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CHAPTER 1
GENERAL INTRODUCTION
Chapter 1
General Introduction
Introduction
1.
Role of drug metabolism in drug development
4
1.1. Metabolites in safety testing (MIST)
1.2. Enzyme involved in drug metabolism
1.3. Biosynthesis of reactive metabolites
1.4. Biological screening for active metabolites
2.
Microbial conversion of steroid compounds
12
3.
Cytochrome P450s
15
3.1. Human P450s
3.2. Structural organization of P450s
3.3. The catalytic cycle
4.
Cytochrome P450 BM3
20
4.1. Engineering Cytochrome P450 BM3
4.2. P450 BM3 in biocatalysis
4.3. Key residues in the active site of P450 BM3
5.
Spin lattice relaxation NMR
27
6.
Scope and objective of this thesis
29
7.
Outline of this thesis
30
Chapter 1
General Introduction
1.
Role of drug metabolism in drug discovery and development
Most xenobiotic substances such as drugs undergo an array of biotransformation
reactions after internal exposure to humans. Drug metabolism by the host system is one
of the most important determinants of the pharmacokinetic profile of a drug.
Biotransformation of drugs can have different effects, such as the formation of chemically
stable metabolites, which are devoid of pharmacological or toxicological activities, or the
generation of short lived chemically reactive metabolites, which can lead to toxicological
side effects (1). Furthermore, drug metabolism can also lead to formation of
pharmacologically active metabolites which might contribute significantly to drug action in
vivo, or may be responsible for pharmacologically-based adverse drug reactions.
A relatively novel approach in modern drug development is the use of pharmacologically
active metabolites as potential resources for drug discovery and development. There are
several advantages for screening drug candidates for active metabolites during drug
discovery. For example drug metabolites can show superior properties compared to the
lead itself, such as improved pharmacodynamics, improved pharmacokinetics, lower
probability of drug-drug interactions, less variable pharmacokinetics and/or
pharmacodynamics, improved overall safety profile and improved physicochemical
properties (e.g. solubility) (1).
Examples of active metabolites of previously marketed drugs that have been developed
into drugs are listed in Table 1.
Table 1. Pharmacologically active metabolites developed as new drugs (2)
Parent drugs
Metabolite drugs
Biotransformation
Brand name
Allopurinol
Oxypurinol
Oxidation of xanthine
Oxyprim
Amitriptyline
Nortryptyline
N-Demethylation
Aventyl
Bromhexine
Ambroxol
N-Demethylation
& Mucosovan
Hydroxylation
Diazepam
Oxazepam
N-demethylation
& Serax
Hydroxylation
Etretinate
Acitretin
Deesterification
Soriatane
Hydroxyzine
Cetirizine
Carboxylation
Zyrtec
Imipramine
Desimipramine
N-Demethylation
Norpramin
Loratadine
Desloratadine
Descarboethoxylation
Clarinex
Loxapine
Amoxapine
N-Demethylation
Asendin
Phenacetin
Acetaminophen
O-Deethylation
Tylenol
Terfenadine
Fexofenadine
Carboxylation
Allegra
Thioridazine
Mesoridazine
S-Oxidation
Serentil
Chapter 1
General Introduction
For example, fexofenadine, the primary metabolite of terfenadine was developed as antihistamine agent with selective peripheral histamine H1-receptor antagonist activity.
Fexofenadine is a safer drug than terfenadine because it does not has the ability to cause
cardiotoxicity by hERG inhibition.
In some instances, metabolic transformation can also produce reactive or toxic
intermediates or metabolites, with potential toxicological implications (3). Hence, a good
understanding of the metabolism of a new chemical entity is needed early in the drug
discovery process.
1.1. Metabolites in safety testing (MIST)
Drug metabolism and pharmacokinetics are crucial factors for successful drug
development. Inappropriate pharmacokinetics previously was one of the main reasons of
attrition in drug development. Although inappropriate pharmacokinetics has become a
less important reason for attrition, less progress has been made in decreasing the attrition
due to drug toxicity. The preparative synthesis of drug metabolites is currently of primary
importance in industry in order to assess potential toxicity, drug-drug interactions and to
examine metabolic pathways (4). In fact, testing the toxicities and biological activities of
human metabolites of drugs is important in drug development to assure effectiveness and
safety (5).
The importance of metabolite identification and quantification is stressed by the
guidelines published in 2008 by the US food and drug administration (FDA). The so-called
Metabolites in Safety Testing (MIST) guidelines describe the studies recommended to
perform on metabolites having more than 10% systemic exposure of the parent drug to
support human safety. A decision diagram of this is depicted in Figure 1.
These guidelines challenged the development of biocatalytic systems for the generation of
large amounts of drug metabolites as well as analytical technologies dealing with their
quantification and identification of drug metabolites in an efficient and intelligent way (7).
Chapter 1
General Introduction
Figure 1. Decision tree flow diagram of the MIST guidelines as determined by the FDA,
based on (6).
1.2. Enzymes involved in drug metabolism
Drug metabolism is typically divided in two phases. Phase I metabolism includes many
types of functionalization reactions, e.g. oxidation, reduction, hydrolysis, hydration and
dehalogenation reactions (9). Cytochrome P450s represent the most important class of
enzymes involved in phase I metabolism, being involved in 75-80% of metabolism of
marketed drugs, Figure 2. Other phase I enzymes include monoamine oxidases, flavincontaining oxygenases, amidases and esterases (9). Phase II metabolism involves
conjugation of polar groups (e.g. glucuronic acid, sulfate, and amino acids) to drugs and/or
phase I drug metabolites to further increase their hydrophilicity and facilitate excretion.
These biotransformation reactions involve glucuronidation, sulfation, GSH conjugation,
acetylation, amino acid conjugation and methylation reaction (10). Phase II enzymes
include UDP-glucuronosyltransferase (UGTs), sulfotransferases (STs), N-acetyl transferases
(NATs), methyl transferases and glutathione S-transferases (GSTs) (10), Figure 2.
