Digital Comprehensive Summaries of Uppsala Dissertations

from the Faculty of Science and Technology 1050

Mutational Analysis and

Redesign of Alpha-class
Glutathione Transferases for
Enhanced Azathioprine Activity

OLOF MODÉN

ACTA
UNIVERSITATIS
UPSALIENSIS ISSN 1651-6214
ISBN 978-91-554-8685-3
UPPSALA urn:nbn:se:uu:diva-167332
2013
Dissertation presented at Uppsala University to be publicly examined in B42, Biomedical
Center (BMC), Husargatan 3, Uppsala, Wednesday, June 5, 2013 at 13:15 for the degree of
Doctor of Philosophy. The examination will be conducted in English.

Abstract
Modén, O. 2013. Mutational Analysis and Redesign of Alpha-class Glutathione Transferases
for Enhanced Azathioprine Activity. Acta Universitatis Upsaliensis. Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1050.
72 pp. . ISBN 978-91-554-8685-3.

Glutathione transferase (GST) A2-2 is the human enzyme most efficient in catalyzing
azathioprine activation. Structure-function relationships were sought explaining the higher
catalytic efficiency compared to other alpha class GSTs. By screening a DNA shuffling
library, five recombined segments were identified that were conserved among the most active
mutants. Mutational analysis confirmed the importance of these short segments as their insertion
into low-active GSTs introduced higher azathioprine activity. Besides, H-site mutagenesis led
to decreased azathioprine activity when the targeted positions belonged to these conserved
segments and mainly enhanced activity when other positions were targeted. Hydrophobic
residues were preferred in positions 208 and 213.
The prodrug azathioprine is today primarily used for maintaining remission in inflammatory
bowel disease. Therapy leads to adverse effects for 30 % of the patients and genotyping of the
metabolic genes involved can explain some of these incidences. Five genotypes of human A2-2
were characterized and variant A2*E had 3–4-fold higher catalytic efficiency with azathioprine,
due to a proline mutated close to the H-site. Faster activation might lead to different metabolite
distributions and possibly more adverse effects. Genotyping of GSTs is recommended for further
studies.
Molecular docking of azathioprine into a modeled structure of A2*E suggested three
positions for mutagenesis. The most active mutants had small or polar residues in the mutated
positions. Mutant L107G/L108D/F222H displayed a 70-fold improved catalytic efficiency with
azathioprine. Determination of its structure by X-ray crystallography showed a widened H-site,
suggesting that the transition state could be accommodated in a mode better suited for catalysis.
The mutational analysis increased our understanding of the azathioprine activation in alpha
class GSTs and highlighted A2*E as one factor possibly behind the adverse drug-effects. A
successfully redesigned GST, with 200-fold enhanced catalytic efficiency towards azathioprine
compared to the starting point A2*C, might find use in targeted enzyme-prodrug therapies.

Keywords: allelic variants, azathioprine, bioactivation, chimeric mutagenesis, directed

evolution, DNA shuffling, enzyme engineering, glutathione transferase, GST, lysate
screening, molecular docking, multiple alignment, multivariate analysis, polymorphism,
principal component analysis, prodrug, prodrug activation, protein engineering, protein
redesign, reduced amino acid alphabet, saturation mutagenesis, semi-rational enzyme
engineering, site-directed mutagenesis, structure-activity relationship, structure-based
redesign

Olof Modén, Uppsala University, Department of Chemistry - BMC, Biochemistry, Box 576,
SE-751 23 Uppsala, Sweden.

© Olof Modén 2013

ISSN 1651-6214
ISBN 978-91-554-8685-3
urn:nbn:se:uu:diva-167332 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-167332)
To my family
List of Papers

This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.

I. Kurtovic S., Modén O., Shokeer A. & Mannervik B. (2008)

Structural determinants of glutathione transferases with aza-
thioprine activity identified by DNA shuffling of alpha class
members. J Mol Biol. 375, 1365−1379.
II. Zhang W., Modén O. & Mannervik B. (2010) Differences among
allelic variants of human glutathione transferase A2-2 in the ac-
tivation of azathioprine. Chem Biol Interact. 186, 110−117.
III. Modén O., Zhang W. & Mannervik B. (2012) Mutational analys-
is of human glutathione transferase A2-2 identifies structural ele-
ments supporting high activity with the prodrug azathioprine.
Protein Eng Des Sel. 25, 189−197.
IV. Zhang W., Modén O., Tars K. & Mannervik B. (2012) Structure-
based redesign of GST A2-2 for enhanced catalytic efficiency
with azathioprine. Chem Biol. 19, 414−421.

Reprints of the papers were made with permission of the publishers.

Contents

Background ................................................................................................ 13
Redesign of enzymes ............................................................................. 13
Enzyme evolution ............................................................................. 13
Directed evolution ............................................................................. 13
Rational approaches .......................................................................... 14
Catalytic mechanisms ....................................................................... 14
Screening throughput ........................................................................ 15
Screening efficiency .......................................................................... 15
Applications ...................................................................................... 17
Comparisons of methods ................................................................... 18
Glutathione transferase (GST) ............................................................... 18
Function ............................................................................................ 18
Glutathione ....................................................................................... 19
Phylogeny ......................................................................................... 19
Polymorphism of human GST A2-2 .................................................. 19
Expression of human alpha class GSTs ............................................. 20
Expression of different genotypes ..................................................... 21
Structure ............................................................................................ 21
Mechanism ........................................................................................ 22
Applications ...................................................................................... 22
Azathioprine .......................................................................................... 23
History .............................................................................................. 23
Immunosuppression .......................................................................... 23
Inflammatory bowel disease ............................................................. 24
Adverse effects .................................................................................. 24
Mechanism of action ......................................................................... 25
Polymorphism in the metabolic pathways ......................................... 28
Antibody directed enzyme-prodrug therapy (ADEPT) ...................... 31
Aims of the thesis .................................................................................. 31
Structure-function relationships of human GST A2-2 ....................... 31
Polymorphism of human GST A2-2 .................................................. 32
Characterization using alternative substrates .................................... 32
Redesign of human GST A2-2 for enchanced azathioprine activity . . 32
Present investigation .................................................................................. 33
Azathioprine activity of shuffled alpha class GSTs (Paper I) ................. 33
Introduction ....................................................................................... 33
 Mutant library design ........................................................................ 33
Screening of bacterial lysates ............................................................ 34
Sequence analysis ............................................................................. 35
The H-site library .............................................................................. 35
Crossover locations ........................................................................... 35
Expression and purification ............................................................... 36
Kinetic characterization .................................................................... 36
Structure-activity relationships ......................................................... 37
Thoughts on exploiting the data for directed recombination ............. 39
Comments on the alpha class GSTs included in the library ............... 40
Comments on the alpha class GSTs excluded from the library ......... 40
Another example using DNA shuffling ............................................. 41
Suggested screening modifications ................................................... 41
Conclusion ........................................................................................ 42
Polymorphism in human GST A2-2 (Paper II) ...................................... 42
Introduction ....................................................................................... 42
Kinetic characterization .................................................................... 43
Alternative substrates ........................................................................ 44
Combining activity and expression in different tissues ..................... 44
Activity and variation of expression ................................................. 45
Bioavailability and activation ........................................................... 45
Genotyping for activity and expression ............................................. 45
Thermal inactivation ......................................................................... 46
Conclusion ........................................................................................ 46
Structural elements supporting high azathioprine activity (Paper III) .... 47
Introduction ....................................................................................... 47
Chimeric mutants .............................................................................. 47
Saturation mutagenesis ..................................................................... 49
Conclusion ........................................................................................ 50
Redesign of GST A2-2 for enhanced azathioprine activity (Paper IV) . . 51
Introduction ....................................................................................... 51
Library design ................................................................................... 51
Screening .......................................................................................... 52
Kinetic characterization .................................................................... 52
Comparison to other alpha class sequences ....................................... 52
Structural interpretation .................................................................... 53
Mutational strategies ......................................................................... 54
Conclusion ........................................................................................ 55
Summary ............................................................................................... 55
Structure-function relationships of human GST A2-2 ....................... 55
Polymorphism of human GST A2-2 .................................................. 56
Characterization using alternative substrates .................................... 57
Redesign of human GST A2-2 for enchanced azathioprine activity . . 57
Summary in Swedish ................................................................................. 59
 Mutationsanalys och förbättring av alfaklass-glutationtransferaser med
avseende på aktivitet med proläkemedlet azatioprin .............................. 59
Bakgrund .......................................................................................... 59
Avhandlingens artiklar ...................................................................... 61
Acknowledgments ...................................................................................... 63
References .................................................................................................. 64
Abbreviations

