 mM NADPH, 20 mM glucose-6-phosphate and 2 units/mL of glucose-6-phosphate dehydrogenase) and were incubated for 60 min at 37°C. Reactions were terminated depending on the substrate as described above and centrifuged at 14000 rpm for 15 min to remove precipitated proteins. Analytical methods

The analyses of the GSH-conjugates were performed by reversed-phase liquid x 150 mm) as stationary phase,

used. For incubations of CLZ and DF, a gradient method was used where two mobile phases were mixed: eluent A (1% acetonitrile, 99% water, 0.2% formic acid) and eluent B (1% water, 99% acetonitrile, 0.2% formic acid). The gradient used was: 0 to 5 min isocratic at 0% eluent B; 5 to 30 min a linear increase to 100% eluent B; 30 to 30.5 min linear decrease to 0% eluent B; 30.5 to 40 min isocratic re-equilibration at 0% solvent B. The flow rate was 0.5 mL/min. For analysis of metabolites of APAP, an isocratic method was used with a mobile phase consisting of 18% methanol, 82% water, and 0.1 % trifluoroacetic acid (TFA) and a flow rate of 1 mL/min. Samples were

wavelength of 243 nm (APAP) or 254 nm (CLZ, DF). Quantification is based on the substrate standard curves, assuming that the extinction coefficients of the GSH adducts are equal to that of the substrate. All metabolites were identified using an Agilent 1200 Series Rapid resolution LC system was connected to a hybrid quadrupole-time-of-flight (Q-TOF) Agilent 6520 mass spectrometer, equipped with electrospray ionization (ESI) source and operating in the positive mode as described in Dragovic et al. (20). The characteristics of the GSH-conjugates analyzed in the incubations are tabulated in Table 1.

CHAPTER 6

Table 1. Characteristics of the GSH conjugates formed by bioactivation of the drugs used in this study. Abbreviation GSH conjugates tret (min) m/z ([M+H]+) Clozapine CG-1 6-(glutathione-S-yl)-clozapine 14.3 632.23 CG-3 9-(glutathione-S-yl)-clozapine 14.0 632.23 CG-4 7-(glutathione-S-yl)-clozapine NI 13.5 632.23 CG-5 (1-4)-(glutathione-S-yl)-clozapine 14.6 632.23 CG-6 8-(glutathione-S-yl)-deschloroclozapine 11.8 598.27 Diclofenac M1 5-hydroxy-4/6-(glutathione-S-yl)-diclofenac 19.7 617.09 M2 4’-hydroxy-3’-(glutathione-S-yl)-diclofenac 20.0 617.09 M3 5-hydroxy-4/6-(glutathione-S-yl)-diclofenac 20.4 617.09 M5 4’-hydroxy-2’-(glutathione-S-yl)-diclofenac 19.7 583.13 DPAB-SG 2-(2,6-dichlorophenylamino)-benzyl- 21.7 557.13 S-thioether glutathione DF-SG 2’-(glutathione-S-yl)-deschlorodiclofenac 19.5 567.14 Acetaminophen APAP-SG 3-(glutathion-S-yl)-acetaminophen 10.3 457.11

Results

Activity of hGSTP1-1 variants in inactivation of clozapine nitrenium ion

Consistent with previous results, addition of hGSTP1-1 to incubations of CLZ with CYP102A1 M11H Phe87 resulted in major changes of the profile and concentrations of GSH conjugates (20). For each of the allelic variants of hGSTP1-1, the increase in formation of GSH conjugates CG-4, CG-5 and CG-6 was proportional with the hGSTP1-1 concentrations when varied from 0.5 to 8 μM (data not shown). Table 2 shows the effects observed at 8 μM of hGSTP1-1 variants. The amount of the total GSH conjugates of CLZ was increased 2.4 to 2.7-fold, when compared to the non-enzymatic conjugation. However, none of the three variants showed a statistically significant difference compared to wild-type hGSTP1*A. As shown in Figure 2A, the variants only showed slightly different profiles of GST-dependent GSH conjugates. For hGSTP1-1*B and hGSTP1-1*C CG-6 was produced at 2-fold higher amount than CG-5. For mutants hGSTP1-1*A and hGSTP1-1*D conjugate CG-6 was produced at 2.7- and 2.5-fold higher amounts, respectively, than CG-5. The minor GSH conjugate, CG-4, was increased with the highest activity by hGSTP1*B and hGSTP1*C being 2 times higher comparing to hGSTP1*A, after subtraction of non-enzymatic conjugation.


As can be seen from Table 2, the variants of hGSTP1-1 enzyme also did not show significant differences in total GSH conjugate formation when bioactivation reactions were performed with HLM which might be due to the almost 4-fold lower amounts of GSH-conjugates formed. However, similar differences in the ratio of GST-dependent conjugates CG-6 and CG-5 were observed. Again, hGSTP1-1*A and hGSTP1-1*D produced conjugate CG-6 at relatively higher amounts than CG-5. The effect on relative amount of CG-4 could not be confirmed because this conjugate is mainly produced by the microsomal GST present in HLM (19). Activity of hGSTP1-1 variants in inactivation of reactive metabolites of diclofenac

As shown in Table 2, all hGSTP1-1 variants showed very high activity in catalysing GSH-conjugation of reactive DF metabolites when using CYP102A1 as bioactivating system. The total amount of GSH-conjugates was increased 7- to almost 9-fold. hGSTP1*A and hGSTP1*D showed the highest activity for the formation of total GSH conjugates. When corrected for the non-enzymatic conjugation hGSTP1*B and hGSTP1*C appeared to have 20% and 30% lower activity, respectively, compared to the other variants. DF is bioactivated by CYP102A1 to different reactive intermediates, of which diclofenac 1',4'-quinoneimine (DF-1'4'-QI), formed via 4'-hydroxylation, and diclofenac 2,5-quinoneimine (DF-2,5-QI), formed via 5-hydroxylation, are quantitatively the major ones. As shown in Table 2, the GSH-conjugation of DF-1'4'-QI was more affected by the polymorphic mutations with hGSTP1*C exhibiting the lowest activity in formation of the corresponding GSH-conjugates M2 and M5. When correcting for the non-enzymatic reaction, hGSTP1*C showed almost 40% lower activity when compared to the wild type hGSTP1*A. Activities of hGSTP1*B and hGSTP1*D were 22% and 17% lower, respectively. No differences in the ratio of M2 and M5 were observed, Figure 3A.

CHAP

TER

6

Tabl

e 2.

Effe

ct o

f rec

ombi

nant

hGS

TP1-

1 va

rian

ts o

n th

e to

tal o

f GSH

conj

ugat

es fo

rmed

in in

cuba

tions

of d

rugs

with

CYP

102A

1-m

utan

ts a

nd h

uman

live

r m

icro

som

es.

Reco

mbi

nant

hum

an G

STP1

-1

Bioa

ctiv

atio

n

D

rug

GSH

-

N

o hG

ST

h

GSTP

1-1*

A

hG

STP1

-1*B

hGS

TP1-

1*C

hG

STP1

-1*D

sy

stem

con

juga

tes

(Ile

105/

Ala1

14)

(Val

105/

Ala1

14)

(V

al10

5/Va

l114

)

(Ile

105/

Val1

14)

N

one

C

DNB

D

NB-

SG

N.D

.

20

.1±

1,0

10

.6 ±

0.5

9.2

± 0

.4

1

7.3

± 0.

9

CYP1

02A1

-mut

ants

C

LZ

T

otal

a

1

3.1

± 0.

6

32.

1 ±

1.3

31

.0 ±

2.8

31.

3 ±

2.9

3

5.3

± 1.

1

DF

Tot

alb

2.2

± 0

.1

1

9.1

± 0.

8

15.9

± 0

.9

1

4.2

± 0.

5

18.

2 ±

0.7

DF-

1',4

’-QI (

M2

+ M

5)

1.7

± 0

.2

1

3.8

± 0.

9

11.1

± 0

.6

9

.2 ±

0.4

11.

7 ±

0.6

DF-

2,5-

QI (M

1 +

M3)

0

.4 ±

0.0

3

5.2

± 0

.6

4.

9 ±

0.4

4

.9 ±

0.3

6.5

± 0

.6

A

PAP

A

PAP-

SG

8

.2 ±

0.7

84.

8 ±

4.3

90

.9 ±

5.1

73.

5 ±

3.4

8

2.3

± 2.

5

HLM

C

LZ

T

otal

a

3

.7 ±

0.1

7.9

± 0

.6

8.

2 ±

0.5

8

.1±0

.3

8

.1 ±

0.2

DF

Tot

alb

0.4

8 ±

0.08

2.02

± 0

.13

1.

90 ±

0.1

7

1.9

4±0.

20

1

.97

± 0.

14

DF-

1',4

’-QI (

M2

+ M

5)

0.4

6 ±

0.04

1.49

± 0

.10

1.

40 ±

0.0

8

1.4

7±0.

09

1

.46

± 0.

10

DF-

2,5-

QI (M

1 +

M3)

0

.02

± 0.

01

0.

53 ±

0.0

7

0.49

± 0

.05

0

.47±

0.04

0.5

1 ±

0.03

APA

P

AP

AP-S

G

1.9

± 0

.1

1

0.5

± 1.

6

11.4

± 1

.7

1

0.0±

1.8

1

0.2

± 2.

4

Met

abol

ites w

ere

form

ed a

fter 1

hou

r inc

ubat

ion

of 5

00 μ

M D

F w

ith 5

00 n

M C

YP10

2A1

M11

L43

7N in

the

pres

ence

of 5

mM

GSH

. Qu

antif

icat

ion

was

bas

ed o

n pe

ak a

reas

in th

e LC

-UV

chro

mat

ogra

ms

usin

g th

e DF

sta

ndar

d cu

rve,

ass

umin

g th

at th

e ex

tinct

ion

coef

ficie

nts

of th

e su

bstr

ate

and

the

met

abol

ites a

t 254

nm

are

the

sam

e.

t ret, r

eten

tion

time

in m

inut

es.

a. O

nly

dete

ctab

le in

incu

batio

ns su

pple

men

ted

with

hum

an G

ST.


A)

B)

Figure 2. Effect of 8 μM of hGSTP1*A, hGSTP1*B, hGSTP1*C, and hGSTP1*D on the regioselectivity of GSH-conjugation of the nitrenium ion of CLZ. CLZ was bioactivated to reactive metabolites using CYP102A1 M11H Phe87 mutant (A) or HLM (B). What concerns the GSH-conjugation of DF-2,5-QI, only hGSTP1*D appeared to have 25% higher activity than wild-type hGSTP1*A, whereas hGSTP1*C and hGSTP1*B showed similar activity. As shown in Figure 3B, small changes were observed in the regioselectivity of GSH-conjugation of DF-2,5-QI since the relative amount of GSH-conjugate M1 was slightly higher in variants hGSTP1*A and hGSTP1*D. No significant differences were observed between the hGSTP1-1 polymorphic variants for the formation of the minor GSH conjugates DF-SG (m/z 567) and DPAB-SG (m/z 557) which are formed by the two minor bioactivation pathways (data not shown) (32,33). In case of HLM as bioactivation system, addition of 8 μM of hGST-variants increased the total GSH-conjugation of reactive metabolites of DF about 4-fold, Table 1. No significant differences were observed between the activities and regioselectivities of the hGSTP1-1 variants, which may be explained by the lower levels of reactive

CHAPTER 6

0

20

40

60

80

100

no GST hGSTP1*A hGSTP1*B hGSTP1*C hGSTP1*D

% M1 m/z 617 M3 m/z 617

intermediates produced and the analytical error, Figure 4. The fact that GSH-conjugation of DF-1'4'-QI is increased to a smaller extent by hGSTP1-1, whereas a significantly different ratio of conjugates M2 and M5 was found, point to contribution of microsomal GST in the HLM incubations, Figure 4B. Again no significant differences were observed between the polymorphic variants of hGSTP1-1 for the formation of DF-SG and DPAB-SG (data not shown). A) B) Figure 3. Effect of 8 μM hGSTP1*A, hGSTP1*B, hGSTP1*C, and hGSTP1*D on the regioselectivity of GSH conjugation of DF-1',4'-QI (A) and DF-2,5-QI (B), using CYP102A1 M11 L437N mutant as bioactivation system. Activity of hGSTP1-1 variants in inactivation of acetaminophen reactive metabolites

-1 mutants to the incubation of APAP with CYP102A1 M11H, in the presence of 100 μM GSH, increased the concentrations of NAPQI-SG about 10-fold, Table 2. Variants hGSTP1*A, hGSTP1*B and hGSTP1*D did not show statistically significant differences in conjugation of NAPQI. Only hGSTP1*C showed a 15% decreased activity compared to hGSTP1*A. When using HLM as bioactivation

0

20

40

60

80

100

No GST hGSTP1*A hGSTP1*B hGSTP1*C hGSTP1*D

% M2 m/z 617 M5 m/z 583


0

20

40

60

80

100

no GST hGSTP1*A hGSTP1*B hGSTP1*C hGSTP1*D

% M2 m/z 617 M5 m/z 583

0

20

40

60

80

100

No GST hGSTP1*A hGSTP1*B hGSTP1*C hGSTP1*D

%

M1 m/z 617 M3 m/z 617

system, however, no significant differences were found in the activity of the hGSTP1-1 variants. A)

B)

Figure 4. Effect of 8 μM hGSTP1*A, hGSTP1*B, hGSTP1*C, and hGSTP1*D on the regioselectivity of GSH conjugation of DF-1',4'-QI (A) and DF-2,5-QI (B), using human liver microsomes as bioactivation system. Discussion

Because hGSTP1-1 is highly expressed in normal extrahepatic tissues and overexpressed in many tumors types, in some cases in response to antitumor drugs. Therefore, a large number of studies have been performed to investigate whether genetic polymorphism of hGSTP1-1 has consequences for the inactivation of carcinogenic diolepoxides of PAHs or alkylating antitumor drugs, such as thiotepa and chlorambucil, since these might be revelant for susceptibility to carcinogenesis and efficacy of antitumor treatment. Table 3 summarizes the relative activities observed for the hGSTP1-1-variants with amino acid substitutions Ile105Val and/or Ala114Val

CHAPTER 6

when compared to wild-type hGSTP1-1*A, which is set at 100% for all compounds. The data shown in Table 3 are based on the kcat/Km-values reported because this enzyme kinetical parameter will most likely the most relevant parameter to describe differences in enzyme activity at the low, physiologically relevant, concentrations of reactive carcinogens and toxicants. Only data is shown of studies in which at least three of the hGSTP1-1 variants were compared; for a more complete list see Table S1. The results of the present study show that the amino acid substitutions Ile105Val and Ala114Val have differential effects on the activity of hGSTP1-1, dependent on the reactive intermediate involved, consistent with the previous studies which have been performed with chemically stable electrophilic compounds, see Table 3. For CLZ no significant differences were observed in the total rate by which the CLZ nitrenium ion is conjugated to GSH by hGSTP1-1. The only significant effect which was observed in both CYP102A1 and HLM incubations was a slight change in regioselectivity in GSH-conjugation to the CLZ nitrenium ion. In both hGSTP1*B and hGSTP1*C, which share the Ile105Val substitution, an increase in the relative amount of GSH-conjugates CG-4 and CG-5 was found while substitution of chlorine by GSH and formation of CG-6 was decreased, Figure 2. Although no association studies have been carried out yet to study the role of hGSTP1-1-polymorphism as risk factor for CLZ-induced agranulocytosis, the present data see to rule out that genetically determined differences in hGSTP1-1 activity in white blood cells might determine susceptibility. As shown in Tables 2 and 3, variant hGSTP1-1*B and hGSTP1-1*C both showed a significant decreased total activity in GSH-conjugation of reactive metabolites of DF, which could be mainly attribited to a decreased GSH-conjugation of DF-1',4'-QI, which is quantitatively the major reactive intermediate formed. Both hGSTP1-1*B and hGSTP1-1*C contain amino acid substitution Ile105Val which was also shown to be detrimental for the GSH-conjugation of the antitumor agents chlorambucil and thiotepa, and for the general GST-substrate CDNB but benificial for the catalysis of GSH-conjugation of ethacrynic acid and several carcinogenic diolepoxides of PAHs, Table 3. According to the available crystal structures of hGSTP1*A, hGSTP1*B and hGSTP1*C (42, 43), amino acid residue 105 is localized in the H-site of hGSTP1-1 and can affect enzyme activity in several ways, dependent on the substrate. First, the Ile105Val substitution can affect the shape of the active-site, because the Val105 has more conformational freedom than Ile105. For large substrates, Val105 can point away from the active site, which allows the accommodating of larger substrates, such as BPDE, for catalysis (42). Also, amino acid residue 105 is close to residue Tyr109, which for some substrates plays an important role in the catalytic mechanism of hGSTP1-1 by stabilizing the intermediate complex (44). Furthermore, substitution Ile105Val has shown to affect the hydrophobicity/hydrophilicity of the H-site of

Role

of h

GSTP

1-1

poly

mor

phis

ms

Tabl

e 3.