Almost all phase I and phase II enzymes can catalyze the formation of stable metabolites,
as well as chemically reactive intermediates that may lead to the generation of adverse
drug reactions (11).
Chapter 1
General Introduction
Figure 2. Phase I and phase II metabolism of currently marketed drugs (adapted from
(8)). The percentage of phase I and phase II metabolism of drugs that each enzyme
contributes is estimated by the relative size of each section of the corresponding chart.
ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; DPD, dihydropyrimidine
dehydrogenase; NQO, NADPH quinine oxidoreductase.
COMT, catechol Omethyltransferase; GST, glutathione S-transferase; HMT, histamine methyltransferase;
NAT, N-acetyltransferase; TPMT, thiopurine methyltransferase; UGTs, uridine 5’triphosphate glucuronosyltransferases.
1.3. Role of reactive metabolites in adverse drug reactions
Adverse drug reactions (ADRs), as defined by the World Health Organization, are noxious,
unintended and undesirable effects of a drug, which occurs at therapeutical doses used in
humans and which require hospitalization of patients to recover (11). Although much
effort has been spent to the development of safer drugs, ADRs still constitute a major
reason for attrition in drug development and withdrawal or restriction of marketed
products. Therefore, prevention of ADRs constitutes a major challenge for the
pharmaceutical industry (3).
Approximate 75% of ADR in patients are classified as Type A ADR which involve lifethreatening exaggerated pharmacological activity (on-target and/or off-target) due to
unanticipated increased plasma concentrations of the parent drug. This class of ADR often
results from drug-drug interactions or genetic deficiency of an enzyme which is involved in
the major pathway of metabolism. For the other classes of ADR formation of reactive drug
metabolites are considered to play a crucial role (12).
The main difficulty for pharmaceutical industry is the fact that ADRs cannot always be
predicted in preclinical animal studies. In particular idiosyncratic drug reactions (IDRs;
Type B ADR) are difficult to predict as they normally have a very low incidence, have a
Chapter 1
General Introduction
delayed time of onset, do not necessarily show classic dose-response relationships and are
not predictable from the known pharmacology of the drug. No predictive animal model is
currently available and IDRs therefore remain poorly understood.
Figure 3 shows a general scheme depicting the role of reactive metabolites in the
development of toxicity. Drugs bioactivation can be catalyzed by both phase I and phase II
enzymes.
Different types of reactive metabolites can be distinguished such as electrophiles, radicals
and reactive oxygen species. Electrophilic metabolites can react with nucleophilic sites in
proteins and/or DNA. Free radicals possess unpaired electrons and can abstract hydrogen
atoms from polyunsaturated fatty acids in membranes which will initiate the destructive
process of lipid peroxidation, and can disrupt the cellular redox-state leading to oxidative
stress and subsequent toxicity (12).
Figure 3. Scheme depicting the role of drug metabolism in determining the toxic
outcome in ADRs.
Table 2 gives examples of drugs whose toxicities are attributed to covalent binding of
electrophilic metabolites to proteins. Bioactivation of xenobiotics which contain certain
functional groups such as tertiary amine, furan ring or acetylene group, can also cause
mechanism-based inactivation of P450, leading to adverse clinical drug-drug interactions
(13-14).
Chapter 1
General Introduction
Table 2. Examples of drugs and reactive metabolites possibly involved in ADRs (12-15).
Drugs
Reactive intermediate
Toxicity
Acetaminophen
Quinone-imine
Hepatotoxicity
Carbamazepine
2-Hydroxy, Quinone-imine
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
Hypersensitivity,
Agranulocytosis,
Hepatotoxicity
Isonizaid
Isonicotinic acid, acetylating species
Hypersensitivity
Phenacetin
p-Nitrosophenetole
Hepatotoxicity
Procainamide
Hydroxylamine, Nitroso
Agranulocytosis
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
Hepatotoxicity
epoxide
Valproic acid
Acyl glucuronides
Hypersensitivity,
Hepatotoxicity
Detection of reactive metabolites is difficult due to the chemical reactivity of the shortlived intermediates and to the usually low amounts present in incubations. The most
common way to screen for formation of reactive metabolites is to trap them with model
nucleophiles such as GSH and to analyze the formed adducts with spectroscopic
techniques. The endogenous tripeptide GSH can trap different types of RIs including
quinones, quinoneimines, iminoquinone, methides, epoxides, areneoxides and nitrenium
ions (15). The corresponding GSH adducts are typically analyzed by liquid-chromatography
mass spectrometry (LC-MS). However, a major disadvantage is that structural information
of the adducts obtained by LC-MS/MS experiments is limited, as it may be insufficient in
identifying the exact position of oxidation, to differentiate stereoisomers, or to provide
the exact structure of the metabolite (16). Complementing MS data with Nuclear
Magnetic Resonance (NMR) experiments is required for complete structural elucidation of
drug metabolites and/or resulting adducts. A limitation of NMR, however, is its relative
insensitivity, as it requires several milligrams of pure metabolite.
Chapter 1
General Introduction
The generation of pure metabolites can be achieved by organic synthesis,
electrochemical oxidation of the parent drug or by biocatalysis using appropriate enzymes.
The synthesis by organic chemistry is often complicated by the lack of appropriate
synthetic routes for specific metabolites and may yield only low amounts of the desired
product that has to be purified subsequently.
Electrochemical oxidation has been used to generate drug metabolites and has the
advantage that it can be easily scaled up. However, with this technique not all the relevant
enzymatic metabolites can be formed. Electrochemistry has been previously used to
generate GSH adducts of clozapine (17) and troglitazone (18).