6-MMP 6-methyl-mercaptopurine
6-MMPR 6-methyl-mercaptopurine ribonucleotide
6-MP 6-mercaptopurine
6-TGN 6-thioguanine nucleotide
A2*E Variant E of human GST A2-2
ADEPT Antibody directed enzyme-prodrug therapy
AO Aldehyde oxidase
Aza Azathioprine
cDNA Complementary DNA
CDNB 1-chloro-2,4-dinitrobenzene
CMNI 5-chloro-1-methyl-4-nitroimidazole
DNA Deoxyribonucleic acid
E. coli Escherichia coli
epPCR Error-prone PCR
G-site Glutathione binding site
GDH Mutant G107/D108/H222 based upon GST A2*E
GSH Glutathione
GST Glutathione transferase
GTP Guanosine triphosphate
H-site Hydrophobic substrate binding site
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HER2 Human epidermal growth factor receptor 2
IBD Inflammatory bowel disease
IMPDH Inosine monophosphate dehydrogenase
IPTG Isopropyl-1-thio-β-D-galactopyranoside
ITPA Inosine triphosphate pyrophosphatase
MeTIMP 6-methyl-thioinosine monophosphate
MRP Multidrug resistance protein
NPTI 1-methyl-4-nitro-5-(4-nitrophenylthio)-1H-imidazole
PCR Polymerase chain reaction
SD (or ±) Standard deviation
SNP Single nucleotide polymorphism
TIMP 6-thioinosine monophosphate
TITP 6-thioinosine triphosphate
TNF-α Tumor necrosis factor α
TPMT Thiopurine methyltransferase
XO Xanthine oxidase
Alpha-class GSTs
bA1 Bovine GST A1-1
hA1 Human GST A1-1
hA2 Human GST A2-2
hA3 Human GST A3-3
rA2 Rat GST A2-2
rA3 Rat GST A3-3

Degenerate codons
N A, C, T or G
B C, G or T
D A, G or T
H A, C or T
V A, C or G
K G or T
M A or C
R A or G
S C or G
W A or T
Y C or T
NNN 64 codons. 20 amino acids represented. Uneven distribution. 3 stop codons.
NNS 32 codons. 20 amino acids represented. Uneven distribution. 1 stop codon.
NDT 12 codons representing Arg, Asn, Asp, Cys, Gly, His, Ile, Leu, Phe, Ser, Tyr and Val.
YHC 6 codons representing His, Leu, Phe, Pro, Ser and Tyr.
MAX Strategy with 20 codons using multiple primers. 20 amino acids represented.

The genetic code

TTT Phe TCT Ser TAT Tyr TGT Cys
TTC Phe TCC Ser TAC Tyr TGC Cys
TTA Leu TCA Ser TAA STOP TGA STOP
TTG Leu TCG Ser TAG STOP TGG Trp

CTT Leu CCT Pro CAT His CGT Arg

CTC Leu CCC Pro CAC His CGC Arg
CTA Leu CCA Pro CAA Gln CGA Arg
CTG Leu CCG Pro CAG Gln CGG Arg

ATT Ile ACT Thr AAT Asn AGT Ser

ATC Ile ACC Thr AAC Asn AGC Ser
ATA Ile ACA Thr AAA Lys AGA Arg
ATG Met ACG Thr AAG Lys AGG Arg

GTT Val GCT Ala GAT Asp GGT Gly

GTC Val GCC Ala GAC Asp GGC Gly
GTA Val GCA Ala GAA Glu GGA Gly
GTG Val GCG Ala GAG Glu GGG Gly
Amino acids
A Ala Alanine
C Cys Cysteine
D Asp Aspartic acid
E Glu Glutamic acid
F Phe Phenylalanine
G Gly Glycine
H His Histidine
I Ile Isoleucine
K Lys Lysine
L Leu Leucine
M Met Methionine
N Asn Asparagine
P Pro Proline
Q Gln Glutamine
R Arg Arginine
S Ser Serine
T Thr Threonine
V Val Valine
W Trp Tryptophan
Y Tyr Tyrosine
Background

Redesign of enzymes

Enzyme evolution
Enzymes are biological macromolecules that catalyze biochemical reactions
under physiological conditions, thereby steering the metabolism of the cell
into useful pathways rather than wasteful side reactions. In nature the genes
encoding the enzymes obtain diversity through duplications, recombinations,
point mutations, insertions and deletions. Modifications can occur both in
the coding and the non-coding sequence and they can have effects for ex-
ample on expression, structure, stability, degradation, chemistry, and sub-
strate selectivity. Organisms that survive and reproduce pass on to the next
generation the code for the enzymes best adapted to the environment.

Directed evolution
Evolution of enzymes can also be performed in the laboratory by mimicking
the natural processes. Directed evolution involves iterative mutagenesis of
genes in successive generations and selecting of enzymes with desired prop-
erties. Redesign of enzymes enhances their efficiencies in pre-existing chem-
ical reactions, modifies their substrate or product selectivities, modifies the
stability of the enzymes and their reactions under new conditions, or fulfills
several conditions simultaneously. Alternatively the ability to catalyze new
reactions can be introduced but preferably using existing protein scaffolds
(Park et al., 2006; Siegel et al., 2010).
Point mutations can be randomly introduced in vitro by error-prone poly-
merases (epPCR) or by chemical mutagens (Wong et al., 2005). DNA shuff-
ling utilizes recombination of similar sequences in order to create chimeric
mutants (Stemmer, 1994). Later other methods have been presented which
have expanded or improved the techniques or produced similar outcomes
while circumventing intellectual property rights (Neylon, 2004; Kazlauskas
and Bornscheuer, 2009).

13
Rational approaches
The stochastic methods are also complemented by rational approaches. Point
mutations can be directed to active sites or other regions of interest. Libraries
obtain sufficient diversity by introduction of several possible amino acids in
each position. The rationality can be based for example upon structural in-
formation and multiple alignments. By using optimized sets of codons for
each position, the bias of the epPCR, where only one base at the time is
mutated, can be avoided. Recombination of sequences from different sources
can also be focused to specific sites in order to minimize structural disrup-
tions while also allowing sequences of less homology to be recombined
(Voigt et al., 2002; Hiraga and Arnold, 2003; Endelman et al., 2004; Meyer
et al., 2006; Otey et al., 2006). Recombination of more distantly related se-
quences generally gives fewer crossover sites and the chimeric proteins eas-
ily become unstable (Goyal et al., 2002; Kim et al., 2006).
The redesign can comprise different phases in which first a diverse set of
mutants is examined followed by a phase of mutant creation based on the in-
formation obtained in the previous phase. Thus beneficial mutations can be
enriched and deleterious mutations removed in a more organized manner
than by simply letting the best mutants become templates in the next genera-
tion (Fox et al., 2003; Gustafsson et al., 2003; Ehren et al., 2008). This de-
velopment including the synthesis of entire genes is possible due to de-
creased costs of DNA synthesis.
One problem that rational or reductionistic design faces is the epistatic in-
teractions of the mutated residues. The higher the number of mutations intro-
duced in the same template, the less accurately the final properties can be es-
timated from single mutations alone. One way to partially capture the co-
operative effects of mutagenesis is to iteratively saturate different sites of
two to three positions each (Reetz et al., 2005, Reetz et al., 2006). Mutagen-
esis of too many residues at the same time might on the other hand hide any
beneficial effects among the deleterious ones.
Computational chemistry can also be of use, for example in estimating en-
antioselectivity of mutants in silico. In one example (Frushicheva and
Warshel, 2012), the rate of free-energy calculations performed using 400
processors would equal the rate of bacterial lysates screened manually in our
laboratory. However, subtle structural alterations introduced by mutations
can have effects not always captured by modeling.

Catalytic mechanisms
Some enzymes have evolved to have a catalytic efficiency up to about 10 8
M-1s-1 and only be limited by substrate diffusion. Enzyme catalyzed reactions
can be many orders of magnitude faster than the corresponding uncatalyzed
reactions. Mechanisms of enzymatic reactions can be explained by different

14
kinds of contributing effects like acid-base, nucleophilic, electrophilic and
covalent catalysis as well as redox reactions, hydrogen bonding, desolvation,
immobilization, transition state stabilization, ground state destabilization,
preorganization of the active site, binding of near attack conformations, con-
formational mobility and quantum tunneling (Benkovic and Hammes-Schif-
fer, 2003; Frey and Hegeman, 2007). Another view is that the combination
of different effects is dominated by the preorganization of polar or charged
groups in the active site stabilizing the transition state (Warshel, 2003). The
characterization of libraries with point mutations in the active site of the en-
zyme might provide knowledge regarding the underlying mechanism while
the stochastic nature of the methodology allows for improved variants to be
found without explicit mechanistic knowledge.