Rel

ativ

e ac

tivity

of p

olym

orph

ic v

aria

nts o

f hGS

TP1-

1 in

GSH

-con

juga

tion

of e

lect

roph

ilic d

rugs

and

reac

tive

drug

met

abol

ites.

Elec

trop

hilic

dru

g /

hGS

TP1*

A

hGS

TP1*

B h

GSTP

1*C

hG

STP1

*D

Re

activ

e in

term

edia

te

(Ile

105/

Ala1

14)

(Va

l105

/Ala

114)

(V

al10

5/Va

l114

) (I

le10

5/Va

l114

) L

itera

ture

Cl

ozap

ine

nitr

eniu

m io

n /

CLZ

NI

10

0 ±

10

94 ±

22

9

6 ±

30

117

± 2

1

Thi

s stu

dy a

Dicl

ofen

ac-1

',4'-q

uino

neim

ine

/ DF

-1',4

'-QI

100

± 9

78 ±

7‡

6

2 ±

5‡

8

3 ±

7

Thi

s stu

dy a

Dicl

ofen

ac-2

,5-q

uino

neim

ine

/ DF

-2,5

-QI

10

0 ±

13

94 ±

9

9

4 ±

7

127

± 1

3

Thi

s stu

dy a

N-A

cety

l-p-b

enzo

quin

onei

min

e /

NAP

QI

100

± 7

1

08 ±

8

8

5 ±

5‡

97

± 4

Thi

s stu

dy a

Etha

cryn

ic a

cid

10

0 ±

13

1

50 ±

20‡

253

± 1

3‡

93

± 13

(43

) Th

iote

pa

10

0 ±

8

5

3 ±

9‡

38

± 16

‡

(

48)

Chlo

ram

buci

l

1

00 ±

7

13

± 4‡

7

± 2

‡

40

± 1

0‡

(49)

At

razi

ne

1

00 ±

4

1

03 ±

14

1

00 ±

11

9

7 ±

14

(

50)

1-ch

loro

-2,4

—di

nitr

oben

zene

/ C

DNB

10

0 ±

5

5

3 ±

3‡

46

± 2‡

86 ±

4

T

his s

tudy

a

1

00 ±

14

3

7 ±

7‡

44

± 9‡

56 ±

10‡

(51

)

1

00 ±

5

27

± 3‡

3

2 ±

5‡

85

± 3

(43

)

1

00 ±

8

30

± 3‡

2

4 ±

3‡

(14

)

1

00 ±

35

21

± 4‡

6

4 ±

19‡

157

± 2

6

(

52)

7-ch

loro

-4-n

itrob

enz-

2-ox

a-1,

3-di

azol

e /

NBD

-Cl

100

± 5

15

± 2‡

104

± 4

47

± 3

‡

(43

) Ac

role

in

1

00 ±

2

99

± 3

7

1 ±

2‡

90

± 2

(53

) Cr

oton

alde

hyde

1

00 ±

13

106

± 1

3

7

5 ±

13‡

75

± 6

‡

(53

) Di

ol e

poxi

des o

f pol

ycyc

lic a

rom

atic

hyd

roca

rbon

s (+

)-an

ti-be

nzo[

a]py

rene

dio

lepo

xide

/ (+

)-an

ti-BP

DE

100

± 1

2

1

30 ±

20‡

500

± 1

00‡

150

± 70

(5

2)

(+/-

)-ch

ryse

ne d

iole

poxi

de /

(+/-

)-an

ti-CD

E

100

± 2

5

767

± 2

19‡

438

± 1

10‡

151

± 27

(5

2)

(+)-

anti-

5-m

ethy

lchr

ysen

e di

olep

oxid

e /

(+)-

anti-

5-M

eCDE

1

00 ±

11

109

± 4

82

± 16

(54)

(-

)-an

ti-5-

met

hylc

hrys

ene

diol

epox

ide

/ (-

)-an

ti-5-

MeC

DE

100

± 8

0

200

± 4

0‡ 1

80 ±

100

‡

(54)

(±

)-an

ti-be

nzo[

c]ch

ryse

ne d

iole

poxi

de-1

/ (+

/-)-

anti-

B[c]

CDE-

1

100

± 17

233

± 3

3‡ 1

67 ±

17‡

(5

5)

(±)-

anti-

benz

o[c]

chry

sene

dio

lepo

xide

-2 /

(+/-

)-an

ti-B[

c]CD

E-2

10

0 ±

10

61

± 5‡

2

3 ±

4‡

(5

5)

Num

bers

of p

ublis

hed

data

are

bas

ed o

n re

port

ed k

cat/

K m-v

alue

s. a. D

ata

of in

cuba

tions

with

CYP

102A

1 M

11H

-mut

ants

as b

ioac

tivat

ion

syst

em. ‡ S

igni

finan

tly

incr

ease

d or

dec

reas

e

CHAPTER 6

hGSTP1-1 by influencing the number of active-site water molecules which can affect substrate binding and/or product release (45). Which of these mechanisms contribute to the decreased GSH-conjugation of DF-'1,'4-QI and the slight change in regioselectivity of GSH-conjugation of CLZ-nitrenium ion is not known. A recent association study has shown that the frequency of hGSTP1*C-allele was significantly lower in APAP-poisoned patients than in insensitive patients, which suggests that this genotype may reduce the risk of APAP-hepatotoxicity (23). The results of the present study shows that the hGSTP1*C variant had the lowest activity in catalysing the GSH-conjugation of NAPQI, Table 1. However, this activity was only 15% lower than that of hGSTP1-1*A, which is not likely to be of toxicological relevance, also considering the fact that the current study was performed at reduced GSH-concentration, to minimize non-enzymatic GSH-conjugation, and that hGSTP1-1 is expressed in the human liver only to a significant extent in the biliary tree (11). In conclusion, the results of the present study show that the amino acid substitutions Ile105Val and Ala115Val in hGSTP1-1 have only relatively minor effect on the activity of GSH-conjugation of the reactive metabolites of APAP, CLZ and DF. Differences in the rate of inactivation of their reactive intermediates are, therefore, unlikely to play a role in the susceptibility to their toxic side-effects. However, in recent years it has been shown that GSTP1-1 also can directly influence signalling pathways underlying stress response, apoptosis, inflammation, and cell proliferation by protein-protein interaction and protein glutathionylation (46). Interestingly, it was recently shown that only variants hGSTP1-1*A and hGSTP1-1*C were able to reactivate the antooxidant enzyme peroxiredoxin VI, whereas variants hGSTP1-1*B and hGSTP1-1*D were without effect, suggesting that these polymorphisms may influence human population susceptibility to oxidant stress (47). Therefore, these factors rather than interindividual differences in the rate of GSH-conjugation of reactive drug metabolites by hGSTP1-1 can play a role in susceptibility to adverse drug reactions induced by the drugs studied. References (1) Hayes JD, Flanagan JU, Jowsey IR. (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45, 51-88. (2) Hayes JD and Pulford DJ. (1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol 30, 445-600. (3) Mannervik B, Board PG, Hayes JD, Listowsky I, Pearson WR. (2005) Nomenclature for mammalian soluble glutathione transferases. Methods Enzymol 401, 1-8.

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CHAPTER 6

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Role of hGSTP1-1 polymorphisms (40) Dieckhaus CM, Roller SG, Santos WL, Sofia RD, Macdonald TL. (2001) Role of glutathione S- transferases A1-1, M1-1, and P1-1 in the detoxification of 2-phenylpropenal, a reactive felbamate metabolite. Chem Res Toxicol 14, 511-6. (41) Joshi EM, Heasley BH, Macdonald TL. (2009) 2-ABT-S-oxide detoxification by glutathione S- transferases A1-1, M1-1 and P1-1: implications for toxicity associated with zileuton. Xenobiotica 39, 197-204. (42) Ji X, Blaszczyk J, Xiao B, O'Donnell R, Hu X, Herzog C, Singh SV, Zimniak P. (1999) Structure and function of residue 104 and water molecules in the xenobiotic substrate-binding site in human glutathione S-transferase P1-1. Biochemistry 38, 10231-8. (43) Parker, L. J., Ciccone, S., Italiano, L. C., Primavera, A., Oakley, A. J., Morton, C. J., Hancock, N. C., Bello, M. Lo, and Parker, M. W. (2008) The anti-cancer drug chlorambucil as a substrate for the human polymorphic enzyme glutathione transferase P1-1: kinetic properties and crystallographic characterisation of allelic variants., J.Mol.Biol. 380, 131–44. (44) Oakley, a J., Lo Bello, M., Battistoni, a, Ricci, G., Rossjohn, J., Villar, H. O., and Parker, M. W. (1997) The structures of human glutathione transferase P1-1 in complex with glutathione and various inhibitors at high resolution., J.Mol.Biol. 274, 84–100. (45) Hu X, Ji X, Srivastava SK, Xia H, Awasthi JC, Nanduri B, Awasthi JC, Zimniak P, and Singh SV (1997c) Mechanism of Differential Catalytic Efficiency of Two Polymorphic Forms of Human Glutathione S-Transferase P1-1 in the Glutathione Conjugation of Carcinogenic Diol Epoxide of Chrysene. Arch. Biochem.Biophys. 345, 32–38. (46) Tew KD, Townsend DM. (2011) Regulatory functions of glutathione S-transferase P1-1 unrelated to detoxification. Drug Metab Rev. 43, 179-93. (47) Manevich Y, Hutchens S, Tew KD, Townsend DM. (2013) Allelic variants of glutathione S- transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation. Free Radic Biol Med. 54, 62-70. (48) Srivastava SK, Singhal SS, Hu X, Awasthi YC, Zimniak P and Singh SV (1999) Differential Catalytic Efficiency of Allelic Variants of Human Glutathione S-Transferase Pi in Catalyzing the Glutathione Conjugation of Thiotepa1 Arch Biochem Biophys 366, 89–94. (49) Pandya U, Srivastava SK, Singhal SS, Pal A, Awasthi S, Piotr Zimniak, Awasthi YC and Singh SV (2000) Activity of Allelic Variants of Pi Class Human Glutathione S-Transferase Toward Chlorambucil. Biophys Res Commun 278, 258–262. (50) Abel EL, Opp SM, Verlinde CL, Bammler TK, Eaton DL. (2004) Characterization of atrazine biotransformation by human and murine glutathione S-transferases. Toxicol Sci. 80, 230-8. (51) Goodrich JM, Basu N. (2012) Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals. Toxicol In Vitro. 26, 630-5. (52) Hu, X., Xia, H., Srivastava, S. K., Herzog, C., Awasthi, Y. C., Ji, X. (1997a). Activity of four allelic forms of glutathione S-transferase hGSTP1-1 for diol epoxides of polycyclic aromatic hydrocarbons. Biochem Biophys Res Commun 238, 397–402. (53) Pal A, Hu X, Zimniak P, and Singh SV. (2000a) Catalytic efficiencies of allelic variants of human glutathione S-transferase Pi in the glutathione conjugation of a,b-unsaturated aldehydes. Cancer Lett 154 39-43. (54) Hu X, Pal A, Krzeminski J, Amin S, Awasthi YC, Zimniak P, Singh SV. (1998b) Specificities of human glutathione S-transferase isozymes toward anti-diol epoxides of methylchrysenes. Carcinogenesis. 19, 1685-9. (55) Pal A, Desai DH, Amin S, Srivastava SK, Hu X, Herzog C, Zimniak P, Singh SV. (2000b) Location of the epoxide function determines specificity of the allelic variants of human glutathione transferase Pi toward benzo[c]chrysene diol epoxide isomers. FEBS Lett 486, 163-6.

CHAP

TER

6

Tabl

e S1

. Cat

alyt

ic e

ffici

ency

(kca

t/K m

) of p

olym

orph

ic v

aria

nts o

f hum

an g

luta

thio

ne S

-tran

sfer

ases

P1-

1 (h

GSTP

1-1)

hGST

P1-1

var

iant

*A

*B

*

C

*

D

Subs

trat

e

Abb

revi

atio

n

Ile10

5/Al

a114

V

al10

5/Al

a114

V

al10

5/Va

l114

I

le10

5/Va

l114

Unit

Refe

renc

e

1-

chlo

ro-2

,4--d

initr

oben

zene

CDN

B

55,1

± 4

,6

16

,7 ±

1,4

13,2

± 1

,7

(mM

-1.s-1

)

(a)

1-

chlo

ro-2

,4--d

initr

oben

zene

CDN

B

70 ±

20

13

± 2

11

0 ±

15

4

0 ±

12

(mM

-1.s-1

)

(b)

1-

chlo

ro-2

,4--d

initr

oben

zene

CDN

B

98,2

± 7

,5

4

2,5

± 4,

2

(m

M-1

.s-1)

(

c)

1-ch

loro

-2,4

--din

itrob

enze

ne

CD

NB

66

,0 ±

3,0

18,

0 ±

2,0

21

,0 ±

3,0

56,

0 ±

2,0

(mM

-1.s-1

)

(d)

1-

chlo

ro-2

,4--d

initr

oben

zene

CDN

B

98,2

± 1

4,0

3

5,9

± 6,

8

43,4

± 9

,3

5

5,0

± 9,

8 (m

M-1

.s-1)

(

e)

Etha

cryn

ic a

cid

15,0

± 2

,0

22

,5 ±

3,0

38,0

± 2

,0

1

4,0

± 2,

0 (m

M-1

.s-1)

(

d)

Etha

cryn

ic a

cid

45,5

± 9

.6

5

4,6

± 9,

0

(m

M-1

.s-1)

(

c)

Thio

tepa

1

1,8

± 1,

0

6,3

± 1

,1

4,

5 ±

0,7

(M-1

.s-1)

(f)

Chlo

ram

buci

l

15

,0 ±

1,0

2,0

± 0,

6

1,

0 ±

0,3

6

,0 ±

1,6

(m

M-1

.s-1)

(

g)

Atra

zine

18,

0 ±

0,8

18,

5 ±

2,5

18

,0 ±

2,0

17,

5 ±

2,5

(nm

ol/m

in/m

g)

(h)

7-ch

loro

-4-n

itrob

enz-

2-ox

a-1,

3-di

azol

e N

BD-C

l

250

± 12

37

± 5

26

0 ±

10

118

± 7

(mM

-1.s-1

)

(d)

7-

chlo

ro-4

-nitr

oben

z-2-

oxa-

1,3-

diaz

ole

NBD

-Cl

36

2 ±

37

1

77 ±

19

(m

M-1

.s-1)

(

c)