Biocatalysis by human or bacterial P450s can also be used for the scaling up of metabolite
production (19). For example, human relevant diclofenac metabolites were synthesized by
microbial fermentation in large amount (up to 170 mg) and their structures were
elucidated by MS and NMR (20).
More recently the use of highly active bacterial P450 BM3 mutants for the generation of
reactive metabolites of drugs has been explored. Damsten et al. showed that BM3
mutants could be used for the generation of human relevant reactive metabolites of
clozapine, acetominophen, diclofenac (21), and trimethoprim (22). Boerma et al. showed
that P450 BM3 mutants with high capacity to activate drugs (clozapine, acetaminophen
and troglitazone) into relevant reactive metabolites can be employed to produce protein
adducts to study the nucleophilic selectivity of highly reactive electrophiles (23). Dragovic
et al. applied highly active BM3 mutant as bioactivation system to study the role of human
GSTs in the inactivation of reactive intermediates of clozapine (24).
1.4. Biological screening for active metabolites
To assess the biological activity of drug metabolites there are several approaches
available. The traditional approach is to isolate and purify all the metabolites from in vitro
incubations with subcellular fractions such as liver microsomes or intact cellular systems
(e.g. epatocytes) containing a full complement of drug metabolizing enzymes.
An alternative approach is to use bioassay-guided methods where biological samples
containing biotransformation products are first evaluated for their pharmacological
activity prior to isolation of the metabolites. The bioassay methods may be based on the
assessment of the pharmacological activity using in vitro ligand binding (25, 26), cell based
assays (1), or in vivo pharmacological assays (1).
One of the major bottlenecks in in vitro assays is the lack of possibility to screen affinity or
activity of individual compounds in complex mixtures.
In general, two approaches can be used to measure the effect of individual
compounds in a mixture. One strategy is to combine HPLC with fractionation techniques
to enable the compounds in the mixture to be separated and allow screening of the
individual components using plate reader assays.
Chapter 1
General Introduction
The other available strategy is the on-line high resolution screening technique (HRS)
depicted in Figure 4 (27-29).
HRS enables the screening of individual compounds in complex mixtures by coupling a
separation technology, usually gradient HPLC, to post-column biochemical detection
assays on-line. This technique has already been successfully used to develop a number of
antibody- (30) and biotin- (31), receptor- (32), enzyme- (33) (34) (35), and MS-based (36)
screening assays. The HRS platforms are especially attractive when complex mixtures such
as metabolic incubations (37), combinatorial mixtures (38), or natural extracts (39) are
screened. In contrast to conventional high-throughput screening (HTS), isolation of
individual metabolites not showing affinity can be avoided. Thus, the laborious
development of high-yield synthesis and isolation methods can be limited to compounds
with the desired affinity.
Figure 4. Scheme of the online setup. The system enables the separation of mixtures and
subsequent parallel detection of enzyme binding and accurate mass. The samples are
injected into and separated by an HPLC system. The eluent is split between HR-MS and
enzyme binding detection. The fraction which enters the enzyme binding detection is
mixed with the target (e.g. p38) and the tracer and incubated after each mixing step,
24 s for the target–ligand interaction and 11 s for the target–tracer interaction. Finally,
the fluorescence is measured as readout of affinity towards the target. In parallel, the
second part of the eluent is analyzed by HR-MS delivering structural information to
identify the small molecules tested. Adapted from (28).
2.
Microbial conversion of steroid compounds
Chapter 1
General Introduction
Totally, about 300 approved steroid drugs are known to date, and this number tends to
grow (40). Steroid pharmaceuticals are ranked among the most marketed pharmaceuticals
and represent the second largest category next to antibiotics.
The research on steroid drugs started in 1950, with the discovery of the pharmacological
effects of cortisol and progesterone, two endogenous steroids, and with the identification
of the 11-hydroxylation activity of a Rhizopus species (41-42). Microbial steroid
transformation is a powerful tool for generation of novel steroidal drugs, as well as for
efficient production of steroid-containing active pharmaceutical ingredients (APIs) and key
intermediates.
Steroid hydroxylation is a key tool to develop steroid drugs with improved potency, longer
half-lives in the blood stream, simpler delivery methods and reduced side effects (43). The
therapeutic area of hydroxy-steroids is very wide: they are used as anti inflammatory,
immunosuppressive, progestational, diuretic, anabolic and contraceptive agents (41, 4447). Applications are found in the treatment of adrenal insufficiencies, in the prevention of
coronary heart disease, as anti-fungal agents, as anti-obesity agents, and in the inhibition
of HIV integrase, the prevention and treatment of infection by HIV and in the treatment of
declared AIDS (43).
The complex structure of the steroid molecule requires complicated, multi-step schemes
for the chemical synthesis of steroid compounds. The selective oxidation of an
unactivated, aliphatic C-H bond to an alcohol is a challenging problem in synthesis (48).
Chemical synthesis requires the use of reagents as pyridine, sulfur trioxide or selenium dioxide, whose disposal constitutes an environment issue (43). Moreover, the chemical
catalysts used often show very low regioselectivity.
Steroid hydroxylation by microorganisms represents a powerful alternative to chemical
synthesis. Current trends in microbial hydroxylation studies cover the search for novel
biocatalysts capable of performing reactions targeting specific positions in the steroid
molecule that are particularly important for the pharmaceutical industry (7α, 9α, 11α,
11β, 16α, 17α), production of novel hydroxysteroids with potent therapeutic properties,
and the construction of novel biocatalysts by genetic engineering (40).
Examples are given in Table 3.
Cytochrome P450s play a very important role in endogenous steroid metabolism.
Testosterone hydroxylation in particular has been studied extensively with human liver
microsomal P450s. For example, CYP3A4, the most abundant P450 in human liver and
small intestine, catalyzes the monohydroxylation at 10 different positions of testosterone
(8). Hence testosterone hydroxylation patterns have been utilized as probes for the
presence and characterization of P450s (49) (50).