Screening throughput
Display methods that link the protein with the encoding DNA are useful
when improving the affinity of a target protein (Scott and Smith, 1990;
Roberts and Szostak, 1997). In vitro compartmentalization by water-in-oil
emulsions provides one way to link genes and enzymatic activity in a selec-
tion process (Tawfik and Griffiths, 1998). Some enzymatic reactions, like
antibiotic resistance, can be selected for using living systems. The mentioned
selection methods allow for a large part of the libraries to be assayed simul -
taneously. Many cases however require a screening assay where the enzyme
variants are tested one at the time, making the process of finding improved
mutants slow. Depending on screening methodology and automation up to
several hundreds of thousands of mutants can be screened for a certain prop-
erty (Jones et al., 2008). If alternative substrates are screened for instead of
the desired property itself, then you simply get what you screen for (You and
Arnold, 1996), and that can be a reason for screening fewer mutants using a
more sophisticated method.

Screening efficiency

Library quality
Design, quality, and coverage of the library are also of importance for the ef-
ficiency of the screening process, aside from screening for the desired prop-
erty. The quality factor involves minimizing or avoiding stop codons,
frameshifts and template sequences in the library. It is it also useful to design
the mutated positions to have equal codon frequencies for the introduced
amino acids, as well as to minimize the possibilities of introducing or ampli-
fying bias during the cloning of the library.

15
Codon distribution
While methods like epPCR are based on point mutations of single bases and
not complete codons, saturation mutagenesis using degenerate primers with
codons like NNN or NNS can yield all 20 amino acids in each targeted posi-
tion. Using reduced sets of codons, like NDT, is one way to get an even
codon distribution but also to make smaller libraries while still keeping the
diversity of the amino acids or alternatively focusing them towards a specific
property. To narrow the choice of amino acids to fewer than 20 is not neces -
sarily disruptive to the function or stability of the enzyme since functional
enzymes can be created using down to only nine types of amino acids (Wal-
ter et al., 2005). One paper compared the result of screening of 5000 mutants
from two libraries with three positions mutated with either NNK or NDT
codons (Reetz et al., 2008). There were more hits found in the library with
NDT codons and that could probably be explained by the even distribution
of amino acid probabilities. To have an equal codon distribution with all 20
amino acids included, the MAX strategy (Hughes et al., 2003) can be used.
Alternatively four degenerate primers can be combined in the right propor-
tions to obtain one codon for each amino acid or the DNA can be synthes -
ized using trinucleotide phosphoramidites.

Coverage
The coverage of a library depends on the size of the library, the codon distri-
bution in each position and the number of mutants screened (Figure 1). Lib-
rary coverage is sometimes discussed and it is even mentioned that lower
coverage might be enough in particular experiments (Reetz et al., 2008) but
still the general discussion is focused on three times oversampling which
gives approximately 95 % coverage. Oversampling of a library will give
higher coverage, which can be beneficial when characterizing a specific lib-
rary or specific positions in the active site, or when the cost of the library
construction was significantly larger than the cost of screening additional
mutants.

16
Figure 1. (Paper III: Figure 5f). Theoretical coverage of a library with two positions
mutated with NNS codons, showing both the mean and the 95 % confidence level.
About 2100 colonies must be picked to reach a coverage of 95 %.

However, enzyme mutant activities are like rugged multidimensional energy

landscapes, with each dimension being represented by one position with 20
possible amino acids. When traversing such an energy landscape it is diffi-
cult to predict which mutations will be beneficial. A screening strategy with
higher coverage most likely have higher redundancy, with the same mutants
screened multiple times. For example, three times oversampling means that
only one out of three samples screened will be unique mutants and thus the
screening efficiency is already quite low. Since no one knows which mutant
combinations are desirable, it might be better to just avoid the oversampling
and reduce the redundancy. After screening a smaller part of a larger library
using a higher screening efficiency one could rather make new libraries with
better starting points in order to benefit from the enhanced mutants. It is also
preferable to continue screening in several directions of the energy landscape
with the possibility of returning to previous amino acids in order to avoid
getting trapped in local energy minima (Gumulya and Reetz, 2011).

Applications
Engineered enzymes have many applications in molecular biology and dia-
gnostics. Enzymes are also used in industrial applications regarding deter-
gent, starch and fuel, food, baking, animal feed, beverage, textile, pulp and
paper, fats and oils, organic synthesis, leather, and personal care (Kirk et al.,
2002). In medicine enzymes conjugated with antibodies can activate prod-
rugs locally (Bagshawe, 1987). Directed evolution can also be used to engin-
eer alternative variants of drug targets in order to identify structural features
that are conserved among the active target mutants or to proactively screen
for drugs resistant to the possible target mutants. For numerous examples of
applications of protein engineering for therapeutics, see Methods in En-

17
zymology volumes 502−503 (Wittrup and Verdine, 2012a; Wittrup and
Verdine, 2012b).

Comparisons of methods
One paper suggested that substrate specificity is faster changed using site
directed mutagenesis than DNA shuffling (Parikh and Matsumura, 2005).
Another paper compared several approaches for change of substrate spe-
cificity, and reported that the strategies which had rationally chosen large
active site residues in order to accommodate smaller substrates were more
successful (Chen et al., 2012). In one case with optimization of enantiose-
lectivity, iterative saturation mutagenesis performed better than both epPCR,
some forms of saturation mutagenesis as well as DNA shuffling (Reetz et al.,
2010). Reduced sets of codons and two to three positions mutated per library
were recommended.
One meta-analysis suggested that mutational efforts should be focused to
the active site when optimizing enantioselectivity, substrate selectivity or al-
ternate catalytic activity, while there was no preference when optimizing
thermostability and catalytic activity (Morley and Kazlauskas, 2005). A diffi-
culty with such a comparison is of course the bias of what types of experi-
ments are mostly performed, what parts of the enzymes different strategies
usually target, that only successful attempts are reported, and the fact that
whole enzyme engineering processes seldom are repeated with the same or
different methods using the same enzyme, conditions and targeted proper-
ties. Both DNA shuffling and directed mutagenesis of active site residues
and other positions of interest were performed in papers I−IV. Thus, in this
thesis different approaches to enzyme engineering were compared by screen-
ing for the same properties using several mutant libraries. The target enzyme
of the mutational analysis will be the subject of the next chapter.

Glutathione transferase (GST)

Function
The main biological role of GSTs (EC 2.5.1.18) is detoxification and protec-
tion against oxidative stress. By conjugating the antioxidant glutathione
(GSH) to toxic electrophilic substrates the resulting molecules become less
reactive and more soluble and can be excreted from the cell and the organ-
ism. The GSTs are promiscuous in their substrate acceptance and together
they can deal with various xenobiotics or metabolic byproducts that other-
wise could be harmful and for example damage the DNA (Josephy and Man-
nervik, 2006).

18
 GSTs and other genes involved in cellular detoxification are upregulated
in long lived individuals in different species (McElwee et al., 2007). The
protective effects of GSTs can also be found for example as herbicide resist-
ance in plants (Dixon et al., 2002), as insecticide resistance in insects (Low
et al., 2007) or as drug resistance in tumors (Morgan et al., 1998).

Figure 2. Glutathione (γ-L-Glu-L-Cys-Gly).

Glutathione
The tripeptide glutathione (Figure 2) is synthesized inside every cell while
degradation of glutathione conjugates is initiated by γ-glutamyl transferase
on the cell surface (Ballatori et al., 2005). Glutathione might have evolved as
a stable storage of cysteine (Fahey and Sundquist 1991). The concentration
of reduced glutathione has been estimated to be about 1−2 μM in human
plasma and about 1 mM in whole blood (Andersson et al., 1999), while as-
sumed to be in the range 0.5−10 mM inside mammalian cells (Meister and
Anderson, 1983).

Phylogeny
There are four families of GSTs; cytosolic, mitochondrial, microsomal/mem-
brane-associated and fosfomycin/glyoxalase (Pearson 2005). In human there
are 17 different cytosolic GSTs, divided into seven classes (alpha, mu,
omega, pi, sigma, theta and zeta) based on sequence similarity. The alpha
class have five members in human, A1-1 to A5-5, and their genes are located
together on chromosome 6.

Polymorphism of human GST A2-2

Alpha class GSTs and especially human A2-2 will be the focus of the thesis.
Four single nucleotide polymorphisms (SNPs) have been described for hu-
man A2-2 leading to five possible genotypes. Based upon A2*A (P110,
S112, K196, E210) the other variants differ in positions E210A (A2*B),
S112T (A2*C), K196N/E210A (A2*D) and P110S (A2*E). A2*D was only
found in an expressed sequence tag database and not in population studies.