Acro

lein

129,

0 ±

3,0

1

28,0

± 4

,0

92

,0 ±

3,0

11

6,0

± 3,

0 (m

M-1

.s-1)

(

i) Cr

oton

alde

hyde

16

,0 ±

2,0

17,

0 ±

2,0

12

,0 ±

2,0

12,

0 ±

1,0

(mM

-1.s-1

)

(i)

4-

nitr

ocin

nam

alde

hyde

4-N

CA

19

,5 ±

3,0

16,

6 ±

2,0

(m

M-1

.s-1)

(

c)

4-

viny

lpyr

idin

e

20

2,0

± 31

234,

7 ±

33,8

(m

M-1

.s-1)

(

c)

Ph

enet

hylis

ocya

nate

463

± 3

9

5

03 ±

51

(mM

-1.s-1

)

(c)

(+)-

anti-

benz

o[a]

pyre

ne d

iole

poxi

de

(+)-

anti-

BPDE

7,

5 ±

1,9

20,

0 ±

2,2

(mM

-1.s-1

)

(j)

(+

)-an

ti-be

nzo[

a]py

rene

dio

lepo

xide

(+

)-an

ti-BP

DE

10 ±

1

13

± 2

5

0 ±

10

15 ±

8

(m

M-1

.s-1)

(

b)

(+

)-an

ti-be

nzo[

a]py

rene

dio

lepo

xide

(+

)-an

ti-BP

DE

11 ±

1

14

± 2

(m

M-1

.s-1)

(

k)

(-

)-an

ti-be

nzo[

a]py

rene

dio

lepo

xide

(-

)-an

ti-BP

DE

2,7

± 0

,3

3,0

± 0

,3

(m

M-1

.s-1)

(

k)

(+

)-sy

n-be

nzo[

a]py

rene

dio

lepo

xide

(+

)-sy

n-BP

DE

64,0

± 0

,3

1

00,0

± 1

1,0

(m

M-1

.s-1)

(

j)

(+)-

anti-

Chry

sene

dio

lepo

xide

(+

)-an

ti-CD

E 3,

6 ±

1,0

9,9

± 1

,8

(m

M-1

.s-1)

(

j)

Role

of h

GSTP

1-1

poly

mor

phis

ms

(+/-

)-Ch

ryse

ne d

iole

poxi

de

(+/-

)-an

ti-CD

E

4

,0 ±

1.0

2

8 ±

4

(mM

-1.s-1

)

(l)

(+

/-)-

Chry

sene

dio

lepo

xide

(+

/-)-

anti-

CDE

3 ±

1

28 ±

8

16 ±

3

6 ±

2

(

mM

-1.s-1

)

(b)

(+)-

syn-

Chry

sene

dio

lepo

xide

(+)-

syn-

CDE

11,

0 ±

3,2

11,0

± 1

,0

(m

M-1

.s-1)

(

j)

(+)-

anti-

5-m

ethy

lchr

ysen

e di

olep

oxid

e

(+)-

anti-

5-M

eCDE

31

± 3

18 ±

3

26 ±

3

(mM

-1.s-1

)

(m)

(-)-

anti-

5-m

ethy

lchr

ysen

e di

olep

oxid

e

(-)-

anti-

5-M

eCDE

17

± 2

2

5 ±

3

11

± 1

(m

M-1

.s-1)

(m

) (-

)-an

ti-Be

nzo[

c]ch

ryse

ne d

iole

poxi

de

(-

)-an

ti-B[

c]CD

E

1

,9 ±

0,3

1

,8 ±

0,3

(mM

-1.s-1

)

(n)

(+

)-sy

n-Be

nzo[

c]ch

ryse

ne d

iole

poxi

de

(+

)-sy

n-B[

c]CD

E

0

,53

± 0,

09

0

,87

± 0,

13

(m

M-1

.s-1)

(n

)

(±)-

anti-

benz

o[c]

chry

sene

dio

lepo

xide

-1

(+/-

)-an

ti-B[

c]CD

E-1

0,

12 ±

0,0

2 0

,28

± 0,

04

0,20

± 0

,02

(m

M-1

.s-1)

(o

) (±

)-an

ti-be

nzo[

c]ch

ryse

ne d

iole

poxi

de-2

(

+/-)

-ant

i-B[c

]CDE

-2

0,0

92 ±

0,0

10

0,0

55 ±

0,0

08

0,02

2 ±

0,00

8

a.

Ali-

Osm

an e

t al.(

1997

). J.

Biol

. Che

m. 2

72, 1

0004

–100

12; b

. Hu

et a

l. (1

997)

Bio

chem

. Bio

phys

. Res

. Com

mun

. 238

, 397

–402

; c. J

ohan

sson

et a

l. (1

998)

J M

ol B

iol.

278,

68

7-98

; d. P

arke

r et

al.

(200

8) J

Mol

Bio

l. 38

0, 1

31-4

4; e

. Goo

dric

h an

d Ba

su N

. (20

12) T

oxic

ol In

Vitr

o. 2

6, 6

30-5

; f. S

riva

stav

a et

al.

(199

9) A

rch.

Bio

chem

.Bio

phys

. 36

6, 8

9–94

; g. P

andy

a et

al.

(200

0) B

ioch

. Bio

phys

.Res

Com

mun

278

, 258

–262

; h. A

bel e

t al.

(200

4) T

oxic

ol S

ci. 8

0, 2

30-8

; i. P

al e

t al.

(200

0) C

ance

r Let

t. 15

4 39

-43;

j.

Sund

berg

et a

l. (1

998)

Car

cino

gene

sis. 1

9, 4

33-6

; k. H

u et

al.

(199

7) B

ioch

. Bio

phys

.Res

Com

mun

. 235

, 424

–428

; l. H

u et

al.

(199

7) A

rch.

Bio

chem

.Bio

phys

. 345

, 32–

38;

m. H

u et

al.

(199

8b) C

arci

noge

nesis

. 19,

168

5-9;

n. S

undb

erg

et a

l. (1

998a

) FEB

S Le

tt. 4

38, 2

06-1

0; o

. Pal

et a

l. (2

000b

) FEB

S Le

tt. 4

86, 1

63-6

;

Chapter 7

CYTOCHROME P450 MEDIATED BIOACTIVATION OF MEFENAMIC ACID TO REACTIVE BENZOQUINONEIMINES AND THEIR

INACTIVATION BY HUMAN GSTs Adapted from Harini Venkataraman, Michiel W. den Braver, Nico P. E. Vermeulen, Jan N. M. Commandeur (2014) Chemical Research in Toxicology

Bioactivation Of Mefenamic Acid By Cytochrome P450s

Abstract

Mefenamic acid (MFA) has been associated with rare but severe cases of hepatotoxicity, nephrotoxicity and hypersensitivity reactions which are believed to result from the formation of reactive metabolites. Although formation of protein-reactive acylating metabolites by phase 2 metabolism has been well-studied and proposed as cause of these toxic side effects, the oxidative bioactivation of MFA has not yet been competely characterized. In the present study, the oxidative bioactivation of MFA was studied using human liver microsomes (HLM) and recombinant human P450 enzymes. In addition to the major metabolite 3’-OH-methyl-MFA, resulting from the benzylic hydroxylation by CYP2C9, 4'-hydroxy-MFA and 5-hydroxy-MFA were identified as metabolites resulting from oxidative metabolism of both aromatic rings of MFA. In presence of GSH, three GSH conjugates were formed which appeared to result from GSH-conjugation of the two quinoneimines formed by further oxidation of 4'-hydroxy-MFA and 5-hydroxy-MFA. The major GSH-conjugate was identified as 4’-OH-5’glutathionyl-MFA and was formed at the highest activity by CYP1A2 and to a lesser extent by CYP2C9 and CYP3A4. Two minor GSH-conjugates resulted from secondary oxidation of 5-hydroxy-MFA and were formed at the highest activity by CYP1A2 and to a lesser extent by CYP3A4. Additionally, the ability of seven human glutathione S-transferases (hGSTs) to catalyze the GSH conjugation of the quinoneimines formed by P450s was also investigated. The highest increase of total GSH-conjugation was observed by adding hGSTP1-1, followed by hepatic hGSTs hGSTA2-2 and hGSTM1-1. The results of this study show that next to phase 2 metabolites, also reactive quinoneimines formed by oxidative bioactivation might contribute to the idiosyncratic toxicity of MFA.

160

CHAPTER 7

Introduction

Mefenamic acid (MFA) belongs to the class of N-aryl-anthranilic acid containing non-steroidal anti-inflammatory drugs (NSAIDs) and is widely used in the treatment of pain, arthritis and dysmenorrhea (1, 2). The anti-inflammatory effect of MFA is due to the inhibition of the production of prostaglandins formed by cyclooxygenase (COX) enzymes (3). Use of MFA has been associated with gastrointestinal toxicity which is a common adverse effect of carboxyl-group containing NSAIDs (4, 5). Additionally, MFA has also been shown to produce rare cases of nephrotoxicity and hepatotoxicity in patients (6-8). A proposed mechanism for the NSAID-mediated toxicity is the bioactivation of the drug to chemically reactive metabolites which can then covalently bind to proteins (9). Both formation of acyl glucuronides and reactive oxidative metabolites, such as arene oxides and quinoneimines, have been shown to contribute to irreversible protein binding of NSAIDs. In case of MFA, glucuronidation of the parent drug and its oxidative metabolites appear to be the major pathways of metabolism. Analysis of the urine samples of patients undergoing MFA therapy showed that glucuronides of 3’-hydroxy-methyl-MFA, 3’-carboxy-MFA and MFA itself were excreted as metabolites (10). MFA is mainly glucuronidated to form 1-O-acyl-MFA glucuronide (MFA-1-O-G) which can bind irreversibly to proteins in vitro and ex vivo. The covalent binding of MFA to cellular protein was increased in Chinese hamster V79 cells expressing UGT 1A9 (2). Also the involvement of UGT1A9 and UGT2A7 in the glucuronidation of MFA by human kidney cortical microsomes has been reported (11). More recently, MFA-acyl-adenylate (MFA-AMP), MFA-S-acyl-coenzyme A (MFA-CoA) and MFA-S-acyl-glutathione (MFA-GSH) were shown to be additional reactive acylating metabolites of MFA which appeared to be even more reactive than MFA-1-O-G (1, 12). Although the bioactivation of MFA by phase II-enzymes to acylating metabolites has been well characterized, the possible role of oxidative bioactivation is much less defined. In the case of diclofenac, protein modification by oxidative metabolites were suggested to be more critical for acute cytotoxicity than modification by its acyl glucuronide (13). It has been shown previously that MFA is mainly metabolized via benzylic hydroxylation to 3’-hydroxymethyl-MFA in human liver microsomes (HLM) (14, 15) (Figure 1). Inhibition studies showed that cytochrome P450 2C9 (CYP2C9) is the major enzyme involved. 3’-Hydroxymethyl-MFA is further metabolized to form the 3’-carboxy-MFA. Zheng et al. showed that incubation of MFA with HLM in the presence of NAPDH and GSH resulted in the formation of GSH conjugates with m/z

161

Bioactivation Of Mefenamic Acid By Cytochrome P450s

563 and m/z 547 which were proposed to originate from quinoneimine and epoxide metabolites, respectively (16). A corresponding GSH conjugate, with m/z 561 when detected using negative electrospray ionization, was recently found in incubations of MFA with rat liver microsomes in the presence of GSH (17). The specific enzymes involved in these oxidative bioactivation reactions and the structures of the GSH-conjugates, however, have not yet been investigated. The conjugation of reactive quinoneimines to GSH can be spontaneous and/or mediated by glutathione S-transferases (GSTs) (18). Because several human GSTs are known to be genetically polymorphic, interindividual variations in GSTs have been proposed as one of the factors determining susceptibility to adverse drug reactions (19, 20). For the structurally similar drug diclofenac,it has been shown that hGSTs play an important role in the inactivation of diclofenac quinoneimines (21). In a recent study, hGSTP1-1 was shown to catalyze GSH conjugation which indicated the importance of hGST in the inactivation of quinoneimines (22). However, for MFA, the effect of other human hGSTs has not yet been reported. The aim of this study is to characterize the P450 dependent pathways that are involved in the oxidative bioactivation of MFA. To identify the specific P450s involved in bioactivation, incubations were performed with HLM, with and without isoenzyme-specific inhibitors, and recombinant human P450 enzymes. To enable structural elucidation of the metabolites by NMR, the drug metabolizing cytochrome P450 BM3 mutant CYP102A1 M11H was used to produce human relevant metabolites of MFA in large amounts (23-25). The role of a panel of seven human GSTs in the GSH conjugation of reactive quinoneimine metabolites was studied in incubations of MFA using drug-metabolizing bacterial P450 BM3 mutant (CYP102A1M11H) as bioactivation system.

162

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)

163

Bioactivation of mefenamic acid by cytochrome P450s

Materials and methods

Materials

Human liver microsomes (HLM; Lot No. 0710619), pooled from 50 donors, were obtained from Xenotech (Lenexa, USA) and contained 20 mg protein/mL. Supersomes containing cDNA-expressed human cytochrome P450 enzymes were purchased from BD Biosciences (Breda, Netherlands). The enzymes used were CYP1A1(Lot No. 35400), CYP1A2 (Lot No. 21667), CYP2A6 (Lot No. 33769), CYP3A4 (Lot No.38275), CYP3A5 (Lot No. 44743), CYP1B1 (Lot No. 26314), CYP2B6 (Lot No. 62543),CYP2C8 (Lot No. 62556), CYP2C9*1(Arg144) (Lot No. 41274), CYP2C18 (Lot No. 11301),CYP2C19 (Lot No. 62542), CYP2D6*1 (Lot No. 38273), CYP2E1 (Lot No. 44748) and CYP2J2 (Lot.No. 456264). Cytochrome P450 BM3 mutant CYP102A1M11H was expressed and purified as described previously (24). MFA was purchased from Sigma Aldrich. Escherichia coli XL-1 Blue cells containing the expression plasmid for human GSTP1-1, M1-1, A1-1 was a kind gift from Prof. Mannervik (Department of Biochemistry and Organic chemistry, Uppsala University, Sweden). Expression plasmid for GST T1-1 was a kind gift from Prof. Hayes (Biomedical Research Centre, University of Dundee, Scotland, United Kingdom). All other chemicals and reagents were of analytical grade and obtained from standard suppliers. Construction of expression plasmids for hGST A2-2, hGST M2-2 and hGST M3-3

The coding region of GSTA2-2, GSTM2-2 and GSTM3-3 was amplified by polymerase chain reaction from human GST cDNA clones obtained from Origene, Rockville, USA (SC119650 for A2-2, SC119651 for M2-2, SC319401 for M3-3) using the following gene specific primers flanked by upstream restriction site NdeI and downstream site BamH1. GSTA2-2 (F) : 5’- GGA ATT CCA T ATG GCA GAG AAG CCC AAG CTC C - 3’ GSTA2-2 (R) : 5’- CG GGA TCC TTA AAA CCT GAA AAT CTT CCT TGA TTC -3’ GSTM2-2 (F) : 5’- GGA ATTC CAT ATGCCCATGACACTGGGGTAC-3’ GSTM2-2 (R) : 5’-CG GGA TCC CTA CTT GTT GCC CCA GAC AGC CAT C -3’ GSTM3-3 (F) : 5’ -GGA ATTC CAT ATGTCG TGC GAG TCG TCT ATG G-3’ GSTM3-3 (R) : 5’-CG GGA TCC TCA GCA TAC AGG CTT GTT GCC CC-3’ The amplified gene was subcloned into the NdeI and BamHI sites of the pET-20b(+) vector (Novagen, Madison, WI), and introduced into E. coli BL21(DE3). The subcloned cDNAs were confirmed by sequencing (Macrogen, Amsterdam, The Netherlands).