Hydroxylation of testosterone at 1-, 2-, 2-, 6-, 6-, 7-, 7-, 15-, 15-, 16-, 16and 17-positions has been reported in human and rat liver microsomes (Figure 5).
Table 3. Examples of steroid hydroxylation in microrganisms
Substrate
Product
Microrganism
Ref.
Chapter 1
7-Hydroxylation
Testosterone
Pregnenolone
Epiandrosterone
7-Hydroxulation
DHEA
7-15Dihydroxylation
DHEA
General Introduction
7-OH-testosterone
7-OH-pregnenolone
7-OH-epiandrostenone
Botrytis cinerea AM235
Fusarium oxysporum var.cubense
Mortierella isabellina AM212
(49)
7- and 7-derivatives
7-OH DHEA
M.racemosus
Botryodiplodia malorum CBS 134.50
(51)
(52)
7-15-diOH-DHEA (3,7,15- C.lini CBS 112.21
triOH-androst-5-ene-17-one)
7-15Dihydroxylation
Corynespora cassiicola CBS 161.60
17-OH-progesterone 8-OH-derivative, along with
15-hydroxy derivative
9-hydroxylation
Testosterone
9-OH-AD, 9-OH-testosterone R.equi ATCC 14887
Progesterone
9-OH-progesterone
Expression of 9-hydroxylase
from M.smegmatis in E.coli BL21
Androsterone
9-OH-adrenosterone, 9-OH- Cunninghamella elegans TSY-0865
11-keto-testosterone
11-Hydroxylation
Testosterone
Rhizopus stolonifer
11-OH-testosterone
ATCC 10404, Fusarium lini
NRRL 68751
11-hydroxylation
Progesterone
T.harzianum, T.hamatum
11-OH-progesterone
14-Hydroxylation
Testosterone
A.wentii MRC 200316
14-OH-testosterone
15-Hydroxylation
Testosterone
F.oxysporum var. cubense
15-OH testosterone
Progesterone
15-OH progesterone
15-Hydroxylation
Testosterone
Solvent tolerant
15-OH-testosterone
Pseudomonas putida S12
expressing B.megaterium
steroid hydroxyase CYP106A2
16-Hydroxylation
Testosterone
2,16,17-triOH-androst-4-en- Whetzelinia sclerotiorum
ATCC 18687
3-one
17-Hydroxylation
Progesterone
B.sphaericus ATCC 245,
17-OH-progesterone
B.sphaericus ATCC 7063,
B.sphaericus ATCC 13805
(50)
(52)
(53)
(54)
(55)
(56)
(57)
(58)
(59)
(60)
(61)
(62)
(63)
Chapter 1
General Introduction
Figure 5. Hydroxylation of Testosterone by human P450s (adapted from (51))
Figure 6. Testosterone hydroxylation by bacterial P450s (adapted from (40)).
Chapter 1
General Introduction
Hydroxylation of Testosterone by bacterial P450s has also been extensively studied (Figure
6) (51). Profiling this hydroxylation provides useful information for characterizing bacterial
P450s as monogenic traits and for comparing bacterial P450s with human P450s.
The features and functionality of steroid hydoxylases have been recently reviewed by, e.g.
Bernhardt (2006) (52), Hannemann et al (2007) (53), Novikova et al. (2009) (54) and
Kristan and Lanisnik-Rizner (2012) (55).
3.
Cytochrome P450s
Cytochrome P450s (P450s or CYPs) constitute a family of monoxygenases involved in the
biotransformation of drugs, xenobiotics, alkanes, terpenes, and aromatic compounds, in
the metabolism of chemical carcinogens, the biosynthesis of physiologically relevant
compounds as steroids, fatty acids, eicosanoids, fat-soluble vitamins, bile acids and in the
degradation of pesticides (52).
They are able to catalyze a number of difficult oxidative reactions, such as C-H bond
hydroxylation, N-dealkylation, N-hydroxylation, O-dealkylation, S-oxidation and
epoxidation of numerous endogenous and exogenous compounds (3). An overview of the
different reactions catalyzed by P450s is listed in Table 3.
The broad substrate acceptance and this huge diversity in catalyzed reactions make the
class of P450 very promising biocatalysts in industrial processes. In fact, in recent years
there has been an increasing interest in the application of CYP biocatalysts for the
industrial synthesis of bulk chemicals, pharmaceuticals, agrochemicals, and food
ingredients, especially when a high grade of stereo and regioselectivity is required (57).
Moreover P450s are of great interest in drug metabolism. They are ubiquitous enzymes
often involved in the oxidation of drugs resulting in the generation of metabolites that are
more easily excreted, thus modulating the toxicity of these compounds (57).
3.1. Human P450s
The human genome encodes for 57 functional P450s (58) of which approximately a dozen
are involved in drug metabolism. The P450s mostly involved in metabolism of drugs in
humans are listed in Table 5, where the main reaction and substrate specificities are
indicated. Genetic polymorphisms that may alter the enzyme activity have been reported
for a number of P450s (59). While for the P450s 1A2, 2A6, 2B6 and 2C8 the clinical effects
due to these polymorphisms are minor, major effects are observed for 2C9, 2C19, and 2D6
(60). Polymorphisms in these isoenzymes affect metabolism of 20% to 30% of the clinically
used drugs and this has become a major issue in the discovery and development of drugs
and other NCEs (61).
Table 4. List of different reactions catalyzed by P450s (56)
Reaction type
Example
Chapter 1
General Introduction
Aliphatic
hydroxylation
Aromatic
hydroxylation
Epoxidation
Heteroatom
dealkylation
Alkyne
oxygenation
Heteroatom
oxygenation
Aromatic
epoxidation and
NIH-shift
Dehalogenation
Dehydrogenation
Reduction
Cleavage of esters
Table 5.Substrate specificity of human CYPs
CYP
CYP1A2
Substrate properties
Drug/Substrates
Planar, polyaromatic
compounds
Clozapine
Phenacetine
Olanzepine
Imipramine Propranolol
Theophylline
Reaction
Oxidation
O-deethylation
Oxidation
Demethylation
4-hydroxylation
Chapter 1
3.2.