19
The others have allele frequencies reported for different populations to be
30−86 % of variant A2*A, 8−43 % of A2*B, 1−57 % of A2*C and 1−13 %
of A2*E (Tetlow et al., 2001; Ning et al., 2004; Tetlow and Board, 2004;
Coles and Kadlubar, 2005; Silva et al., 2009). Variant A2*C is the most com-
mon allele in Caucasians while A2*A is the most common in Chinese and
Australian populations. The allele frequency of A2*E is 1 % in Bantu Afric-
ans, 4 % in Portuguese Caucasians, 5 % in Australians, 12 % in Chinese and
13 % in Creole Africans.

Expression of human alpha class GSTs

Human GSTs A1-1 and A2-2 are expressed at high levels in liver, small in-
testine, testis, kidney, adrenal gland and pancreas and at lower levels in other
tissues (Coles and Kadlubar, 2005). A3-3 is expressed in steroidogenic tis-
sue, often at lower concentrations than A1-1 and A2-2 (Johansson and Man-
nervik, 2001; Larsson et al., 2011). A4-4 is expressed in a wide range of tis-
sues while no expression of A5-5 has been identified. The variations of GST
concentrations expressed as μg GST per mg cytosolic protein have been re-
ported for alpha class GSTs in several tissues, see Table 1. The ratios
between the minimum and maximum expression levels were at the most 50-
fold for A1-1 and 15-fold for A2-2.
Table 1. Variation of expression levels in various human tissue types. N = Number
of subjects.
Liver (N = 35, Mulder et al., 1999)
GST alpha class mean 25.1 ± 9.4 μg/mg

Liver (N = 52, Coles and Kadlubar, 2005)

GST A1-1 mean 6 μg/mg, 10th−90th percentiles 3−10 μg/mg
GST A2-2 mean 3 μg/mg, 10th−90th percentiles 1−7 μg/mg

Pancreas (N = 43, Coles et al., 2000)

GST A1-1 mean 1.4 μg/mg, range 0.2−5.1 μg/mg
GST A2-2 mean 4.3 μg/mg, range 0.7−10.8 μg/mg

Gastrointestinal tract, first part of the small intestine (N = 15, Coles et al., 2002)
GST A1-1 mean 7.7 ± 3.4 μg/mg, range 0.3−12.6 μg/mg
GST A2-2 mean 1.5 ± 0.6 μg/mg, range 0.3−2.7 μg/mg

20
Expression of different genotypes
The expression can also depend on the genotype of the GSTs. For human
A1-1, polymorphism in the 5' non-coding region gave rise to altered expres-
sion levels in liver, with median 8 μg/mg, range 1−13 μg/mg, for A1*A
versus median 4 μg/mg, range 2−7 μg/mg, for A1*B (Coles and Kadlubar,
2005). There were some inverse relationships between the expression of A1-
1 and A2-2 due to this polymorphism, with A2-2 expression having median 2
μg/mg (range 0−5 μg/mg, N = 20) versus median 7 μg/mg (range 5−9
μg/mg, N = 10) for the two different homozygous genotypes A1*A and
A1*B respectively. The differences in expression in relation to the genotypes
were more pronounced in A2-2 than in A1-1, where the individual variation
is larger.
For the hepatic expression levels there was a similar result for the A2-2
polymorphism. The homozygous genotype A2*C (median 2 μg/mg, range
0−5 μg/mg, N = 20) had 3-fold lower expression than combinations of the
genotypes A2*A/B/E (median 7 μg/mg, range 3−9 μg/mg, N = 11), (Ning et
al., 2004). In pancreas there was however no significant difference between
the median expression levels of the different A2-2 genotypes. The variability
within each genotype was larger than for the liver samples.

Structure
The cytosolic GSTs are dimeric proteins with one catalytic center in each
subunit, located near the interface between the subunits (Figure 3). The G-s-
ite binds and activates glutathione while the H-site accommodates the hydro-
phobic second substrate. The amino acids forming the H-site are located in
three specific regions in the primary structure while each G-site is composed
of amino acids from both subunits. Each subunit consists of an N-terminal
thioredoxin domain with secondary structure βαβαββα and an all-helical C-
terminal domain. Most alpha class genes encode GSTs with 222 amino acids
and each subunit has a molecular mass of around 25 kDa.

21
Figure 3. Structure of human GST A2-2 in complex with glutathione determined by
X-ray crystallography (PDB: 2WJU).

Mechanism
Glutathione is first activated by deprotonation of its thiol group. The
glutamyl α-carboxylate group of the glutathione receives the proton while an
active site water assists as a bridge (Dourado et al., 2008). The formed thi-
olate is stabilized by hydrogen bonding to a conserved tyrosine (Tyr9) and
thus ready for nucleophilic attack on the second substrate. The reactions
catalyzed by GSTs can be substitutions, additions or isomerizations, depend-
ing on the enzyme and the second substrate.

Applications
The presence of alpha class GSTs in blood plasma is low in healthy individu-
als (median 2.6 μg/l, range 0.2−20.4 μg/l, N = 350, Mulder et al., 1999). Al-
pha class GSTs can be used as a marker of liver disease due to raised plasma
levels during liver damage (Mulder et al., 1996). Expression of alpha and pi
class GSTs is localized to different parts of the kidney and their urinary ex -
cretion can be used as markers during various pathological conditions (Sund-
berg et al., 1995).
GSTs can also be used as fusion partners for coexpression and affinity
purification of proteins. Engineered GSTs with altered substrate selectivities
can for example be designed to detoxify herbicides or to specifically activate
prodrugs. One prodrug activated by GSTs, azathioprine, will be presented in
the next chapter.

22
Azathioprine

History
Hitchings had theorized that antagonists of DNA bases might be able to in-
hibit the growth of rapidly dividing cells and in the 50's Elion screened sev-
eral purine variants for inhibition of purine utilization (Elion, 1989). 6-mer-
captopurine (6-MP) turned out to be active against tumors in animal models
and acute leukemia in children. Azathioprine was designed as a prodrug that
should release 6-MP slowly and preferably by a tumor-specific enzyme. For
childhood leukemia the chemotherapeutic index was similar between aza-
thioprine and 6-MP while for another carcinoma in mice azathioprine was as
active but less toxic.
Schwartz proposed that since the immunoblastic lymphocytes formed dur-
ing an immune response were similar to leukemic lymphocytes the purine
analogs might inhibit an immune response. 6-MP given to rabbits simultan-
eously with antigen administration was indeed effective. Calne proved that
kidney transplantation in dogs was possible with immunosuppression by 6-
MP and later studies showed that azathioprine was superior for preventing
rejection. In the early 60's kidney transplantations in humans became suc-
cessful by administration of azathioprine and anti-inflammatory corticoster-
oids.

Figure 4. (Paper III: Figure 1a). The reaction of glutathione with Aza. The electro-
philic carbon of Aza is attacked by the sulfur of the glutathionylate, forming the
glutathione-imidazole conjugate and 6-MP.

Immunosuppression
The thiopurines azathioprine and 6-MP have been widely used as immun-
osuppressive and anti-inflammatory agents in organ transplantation and in
treatment of various autoimmune and chronic inflammatory diseases, such as
multiple sclerosis, rheumatoid arthritis and inflammatory bowel disease
(IBD), (Tiede et al., 2003). Regarding organ transplantation there are today

23
drugs that act faster than the thiopurines but also with more adverse effects
(Barba et al., 2012), while the results for organ function and cancer incid-
ence are similar in the longer perspective (Clayton et al., 2012).

Inflammatory bowel disease

Thiopurine treatment
IBD is a class of chronic inflammatory disorders of the gastrointestinal tract
including Crohn’s disease and ulcerative colitis. Azathioprine have proven
efficacy in induction of remission, maintaining remission and reducing ster-
oid dependence (Pearson et al., 1995; Chouchana et al., 2012). Stronger anti-
inflammatory corticosteroids are often used for induction of remission fol-
lowed by administration of immunosuppressive thiopurines as steroid spar-
ing agents or when the steroids no longer work or produce adverse effects
(Prefontaine et al., 2009; Lakatos and Kiss, 2011). According to a meta-ana-
lysis of relapse rates, it is significantly better to continue than to stop the
treatment, at least up to 18 months after induction of remission (French et
al., 2011). For patients continuing with azathioprine treatment after achiev-
ing remission, 62 % were after five years still in remission and 19 % had
only had a short relapse (Fraser et al., 2002).

Monoclonal antibodies
Monoclonal antibodies against tumor necrosis factor α (TNF-α) represent an-
other alternative for inducing or maintaining remission in IBD and are some-
times used in conjunction with azathioprine or as a third option in patients
when steroids and azathioprine have failed (Lakatos and Kiss, 2011). For ex-
ample infliximab (Knight et al., 1993) binds all forms of TNF-α, even re-
ceptor-bound, leading to caspase-3 activation and apoptosis (Van den
Brande, 2003).