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Expression and purification of hGST enzymes

Human GST enzymes were expressed and purified as described previously (25). Protein concentrations were determined according to the method of Bradford (26) with reagent obtained from Bio-Rad. Purity of the enzymes was checked by SDS polyacrylamide gel electrophoresis on a 12% gel with Coomassie staining. The specific activity of the purified hGSTs was assayed according to Habig et al., (27) . The specific activities of the purified recombinant hGSTs using 1 mM CDNB as a substrate were: 27.6 μmol/min/mg protein for GSTA1-1, 11.5 μmol/min/mg protein for GSTA2-2, 58.9 μmol/min/mg protein for GSTM1-1, 35.7 μmol/min/mg protein for GSTM2-2, 4.2 μmol/min/mg protein for GSTM3-3 and 47.7 μmol/min/mg protein for GSTP1-1. The specific activity of hGSTT1-1 was determined using 1,2-epoxy-3 (pnnitrophenoxy)propane as a substrate as described (28) and was 1.83

otein. Incubation of MFA with HLM and recombinant human cytochrome P450s

Incubations containing 100 M MFA with and without 5 mM GSH were performed in 100 mM potassium phosphate buffer (pH 7.4) at a final volume of 250 L containing 2 mg/ml HLM. Reactions were initiated by the addition of a NADPH regenerating system (0.5 mM NADPH, 20 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, final concentrations) and performed for 60 min at 37 ºC.For bioactivation of MFA by recombinant human P450s, incubations were performed at a

of 100 nM. Final DMSO concentrations in the incubations were always below 1%. For 3 of the most active human CYPs (CYP1A2, CYP2C9, CYP3A4) and HLM, incubations were also performed as described above at low MFA concentration of 5 μM. Reactions were initiated by the addition of NADPH regenerating system and continued for 60 min at 37 ºC. All reactions were terminated by addition of an equal volume of ice-cold methanol containing 2% (v/v) of aqueous 50 mM ascorbic acid and centrifuged for 15 min at 14000 rpm. The supernatants were analyzed by reversed-phase liquid chromatography and LC-MS/MS as described below. Inhibition of metabolite formation in incubations of MFA with pooled human liver microsomes by isoenzyme-specific inhibitors of P450s

The contribution of individual human P450s to metabolite formation in incubation of MFA with pooled HLM was determined by performing incubations in the presence or absence of specific inhibitors of individual P450 enzymes. The final concentration of HLM was 2 mg/mL. Incubations were performed in 100 mM potassium phosphate

165


buffer (pH 7.4) and at a final volume of 100 μL. The concentration of MFA was 100 μM and the final concentration of DMSO in incubations was less than 1%. P450 selective inhibitors, furafylline (FURA,10 μM), ketoconazole (KTZ, 2 μM), sulfaphenazole (SPZ, 10 μM) and quinidine (QND, 2 μM) were used to investigate the involvement of CYP1A2, CYP3A4, CYP2C9 and CYP2D6 respectively (29-31). All inhibitors were dissolved in methanol and the final concentration of methanol in the incubations did not exceed 1%. Reactions were initiated by the addition of NADPH regenerating system and incubated for 60 min at 37 ºC. Incubations containing the mechanism-based inhibitor FURA were first pre-incubated for 15 min in the presence of NADPH before addition of MFA. The reactions were terminated by the addition of 100 μL of ice-cold methanol containing 2% (v/v) of 50 mM aqueous ascorbic acid and centrifuged for 15 min at 14000 rpm. The supernatants were analyzed by HPLC and LC-MS/MS, as described in section 2.7. Control incubations without MFA were performed under the same conditions to ensure that the presence of inhibitors did not interfere with the quantification of formed metabolites. Bioactivation of 4’-OH and 5-OH mefenamic acid in the presence of GSH and human recombinant hGSTs

Incubations in 100 mM potassium phosphate buffer (KPi buffer, pH 7.4) containing 4’-OH-MFA or 5-OH-MFA (final concentration 50 μM) were performed using purified CYP102A1 M11H (20 nM) as bioactivation system in the presence of 0.1 mM GSH for 60 minutes at 24 °C. The concentration of CYP102A1 M11H was chosen such that bioactivation rates similar to that of HLM (2 mg/ml) were obtained. Enzymatic GSH

(final concentration) of hGSTs A1-1, A2-2, M1-1, M2-2, M3-3, P1-1 or T1-1 to the incubations in the presence of 0.5 mM GSH. The reactions were initiated and stopped as described above. Samples were analyzed by an LC-MS method described below. Analytical methods

The analyses of metabolites were performed by reversed-phase liquid chromatography on a Shimadzu HPLC equipped with two LC-20AD pumps, a SIL20AC

as stationary phase. For separation of parent compound and metabolites, a gradient method was used with two mobile phases: solvent A (1% acetonitrile, 99% water, 0.2% formic acid) and solvent B (1% water, 99% acetonitrile, 0.2% formic acid). The first 5 min was isocratic at 20% solvent B. From 5 till 30 min the concentration of solvent B linearly increased to 100%, followed by linear decrease back to 20% until

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30.5 min. Isocratic re-equilibration at 20% solvent B was maintained until 45 min. The flow rate was 0.5 mL/min and the detection was at 254 nm. For MS analysis, samples were analyzed by using an Agilent 1200 Series Rapid resolution LC system connected to a time-of-flight (TOF) Agilent 6230 mass spectrometer equipped with electrospray ionization (ESI) source and operating in the positive mode. The MS ion source parameters were set with a capillary voltage at 3500 V. Nitrogen was used as drying gas (10 L/min) and nebulizing gas (pressure 50 psig) at a constant gas temperature of 350 °C. MS spectra were acquired using Agilent TOF system. Data analysis was performed using Agilent MassHunter Qualitative analysis software. For the product ion spectrum, samples were analyzed by automated MS/MS analysis using an Agilent 1200 Series Rapid resolution LC system connected to a hybrid quadrupole-time-of-flight (Q-TOF) Agilent 6520 mass spectrometer (Agilent Technologies, Waldbronn, Germany). Large scale incubation of MFA with cytochrome P450 BM3 mutant CYP102A1 M11H

To obtain the oxidative metabolites and major GSH conjugate MG1 of MFA on a preparative scale, large scale incubations were performed with CYP102A1 M11H as biocatalyst. A 20 mL reaction volume containing 250 nM purified CYP102A1 M11H, 500 μM MFA, 5 mM GSH and an NADPH regenerating system (0.5 mM NADPH, 20 mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase) was prepared in 100 mM potassium phosphate buffer pH 7.4. The reactions were allowed to continue for 4 h at 25°C. The reactions were terminated by adding an equal volume of ice cold methanol containing 2 % (v/v) of aqueous 50 mM ascorbic acid and centrifuged for 15 min at 14000 rpm. After precipitation of the proteins, the sample was evaporated and reconstituted in 20% B. A series of 1 mL injections of the sample were loaded on a preparative HPLC column Luna 5 μm C18(2) column (250 mm x 10 mm i.d.) from Phenomenex (Torrance, CA, USA) which was initially equilibrated with 20% eluent B. A flow rate of 1.5 mL/min and a gradient using the eluent A (0.8% acetonitrile, 99% water, and 0.2% formic acid) and B (99% acetonitrile, 0.8% water, and 0.2% formic acid) was applied for separation of formed MFA metabolites. The first 10 min was isocratic at 20% eluent B: from 10 to 45 min, the percentage of eluent B increased linearly to 100% followed by linear decrease back to 20% until 45.5 min. Isocratic re-equilibration at 20% solvent B was maintained until 60 min. Metabolites were detected using UV detection (254 nm) and collected by automated fraction collector. Collected fractions were first analyzed for purity and identity by the analytical HPLC and LC-MS/MS methods as described above. The samples were evaporated to dryness under a nitrogen stream and redissolved in 500 μL of methyl alcohol-d4. 1H-NMR-

167


analysis was performed on Bruker Avance 500 (Fallanden, Switzerland), equipped with cryoprobe. 1H-NMR measurements were carried out at 500 MHz. The structural assignment of the metabolites was done by using COSY spectrum. Quantification of isolated metabolites of mefenamic acid by 1H NMR

To enable absolute quantification of the major metabolites of mefenamic acid by NMR, dimethyl formamide (DMF) was used as an internal standard. A final concentration of 3.2 mM DMF was added to dmso-d6 solutions of M1, M2 and MG1 and analyzed by 1H NMR. All NMR data were acquired on Bruker 500 MHz spectrometer equipped with 5 mm cryoprobe. DMF was selected as internal standard because its signals (singlets at 2.73 ppm and 2.89 ppm corresponding to 2 methyl groups) do not interfere with the aliphatic proton signals of MFA metabolites. By integrating the signals of aliphatic methyl group protons of M1 [5H], M2 [6H] and MG1 [6H] and comparing to that of DMF ([6H]), the concentration of the metabolites were determined. Standard curves of 3’-OH-MFA, 4’-OH-MFA and the major GSH conjugate were prepared using these solutions quantified by 1H NMR spectroscopy. The samples were analyzed by HPLC-UV and LC-MS methods as described above. Linear standard curves were obtained for each of the metabolites by HPLC UV- - - -

M2 and MG1 were used to estimate the amount of M3 and minor GSH conjugates (MG2 and MG3) respectively as high amount of material could not be generated to enable NMR quantification.

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Results Oxidative metabolism of MFA by HLM MFA was first incubated with HLM in the presence of NAPDH and GSH to identify all the oxidative metabolites and GSH conjugates. As shown in Figure 2A, incubation of MFA with HLM produced 3 metabolites (M1, M2, M3) with an m/z value of 258.11 (Figure 2B) corresponding to mono-oxygenated products. The major product M1 contributed to about 84 % of the total mono-hydroxylated product formed while M2 and M3 accounted for about 13% and 3% respectively. The major product of MFA formed by human P450s was previously identified as 3’-hydroxy-methyl-MFA (15, 32). As shown in Figure 2C, in the presence of 5 mM GSH, three GSH conjugates were observed with m/z values of 563, indicating the incorporation of both an oxygen atom and GSH into the MFA moiety. These products implicate the involvement of quinoneimine intermediates. The product ion spectrum of the major conjugate MG1 showed the characteristic fragments arising from the cleavage of the GSH moiety such as the loss of glycine, loss of pyroglutamate, loss of GSH, cleavage between the cysteinyl C-S bond with charge retention on the aromatic moiety and further loss of a water molecule from the fragment ion (Supplementary Figure S1). Table 1 Oxidative metabolites and GSH conjugates formed in incubation of MFA with HLM and CYP102A1 M11H

a Retention time tr in minutes is based on HPLC-UV chromatogram b Retention time tr in minutes is based on MS analysis c m/z values correspond to the singly protonated molecule [(M+H)]+

Metabolites tr(min) Exact mass m/zc

Elemental composition Proposed structure

Oxidative metabolites a

M1 21.58 258.117 C15H15NO3 MFA+O M2 M3

23.35 23.68

258.117 258.117

C15H15NO3 C15H15NO3

MFA+O MFA+O

GSH-conjugates b

MG1 16.41 563.189 C25H31N4O9S MFA+O+SG MG2 15.97 563.189 C25H31N4O9S MFA+O+SG MG3 15.27 563.189 C25H31N4O9S MFA+O+SG

169


Figure 2.A. Representative HPLC chromatogram obtained after incubation of mefenamic acid (100 μM) with human liver microsomes (2 mg/ml) in presence of NADPH and reduced glutathione (GSH). B. Extracted ion chromatogram (EIC) at m/z 258.11 of mono-hydroxylated metabolites C. Extracted ion chromatogram (EIC) at m/z 563.18 of GSH conjugates The product ion spectrum of MG1 was identical to the GSH conjugate observed by Zheng et al.,(2007) which was attributed to GSH conjugation to a quinoneimine metabolite. However, it was not yet elucidated which ring of MFA was bioactivated to the quinoneimine. The product ion spectrum of the two minor GSH conjugates is shown in Supplementary Figure S2. The product ions with m/z 244, m/z 444 and m/z 519 show that the GSH conjugates MG2 and MG3 are most likely to be derived from 5-OH-MFA pathway, corresponding to aromatic hydroxylation on carboxy phenyl ring. These two minor conjugates were not reported previously in the study of Zheng et al. (2007).

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Figure 3. A. Representative HPLC chromatogram obtained after incubation of mefenamic acid (500 μM) with CYP102A1 in presence of NADPH and reduced glutathione (GSH). S1, S2 and S3 denote secondary oxidation products with m/z 274 , m/z 272 and m/z 256 respectively. B. Extracted ion chromatogram (EIC) at m/z 258.11 of mono-hydroxylated metabolites C. Extracted ion chromatogram (EIC) at m/z 563.18 of GSH conjugates Large scale production of MFA metabolites by CYP102A1 M11H and structural elucidation by NMR Incubation of CYP102A1 M11H with MFA resulted in the formation of all relevant metabolites as observed in HLM incubation (Figure 3). The retention times and the fragmentation patterns of the products formed by CYP102A1 M11H and HLM were identical (Table 1). In addition, three metabolites with m/z values of 274 (S1), 272 (S2) and 256 (S3) were observed in the CYP102A1 M11H incubation, possibly due to secondary oxygenation and dehydrogenation. In the case of CYP102A1 M11H incubation, M1 contributed to about 20% of the total mono-hydroxylated product formed whereas M2 and M3 contributed to about 70% and 10% respectively. Although the product ratio of the hydroxylated metabolites was different between CYP102A1 M11 and HLM, cytochrome P450 BM3 mutant was highly active and converted more than 70% of MFA to the oxidative metabolites.

171


As shown in Figure 3C, CYP102A1 M11H was also able to produce all 3 human relevant GSH conjugates, with the highest production of the major GSH conjugate MG1. The retention time and fragmentation pattern of the GSH conjugates were identical to those observed in HLM incubations. Large scale incubations were performed using CYP102A1 M11H as biotransformation system to generate the metabolites on large scale for the structural information of the metabolites by NMR. The 1H NMR spectrum of M1 was identical to that of 3’-OH-methyl MFA, as previously described (33). The 1H NMR spectra of M2 and M3, showed that the two benzylic methyl groups were unchanged implicating that the hydroxylation has taken place on any one of the aromatic rings. Table 2. 1H NMR shifts of aromatic region for the mefenamic acid metabolites M2, M3 and major glutathione conjugate MG1 formed by CYP102A1 M11H.