Structural organization of P450s
General Introduction
Chapter 1
General Introduction
All P450s are heme-containing proteins containing an iron atom at the center of the heme
which is also complexed by a cysteine residue as axial ligand. Based on their redox partner,
P450s can be classified in different classes (Figure 7):
Figure 7. Structural organization of P450s: (a) Class I (mostly bacterial P450s), (b) Class II
(mostly microsomal P450s) and (c) P450 BM3. (Adapted from (62))
Class I P450s (a) require two redox proteins: a small redox 2Fe-2S iron-sulfur ferredoxin
and a FAD or FMN containing reductase. Mitochondrial P450s and most soluble bacterial
P450s belong to this class (63).
In class II (b), electrons are transferred via only one reductase having a FAD and a FMN
domain. Microsomal P450s that are attached to the endoplasmatic reticulum belong to
this class (63).
Most bacterial P450s belong to class I. However, there are exceptions: in 1981
Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium was identified. P450 BM3
(c) is soluble and uses a Class II redox system. This soluble bacterial enzyme contains the
P450 monoxygenase domain fused to the electron transfer flavin mononucleotide
(FMN)/flavin adenine dinucleotide (FAD) reductase domain in a single polypeptide (63). In
contrast, mammalian CYPs require additional redox partners as cytochrome P450
reductase (CPR) and often cytocrome b5 and lipids (64, 65).
3.3. The catalytic cycle
The general catalytic cycle for substrate hydroxylation by P450s is depicted in Figure 8.
Chapter 1
General Introduction
Figure 8. The catalytic cycle of P450s, adapted from (66)
In the resting state, in absence of substrate, the ferric iron atom is six-coordinated,
complexed with a water molecule (I). Binding of a substrate displaces the water molecule
(II). This causes the ferric spin state to convert from low spin to high spin, a transition that
can be monitored spectrophotometrically since the Soret band undergoes a “type I” shift
from ca. 418 nm to ca. 390 nm. A single electron supplied by a reduced pyridine
nucleotide (NADPH or NADH) reduces the heme iron to the ferrous state (III).
Subsequently, dioxygen binds to the ferrous iron generating the oxy-complex (IV). A
second electron is then transferred, creating a ferric peroxy complex (V). Protonation of
the terminal oxygen atom gives “Compound 0”, a hydroxyperoxy adduct (VI). Addition of a
second hydrogen atom followed by loss of water gives “Compound I” (VII), a ferryl species
thought to be the active entity in most P450 oxidations, which has been characterized
recently (67). This complex abstracts hydrogen from the substrate (VIII) after which the
hydroxyl group formed rebounds to the substrate radical, resulting in formation of the
hydroxylated product which releases from the active site. NAD(P)H consumption is not
necessarily fully coupled to product formation (68). If dioxygen binding is hindered, the
second electron transfer will be too slow, resulting in the loss of superoxide from (IV)
(superoxide uncoupling). If substrate fitting in the active site cannot prevent water
encroachment, (VI) will be protonated at the iron-bound oxygen, resulting in the loss of
Chapter 1
General Introduction
hydrogen peroxide (peroxide uncoupling). If a substrate binds with no hydrogen atom
conveniently positioned for abstraction, the oxygen atom in (VII) could be reduced to
water (oxidase uncoupling). Those alternative pathways can hamper the use of P450s in
biocatalysis because the expensive electron donating cofactor is used in futile redox
cycling instead of product formation. Moreover the peroxide and superoxide formed by
uncoupling can damage the biocatalyst.
4.
Bacterial Cytochrome P450 BM3
Bacterial P450 systems often exhibit much higher catalytic activities than membranebound eukariotic P450s and are easy to handle in the laboratory due to their solubility and
high expression level in heterologous hosts (69). In particular Cytochrome P450 BM3
(BM3) is considered as one of the most promising monoxygenases for biotechnological
applications as it possesses the highest activity ever recorded for a P450 (63, 70, 71) and
can be easily expressed at high yield in Escherichia coli.
The natural substrates of BM3 are C12-C18 fatty acids which are hydroxylated at very high
activity at subterminal positions (72). However, over the recent years, several research
groups have succeeded in expanding the substrate selectivity of P450 BM3 and improving
its catalytic properties by site-directed and/or random mutagenesis. Through rational
redesign and directed evolution, BM3 mutants have been obtained that are able to oxidize
aromatics (73), alkanes (74), hydrocarbons (75), carboxylic acids (76) and pharmaceuticals
(21, 22, 77-84).
Recently a large body of research has been conducted to broaden and alter the substrate
specificity of bacterial CYPs in an effort to generate “human like” P450 activities (57). The
demand of “humanized” bacterial P450 with high activity is constantly growing to meet
the needs of the fine-chemical synthesis, pharmaceutical production and bioremediation
industries (85).
4.1. Engineering P450 BM3
The application of engineered bacterial P450s also have application in pharmacology and
toxicology as it enable the generation of substantial amounts of drug metabolites as well
as in optimization of lead compounds (86). Figure 9 shows examples for substrates of
engineered cytochrome P450 BM3 variants.
In order to improve the selectivity of oxidation to a specific product, the substrate from
which it is formed must be constrained to bind in a particular orientation (87). This can be
obtained by rational redesign of the active site, for example introducing polar residues or
bulky side-chains (88-90).
Chapter 1
General Introduction
Figure 9. Examples for substrates of engineered cytochrome P450 BM3 variants.
(Adapted from (62)).