Adverse effects

Dose adjustment
Dose is often adjusted during therapy in order to reach therapeutic effect or
to decrease adverse effects and can be adjusted before administration due to
thiopurine methyltransferase (TPMT) status (Stocco et al., 2007). Among
IBD-patients that had started azathioprine therapy, about 9 % had stopped
because of lack of effect and 11 % because surgery was needed (Barabino et
al., 2002; Fraser et al., 2002; Zabala-Fernández et al., 2011).

24
Different adverse effects
About 28 % of patients with IBD receiving azathioprine experienced adverse
effects and the severity led to withdrawal in about 20 % of the patients under
treatment (Connell et al., 1993; Barabino et al., 2002; Schwab et al., 2002;
Weersma et al., 2004; van Dieren et al., 2005; Stocco et al., 2007; Gardiner
et al., 2008; Xiong et al., 2010; Zabala-Fernández et al., 2011; Wroblova et
al., 2012). Reported adverse effects were bone marrow suppression (7 %),
gastrointestinal toxicity (6 %), hepatotoxicity (4 %), pancreatitis (4 %), flu-
like symptoms (3 %), rash (2 %) and other (2 %).

Cancer
The use of DNA base analogs were intended to inhibit the growth of fast di-
viding cells but the mutations introduced could potentially also lead to can-
cer. The risk of developing lymphoproliferative disorder was estimated in
IBD patients receiving thiopurines to be 0.1 % per year, which was 5 times
higher than for IBD patients not receiving thiopurine treatment (Beaugerie et
al., 2009). Since thiopurines absorb ultaviolet A radiation, leading to oxidat-
ive stress and DNA damage, the combination of sunlight and thiopurine
treatment might synergistically increase the risk of skin cancer (O'Donovan
et al., 2005; Maddox and Soltani, 2008).

Selectivity
About 60 % of azathioprine intolerant patients could tolerate 6-MP instead
(Lees et al., 2008). Several of those that did not tolerate the switch to 6-MP,
experienced different types of adverse effects compared to during the stage
of azathioprine intolerance. Selectivity of the toxicity depends on the levels
of individual metabolic enzymes, mitotic rate, drug transport and metabolite
pool sizes (Elion, 1989). More knowledge about the enzymes involved in the
metabolism can provide insight relevant to dosing adjustment, efficacy and
adverse effects during azathioprine therapy.

Mechanism of action

Azathioprine metabolism
The metabolism of azathioprine is complex, involving multiple pathways of
activation or inactivation, variation among patients as well as uncertainty of
which metabolites actually exert effects and adverse effects and by what
mechanisms. An overview is presented in Paper II: Figure 4.

Activation
The prodrug azathioprine is activated by conjugation with thiols, mainly
glutathione, and it was widely considered to be activated non-enzymatically

25
even though it was known that GSTs could be involved. Recently it was
shown that the activation in human was mainly performed enzymatically by
GSTs A2-2, A1-1 and M1-1 (Eklund et al., 2006). Upon conjugation with
glutathione, 6-MP is released (Figure 4) and its metabolites are most likely
responsible for the therapeutic effects.

Cytotoxicity
After the initial GST-catalyzed activation, 6-MP is metabolized in several
steps into 6-thioguanine nucleotides (6-TGNs). Incorporation of 6-TGNs
into DNA can lead to methylation, DNA strand breaks, erroneous base pair-
ing and can with an active mismatch-repair system result in cell death
(Swann et al., 1996; Karran, 2007).

Inactivation
There are two major inactivating enzymes involved (Roberts and Barclay,
2012). Xanthine oxidase (XO) catalyzes the formation of 6-thiouric acid
which is excreted. TPMT methylates 6-MP and 6-thioinosine monophos-
phate (TIMP) into 6-methyl-mercaptopurine (6-MMP) and 6-methyl-thioin-
osine monophosphate (MeTIMP) respectively. Higher activities of these in-
activating enzymes lead to lower concentrations of active 6-TGN. Aldehyde
oxidase (AO) also acts on azathioprine, 6-MP and other metabolites but the
functional significance of the products is poorly understood (Smith et al.,
2009).

Purine starvation
An additional effect of the thiopurines is the fact that MeTIMP is a potent in-
hibitor of phosphoribosylpyrophosphate amidotransferase, leading to block-
ing of the primary pathway of purine nucleotide biosynthesis (Tay et al.,
1969). It is possible that the effect of the purine starvation has been exagger -
ated by in vitro studies involving cell lines with mismatch-repair defects and
it is not clear to which extent the purine starvation contribute to the cellular
toxicity (Karran, 2007). On the other hand patients taking 6-thioguanine re-
quire higher levels of 6-TGN in order to reach therapeutic efficiency com-
pared to azathioprine and 6-MP, indicating either that the distribution and ac-
tivation profiles are different or that there are other effects additional to that
of 6-TGN (Cara et al., 2004).

Other 6-MP metabolites

The metabolite TIMP can be phosphorylated twice leading to 6-thioinosine
triphosphate (TITP). Inosine triphosphate pyrophosphatase (ITPA) then con-
verts it back to TIMP. In the case of ITPA deficiency, accumulation of TITP
could potentially lead to toxicity, perhaps via incorporation into DNA
(Chouchana et al., 2012). The methylated 6-MMP as well as methylated

26
mercaptopurine ribonucleotides, 6-MMPR, might also contribute to the tox-
icity of the thiopurines.

Changes in metabolite levels

When non-responders with high 6-MMP levels were given reduced aza-
thioprine doses and coadministration of the XO inhibitor allopurinol, the
mean levels of 6-MMP decreased from 10600 to 2000 pmol/8·108 red blood
cells while the mean levels of 6-TGN increased from 190 to 400 pmol/8·108
red blood cells (Sparrow et al., 2007). For patients given a 50 % dose in-
crease, the mean 6-TGN levels increased almost proportionally, 150 to 260
pmol/8·108 red blood cells, while the mean 6-MMP levels increased from
280 to 2260 pmol/8·108 red blood cells (Kennedy et al., 2013). When using
split dosing for a group of mostly 6-MP users, half the dose was admin -
istered twice a day. The mean levels of 6-MMP decreased with 55 % from
11800 to 5300 pmol/8·108 red blood cells while the mean levels of 6-TGN
did not change significantly, only 239 to 216 pmol/8·108 red blood cells
(Shih et al., 2012). The changes in metabolite levels were accompanied by a
reduction in adverse effects. The notion that the dose or the rate of accumu-
lation of early metabolites can influence the distribution of the metabolites
later along the pathways suggests that differences in activation in the first
step of azathioprine metabolism by GSTs could influence metabolite concen-
trations in later steps.

Metabolite concentration thresholds

A 6-TGN concentration threshold of 235 pmol/8·108 red blood cells has been
suggested, with higher proportion of response for patients with higher 6-
TGN concentration (Dubinsky et al., 2000). It was also reported that the
mean level of 6-MMP in patients with hepatotoxicity was elevated and that
5700 pmol/8·108 red blood cells could be a suitable threshold for 6-MMP re-
lated liver damage. However, others have failed to find a difference in 6-
TGN concentrations between subjects with active IBD and subjects in remis-
sion (Lowry, 2001). An analysis of different possible thresholds noticed no
correlations between levels of 6-TGN, 6-MMP or the ratio 6-MMP/6-TGN
on the one hand and efficacy of treatment or adverse effects on the other
(González-Lama et al., 2011). In a large study, TPMT activity correlated
with 6-MMP levels and the 6-MMP/6-TGN ratio but not with 6-TGN levels
(van Egmond et al., 2012). However, the distributions of TPMT activities for
the group with high or low 6-MMP/6-TGN ratio were almost overlapping
with a small but significant difference in mean. Even though there was a
small difference in mean there could be no clinically relevant threshold level
that could separate the metabolite level groups based on TPMT activity.
Most of the variations in these metabolite concentrations are thus not ex-
plained by the TPMT status.

27
Signaling pathway
Upon CD28 costimulation of T lymphocytes, the small GTPase Rac1 medi-
ates anti-apoptotic signals by activating bcl-x L. 6-thio-GTP binds to Rac1 but
blocks the corresponding Vav guanosine exchange activity leading to accu-
mulation of inactive 6-thio-GDP-Rac1 complexes (Poppe et al., 2006). The
molecular target of azathioprine and 6-TGN in immunosuppression is thus
blocking of the Vav guanosine exchange activity on Rac1, thereby inhibiting
antigen presentation and antigen-specific immune responses, as well as indu-
cing apoptosis via the mithochondrial pathway and caspase-9 activation
(Tiede et al., 2003).