Proton 4’-OH-MFA (M2) 5-OH-MFA (M3) (MG1)

2’-CH3 2.12(1H,s) 2.17(1H,s) 2.13(1H,s)

3’-CH3 2.18(1H,s) 2.32(1H,s) 2.26(1H,s)

4’-H - 7.09(1H,d) J=7.9Hz

-

5’-H 6.88 (1H,d) J=8.5 Hz

7.03(1H,t) J= 7.7 Hz

-

6’-H 6.67 (1H,d) J=8.5 Hz

6.92(1H,d) J=7.3 Hz

7.22 (1H,s)

3-H 6.41 (1H,d) J=8.2 Hz

6.74(1H,d) J=8.8 Hz

6.48(1H,d) J=8.5 Hz

4-H 7.11(1H,ddd) J=8.5, 7.6, 1.6 Hz

6.83(1H,dd) J=8.9, 3.0 Hz

7.25(1H,ddd) J=8.8, 6.9, 1.6 Hz

5-H 6.57(1H,t) J=7.6 Hz

- 6.63(1H,t) J=7.6 Hz

6-H 7.91(1H,dd) J=7.9, 1.6 Hz

7.42(1H,d) J=2.8 Hz

7.94(1H,dd) J=7.9, 1.6 Hz

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Figure 4. Comparison of aromatic regions of 1H HMR spectrum of A. 4’-OH-MFA B. Major GSH conjugate 4’-OH-5’-(glutathion-S-yl)-MFA (MG1) formed via 4’-OH pathway. The aromatic region of 1H NMR spectrum of M2 showed 6 signals with an integral of 6 protons. The presence of a doublet of doublet signal with ortho coupling of 7.9 Hz and meta coupling of 1.6 Hz corresponding to proton H-6 in the carboxy phenyl ring, indicated that protons H-5 and H-4 were still present (Table 2). Also the presence of doublet of doublet of doublet multiplicity pattern (ddd) with 2 ortho couplings and one meta coupling corresponding to H-4 was found. This indicates that the hydroxyl group is localized on the dimethyl phenyl ring. The aromatic protons of this ring were assigned with help of COSY spectra (Supplementary Figure S3). The presence of doublets at 6.88 and 6.67 ppm with strong ortho couplings corresponding to H-5’ and H-6’, respectively and the absence of a signal corresponding to H-4’ indicated that the metabolite M2 results from hydroxylation at 4’ position on the dimethyl phenyl ring. Metabolite M3, which is also hydroxylated on an aromatic ring, did not show the characteristic meta and ortho coupling arising from H-6, clearly showing that the hydroxylation took place on the carboxy phenyl ring. The H-6 proton of metabolite M3 is strongly shifted 0.5 ppm up-pattern from a double doublet (dd) to a doublet (d) with a coupling constant of 2.8 Hz

173


corresponding to a meta coupling. Also, H-4 proton showed only one ortho coupling and one meta coupling indicating that proton H-5 was not present anymore confirming that the position of hydroxylation was C-5 thus assigning M3 to be 5-OH-MFA. Figure 4B shows the aromatic region of 1H NMR spectra of the major GSH conjugate MG1 isolated by preparative HPLC from the large scale incubation. The signal at 7.94 ppm corresponding to H-6 proton gives rise to doublet of doublet with both ortho and meta coupling with the neighbouring protons. This confirms that the major GSH conjugate formed is via the hydroxylation on dimethyl phenyl ring. Comparison of the 1H NMR spectrum of 4´-OH-MFA and the GSH conjugate MG1 clearly shows all signals from carboxy phenyl ring protons were identical (Figure 4). Additionally, the dimethyl phenyl ring showed only a singlet from one proton. Based on COSY spectra, this proton was assigned to be H-6’ (Supplementary Figure S4). This further confirms that the addition of GSH has taken place at C-5’ position in the dimethyl phenyl ring. Therefore the major GSH conjugate was assigned as 4´-hydroxy-5´-glutathionyl-MFA (4´-OH-5´-GS-MFA) which can be rationalized by GSH-conjugation of MFA-1',4'-quinoneimine (MFA-1',4'-QI). NMR characterization of the minor GSH conjugates was not possible because of the low amount of products formed. However, incubation of purified 5-OH-MFA with HLM and GSH resulted in the formation of both MG2 and MG3 (Supplementary Figure S5). This indicates that these two GSH conjugates are derived from MFA-2,5-quinoneimine (MFA-2,5-QI) which is formed by secondary oxidation of 5-OH-MFA. Metabolism and bioactivation of MFA by recombinant P450s To investigate the contribution of individual human cytochrome P450 enzymes to the identified metabolites of MFA, incubations were performed with Supersomes containing human recombinant P450s at MFA concentration of 100 μM. Figure 5 shows the activities relative to the most active CYP. The major metabolite of MFA, M1, was produced mainly by CYP2C9 and to a lesser extent by CYP2C19 (Figure 5A). The relative rates of the formation of 4’-OH-MFA and the major GSH adduct MG1 from 4’-OH pathway with respect to the most active P450 is shown in Figure 5B. CYP1A2 and CYP2C9 showed the highest activity, forming almost equal amounts of 4’-OH-MFA followed by CYP1A1 and CYP3A4 to a lesser extent. CYP1A2 showed the highest activity for MG1 which results from the addition of GSH to MFA-1',4'-QI. CYP2C9 also produced MG1, however the amount of MG1 formed by CYP1A2 was higher than that of CYP2C9, suggesting that CYP1A2 may catalyze the second oxidation step more efficiently.

174

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175


Figure 5C shows the relative rates of the formation of 5-OH-MFA and the minor GSH adducts resulting from MFA-2,5-QI. CYP1A2 catalyzed the formation of 5-OH MFA followed by CYP3A4, CYP2C19 and CYP1A1. The formation of minor GSH conjugates derived from the 5-OH pathway was also predominantly catalyzed by CYP1A2 (Supplementary Figure S6) and to a relatively lower extent by CYP 3A4 and CYP 1A1 (Figure 5B). Similar results were also obtained when incubations were performed at

CYP1A2, 2C9 and 3A4 (Figure 5D,E,F). As shown in Figure 5D, the formation of M1 was primarily catalyzed by CYP 2C9. CYP1A2 was the major P450 isoform involved in the bioactivation pathway forming the highest amount of MG1 (Figure 5E) as well as in the formation of M3 (Figure 5F). 3.4.Inhibition of oxidative metabolism of MFA by HLM by P450-specific inhibitors Figure 6. Effect of CYP450 inhibitors on the metabolism of mefenamic acid (MFA) by HLM to A. 4’-OH mefenamic acid (4’-OH-MFA) and major GSH conjugate MG1 formed via the 4’-OH quinone imine pathway B. A. 5-OH mefenamic acid (5-OH-MFA) and minor GSH conjugates MG2 and MG3 formed via the 5-OH-quinone imine pathway. Data are expressed as % of control activity and represent mean of duplicate determinations.

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Incubations of MFA with HLM were performed with P450 isoenzyme-specific inhibitors to test the effect of the inhibitors on the formation of M2, M3 and the GSH conjugates (MG1, MG2 and MG3) formed by the respective quinoneimine pathways. Figure 6 shows the effect of P450 isoenzyme selective inhibitors on the formation of 4’-OH and 5-OH-MFA and their corresponding GSH conjugates.The results obtained are expressed as percentage of activity of the control HLM incubation in which no inhibitor was added. As shown in Figure 6A, addition of the CYP2C9-inhibitor SPZ resulted in the highest amount of inhibition (75%) for the formation of 4’-OH-MFA, followed by the CYP1A2-inhibitor FUR. FUR and SPZ inhibited the formation of the major GSH conjugate MG1 to almost similar extent which is consistent with the results obtained with recombinant P450s. For the formation of 5-OH-MFA (M3) and the corresponding GSH conjugates derived from this pathway, addition of FUR inhibited formation of M3, MG2 and MG3 about 70% (Figure 6B). The results from the inhibition studies was consistent with the results obtained with the recombinant P450 incubations indicating that CYP1A2 and CYP2C9 play an important role for the 4’-OH pathway while CYP1A2 is the main isoform for the 5-OH pathway. Human glutathione S-transferases mediated inactivation of mefenamic acid quinoneimines In order to study the effect of human glutathione S-transferases in the inactivation of the quinoneimine intermediates of MFA, incubations were performed with 4’- and 5-OH-MFA in the presence of one of the 7 hGSTs and cytochrome P450 M11 as bioactivation system. As shown in Figure 7A, using 4’-OH-MFA as a substrate,

– 1.5 fold increase compared to the non-enzymatic formation of MG1. This increase in GSH conjugation is moderate in comparison to structurally comparable 4’-OH-diclofenac which showed much higher GSH conjugation in the presence of hGSTs (21). This may be attributed to the fact that the non-enzymatic glutathione conjugation is higher for MFA than diclofenac. For 5-OH-MFA, GSH conjugate formation from MFA-2,5-QI was significantly increased by the addition of the different hGSTs. Consistent with previous study, hGSTP1-1 showed the highest increase (about 8.5 fold) towards the formation of GSH conjugates in comparison to the non-enzymatic conjugation (22). The addition of hepatic hGSTs A2-2 and M1-1 also resulted in 3.5-4 fold increase towards the formation of GSH conjugates from 5-OH pathway (Figure 7B).

177


Figure 7. -S-transferases in the inactivation of A. MFA-1’4’-quinoneimine B. MFA-2,5-quinoneimine Discussion Several recent studies have demonstrated that the carboxylic acid-containing NSAID MFA can be bioactivated by phase 2-enzymes to a variety of protein-reactive acylating metabolites, such as MFA-1-O-G, MFA-AMP, MFA-CoA and MFA-GSH (1, 12). The formation of protein adducts from these acylating agents have been suggested to play a role in the onset of the idiosyncratic toxicities of MFA, which include hepatic and renal toxicity and hypersensitivity reactions (8, 34, 35). Although GSH-conjugates derived from oxidative bioactivation of MFA have been detected previously (16, 17), the structures of the reactive intermediates and the P450s involved have not yet been characterized. Therefore, the aim of the present study was to characterize the P450-dependent bioactivation of MFA to quinoneimines and their inactivation by human glutathione S-transferases in more detail. Consistent with previous studies, the major P450 mediated oxidative metabolite formed by HLM was found to be 3’-OH-methyl-MFA (15, 32). In addition to 3’-OH-methyl-MFA, 4'-OH-MFA (M2) and 5-OH-MFA (M3) were identified as metabolites resulting from aromatic hydroxylation, Figure 2. These two hydroxylated MFA-metabolites can be bioactivated by dehydrogenation to reactive quinoneimines, as has

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been previously reported for drugs like diclofenac, meclofenamic acid, glafenine, lumiracoxib (36-39). For the structurally related NSAIDs diclofenac and meclofenamic acid, several GSH conjugates have been identified that result from GSH-conjugation to the quinoneimines formed via the 4’-OH- and 5-OH-hydroxylation pathways (38, 40). Zheng et al., previously reported the formation of one GSH adduct of MFA with m/z 563 which was tentatively assigned to a reactive quinoneimine metabolite (16). In the present incubations with HLM and recombinant P450s, three GSH conjugates with m/z value of 563 could be detected that can be explained by GSH-conjugation to reactive quinoneimine metabolites. The major conjugate MG1 with m/z 563 was unequivocally identified by 1H NMR as a GSH-conjugate formed from MFA-1',4'-QI (Figure 4). The two minor GSH conjugates MG2 and MG3 with m/z 563, which were not identified previously, were identified as regioisomeric GSH-conjugates derived from MFA-2,5-QI. Formation of GSH conjugates with m/z 547 which were reported by Zheng et al., was not observed in our incubations of MFA with HLM and recombinant P450s. This may be explained by the higher sensitivity of the multiple reaction monitoring method used. The extremely low levels of these products may question the toxicological relevance of the short-lived epoxide intermediates of MFA. A proposed mechanism for the formation of the major GSH conjugate as well as the 2 minor GSH conjugates is depicted in Figure 8. The first step involves the oxidation on either one of the aromatic rings to yield 4’-OH-MFA or 5-OH-MFA. This is followed by a P450 mediated dehydrogenation of 4’-OH-MFA or 5-OH-MFA that results in the corresponding reactive quinoneimines. As shown in Figure 5, the incubations of MFA with recombinant human P450s showed that both CYP1A2 and CYP2C9 catalyze the formation of 4’-OH-MFA while the GSH conjugate MG1 was formed at higher amounts by CYP1A2 compared to CYP2C9. Pooled HLM inhibition studies with P450 selective inhibitor showed that both CYP2C9 and CYP1A2 inhibited the formation of MG1 to almost equal amounts (Figure 6). This may be due to the relatively higher abundance of CYP2C9 compared to CYP1A2 in human liver (41, 42). Aromatic oxidation to 5-OH and the formation of GSH conjugates MG2 and MG3 was mainly catalyzed by CYP1A2. Previously, it was shown that MFA inhibited phenacetin O- deethylation catalyzed by CYP1A2 with IC50 value < 10 μM, indicating that it has a high affinity for CYP1A2 (43). However, prior to this study, there is no information available about the involvement of CYP1A2 or other human P450s in the bioactivation pathway of MFA. CYP2C9 is polymorphic in human population (44) and polymorphic variants of CYP2C9 have been shown to exhibit differences in metabolic clearance for NSAIDs (45). Additionally, CYP1A2 is an inducible P450 enzyme (46) and therefore variations in CYP1A2 and CYP2C9 enzyme activities between individuals can be a risk factor for adverse drug reactions caused by MFA.

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Figure 8. Proposed bioactivation pathway of mefenamic acid in HLM mediated via quinone imine pathway. Dashed arrows indicate the shifting of metabolic pathway towards bioactivation due to CYP2C9 polymorphism or CYP1A2 enzyme induction. Quinoneimines formed from the diphenyl amine moiety of NSAIDs have been shown to play an important role in hepatotoxicity (47). In the present study, the capacity of human hGSTs to inactivate MFA quinoneimines was also studied. Based on our study, the major GSH pathway towards the formation of 4’-OH-5’-SG-MFA was not significantly increased by the addition of hGSTs consistent with previous report (22). However, the minor GSH pathway towards the formation of 5-OH-4/6-SG-MFA was significantly increased by the addition of hepatic hGSTs A1-1, A2-2 and M1-1 and P1-1. For the total GSH conjugation, hGST P1-1 was found to be the most active GST catalyzing the formation of GSH conjugates. A recent study reported that among the human hepatic drug metabolizing enzymes, hGST A1 and A2 enzymes were shown to exhibit one of the highest differences in expression among individuals (48). Therefore, polymorphism of cytochrome P450s at the bioactivation level combined with interindividual variations in the hGST expression at the detoxification level may be a potential risk factor in susceptibility to adverse drug reactions. Although hGSTP1 is expressed in low quantities in liver, it has high extrahepatic tissue expression. For example, hGSTP1-1 is highly expressed throughout the gastrointestinal (GI) tract with a slight decrease of expression from stomach to colon (49). Apart from hepatotoxicity, MFA has also been associated with GI tract toxicity (4, 5, 50). Therefore, reduced protective effect of hGSTP1 due to genetic polymorphism may be an aggravating factor for the adverse effects. The contribution of MFA quinoneimines to MFA induced

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toxicity still remains to be established in cellular context. Cellular toxicity studies using transgenic knock out models lacking a specific hGST or P450 can provide more insights into the mechanism of bioactivation/bio-inactivation. In conclusion, MFA undergoes P450-mediated aromatic ring hydroxylation as a minor pathway which is subsequently involved in bioactivation to two different reactive quinoneimines and formation of MFA-GSH conjugates in human liver microsomes. The major GSH conjugate was found to be arising from the 4’-OH quinoneimine pathway and subsequent GSH conjugation on C-5’ position. Our study shows that in addition to other Phase II metabolites, reactive quinoneimines formed from aromatic hydroxylation of MFA and reduced hGST activity may also contribute to the adverse effects of MFA.

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Supplemental Data

Figure S1. Product ion spectra of the major GSH conjugate MG1 (m/z 563) formed via quinoneimine pathway obtained after incubation of HLM with 5 mM glutathione.

Figure S2: Product ion spectra of the minor GSH conjugates A. MG3 and B. MG2 (m/z 563) formed via quinoneimine pathway obtained after incubation of HLM with 5 mM glutathione.

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8.0 7.5 7.0 6.5F2 Chemical Shift (ppm)

6.5

7.0

7.5

8.0 F1 C

hem

ical

Shi

ft (p

pm)

Figure S3: COSY spectrum of 4’-OH-MFA (M2)

7.5 7.0 6.5F2 Chemical Shift (ppm)

6.5

7.0

7.5

F1 C

hem

ical

Shi

ft (p

pm)

Figure S4: COSY spectrum of 4’-OH-5’-SG-MFA (MG1)

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Figure S5: Extracted ion chromatogram (EIC) of m/z 563.18 obtained from incubations of human liver microsomes (2 mg/mL) with A. 4’-OH-MFA and B. 5-OH-MFA. The GSH conjugates MG1 and MG2, MG3 are labelled.