Directed evolution, in which mutations are introduced at random positions using errorprone PCR, is a valuable complementary tool to site-directed mutagenesis. This approach
has the advantage of being able to generate unpredictable mutations with unanticipated
beneficial properties. The benefits of the two strategies can be combined using rational
evolution, in which selected residues are subjected to random or semi-random
mutagenesis. Alternatively, directed evolution can be used to identify sites suitable for
targeting, followed by site-saturation mutagenesis to establish which mutations are most
effective in those positions.
For example, Dietrich et al. (91) developed a novel semi biosynthetic route for the
production of artemisinin, a highly important antimalarian agent. By rational redesign of
Chapter 1
General Introduction
the active site they introduced mutations F87A/A328L in the active site of substrate
promiscuous P450 BM3: mutation F87A appeared to relieve the steric hindrance imposed
upon the substrate amorphadiene and allowed an increased access to the heme group,
while mutation A328L decreased the mobility of amorphadiene in the active site,
promoting epoxidation.
A similar approach has been used by Seifert et al. (75), who focused on the same hotspots
F87 and A328, creating a minimal and highly enriched P450 BM3 mutant library, by
mutating those two positions to all possible combinations of five hydrophobic aminoacids.
In this way, by systematically altering the size of the side chains in those two positions, a
broad range of binding site shapes was generated to convert a range of differently sized
and shaped terpenes (75). Recently, Seifert et al. (92) set up an iterative approach
comprising successive rounds of modeling, designing of focused mutant libraries and
screening, to identify a mutant for the production of the highly valuable product perillyl
alcohol.
It is widely accepted that increased regio-, stereoselectivity is the result of a reduced
number of substrate orientations close to the heme, as discussed in chapter 4 of the
present thesis (82, 90, 93).
Considerable effort has been invested for improving the coupling efficiency (ratio of
product formed to NAD(P)H cofactor consumed) of P450 BM3 by rational mutagenesis.
However the efficiencies obtained are still far lower than with native enzyme-substrate.
Detailed information on the structural determinants of coupling with novel substrates are
missing, therefore it is difficult to rationalize mutations to improve coupling efficiencies.
However, cost-effective, high-throughput screening methods will need to be developed to
assess relative coupling efficiency among P450s in mutant libraries (86).
Properties that need to be improved to enable the application of P450s in industrial
processes are: TTN (total turnover number), substrate acceptance, regio-and stereoselectivity, activity (kcat, KM), inhibition and coupling efficiency (70). Also improvement of
physical properties as thermostability, solvent tolerance, oxidative stability and substrate
and product tolerance should be addressed (94).
4.2. P450 BM3 in biocatalysis
One emerging application of P450s for the pharmaceutical industry is the generation of
large amount of drug metabolites, as discussed above (86). Several groups showed drug
metabolite preparation using human P450s expressed in Escherichia coli and in insect cells
(95). The Arnold group showed that human metabolites of propranolol could be produced
by mutants of bacterial P450 BM3 heme domain in a reaction driven by hydrogen
peroxide (5). Yields obtained were comparable to those produced by recombinant human
P450s in bacterial or baculovirus bioreactors (96). Recently, Dubey et al. set up a
biotechnological production of anticancer drug (colchicine derivatives) using P450 BM3 as
biocatalyst obtaining a yield of 7.3 g/L in 70 L fermentor (97). Kim at al. applied BM3 wt
Chapter 1
General Introduction
and a set of mutants for the generation of the human metabolite piceatannol from the
anticancer-preventive agent resveratrol (77), for the generation of human chiral
metabolites of simvastatin and lovastatin (79) and for the generation of human
metabolites of 7-ethoxycoumarin (78). The Arnold group compiled a panel of 120 mutants
to prepare nearly all human metabolites and a number of novel hydroxylated derivatives
for the drugs verapamil and astemizole (81). This panel of enzymes could also be applied
for the generation of new chemical entities (NCEs) to diversify lead compounds. Within
any catalyst panel both extremes of regioselectivity can be useful: enzymes selectively
producing individual metabolites and less selective enzymes to survey metabolite
possibilities (81).
A recent area of interest is the design and development of novel P450s as biocatalysts of
reactions of commercial interest, such as in pharmaceutical or fine chemical synthesis (86)
as well as for the optimization of lead compounds.
An example of the application of BM3 mutants in chemical synthesis is the
chemoenzymatic elaboration of monosaccharides applied by the Arnold group (98).
Regioselective deprotection of monosaccharide substrates using engineered P450 BM3
demethylases provides a highly efficient method to obtain valuable intermediates that can
be converted to a wide range of substituted monosaccharides and polysaccharides (98).
Engineered Cytochrome P450 BM3s were also applied for the enantioselective hydroxylation of 2-arylacetic acid derivatives and could produce an authentic human
metabolite of buspirone with high activity and selectivity (80).
Rentmeister et al. showed that products hydroxylated by BM3 can be subsequently
fluorinated in a chemo-enzymatic process that greatly simplifies the insertion of fluorine
into these structures at specific positions (99). Fluorination has become an important tool
in drug discovery and development as it can modulate the pharmacokinetic and the
pharmacological properties of drugs and lead compounds. In particular, fluorination can
improve membrane permeability, metabolic stability and/or receptor-binding properties
of bioactive molecules (99).
4.3. Key residues in the P450 BM3 active site
Control of regio- and stereoselectivity of biocatalysts is one of the major challenges in
biotechnology, as a limited number of residues in the substrate pocket appear to possess
significant selectivity-influencing powers. Ala82 and Phe87 are among those.
In Figure 10 key residues of the substrate channel and the active site of substrate-bound
P450 BM3 are shown. P450 BM3 crystallizes in a “precatalytic conformation” in which the
substrate is located too far to the heme iron for the oxidation to take place (87). Therefore
it is unclear whether the residues located in the substrate access channel are also
catalytically relevant. Also the spin relaxation NMR experiments of reduced P450 BM3
suggested that the substrates are 6 Å closer to the porphyrin ring than in the oxidized
form, indicating that structural rearrangements occur in order to allow the substrate to
Chapter 1
General Introduction
move closer to the heme iron (100). In the present thesis mutagenesis studies at position
87 (101) (84) and position 82 (89) will be presented.