Glutathione
Aside from the 6-MP metabolites it is possible that the glutathione conjugate
of the reaction with azathioprine exerts effects during therapy. When the 6-
MP component of azathioprine was replaced by different non-toxic thiols,
several of the prodrugs were more potent than azathioprine in the human
mixed lymphocyte reaction, which tests inhibitory potency (Crawford et al.,
1996). Two of the variants performed better than azathioprine in prolonging
graft survival in mice, without showing toxicity like azathioprine. Immun-
osuppressive effects were later confirmed in rabbits by determining antibody
titer response (Al-Safi et al., 2003).
Glutathione depletion is also a possible cause of toxicity during therapy
with azathioprine or the above mentioned prodrugs. Cell studies often show
changes in viability after a few days of incubation with azathioprine while
therapeutic results usually appear on a larger time scale (Chouchana et al.,
2012). It is possible that the glutathione depletion can be the cause of some
of these effects, especially in the in vitro experiments (DeLeve et al., 1996;
Lee and Farrell, 2001).

Polymorphism in the metabolic pathways

GST polymorphism
The specific activity towards azathioprine was estimated in liver samples
from eight donors to be 4.3 ± 2.1 min-1 mg-1, with a range of 1.6−7.6 nmol
min-1 mg-1 (von Bahr, 1980). The urinary excretion of thiouric acid reached a
maximum within the first 4 hours after oral administration of azathioprine (1
mg/kg body mass) in three individuals (Chalmers et al., 1967). The time to
maximum plasma concentrations of azathioprine and 6-MP was determined
in eight subjects receiving 100 mg azathioprine orally to be 75 ± 77 min and
114 ± 83 min respectively, with a range of 15−240 min for both azathioprine
and 6-MP (Odlind et al., 1986). The time to azathioprine and 6-MP maxim-

28
um coincided for some subjects and were separated up to 4 hours in other
subjects.
With such rate differences in the first step of the activation of aza-
thioprine, it is reasonable to believe that the activity and expression of the
relevant GSTs might affect the distributions and toxicities of the prodrug
metabolites. When examining the GST M1-null genotype in relation to ad-
verse effects during azathioprine treatment there was a significant under rep-
resentation of homozygous M1-null individuals among patients suffering
from adverse effects (Stocco et al., 2007). Since GST A2-2 has even higher
specific activity towards azathioprine than M1-1, the polymorphism of A2-2
was further investigated in Paper II.

TPMT polymorphism
Polymorphism in the gene for TPMT results in 89 % of subjects having high
TPMT activity, 11 % being heterozygous with intermediate activity and 1
out of 300 being TPMT deficient (Weinshilboum and Sladek, 1980). Several
variant genes have been described leading to absence of TPMT activity
(Yates et al., 1997). Determination of TPMT genotype was one of the first
pharmacogenetic tests to be incorporated into routine clinical practice, in or-
der to avoid potentially fatal myelotoxicity in double null mutant patients
(Gearry and Barclay, 2005). They generally receive a 10-fold dose reduction
or avoid these thiopurines completely (Roberts and Barclay, 2012). Of sever-
al examined TPMT variants with non-synonymous SNPs, only one had lost
its enzymatic activity, one had no expression at all and the rest had about the
same kinetical properties but lower expression levels than the wild-type
(Wang and Weinshilboum, 2006). The difference in expression levels was
probably caused by faster degradation involving ubiquitination and protea-
some degradation or misfolding and aggregation.
A meta-analysis showed that bone marrow toxicity was 6 times more com-
mon in patients with at least one TPMT mutation compared to patients ho-
mozygous for functional TPMT (Dong et al., 2010). Still only 30 % of the
cases of bone marrow toxicity could be explained by the TPMT mutation
and only 21 % of patients with the TPMT mutation had bone marrow tox-
icity compared to 5 % of the control population, indicating that other factors
than TMPT status are influencing the outcome.
S-adenosyl-L-methionine is involved in the reaction and stability of
TPMT and polymorphism in enzymes affecting S-adenosyl-L-methionine
concentrations might decrease the TPMT activity of subjects with wild-type
TPMT genotype (Arenas et al., 2005; Roberts and Barclay, 2012). On the
other hand, polymorphism in inosine monophosphate dehydrogenase (IMP-
DH) can in individuals with wild-type TPMT give metabolism preferentially
into 6-MMPR, which might lead to toxicity (Roberts and Barclay, 2012).

29
ITPA polymorphism
One polymorphic variant, C94A, results in loss of ITPA activity. Some stud-
ies have indicated a correlation between the occurrence of this mutation and
adverse effects or lack of response (Marinaki et al., 2004; von Ahsen et al.,
2005), while others have not been able to confirm these findings (Gearry et
al., 2004; Allorge et al., 2005; Breen et al., 2005; van Dieren et al., 2005).
When examining ITPA activity instead of genotype, it seems that lower
activity could be correlated with adverse effects (Xiong et al., 2010; Ship-
kova et al., 2011). Few homozygous C94A individuals are included in the
mentioned studies and they are often suffering from adverse effects (Mar-
inaki et al., 2004; Xiong et al., 2010; Shipkova et al., 2011) suggesting that
homozygocity with the deficient allele is a risk factor while heterozygous or
wild-type individuals have adverse effects not directly related to the IPTA
status. The adverse effects of azathioprine therapy are a multifactorial issue
with more than just differences in TPMT and ITPA contributing (Zabal-
a-Fernández et al., 2011).

Polymorphism in transporters
6-MP metabolites can be transported by multidrug resistance protein 4
(MRP4) and multidrug resistance protein 5 (Wielinga et al., 2002). One
known MRP4 mutant exhibits decreased cell surface expression leading to
enhanced sensitivity to 6-MP (Roberts and Barclay, 2012). Its allele fre-
quency is 15−22 % in Japanese, 8 % in Han Chinese and less than 1 % in
Caucasians and Africans. Concentrations of 6-TGN were higher and adverse
effects in the form of leucopenia tended to be more common in mutant carri-
ers (Ban et al., 2010).

XO and AO polymorphism
One case report indicated that high XO activity could prevent response to
azathioprine therapy, including no detectable levels of 6-TGN or 6-MMP,
while administration of allopurinol and 6-MP instead could result in high
metabolite levels and toxicity (Wong et al., 2007). Different genetic variants
of XO have been described and deficiency in XO activity is very rare (Kudo
et al., 2008). One SNP of AO with 11−16 % allele frequency have been re-
ported to be involved in improved response to azathioprine therapy (Smith et
al., 2009). They also reported that polymorphisms in XO and the enzyme ad-
apting the molybdenum cofactor for AO and XO might contribute to fewer
adverse effects.

Rac1 polymorphism
Polymorphism in the promotor region of Rac1 can influence the expression
of Rac1 but there was no significant correlation between occurrence of poly-
morphism and therapy response or leucopenia (Bourgine et al., 2011).

30
Polymorphism summary
Several factors have been examined that could influence metabolite levels,
therapeutic response and adverse effects during azathioprine treatment, espe-
cially in the case of enzyme deficiency and homozygosity of mutant alleles.
Still only a minority of adverse effects can be explained or predicted using
existing knowledge from genotyping and metabolite monitoring and some of
the indications need to be replicated and confirmed by other studies.

Antibody directed enzyme-prodrug therapy (ADEPT)

The usage of prodrugs is expanded by the possibility of making fusion pro-
teins of the activating enzyme and an antibody against a suitable target (Bag-
shawe, 1987). The ligand does not necessarily have to be an antibody
(Grabulovski et al., 2007). One potential benefit aside from the targeted ac-
tivation and the improved distribution profile is that a smaller drug might
penetrate the surrounding tissue or tumor better than the antibody (Senter
and Springer, 2001).
Azathioprine is a prodrug that has been in clinical use for cancer and cur-
rently is for IBD. Localized activation by targeted GSTs with improved cata-
lytic efficiency towards azathioprine might improve its therapeutic index and
provide efficacy at lower doses or decrease adverse effects. Suitable targets
that today are targeted by antibodies could be for example TNF-α during
IBD (Lakatos and Kiss, 2011) or the human epidermal growth factor recept-
or 2 (HER2) which is over-expressed in certain tumors (Orlova et al., 2006;
Whittington et al., 2008; Zielinski et al., 2011). Immunogenicity is often a
problem with protein drugs (Holliger and Hudson, 2005; Hosse et al., 2006;
Afshar et al., 2009) but combination therapy of azathioprine together with
infliximab has resulted in a decrease in antibody formation against inflixim-
ab (Baert et al., 2003), suggesting that azathioprine could be well suited for
ADEPT.