Figure S6: HPLC chromatograms showing mefenamic acid metabolites formed in vitro by CYP1A2 (lower trace) and CYP1A2 in the presence of GSH (upper trace).100 M MFA was incubated for 60 minutes with 100 nM of CYP1A2. M1 (3’-OH-methyl-MFA) M2 (4’-OH-MFA) M3 (5-OH-MFA). MG1 is the major GSH conjugate derived from 4’-OH pathway. MG2 and MG3 are GSH conjugates derived from 5-OH pathway. Background peak ‘X’ was also present in the control incubations.

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SUMMARY, CONCLUSIONS AND PERSPECTIVES

Summary, Conclusions And Perspectives

Summary The application of enzymes as biocatalysts in chemical and pharmaceutical industry has gained considerable importance over the years. Enzymes are capable of catalyzing a wide range of reactions with high selectivity under mild conditions. Oxidative biocatalysts are especially of great significance because of the ease with which they are capable of catalyzing many reactions including the selective oxidation of unactivated C-H bond (1, 2). Regio- and stereoselective oxidation of compounds ranging from fine chemicals to drug like compounds is of high value in the chemical industry. This thesis covers the potential of cytochrome P450 BM3 mutants for metabolite production for different applications. The main goal of this thesis was to engineer bacterial cytochrome P450 BM3 enzyme for regio- and stereoselective hydroxylation of fine chemicals, steroids and drugs. Novel site-directed mutants specific for different applications have been developed. Mutants M01 and M11 which were generated prior to this study (3), were used as templates for additional site-directed mutagenesis of active site residues guided by computational modelling of the active site of P450 BM3. The engineered P450 BM3 mutants mimicking human liver mcirosomes were used for the synthesis of drug metabolites for toxicological studies. Additionally, engineered P450 BM3 mutants were used for the large scale production of GSH conjugates to enable structural elucidation by NMR and for bioactivation of drugs to study the effect of human glutathione-S-transferases. The work described in chapters 2, 3 and 4 of this thesis was performed as a part of an IBOS project titled “Biocatalytic exploitation of monooxygenases” (BIOMOX). The aim of this part was to engineer and characterize P450 BM3 enzymes for selective oxidation of fine chemicals and steroids. The last 3 chapters (5, 6 and 7) describe the use of engineered cytochrome P450 BM3 variants for drug metabolite production and their potential use in toxicological studies. Chapter 1 gives a general introduction to the various scientific aspects covered in this thesis. In this chapter, the potential of oxidative biocatalysts and in particular monooxygenases is discussed. Cytochrome P450s are a class of heme-dependent mono-oxygenases which metabolize more than 75 % of the drugs in the market. Although human P450s are widely known for their biotransformation of xenobiotic compounds, their use at preparative scale is limited because of their low stability and membrane bound nature. Microbial P450s are soluble and easier to express and therefore are an attractive alternative for metabolite synthesis compared to their

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human counterparts (4). Protein engineering of bacterial P450s and specifically P450 BM3 has proved to be an excellent means to broaden their substrate scope, specificity and to improve their activity. Recent examples of protein engineering from literature to illustrate the tuning of bacterial P450s for selective oxidations are discussed in this chapter (5, 6). The preparative scale synthesis and isolation of drug metabolites is of utmost importance in light of the recent regulatory guidelines of FDA (7). There is a growing need to find green alternatives for synthesis of drug metabolites (8). The optimization of P450 BM3 for improved production of drug metabolites is also discussed in this chapter. Apart from the metabolism of drugs, P450s are also involved in bioactivation reactions leading to the formation of toxic reactive intermediates. These reactive metabolites can lead to covalent modifications of proteins thus resulting in adverse drug reactions. Development of effective methods for detection and structural elucidation of reactive metabolites early in drug development is important for the development of safer drugs (9). Different strategies employed for reactive metabolite and glutathione (GSH) conjugates generation are discussed in this chapter. The conjugation of a reactive metabolite to GSH can be spontaneous or catalyzed by human glutathione-S-transferases (hGSTs) (10). The role of hGSTs in the detoxification of the reactive metabolites by catalyzing GSH conjugation reactions is also covered in this chapter.

Figure 1. Examples of compounds used in this thesis as substrates for engineered P450 BM3 variants Enzymes capable of selective oxidation of fine chemicals are of high value in the chemical industry (11). -ionone and its hydroxylated variants have organoleptic properties and are of commercial interest in the flavour and fragrance industry. In chapter 2, a library of P450 BM3 mutants was screened for their potential to selectively hydroxylate -ionone. Initial screening with racemic -ionone revealed that mutants M01 A82W, M11 A82W and M11 V87I selectively hydroxylated -ionone


at C-3 with trans stereoselectivity while mutants of M11 with mutation at 437 position formed almost equal amounts of both cis and trans diastereomers of 3-OH- -ionone. Interestingly, incubation with individual enantiomers (6R)- and (6S)- -ionone showed that the mutant M11 L437N showed opposite stereoselectivity resulting in the formation of (3S,6R)-OH and (3S,6S)-OH- -ionone respectively with >90% d.e. One of the mutants tested M11 V87F L437N showed moderate selectivity (40%) for the formation of (3R, 6S)-OH diastereomer which could be used as a starting template for further optimization.Docking studies identified the importance of residue at position 82 in the stereoselective hydroxylation of -ionone enantiomers. These engineered P450 BM3 mutants with high total turnover numbers can be used for preparative scale oxidation of small molecules like -ionone. In chapter 3, the potential of Rhodococcus erythropolis as a host for P450 BM3 mediated whole cell biocatalysis was explored using norandrostenedione (nordione) as a model substrate. The wildtype Rhodococcus strain breaks down steroids because

-hydroxylase and 3-ketosteroid dehydrogenase activities, thus hampering its use as a host for steroid bioconversion. By distrupting the genes encoding these catabolic enzymes, several mutants of Rhodococcus erythropolis do not degrade steroids. One such mutant RG9 was used in this chapter to heterologously express P450 BM3 mutant M02 and study the biotransformation of nordione. P450 BM3 variant M02 has been shown to convert steroids with high selectivity and therefore was chosen for this study. Using in vitro biotransformation, the structure of the product was first determined by NMR to be 16- -OH-nordione. The BM3 variant M02 was then heterologously expressed in RG9 and the biotransformation was studied. About 0.35 g/L of the product was formed which corresponded to the product 16- -OH-nordione formed in vitro. Therefore, the product conversion in RG9 is P450 BM3 mediated. This study shows that the combination of mutant Rhodococcus strain and a highly active engineered P450 BM3 variant is a promising option for whole cell biocatalysis. In chapter 4, a semi-rational approach was used to identify the key active site residues involved in the binding of testosterone in P450 BM3. Residues 72, 82, 87 and 437 were identified to have an effect on the binding orientation of testosterone. A minimal library consisting of combinatorial single and double mutations at these residues was constructed. At the time of this study, P450 BM3 mutants capable of hydroxylation of only A or D ring on the -face of testosterone were reported. Screening of the minimal library revealed that two mutants were able to produce a new metabolite which was not reported before. One of these BM3 mutants, M01 A82W S72I was to used to scale up the reaction and the new metabolite was identified

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by a combination of HPLC co-elution, GC-MS and NMR analysis to be 16- -hydroxy-testosterone. The introduction of S72I mutation led to about four fold decrease in the catalytic activity in comparison to its parent M01 A82W. Nevertheless, this mutant could invert the stereoselectivity of testosterone C-16 hydroxylation by only one mutation. The parent mutant M01 A82W was known to catalyze C-16 hydroxylation with 100% -enantioselectivity. Introduction of this mutation in another 16- -hydroxylase variant M11 V87I also resulted in a similar change in enantioselectivity. In-silico modelling studies provided insights into active site interactions responsible for this change in selectivity. By replacing the polar serine with a hydrophobic isoleucine at position 72, the hydrogen bond formation with the A-ring carbonyl group of testosterone which was necessary for 16- was removed. In addition, different rotamers of isoleucine showed steric hindrance of its side chain with testosterone bound in a conformation suitable for 16- hydroxylation. This study showed that small differences in the key substrate interaction residues in P450 BM3 can have a profound effect on the stereoselectivity of hydroxylation. In chapter 5, the applicability of P450 BM3 mutants for the generation of human relevant metabolites of fenamic acid NSAIDs was studied. A library of mutants was screened with meclofenamic acid to identify catalytically active and selective mutants. Mutants of M11 with isoleucine and phenylalanine at position 87 were found to selectively hydroxylate at benzylic and aromatic positions of meclofenamic acid. Engineered P450 BM3 variants were used as biocatalysts to enable structural elucidation of metabolites by NMR. The isolated metabolites were found to be 3’-hydroxy-methyl, 4’-hydroxy and 5-hydroxy-meclofenamic acid. These mutants were also found to hydroxylate structurally similar compounds mefenamic acid and tolfenamic acid with high selectivity and total turnover numbers ranging from 4000-6000. It was found that while both mefenamic acid and meclofenamic could undergo benzylic as well as aromatic hydroxylation by engineered P450 BM3 variants, tolfenamic acid was hydroxylated only on aromatic rings. Computational studies helped to rationalize the observed aromatic ring selectivity of P450 BM3 mediated hydroxylation. The enzymatically synthesized metabolites can be used as references for cellular studies as well as for further toxicological evaluation. In chapter 6, engineered P450 BM3 mutants and human liver microsomes were used for the in vitro bioactivation of drugs like clozapine, acetaminophen and diclofenac to their corresponding reactive metabolites in order to study the ability of four allelic variants of hGSTP1-1, namely hGSTP1*A (Ile105/Ala114), hGSTP1*B (Val105/Ala114), hGSTP1*C (Val105/Val114) and hGSTP1*D (Ile105/Val114), to catalyze the GSH conjugation of the reactive metabolites. Differences in activity


between the proteins could not be attributed to a general decrease in catalytic efficiency. Rather, the differences reflected the effect of residue 105 and 114 specific for any given substrate. Single substitutions at residue 105 or 114 did affect the ability to catalyze GSH conjugation. However, when both residue 105 and 114 were substituted the effect could be enhanced or diminished. Based on the results in this chapter, we suggest that the binding orientation of substrates in the active site of GSTP1 mutants is changed and has effect on GSH conjugation. Chapter 7 describes the use of cytochrome P450 BM3 as a tool to bioactivate mefenamic acid (MFA) to reactive benzoquinoneimine intermediates and for the structural elucidation of the GSH conjugates. The high activity of BM3 mutant M11 resulted in the convenient large scale production and isolation of the major GSH conjugate. The structure of the major conjugate was elucidated by NMR analysis and was found to be derived from MFA-1’,4’- quinoneimine followed by subsequent GSH conjugation at 6’ position. Two more minor conjugates were found to be derived from the MFA-2,5-quinoneimine pathway. MFA was incubated with fourteen different recombinant human CYPs to elucidate enzymes responsible for its bioactivation. While P450 mediated benzylic hydroxylation was catalyzed by CYP 2C9, the aromatic hydroxylation leading to the formation of 1’,4’-quinoneimine and the major GSH conjugate was catalyzed by both CYP 1A2 and CYP2C9. The formation of 5-OH-MFA and the minor GSH conjugates was mainly catalyzed by CYP1A2. Inhibition of metabolite formation by addition of selective inhibitors of individual CYP enzymes to human liver microsomes incubations confirmed these results. Since P450 BM3 could bioactivate MFA to the same human relevant intermediates, it was used as a tool to study the bioactivation and subsequent inactivation of the reactive quinoneimines by glutathione-S-transferases. The effects of a panel of seven major recombinant human GSTs on the formation of GSH conjugates of MFA were studied. The formation of the major GSH conjugate was not significantly increased by the addition of hGSTs. However, the formation of minor conjugates was catalyzed by hGSTs, in particular the addition of hGST P1-1 led to a ten-fold increase in the formation of one of the regioisomeric conjugates derived from 5-OH pathway. Conclusions During the last ten years, several labs around the world have worked on developing novel mutants of cytochrome P450 BM3 for a variety of applications (6). In the beginning, focus was laid on selective oxidation of small molecules and aromatics which over the years has shifted to exploit the catalytic potential of P450 BM3 enzymes for pharmaceutical applications (12) and novel reactions like

CHAPTER 8

cycloproponation and C-H amination not known thus far for P450s (13, 14). Figure 2 summarizes the powerful tool of protein engineering used in context of P450 BM3 and the various novel applications of the engineered P450 BM3 variants.

Figure 2. The figure shows protein engineering approach (top panel), and applications of engineered P450 BM3 mutants (bottom panel). Pictures depicting the various approaches to engineer P450s are taken from references (15-18). Pictures depicting the applications of the engineered variants were reproduced from references (13, 19-22). The general template of this figure is adapted from (18). Future prospectives based on this thesis Some aspects described in this thesis that be applied in future studies are discussed below.

Drug metabolite

Appl

icat

ions

Prot

ein

engi

neer

ing

Site directed mutagenesis Directed evolution

CASTing mutagenesisScanning chimeragenesis

Noncanonical amino acid incorporation

Reinen et al., 2011 Chen et al., 2001 Urlacher et al., 2014Shapiro et al., 2010Coelho et al., 2013

P450 BM3


Chimeragenesis as an approach for B or C ring steroid hydroxylation The regio- and stereoselectivity of P450-mediated reactions depends upon the orientation of the substrate relative to the reactive iron-oxo species, which is in turn determined by the active-site configuration of the P450 enzyme. (23). P450 BM3 mutants engineered by combinatorial saturation mutagenesis or alanine scanning mutagenesis can perform only A or D ring hydroxylation of steroids (16, 24). It still remains a challenge to achieve B or C ring hydroxylation such as 6 -hydroxy-testosterone which is the major human metabolite of testosterone. These observations reiterate the fact that more rigorous methods may be required to re-shape the active site to enable B or C ring hydroxylation. One approach is to create chimera of cytochrome P450s by domain swapping or by scanning chimeragenesis which has been successfully employed to for other substrates (15, 25). For example, in a recent study, the substrate recognition sites of insect CYP4C7, which hydroxylates farnesol at the terminal C12 position with high selectivity but low activity, were introduced into P450BM3 to yield successful chimeras capable of producing 12-hydroxy-farnesol not observed with wild-type P450BM3(15). Computational and structural studies In chapters 2 and 4, we show that in silico docking studies can provide insights into the structural rationalization of the experimentally observed regio- and stereoselective hydroxylation -ionone and testosterone. In these studies, computational methods were used to identify critical residues involved in substrate recognition and to rationalize the experimentally observed selectivity. More recently, free energy calculations were applied to study the stereoselective hydroxylation of -ionone enantiomers by engineered BM3 mutants (26). In chapter 5 of this thesis, the regioselective benzylic and aromatic hydroxylation of fenamic acid NSAIDs is described. In silico modelling studies were used to get preliminary insights into the ring preference of fenamic acid hydroxylation by P450 BM3 mutants. In future studies, the observed benzylic and aromatic hydroxylation selectivity in different mutants can be further rationalized in extensive molecular dynamics (MD) simulation based studies by including protein plasticity and induced fit effects. This will be in line with the previous studies in which it was shown that predicting the effect of P450 BM3 small active site mutations on regio-or stereoselectivity of substrates was found to be difficult based on (rigid) docking and scoring only (27). Moreover, crystallization of the selective P450 BM3 variants in the ligand bound and ligand free forms can potentially help to unravel the conformational changes caused by the mutation and get insights into the structural basis of selective oxidation.