Figure 10. The substrate access channel (A) and active site (B) of NPG-bound P450BM3
(Adapted from (66))
Phe 87. Phe 87 is one of the most studied residues of the active site of BM3 and it is object
of chapter 2 and 3 of the present thesis. In the substrate-free crystal structure, this
residue lies perpendicular to the porphyrin system, but in the substrate-bound crystal, the
Chapter 1
General Introduction
orientation of the aromatic ring changes to parallel to the plane of the heme (102) (103)
(63). It has been proposed that this motion influences the orientation of the substrate
relative to the catalytic center and it is responsible for the displacement of the axial water
ligand from its coordination site (104).
Phe87 forms with Phe81 a small hydrophobic pocket within the active site that sequesters
the -end of fatty acids. It seems likely that small hydrophobic substrates could
preferentially bind in the pocket generating non-productive complexes (105).
Phe87 is the most commonly mutated residue of P450 BM3 (Table 6).
Site-directed mutagenesis studies proved that Phe87 plays a key role in the control of
reaction selectivity (106) (107).
Typically, Phe87 was mutated to aminoacids with less bulky side chains, in order to
destroy the hydrophobic pocket, creating more space close to the heme, to accomodate
larger substrates (73-75, 77, 80, 103, 105, 108-116). Such substitutions, however, lead to a
decreased coupling efficiency of NADPH consumption to product formation due to a less
efficient exclusion of water from the active site (105).
The F87V mutation is a common component of several drug metabolizing variants (69)
(117). Moreover, it has been shown that mutation F87V significantly affected the
stereoselectivity of arachidonic acid epoxidation (106) and the activity towards indoles
(118). Li et al. also showed that mutation F87V is beneficial for oxidation of polycyclic
aromatic hydrocarbons, such as naphthalene, fluorene, acenaphthene, acenaphthylene,
and 9-methylanthracene (119).
In the present thesis saturation mutagenesis was applied to evaluate the effect of all
possible amino acids in this position on the metabolism of alkoxyresorufins, testosterone
and clozapine (84, 101).
Ala 82. The existence of a hydrophobic pocket between Phe81 and Phe87 clearly affects
the attempts to engineer BM3 towards novel target substrates. Huang et al. (90) were the
first to try to “fill” the hydrophobic pocket rather than destroying it, by mutating Ala82 to
large hydrophobic residues like Phe and Trp.
The mutants A82F and A82W showed a remarkable increase in affinity (800-fold) for fatty
acids. Moreover, the efficiency of indole hydroxylation increased, due both to increased
Kcat/KM values and to increased coupling efficiency, achieved by a more efficient exclusion
of water from the active site. Mutation at this position will be object of chapter 4 of the
present thesis.
Table 6. Reported mutations at position Phe87:
ALA (29 )(73-75) ( 81) (98, 99) (107 )(120-134)
ARG (101, 135)
ASN (101)
ASP (101)
Chapter 1
CYS
GLN
GLU
GLY
HIS
ILE
LEU
LYS
MET
PRO
SER
THR
TRP
TYR
VAL
SS
General Introduction
(101, 133)
(101, 135)
(101, 135)
(5, 74, 81, 98, 101, 122, 130, 135-143)
(101, 135)
(75, 81, 92, 98, 99, 135, 144, 145)
(75, 81, 92, 121, 124, 135, 144, 145)
(101, 135)
(101, 135)
(101, 135)
(101, 135)
(101, 135)
(101, 135)
(101) (106) (135) (136) (140) (146-147)
(69,74, 75, 81, 82, 98, 99, 106, 118, 119, 142, 144, 145,148-153)
(5, 91, 101, 118, 144, 145, 154, 155)
Table 7. Reported mutations at position Ala82:
CYS (81, 135, 144)
GLY (81, 99, 144, 155)
ILE
(81, 90, 144, 156)
LEU (5, 80, 81, 99, 121, 134, 144, 156, 157)
PHE (81, 90, 144, 156, 158)
PRO (5, 81)
SER (81, 144, 155)
THR (81, 144)
TRP (90, 99, 135, 159, 160)
VAL (156)
SS
(5, 144, 155)
5.
Spin lattice relaxation NMR (or T1 paramagnetic relaxation NMR)
In order to understand the structural basis that governs the regioselectivity of the
metabolism of a certain substrate or the specificity of a given P450 for a particular drug, it
1
is important to determine the structure of the substrate binding sites. For that purpose H
NMR appears as a powerful and attractive technique, since measurements of
Chapter 1
General Introduction
paramagnetic relaxation can be used to determine the distances between the heme iron
and protons of the bound substrate in P450-substrate complexes (161).
The heme iron atom of a CYP is paramagnetic when in oxidized state. Hydrogen atoms in a
magnetic field will re-align in the direction of this field after their orientation has been
flipped with a radio-frequency pulse. The velocity of this re-alignment (relaxation rate) is
dependent on the local strength of the magnetic field in the direct proximity of the
hydrogen atom. So a hydrogen atom close to the heme in the active site of a CYP, will
relax faster due to the magnetic moment of the heme iron atom.
The relationship between the iron atom to hydrogen atom distance and the rate of
relaxation has been established by Solomon and Bloembergen (162) and can be used to
measure substrate orientations in CYP active sites. A disadvantage of the technique is that
high concentrations (~mM) of ligands need to be present to measure NMR spectra.
Furthermore, average substrate active site orientations do not always match the
metabolic profile (163).
An overview of reported spin lattice relaxation studies using CYPs is given in Table 6.