Aims of the thesis

Structure-function relationships of human GST A2-2

Before this project started in became clear that the bioactivation of the prod-
rug azathioprine was mainly catalyzed by human GST A2-2, A1-1 and M1-1,
and that the uncatalyzed reaction of azathioprine with glutathione was estim-
ated to contribute with less than 1 % of the enzymatically catalyzed biotrans-
formation (Eklund et al., 2006). GST A2-2 had the highest catalytic effi-

31
ciency of the these three enzymes and one aim of the thesis was to under-
stand why by gathering structure-function relationships from characteriza-
tion of different GST mutant libraries (Papers I, III).

Polymorphism of human GST A2-2

There is polymorphism in the genes encoding these enzymes and there is
particularly one GST M1-null genotype which does not produce any en-
zyme. Its presence seems to be negatively correlated with adverse effects in
patients receiving azathioprine (Stocco et al., 2007). Since GST A2-2 has an
even higher azathioprine activity it was of interest to examine the effects of
polymorphism in this enzyme (Paper II).

Characterization using alternative substrates

Previously, an azathioprine analogue was developed for colorimetric screen-
ing of azathioprine activity (Kurtovic et al., 2008) and throughout this study
the activities of the mutated GSTs with substrates other than azathioprine
were determined. By such analyses further information about structure-func-
tion relationships could be gained (Papers II, III).

Redesign of human GST A2-2 for enchanced azathioprine

activity
The high GST activity with other substrates also led to the question whether
GST mutants could obtain a similarly high activity with azathioprine and this
was approached by further mutational strategies (Paper IV). The applications
of such an enzyme, that could catalyze the bioactivation of azathioprine
faster than the endogenous GSTs, would be as a selectable marker in tempor-
al gene therapy or in ADEPT, for example against the HER2 receptor on tu-
mor cells. The use of different mutational approaches allowed for a compar-
ison of several techniques for enzyme engineering (Papers I−IV).

32
Present investigation

Azathioprine activity of shuffled alpha class GSTs

(Paper I)

Introduction
As previously mentioned GSTs A2-2, A1-1 and M1-1 are the human en-
zymes contributing the most to the azathioprine bioactivation. In order to
learn more about the structure-function relationships regarding azathioprine
activation by GSTs, mutant enzymes were produced by DNA shuffling. In
this case family shuffling was performed using human A2-2 and four other
alpha class GSTs from different species. In this project one generation of
mutants produced by shuffling was examined thoroughly. By this method
segments in A2-2 that are of importance for the particular activity would be
present in the mutants with high activity, while other parts of these mutants
would show more variation.

Mutant library design

There are five reported polymorphic variants of human A2-2 (Coles and
Kadlubar, 2005). Variant A2*C is the most common genotype in Caucasian
populations and it was used throughout paper I. The other alpha class en-
zymes included were human A3-3, bovine A1-1, rat A2-2 and rat A3-3 and
expression clones of them were available in the laboratory. Their specific
activities with azathioprine were very low compared to the specific activity
of human A2-2. To introduce additional variability a previously created
mutant library of human A1-1 was included in the shuffling. This library had
ten potential H-site residues mutated with the degenerate codon NNN to en-
code all 20 amino acids (Hansson et al., 1997). The shuffled chimeras also
had point mutations based on the infidelity of the Taq polymerase. Point
mutations in the active site have probably a higher chance of drastically al-
tering the function of the enzyme compared to mutations further away.

33
Shuffling of homologous sequences introduces mutations in block-wise seg-
ments and affects both the active site and more distant parts of the enzymes.
The construction of the shuffled library (Paper I: Figure 2) started by
cleaving the DNA with DNAse I to segments of 50−100 bp. During cycles of
primerless PCR, pieces of homologous single stranded DNA were annealed
followed by elongation and denaturation. In the end full-length sequences
were amplified using end primers. The resulting chimeric sequences had mo-
saic patterns of segments with different origins. However, these final seg-
ments could be shorter than 50 bp if two products were annealed that in
earlier cycles ended up with crossovers located 1−49 bp away from each oth-
er.

Screening of bacterial lysates

The DNA vectors were transformed into E. coli by electroporation. Bacterial
colonies containing the desired vector encoding ampicillin resistance were
grown on agar plates. The estimated size of the library was 1.3 x 10 6. A test
round of 456 colonies was grown in 2 ml LB medium overnight at 37 ˚C fol-
lowed by dilution 1:100 into 10 ml 2TY medium. After 2 h protein expres-
sion was induced by addition of IPTG. Cell lysates were prepared by centri-
fugation, pellet resuspension, lysozyme treatment for 1 h and repeated
freeze-thaw cycles to break the cell walls. No hits of interest with elevated
azathioprine activity were found in the test set and higher throughput was
desired. A second round of screening of 1114 colonies was performed in 96-
well microtiter plates using 300 μl culture medium. Expression without
IPTG induction was in this case enough for proper detection of the positive
control, human A2-2.
The lysates were either stored in -80 °C or used directly for azathioprine
measurements. The GST-catalyzed reaction of 0.2 mM azathioprine with 1
mM glutathione was monitored using a microtiter plate spectrophotometer.
The absorbance spectrum of the product formation has a maximum at 320
nm and the reaction was followed by an increase in absorbance at this
wavelength (Eklund et al., 2006). The sodium phosphate buffer had pH 7.0
in order to get a lower uncatalyzed reaction rate.
The lysate activity of human A2-2 was 27.0 ± 5.5 (10−3 ΔA320/min) using
29 replicates as positive controls on different plates, with a range of 14.6 to
37.8 (10−3 ΔA320/min). Lysate activity depends on the catalytic activity but
also on the expression level, solubility and stability of the enzyme. Mutant
lysate activities in relation to human A2-2 were sometimes in agreement
with later measurements on the purified enzymes but could also deviate up
to three-fold for active enzymes. Mutants with low activity can also deviate
because of the detection level of the reaction. The uncatalyzed reaction rate
in absence of lysate was determined to be 7.4 ± 0.4 (10−3 ΔA320/min) while
the corresponding value with inactive lysate was 7.0 ± 1.0 (10−3 ΔA320/min).

34
Sequence analysis
In total 1570 bacterial lysates were screened; the majority of them with no
measurable activity with azathioprine (Paper I: Figure 5). A total of 62
colonies were cultivated and their DNA sequenced, some of them because of
their activity with azathioprine in lysate. 13 sequences were identical to hu-
man A2-2. Since it was the most active parental enzyme all of the colonies
expressing human A2-2 were most likely found as hits in the screening. By
assuming that wild type occurrence in the final library was roughly the same
for the different wild type templates, the total occurrence of wild type se-
quences in the library was estimated to be 5 · 13/1570 ≈ 4 %. A frame shift
in the N-terminal sequence of the bovine A1-1 template sequence led to 1/6
of the library not encoding full length GSTs.
Aside from the wild type sequences found the number of crossover points
at the DNA level was 1−13, with a mean of 5.9 ± 2.6. The chimeric enzymes
had sequences originating from on average 4.1 different templates (Paper I:
Table 1). The library additionally had about 0.6 non-silent point mutations
per mutant. A high number of point mutations could lead to deleterious
mutations hiding the effect of beneficial recombinations as well as making it
more difficult to draw conclusions about structure-function relationships.

The H-site library

The human A1-1 library with point mutations in the H-site also increased the
structural diversity of the library. However, no such segment was found
among the chimeric mutants active with azathioprine in the lysate screening.
Perhaps the simultaneous mutation of four positions in the H-site was too
much to tolerate in regions B and C while on the other hand only two posi-
tions were mutated in region A (Paper I: Figure 6 and Paper I: Figure 8).
Screening of 384 colonies of that library itself showed no azathioprine activ-
ity in lysates. That was however not surprising considering that a library
with ten positions with NNN codons would have a premature stop codon in
38 % of the mutants and 7−10 non-wild-type H-site amino acids in 62 % of
the mutants, making the library more suitable for selection methods like
phage display (Hansson et al., 1997).

Crossover locations
A multiple alignment of 30 alpha class sequences from different species
showed regions of more or less sequence conservation (Paper I: Figure 9).
Especially the three regions with H-site residues were among the more vari-
able regions, as well as two regions on the outside of the dimer; one region
around amino acids 35−50, containing helix 2, and one region around amino
acids 170−190, containing helix 7. The crossover locations in the chimeric

35
mutants were mostly located in the conserved regions (Paper I: Figure 3).
The conserved regions were also visualized in an alignment where segments
of amino acids were color coded depending on their corresponding wild type
source (Paper I: Figure 4).