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Engineered P450 BM3 as a bioactivation tool in mammalian cellular studies The detection of the possible bioactivation pathways of drugs to reactive metabolites in early stage drug discovery is often difficult because of the problem of characterizing low levels of reactive metabolites. Therefore, efficient methods for the generation and characterization of reactive metabolites are necessary (28). In chapter 6 and 7, P450 BM3 mutants are used as biocatalytic tools to generate high amounts of reactive intermediates. Recently, Boerma et.al used engineered P450 BM3 mutants to generate protein adducts resulting from the bioactivation of many drugs (29). Moreover, engineered P450 BM3 mutant F87V which metabolized arachidonic acid only to 14,15- epoxyeicosatrienoic acid (14,15-EET) was transfected in pig renal epithelial cell lines (LLCPKcl4) as a tool to study the role of arachidonic acid epoxygenase pathway. The stable transfectants of of LLCPKcl4 cells with P450 BM3 mutant F87V as well as empty vector-transfected LLCPKcl4 cells were used to investigate the cell survival effects of endogenously produced 14,15-EET (20, 30). These studies exemplify the diverse applicability of engineered P450 BM3 which can be extended to mammalian cellular toxicity models in future. To summarize, engineered P450 BM3 mutants have been shown to have the potential as biocatalysts for metabolite production with possible applications ranging from biotechnology to toxicology. Combinatorial active site mutations have been effective to improve the selectivity of the reactions as well as to understand the structure-function relationship of P450 BM3 reflected by the synergy between the experimental and computational studies. Moreover, the combination of P450 BM3 variants with other enzymes has led the development of a potential study system that can be applied for the large scale generation of reactive metabolites for structural elucidation as well as a versatile tool to study the interplay between Phase I and Phase II enzymes.


References (1) Burton, S. G. (2003) Oxidizing enzymes as biocatalysts. Trends Biotechnol 21, 543-549. (2) Hollmann, F., Arends, I. W. C. E., Buehler, K., Schallmey, A. and Buhler, B. (2011) Enzyme-

mediated oxidations for the chemist. Green Chemistry 13. (3) van Vugt-Lussenburg, B. M. A., Stjernschantz, E., Lastdrager, J., Oostenbrink, C., Vermeulen,

P. E. and Commandeur, J. N. M. (2007) Identification of Critical Residues in Novel Drug Metabolizing Mutants of Cytochrome P450 BM3 Using Random Mutagenesis. Journal of Medicinal Chemistry 50, 455-461.

(4) Urlacher, V. B. and Girhard, M. (2012) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends in biotechnology 30, 26-36.

(5) Fasan, R. (2012) Tuning P450 Enzymes as Oxidation Catalysts. ACS Catalysis 2, 647-666. (6) Whitehouse, C. J. C., Bell, S. G. and Wong, L. L. (2012) P450(Bm3) (Cyp102a1): Connecting the

Dots. Chemcial society reviews 41, 1218-1260. (7) Smith, D. A., Obach, R. S., Williams, D. P. and Park, B. K. (2009) Clearing the MIST (metabolites

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(8) Caswell, J. M., O'Neill, M., Taylor, S. J. C. and Moody, T. S. (2013) Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis. Current opinion in chemical biology 17, 271-275.

(9) Fura, A., Shu, Y. Z., Zhu, M., Hanson, R. L., Roongta, V. and Humphreys, W. G. (2004) Discovering drugs through biological transformation: role of pharmacologically active metabolites in drug discovery. J Med Chem 47, 4339-4351.

(10) Mannervik, B. and Danielson, U. H. (1988) Glutathione transferases--structure and catalytic activity. CRC Crit Rev Biochem 23, 283-337.

(11) Clouthier, C. M. and Pelletier, J. N. (2012) Expanding the organic toolbox: a guide to integrating biocatalysis in synthesis. Chem Soc Rev 41, 1585-1605.

(12) Di Nardo, G. and Gilardi, G. (2012) Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites. Int. J. Mol. Sci. 13, 15901-15924.

(13) Coelho, P. S., Brustad, E. M., Kannan, A. and Arnold, F. H. (2013) Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307-310.

(14) McIntosh, J. A., Coelho, P. S., Farwell, C. C., Wang, Z. J., Lewis, J. C., Brown, T. R. and Arnold, F. H. (2013) Enantioselective intramolecular C-H amination catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo. Angew Chem Int Ed Engl 52, 9309-9312.

(15) Chen, C. K., Berry, R. E., Shokhireva, T., Murataliev, M. B., Zhang, H. and Walker, F. A. (2010) Scanning chimeragenesis: the approach used to change the substrate selectivity of fatty acid monooxygenase CYP102A1 to that of terpene omega-hydroxylase CYP4C7. J Biol Inorg Chem 15, 159-174.

(16) Kille, S., Zilly, F. E., Acevedo, J. P. and Reetz, M. T. (2011) Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution. Nat Chem 3, 738-743.

(17) Kolev, J. N., Zaengle, J. M., Ravikumar, R. and Fasan, R. (2014) Enhancing the efficiency and regioselectivity of P450 oxidation catalysts by unnatural amino acid mutagenesis. Chembiochem 15, 1001-1010.

(18) Kumar, S. (2010) Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation. Expert Opin Drug Metab Toxicol 6, 115-131.

(19) Bernhardt, R. and Urlacher, V. B. (2014) Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations. Appl Microbiol Biotechnol 98, 6185-6203.

(20) Chen, J. K., Capdevila, J. and Harris, R. C. (2001) Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis. Mol Cell Biol 21, 6322-6331.


(22) Shapiro, M. G., Westmeyer, G. G., Romero, P. A., Szablowski, J. O., Kuster, B., Shah, A., Otey, C. R., Langer, R., Arnold, F. H. and Jasanoff, A. (2010) Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine. Nat Biotechnol 28, 264-270.

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(23) Jung, S. T., Lauchli, R. and Arnold, F. H. (2011) Cytochrome P450: taming a wild type enzyme.

Curr Opin Biotechnol.(24) -H. and Arnold, F. H.

(2010) Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450BM3 for Selective Hydroxylation of Large Substrates. ChemBioChem 11, 2502-2505.

(25) Kang, J. Y., Ryu, S. H., Park, S. H., Cha, G. S., Kim, D. H., Kim, K. H., Hong, A. W., Ahn, T., Pan, J. G., Joung, Y. H., Kang, H. S. and Yun, C. H. (2014) Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs. Biotechnol Bioeng

(26) de Beer, S. B., Venkataraman, H., Geerke, D. P., Oostenbrink, C. and Vermeulen, N. P. (2012) Free energy calculations give insight into the stereoselective hydroxylation of alpha-ionones by engineered cytochrome P450 BM3 mutants. J Chem Inf Model 52, 2139-2148.

(27) de Beer, S. B., van Bergen, L. A., Keijzer, K., Rea, V., Venkataraman, H., Guerra, C. F., Bickelhaupt, F. M., Vermeulen, N. P., Commandeur, J. N. and Geerke, D. P. (2012) The role of protein plasticity in computational rationalization studies on regioselectivity in testosterone hydroxylation by cytochrome P450 BM3 mutants. Curr Drug Metab 13, 155-166.

(28) Cusack, K. P., Koolman, H. F., Lange, U. E., Peltier, H. M., Piel, I. and Vasudevan, A. (2013) Emerging technologies for metabolite generation and structural diversification. Bioorganic & medicinal chemistry letters 23, 5471-5483.

(29) Boerma, J. S., Vermeulen, N. P. and Commandeur, J. N. (2011) Application of CYP102A1M11H as a tool for the generation of protein adducts of reactive drug metabolites. Chem Res Toxicol 24, 1263-1274.

(30) Chen, J. K., Wang, D. W., Falck, J. R., Capdevila, J. and Harris, R. C. (1999) Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor. J Biol Chem 274, 4764-4769.

Appendices

Nederlandse samenvatting List of Publications

Curriculum vitae Acknowledgements

Nederlandse samenvatting De toepassing van enzymen als biokatalysatoren in de chemische en farmaceutische industrie is door de jaren van steeds groter belang geworden. Enzymen kunnen uiteenlopende reacties katalyseren met hoge selectiviteit en onder milde omstandigheden. Oxidatieve biokatalysatoren zijn vooral van groot belang vanwege het gemak waarmee ze vele reacties kunnen katalyseren waaronder de selectieve oxidatie van niet geactiveerde C-H bindingen (1, 2). Regio- en stereoselectieve oxidaties van verbindingen zoals fijnchemicaliën en (kandidaat)geneesmiddelen met behulp van biokatalysatoren is van grote waarde voor de chemische industrie omdat deze reacties soms moeilijk toegankelijk zijn met organische synthese. Dit proefschrift beschrijft de mogelijkheden van cytochroom P450 BM3 mutanten voor metabolietproductie met verschillende toepassingen. Het belangrijkste doel van het onderzoek dat in dit proefschrift wordt beschreven is om het bacteriële cytochroom P450 BM3 enzym te optimaliseren voor regio- en stereoselectieve hydroxylering van fijnchemicaliën, steroïden en geneesmiddelen. Verschillende nieuwe mutanten specifiek voor verschillende toepassingen zijn ontwikkeld. Mutanten M01 en M11, gegenereerd vóór deze studie (3), werden gebruikt als basis voor verdere mutagenese aan de hand van een computermodel van P450 BM3. P450 BM3 mutanten die de selectiviteit van menselijke levermicrosomen nabootsen werden gebruikt voor de synthese van metabolieten van geneesmiddelen voor toxicologisch onderzoek. Bovendien werden de ontwikkelde P450 BM3 mutanten gebruikt voor de grootschalige productie van GSH-conjugaten, zodat de structuur hiervan kon worden opgehelderd met behulp van NMR. Daarnaast werden deze mutanten gebruikt om het rol van humane glutathion-S-transferases in de inactivatie van reactieve metabolieten te bestuderen. Het werk beschreven in de hoofdstukken 2, 3 en 4 van dit proefschrift werd uitgevoerd in het kader van het NWO-IBOS project getiteld " Biocatalytic exploitation of monooxygenases" (BIOMOX). Het doel van dit project was het ontwerpen en karakteriseren P450 BM3 enzymen voor selectieve oxidatie van fijnchemicaliën en steroïden. De laatste 3 hoofdstukken (5, 6 en 7) beschrijven het gebruik van cytochroom P450 BM3 varianten voor de productie van geneesmiddelmetabolieten en de mogelijke toepassingen in toxicologische studies. Hoofdstuk 1 geeft een algemene inleiding op de verschillende onderdelen die in dit proefschrift worden beschreven. In dit hoofdstuk wordt het potentieel van oxidatieve

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biokatalysatoren en in het bijzonder monooxygenases besproken. Cytochroom P450's vormen een klasse van heem-afhankelijke mono-oxygenasen die meer dan 75% van de geneesmiddelen op de markt metaboliseren. Hoewel de menselijke P450 algemeen bekend staan om rol in de biotransformatie van lipofiele lichaamsvreemde stoffen, is het gebruik van deze membraan-gebonden enzymen voor biosynthese op preparatieve schaal beperkt vanwege hun lage stabiliteit en relatief lage specifieke activiteit. Microbiële P450's zijn oplosbaar en gemakkelijker te expresseren en dus een aantrekkelijk alternatief voor metaboliet synthese vergeleken met hun menselijke tegenhangers (4). Eiwit optimalisatie van bacteriële P450's, en in het bijzonder P450 BM3, heeft bewezen een uitstekend middel te zijn om het substraat gebied en specificiteit te verbreden en om de activiteiten te verbeteren. In dit hoofdstuk worden verschillende recente voorbeelden gegeven van selectieve oxidatieve biokatalysatoren die door middel van optimalisatie van bacteriële P450 zijn verkregen (5, 6). De synthese op preparatieve schaal en de isolatie van geneesmiddelmetabolieten is van groot belang in het licht van de recente richtlijnen van de FDA en EMA die voorschrijven dat ook geneesmiddel metabolieten op hun veiligheid worden getest (7). Er is verder een groeiende behoefte aan groene alternatieven voor de synthese van geneesmiddelmetabolieten (8). De optimalisatie van P450 BM3 voor de verbeterde productie van geneesmiddelmetabolieten wordt ook besproken in dit hoofdstuk. Naast het metabolisme van geneesmiddelen tot stabiele producten zijn P450's betrokken bij bioactiveringsreacties die leiden tot de vorming van zeer reactieve, potentieel toxische intermediairen. Deze reactieve metabolieten kunnen leiden tot covalente binding aan eiwitten hetgeen leidt tot bijwerkingen. Ontwikkeling van effectieve methoden voor detectie en structuuropheldering van reactieve metabolieten vroeg in de ontwikkeling van geneesmiddelen is belangrijk voor de ontwikkeling van veiligere geneesmiddelen (9). Verschillende strategieën die worden gebruikt voor de generatie van reactieve metabolieten en analyse van glutathion (GSH) conjugaten worden besproken in dit hoofdstuk. De conjugatie van een reactieve metaboliet met GSH kan spontaan plaatsvinden of gekatalyseerd worden door glutathion-S-transferases (GSTs) (10). Voorbeelden van de rol van menselijke GSTs in de detoxificatie van de reactieve metabolieten worden ook behandeld in dit hoofdstuk. Enzymen die in staat zijn tot selectieve oxidatie van fijne chemicaliën zijn van hoge waarde voor -Ionon en gehydroxyleerde varianten hebben organoleptische eigenschappen en zijn van commercieel belang in de smaak en geur-industrie. In het in Hoofdstuk 2 beschreven onderzoek werd een bibliotheek

-ionon stereo- en regio -ionon bleek dat mutanten M01 A82W, M11 en -ionon regioselectief

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hydroxyleren op de C-3 positie met trans stereoselectiviteit terwijl mutanten van M11 met mutatie op positie 437 vrijwel gelijke hoeveelheden vormen van zowel cis en trans diastereomeren van 3-OH- -ionon. Interessant is dat uit incubatie met afzonderlijke enantiomeren (6R)- en (6S)- -ionon bleek dat de mutant M11 L437N tegengestelde stereoselectiviteit vertoonde resulterend in de vorming van (3S, 6R)-OH en (3S, 6S)-OH- -ionone respectievelijk bij >90% d.e. M11 V87F L437N, een van de geteste mutanten, vertoonde matige selectiviteit (40%) voor de vorming van (3R, 6S)-OH diastereomeer; deze mutant zou gebruikt kunnen worden als uitgangspunt voor verdere optimalisatie. Door middel van computational docking onderzoek werd het belang van residu 82 voor -ionon enantiomeren aangetoond. Deze P450 BM3 mutanten met hoge omzettingsgetallen

-ionon op preparatieve schaal. In Hoofdstuk 3 werd het potentieel van Rhodococcus erythropolis als gastheer voor P450 BM3 gemedieerde hele cel biokatalyse onderzocht met norandrostenedione (nordione) als modelsubstraat. De wild-type stam van Rhodococcus erythropolis breekt steroïden af -hydroxylase en 3-ketosteroid dehydrogenase activiteiten, wat het gebruik als gastheer voor steroid bioconversie belemmert. Door de genen die coderen voor deze katabole enzymen te ontregelen, zijn verschillende stammen van Rhodococcus erythropolis gegenereerd die niet meer in staat zijn steroïden te af te breken. Een van deze stammen, RG9, werd in dit hoofdstuk gebruikt om P450 BM3 mutant M02 heteroloog te expresseren en de biotransformatie van nordione te onderzoeken. Deze P450 BM3 mutant werd geselecteerd omdat het steroïden omzet met hoge regioselectiviteit; nordione wordt door deze mutant selectief gehydroxyleerd tot 16- -OH-nordione zijn. Door BM3 M02 heteroloog tot expressie te brengen in RG9 kon ongeveer 0,35 g/L van het product 16- -OH-nordione in hele cellen werd gevormd. Deze studie toont aan dat de combinatie van een mutant Rhodococcus stam en een zeer actieve P450 BM3 variant een veelbelovende optie is voor de hele cel biokatalyse. In Hoofdstuk 4 werd een rationele benadering gebruikt om de belangrijke aminozuur residüen te identificeren die betrokken bij de binding en oriëntatie van testosteron in het katalytische centrum van P450 BM3. Residuen 72, 82, 87 en 437 bleken belangrijk te zijn voor de bindingsoriëntatie van testosteron. Een kleine bibliotheek bestaande uit enkele en combinaties van dubbele mutaties van deze residuen werd samengesteld. Ten tijde van dit onderzoek, waren alleen P450 BM3 mutanten bekend die testosteron hydroxyleren aan -zijde van de A of D ring. Screening van deze bibliotheek onthulde twee mutanten die een nieuw, niet eerder beschreven product vormen. Een van deze mutanten BM3 M01 A82W S72I werd gebruikt om dit product