In chapter 4 of the present thesis this technique has been successfully applied to
determine the orientation of testosterone in two BM3 mutants (M11 and M11 A82W) to
rationalize the different regioselectivity obtained in the metabolic profile.
Table 8. Spin lattice relaxation NMR studies used to determine active site ligand
orientations in CYPs.
CYP
102
Ligand
12-Br-lauric acid
1A
Paracetamol
1A2
Caffeine
2B
Paracetamol
Distances (A)*
7.8 (C1H)-16.3 (C12H)
5.9 (phenylH)6.7(methylH)
6.5 (N1methylH)6.7 (N3 and N7 methylH)
5.8 (methylH)- 6.3
(phenylH)
Major reaction
C2-OH
Benzoquinone
formation
Reference
(163)
N1-demethylation
(163)
Inactive
(164)
(164)
Chapter 1
General Introduction
*the minimal and maximal hydrogen atoms to heme iron atom measured are given.
**Measured in the F483I mutant of CYP2D6, the wild type is inactive towards
testosterone.
6.
Scope and objectives
The research presented in this thesis was part of a wider interdisciplinary project
“Metabolic stability assessment as a new tool in the Hit-to-Lead selection process and the
generation of new lead compound libraries” financed by the Dutch Top-Institute Pharma
consortium (grant D2-102). Several industrial (Merck/MSD (Oss, NL) and QPS
(Groeningen, NL)) and academic (Vrije Universiteit Amsterdam and Radboud Universiteit
Chapter 1
General Introduction
Nijmegen) partners participated in this project that led to several publications and four
PhD theses.
The aim of the project was the application of new, more efficient and selective P450 BM3
mutants for the generation of biotransformation products, being new chemical starting
points for lead optimization (lead libraries), followed by the determination of their binding
affinity (e.g. competitive fluorescence detection assay) and chemical structural
characterization (LC-MS/MS-NMR) in a hyphenated approach.
The general objectives of the present thesis are:
 Engineering drug metabolizing P450 BM3 mutants mimicking human P450s and
mutants with unique catalytic properties based on regio- and stereoselective
metabolism of diagnostic substrates.
 Utilization of “humanized” drug metabolizing P450 BM3 mutants for large scale
production of physiologically relevant human metabolites of lead compounds.
Figure 11 shows the general strategy applied in this research: by site-directed, random or
saturation mutagenesis BM3 mutants are engineered to obtain highly active mutants able
to metabolize drugs and drug-like molecules. The metabolic mixtures obtained by
incubation of diagnostic substrates with engineered BM3 mutants are then screened to
verify the acquisition of certain properties: HRS enables the selection of mutants able to
produce metabolites that show pharmacological activity, GSH trapping enables the
identification of mutants able to produce reactive metabolites (subsequently trapped by
GSH), screening by UPLC allows the identification of changes in regio- and stereoselectivity
in a quick and informative way.
Selected mutants that acquired a certain property (e.g. production of high amounts of a
pharmacologically active metabolite, high selectivity for the production of reactive
metabolites, high selectivity of the production of a certain hydroxy-steroid) are then
applied as biocatalysts for the large scale metabolite production in order to enable
pharmacological and toxicological evaluation of drug metabolites and for their structural
elucidation by NMR.
Chapter 1
General Introduction
Figure 11. General strategy applied for the research presented in this thesis, leading to a
“metabolite production and profiling platform”
7.
Outline of this thesis
The work presented in this thesis shows application of BM3 mutants as biocatalyst for
three purposes:
 For the regio- and stereo- selective hydroxylation of lead compounds (e.g.
steroids)
 For the generation of reactive metabolites
 For the generation of pharmacologically active metabolites / lead optimization
Regio- and stereo-selective hydroxylation of unactivated C-H bonds of natural or synthetic
compounds is one of the biggest challenges in synthetic organic chemistry. In particular
steroid hydroxylation is extremely challenging due to the complexity of the steroid
molecule that requires complicated, multi-step schemes involving protecting groups and
subsequent regeneration.
In chapter 2 and 4 of the present thesis cytochrome P450 BM3 mutants are
applied as biocatalysts for the regioselective hydroxylation of testosterone and
norethisterone. In chapter 2 a site saturation mutagenesis library of BM3 mutants was
applied to evaluate the effect of a single mutation in the active site of BM3 on the
regioselective hydroxylation of testosterone and on the coupling efficiency in
alkoxyresorufin oxidation. In chapter 4, a single active site substitution was applied in
order to reduce the substrate mobility of testosterone, thus improving the regioselectivity
Chapter 1
General Introduction
of hydroxylation. Spin lattice relaxation NMR was applied to determine how this single
mutation affected the orientation of the substrate within the active site of two BM3
mutants, M11 and M11 A82W.
The generation of reactive metabolites is important from a toxicological point of
view to be able to identify potentially toxic compounds that may be involved in ADRs or
IDRs.
Chapter 3 of the present thesis focuses on the application of P450 BM3 mutants
for the generation of reactive metabolites of clozapine, which is a drug known to be
responsible of severe ADRs. By screening of a minimal library of BM3 mutants, a mutant
was selected for the generation of large amounts of all major human relevant GSHconjugates of clozapine to enable their structural elucidation by NMR.
The generation of pharmacologically active metabolites is important from a “lead
optimization” point of view. Sometimes drug metabolites possess improved
pharmacological activity and improved physico-chemical properties, compared to the lead
itself.
In chapter 5 of the present thesis a focused library of BM3 mutants has been
applied for the generation of human relevant bioactive metabolites of p38 kinase
inhibitor Tak-715. The HRS screening applied enabled the identification and
pharmacological characterization of the drug metabolites in a quick and informative online
mode. Large scale incubation with BM3 mutants and isolation of active metabolites by
Prep-LC allowed their structural elucidation by NMR.
The last part of this thesis, chapter 6 concerns an overall summary of the work
described in the thesis including general conclusions and perspectives for future work.
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