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op semipreparatieve schaal te produceren. Met behulp van NMR en door vergelijking met de chromatografische eigenschappen en massaspectrum van de referentie-verbinding kon het nieuwe product werd geïdentificeerd als 16- -hydroxy-testosteron, hetgeen de eerste keer is dat hydroxylering aan de -zijde van testosteron is aangetoond. Hieruit blijkt dat door slechts een enkele aminozuur substitutie in M01 A82W, dat C- -enantioselectiviteit katalyseert, de stereoselectiviteit van testosteron 16-hydroxylering verandert van 16- - naar 16- -hydroxylering. Introductie van dezelfde mutatie in een ander 16- -hydroxylase, M11 V87I, resulteerde ook in dezelfde verandering in enantioselectiviteit. In-silico studies gaven inzicht in welke aminozuur interacties mogelijk verantwoordelijk zijn voor deze verandering in selectiviteit. Door het polaire aminozuur serine te veranderen in het hydrofobe isoleucine op positie 72 werd een waterstofbrug met de A-ring carbonyl-groep van testosteron, noodzakelijk voor 16 -hydroxylatie, verwijderd. Bovendien bleek er sprake van sterische hindering met de verschillende rotameren van isoleucine, wanneer testosteron is gebonden in de -hydroxylatie. Deze studie toonde aan dat kleine verschillen in de belangrijkste residüen voor substraat interactie in P450 BM3 een groot effect op de stereoselectiviteit van hydroxylering kunnen hebben. In Hoofdstuk 5 werd de toepasbaarheid van de P450 BM3 mutanten voor de productie van de humaan relevante metabolieten van fenaminezuur NSAIDs bestudeerd. Een bibliotheek van mutanten werd gescreend met meclofenamic acid als substraat om katalytisch actieve en regioselectieve mutanten te identificeren. Mutanten van M11 met isoleucine en fenylalanine op positie 87 bleken in staat tot selectieve hydroxylering op de benzylische en aromatische posities van meclofenamic acid. De meest geschikte P450 BM3 mutanten werden gebruikt voor opschaling van metaboliet-productie om structuuropheldering door middel van NMR mogelijk te maken. De geïsoleerde metabolieten bleken 3'-hydroxy-methyl, 4'-hydroxy en 5-hydroxy-meclofenamic acid te zijn. Deze mutanten bleken ook in staat de structureel vergelijkbare verbindingen, mefenamic en tolfenamic acid, met hoge selectiviteit en omzettingsnummers variërend van 4000-6000 te hydroxyleren. Computationele studies werden uitgevoerd om de waargenomen regioselectiviteit van de hydroxyleringsreacties van de P450 BM3 mutanten te rationaliseren. Deze enzymatisch geproduceerde metabolieten kunnen worden gebruikt voor toxicologische evaluaties in cellulaire studies. In Hoofdstuk 6 werden P450 BM3 mutanten en menselijke levermicrosomen gebruikt voor de in vitro bioactivatie van de geneesmiddelen clozapine, paracetamol en diclofenac naar hun overeenkomstige reactieve metabolieten. Met behulp van deze bioactivatiesystemen werd het activiteit van vier allelische varianten van het

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menselijke glutathione transferase GSTP1-1 in de inactivering van deze reactieve metabolieten onderzocht. De allelische varianten die werden onderzocht waren hGSTP1* A (Ile105 / Ala114), hGSTP1*B (Val105 / Ala114), hGSTP1*C (Val105 / Val114) en hGSTP1*D (Ile105 / Val114). Uit dit onderzoek bleek dat de verschillen in vermogen tot inactivatie van de reactieve metabolieten van de onderzochtte geneesmiddelen relatief beperkt waren. Uit het feit dat de mutaties tot verschillende verhoudingen van de regioisomeren van GSH-conjugaten leiden, blijkt dat bindingsoriëntatie van reactieve metabolieten in het katalytische centrum van GSTP1 mutanten is veranderd. Hoofdstuk 7 beschrijft het gebruik van cytochroom P450 BM3 mutanten voor de bioactivatie van mefenamic acid (MFA) tot reactieve benzoquinoneïmine intermediairen en voor de structurele opheldering van de GSH-conjugaten. De meest actieve BM3 mutant M11 werd gebruikt voor de grootschalige productie en isolatie van het voornaamste GSH conjugaat. De structuur van dit conjugaat werd opgehelderd door NMR-analyse en bleek afkomstig te zijn van MFA-1',4'quinoneimine gevolgd door GSH conjugatie op de 6-positie. Twee kwantitatief minder belangrijke conjugaten bleken te zijn afgeleid uit de MFA-2,5-quinoneimine bioactivatie route. MFA werd geïncubeerd met veertien verschillende recombinante humane CYP enzymen om de enzymen die verantwoordelijk zijn voor zijn bioactivatie op te helderen. De P450 gemedieerde benzylische hydroxylering bleek te worden gekatalyseerd door CYP 2C9, terwijl de aromatische hydroxylering die leidt tot de vorming van 1',4'-quinoneimine en grote GSH conjugaat werd gekatalyseerd door zowel CYP1A2 en CYP2C9. De vorming van 5-OH-MFA en de zijn GSH-conjugaten bleek voornamelijk te worden gekatalyseerd door CYP1A2. Remming van metabolietvorming door toevoeging van selectieve remmers van individuele CYP enzymen aan humane levermicrosomen incubaties bevestigden deze resultaten. Na identificatie van de bij bioactivatie betrokken humane P450s werd de activiteit van zeven recombinant humane GST’s in de inactivatie van de reactieve metabolieten van MFA bestudeerd. De vorming van de GSH-conjugaten die worden gevormd via de 5-OH-route, bleek zeer sterk te worden gekatalyseerd door de GSTs. Vervolgstudies zijn nodig om het belang van erfelijkheid van de beschermende GSTs in de incidenteel voorkomende levertoxiciteit van MFA te onderzoeken. Samengevat, P450 BM3 mutanten blijken potentieel te hebben als biokatalysatoren voor metabolietproductie met mogelijke toepassingen van biotechnologie tot toxicologie. Combinatorische mutaties in het katalytisch centrum zijn effectief om de selectiviteit van een reactie verbeteren, evenals om de structuur-functie relatie van P450 BM3 beter te begrijpen, gereflecteerd door de synergie tussen de experimentele en computationele studies. Bovendien heeft de combinatie van P450 BM3 varianten

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met andere enzymen geleid tot de ontwikkeling van een mogelijk nieuw onderzoek systeem dat kan worden toegepast voor de grootschalige productie van reactieve metabolieten voor structuuropheldering en als een veelzijdig instrument om de wisselwerking tussen fase I en fase II enzymen te bestuderen.

(1) Burton, S. G. (2003) Oxidizing enzymes as biocatalysts. Trends Biotechnol 21, 543-549. (2) Hollmann, F., Arends, I. W. C. E., Buehler, K., Schallmey, A. and Buhler, B. (2011) Enzyme-

mediated oxidations for the chemist. Green Chemistry 13. (3) van Vugt-Lussenburg, B. M. A., Stjernschantz, E., Lastdrager, J., Oostenbrink, C., Vermeulen,

P. E. and Commandeur, J. N. M. (2007) Identification of Critical Residues in Novel Drug Metabolizing Mutants of Cytochrome P450 BM3 Using Random Mutagenesis. Journal of Medicinal Chemistry 50, 455-461.

(4) Urlacher, V. B. and Girhard, M. (2012) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends in biotechnology 30, 26-36.

(5) Fasan, R. (2012) Tuning P450 Enzymes as Oxidation Catalysts. ACS Catalysis 2, 647-666. (6) Whitehouse, C. J. C., Bell, S. G. and Wong, L. L. (2012) P450(Bm3) (Cyp102a1): Connecting the

Dots. Chemcial society reviews 41, 1218-1260. (7) Smith, D. A., Obach, R. S., Williams, D. P. and Park, B. K. (2009) Clearing the MIST (metabolites

in safety testing) of time: The impact of duration of administration on drug metabolite toxicity. Chem-Biol. Interact. 179, 60-67.

(8) Caswell, J. M., O'Neill, M., Taylor, S. J. C. and Moody, T. S. (2013) Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis. Current opinion in chemical biology 17, 271-275.

(9) Fura, A., Shu, Y. Z., Zhu, M., Hanson, R. L., Roongta, V. and Humphreys, W. G. (2004) Discovering drugs through biological transformation: role of pharmacologically active metabolites in drug discovery. J Med Chem 47, 4339-4351.

(10) Mannervik, B. and Danielson, U. H. (1988) Glutathione transferases--structure and catalytic activity. CRC Crit Rev Biochem 23, 283-337.

(11) Clouthier, C. M. and Pelletier, J. N. (2012) Expanding the organic toolbox: a guide to integrating biocatalysis in synthesis. Chem Soc Rev 41, 1585-1605.

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List of publications (1) Venkataraman, H., den Braver, M. W., Vermeulen, N. P. and Commandeur, J.

N. (2014) Cytochrome P450 mediated bioactivation of mefenamic acid to quinoneimine intermediates and inactivation by human glutathione S-transferases. Chem Res Toxicol

(2) Venkataraman, H., Verkade-Vreeker, M. C., Capoferri, L., Geerke, D. P.,

Vermeulen, N. P. and Commandeur, J. N. (2014) Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs. Bioorg Med Chem, 22, 5613-5620

(3) Venkataraman, H

Vermeulen, N. P., Commandeur, J. N. and Dijkhuizen, L. (2014) Bio-synthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme. Appl Microbiol Biotechnol (submitted)

(4) Dragovic, S., Venkataraman, H., Begheijn, S., Vermeulen, N. P. and

Commandeur, J. N. (2014) Effect of human glutathione S-transferase hGSTP1-1 polymorphism on the detoxification of reactive metabolites of clozapine, diclofenac and acetaminophen. Toxicol Lett 224, 272-281.

(5) Venkataraman, H., Beer, S. B. A. d., Geerke, D. P., Vermeulen, N. P. E. and

Commandeur, J. N. M. (2012) Regio- and Stereoselective Hydroxylation of -Ionone Enantiomers by Engineered Cytochrome P450 BM3

Mutants. Adv Synth Catal 354, 2172-2184. (6) Venkataraman, H., Beer, S. B., Bergen, L. A., Essen, N., Geerke, D. P.,

Vermeulen, N. P. and Commandeur, J. N. (2012) A single active site mutation inverts stereoselectivity of 16-hydroxylation of testosterone catalyzed by engineered cytochrome P450 BM3. Chembiochem 13, 520-523.

(7) Vredenburg, G., Elias, N. S., Venkataraman, H., Hendriks, D. F., Vermeulen, N.

P., Commandeur, J. N. and Vos, J. C. (2014) Human NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated inactivation of reactive quinoneimine

metabolites of diclofenac and mefenamic acid. Chem Res Toxicol 27, 576-586.

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(8) de Beer, S. B., Venkataraman, H., Geerke, D. P., Oostenbrink, C. and Vermeulen, N. P. (2012) Free energy calculations give insight into the stereoselective hydroxylation of alpha-ionones by engineered cytochrome P450 BM3 mutants. J Chem Inf Model 52, 2139-2148.

(9) Venkataraman, H., Guerra,

C. F., Bickelhaupt, F. M., Vermeulen, N. P., Commandeur, J. N. and Geerke, D. P. (2012) The role of protein plasticity in computational rationalization studies on regioselectivity in testosterone hydroxylation by cytochrome P450 BM3 mutants. Curr Drug Metab 13, 155-166.

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Curriculum Vitae Harini Venkataraman was born in Chennai, India in 1984. She received her Bachelor’s degree in Chemical Engineering from India and Masters in Biomedical engineering from Aachen University of Applied Sciences, Germany. She performed her master thesis (“Incorporation of non-canonical amino acids in fluorescent proteins”) at Max Planck Institute of Biochemistry, Munich. After her master’s degree, she joined the group of prof. dr. Nico Vermeulen and dr. Jan Commandeur at Vrije Universiteit, Amsterdam to perform her doctorate work on the topic of “Engineered cytochrome P450 BM3 variants as biocatalysts for metabolite production”. A part of this thesis was performed in a NWO-IBOS project titled “Biocatalytic exploitation of monooxygenases” (BIOMOX). Currently, she is working as an associate scientist at DSM biotechnology centre (part of DSM food specialties B.V.), Delft.

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Acknowledgements First of all, I would like to thank my promoter Nico Vermeulen and co-promotor Jan Commandeur for giving me this wonderful opportunity to work in the Moltox group and for their support throughout the course of my PhD. It has been a scientifically enriching and great learning experience. Thanks for all the discussions and constant encouragement which helped me shape up my scientific acumen. Thanks to Chris Vos for your scientific input and great questions during work discussions. Thanks to Daan Geerke for all our “active” P450 BM3 talks and the fruitful collaboration we had. I would like to extend my gratitude to prof.dr. W.J.H.van Berkel, prof.dr. L.Dijkhuizen, prof.dr. P.D.J. Grootenhuis, prof.dr.ir. R.V.A. Orru and prof.dr. V.B.Urlacher for kindly accepting to be a part of the reading committee. During the four years of my PhD, I had a wonderful opportunity to work with many great colleagues. Thanks to the ex-Moltoxers of P240 Galvin, Sanja, Jan-Simon, Jolanda, Cecilia and Vanina. Sanja thanks for our collaboration; Jantje for the interesting scientific and other discussions in-/outside the lab and Galvin for being a great colleague and always having time to discuss about our common favourite compound Mefenamic acid Thanks to Rene, Eduardo and Jeroen for the initial years in the Moltox group.

ates for almost 2 years in P254, for all the fun chats. Thanks to Dr.Reinen (also known as Jelle ) for all the scientific and non-scientific talks and for 3 successful years of Integrated practical course. Ellen thanks for your nice company and late evening chats. Thanks to Stephanie for the fruitful scientific collaboration: great synergy between experimental and computational Moltox groups indeed! Thanks as well to Ruben, Luigi and Mark. Luigi for the nice collaboration we had! Thanks to my IBOS girls Evelien, Stefania and Anette. Evelien, thank you for our collaboration. Thanks to my roommates: Marlies, Michiel and Shalenie in the last 2 years of my PhD for all the great conversations, discussions (scientific/other things), coffee breaks and most of all for making P254 a great place to be in. I wish you guys all the best for your PhD !!! Our room is a proof that science and fun can go hand in hand. Marlies special thanks for agreeing to be my paranymph.

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Thanks to all the current P240 members: Angelina for the lively discussions and pep talks; Stefan for spontaneous coffee breaks and GST talks and for being an unofficial P254 roommate. Yongjie an oltoxers, great addition to the group! All the best for your PhD. Thanks also various MedChem people: Sabrina for the wonderful time we had during the LACDR introductory course and the ULLA Summer school in London, Saskia for your enthusiasm, Ton for valuable NMR discussions. Thanks to the students that I guided: Reggie, Daniel, Jelle, Bita, Alexis. It was a great experience to have worked with you all! Thanks to my friends Anitha for all the great social gatherings. A very special note of thanks to my parents and sister for always being there for me and motivating me throughout my life. Thanks to Aba, Aai, aka,

Priti, Tai, Amit & dearest Shaurya. Finally, big thanks to my husband Dhwajal for your love, support and motivation. You have been and will always be my pillar of strength.

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