Expression and purification

The wild type enzymes and a selection of the sequenced mutants were ex-
pressed in E. coli in 0.5−3 liter scale. The preparations of the bacterial cell
lysates and the enzyme purifications were similar in papers I−IV yielding
several milligrams of purified protein. After over night cultivation the medi-
um was diluted 100-fold and at an optical density of 0.3−0.6, at 600 nm, the
protein production was induced by IPTG. After 12−16 h the cells were har-
vested by centrifugation, dissolved in a sodium phosphate buffer with pro-
tease inhibitors, and lysed by lysozyme treatment followed by sonication or
cell disruption. After centrifugation the enzymes were purified from the lys-
ate using immobilized metal ion affinity chromatography (Porath et al.,
1975).
The N-terminal sequence of the GSTs coded for methionine followed by
six histidines inserted by cloning. The affinity of the hexahistidine tag to Ni2+
allowed for selective absorption of the enzymes. The bound enzymes were
washed and eluted using different concentrations of imidazole. The buffer
was exchanged by dialysis or size exclusion chromatography. The 100 mM
phosphate buffer used for some enzymes could lead to precipitation and was
later replaced by 25 mM HEPES with 150 mM NaCl. Protein purity was
verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Pro-
tein concentration was determined using Bradford assay or absorbance at
280 nm using a previously determined extinction coefficient (Widersten et
al., 1994). Purified enzymes were stored at -80 ˚C.

Kinetic characterization
Reduced glutathione was kept at a constant level of 1 mM in the activity
measurements of the purified enzymes. The concentration of azathioprine
was 0.2 mM for determination of specific activities and varying for determ-
ination of kinetic parameters. The temperature was kept at 30˚C and the so -
dium phosphate buffer had pH 7.4. The specific activities of the wild type
enzymes and the mutants are presented in Paper I: Figure 6. The catalytic ef -
ficiency, kcat/KM, was determined for the three most active enzymes, human
A2-2, human A1-1 and mutant 1362, to be 1.35 s −1mM−1, 0.70 s−1mM−1, and
0.61 s−1mM−1, respectively. Throughout papers I−IV, kcat and kcat/KM were cal-
culated per enzyme subunit.

36
Structure-activity relationships

Some H-site residues conserved among the active chimeric mutants

The analyzed amino acid sequences were divided into 23 segments repres-
enting primary structure regions recombined during the shuffling process.
Segments originating from all wild type GSTs were present among the four
most active chimeric GSTs (1362, 1347, 608 and 1108, see Paper I: Figure 4
and Paper I: Figure 6).
Segments 1−2 and 20−22 were on the other hand conserved among the
four most active chimeras and the most active wild type, human A2-2. Hu-
man A1-1 also had similar azathioprine activity and showed some additional
variation in segments 2 (S10F/I12A) and 22 (S216A). Segments 1−2 were
located in the N-terminus. Segment 2 contained two H-site residues
(S10/I12) and were surrounded by conserved G- and H-site residues
(Y9/G14/R15). Segments 20−22 comprised part of the C-terminus and con-
tained one H-site residue each (M208/L213/S216). Four of these five con-
served segments contained H-site residues and were thus considered the
primary determinants of azathioprine activity in alpha class GSTs.

Other segments of interest

One basic assumption in this study was that, among the most active en-
zymes, variable regions were more tolerant to mutations while conserved re-
gions were more important for the function of interest. Depending on the re-
combination resolution and the number of chimeras sequenced, segments of
importance might in the analysis be linked to other segments that do not ne-
cessarily contribute to the function.
The finding of H-site residues in almost all conserved segments among the
most active enzymes demonstrated the power of family shuffling in identify-
ing important functional elements. However, segment 1, lacking H-site
residues, might still also be of importance even though it might just be
linked to the H-site residues of segment 2 due to the recombination process.
Segment 8, having 5 H-site residues, did not seem to be correlated with aza-
thioprine activity in this alpha class context.

Structural interpretation
Segments 2, 8 and 20−23 form the three parts of the H-site cavity, in paper I
called regions A, B and C (Paper I: Figure 6 and Paper I: Figure 8). Seg -
ments 21−22 are located in the C-terminal helix 9 with segment 20 in the
hinge region and segment 23 at the end of the helix. Human A1-1 crystal
structures have shown that the C-terminal helix is disordered without a lig-
and bound in the active site (Grahn et al., 2006). The flexible C-terminus
forms a lid closing on the H-site, coordinating the binding of both gluta -
thione and the second substrate (Nilsson et al., 2002).

37
 Several of the chimeras with some activity in the screening but low specif-
ic activity with azathioprine when measured with purified enzymes had seg-
ments from human A2-2 in the N-terminal region but not in the C-terminal
region. Mutant 227 had sequences originating from human A2-2 in the C-ter-
minal region but did not have any detectable activity in lysate. Its sequence
was a mix of the active mutants 608 and 1108 aside from segment 1 from rat
A2-2 (A2S/E3G/K6V), segment 2 from human A1-1 as well as mutation
N80D from rat A2-2. This suggests that the residues in segment 1 could in-
fluence the activity, perhaps via the H-site residues in segment 2. However,
the distances from the residues in segment 1 to the active site do not offer
any straightforward structural explanations.

One example of an active GST lacking amino acid 222

With more chimeric enzymes included in the sequence analysis, the resolu-
tion of a crossover pattern becomes more detailed, possibly facilitating a
more detailed understanding of the sequence function relationships. One ex-
ample was that the mutant 1347 differs from human A2-2 only in the last
two amino acids (S221 plus stop codon compared to R221 plus F222). Posi-
tion 222 is in the H-site in segment 23. The mutant still had 42 % of the hu-
man A2-2 specific activity indicating that those two amino acids were not es-
sential for the activity with azathioprine. The division into smaller segments
on the other hand also makes it harder to draw conclusions about the contri-
bution of individual segments to the function, compared to methods using
site-directed recombination.

Five chimeras had specific azathioprine activity > 20 % of human A2-2

Later the last three mutants that showed some azathioprine activity in the
screening were also sequenced and purified. One was very similar in activity
and sequence to A1108. Another one was inactive with azathioprine having
segments from rat A3-3 in the C-terminus. The third was also inactive but
the sequence was basically a mix of the segments present in the active
mutants 608 and 1362, aside from one mutation E3G in segment 1 that was
present in all wild type sequences except human A1-1 and human A2-2. This
could potentially indicate that segment 1 was of more importance than just
as being linked to segment 2 containing the H-site residues in positions 10
and 12. However, other chimeric mutants can also have sequences consisting
of combinations of segments from active mutants without having activity
themselves. It is rather an expected effect of the recombination of segments
to get both increased and decreased activities compared to the starting mater-
ial. With these mutants examined it was concluded that out of 1570 tested
colonies, only five expressed a chimeric GST with more than 20 % of the
specific activity of human A2-2.
Compared to the total number of chimeras with low activity with aza-
thioprine, only a small fraction were sequenced. Thus comparing the active

38
GSTs with the few inactive GSTs that were sequenced is not as relevant as
comparing with the possible sequence space that could be sampled in the
shuffling process. Approximately (1/6) · (1/6) · 1570 - 13 = 31 mutants
would have been expected to have segments originating from human A2-2 in
the N-terminus and C-terminus without being identical to human A2-2 in the
rest of the sequence. Since only five sequences with relatively high activity
were found, inclusion of the five important segments was probably not itself
enough for activity in the sense that mutations elsewhere or specific seg-
ments or combinations of segments in other positions could abolish the
activity with azathioprine.

Thoughts on exploiting the data for directed recombination

Multiple generations of DNA shuffling is basically a genetic algorithm, lead-
ing to linear combinations of the segments that are part of the proteins with
maximum fitness in the previous generation. Semi-rational approaches might
perform one or several exploration steps, gathering information about the ef-
fect on fitness of the mutations, followed by one or several exploitation
steps. In the exploitation steps the beneficial mutations are more strongly en-
riched and the deleterious mutations more often excluded compared to
stochastic methods. If a similar approach would have been used in this pro-
ject the shuffled segments in each template would correspond to the para-
meters while the specific activity would be the output and important seg-
ments would have been identified by multivariate statistics (Fox, 2005).
However, there are not nearly enough sequences determined to paramet-
rize such a system using linear combinations of segments. With simplifica-
tion from 23 segments to N segments, 6N sequences would still be needed.
Besides, the activities in lysates and the specific activities of the GSTs with
low activity were not accurate enough for such a detailed analysis. While the
methods might work with single point mutations, there could likely be epi-
static effects present among longer segments. When taking into account the
combination effects between different segments the number of parameters
increases dramatically. The resulting structure-function relationships in this
study indicated that taking such combination effects into account would have
been necessary.
In another recombination study using three template sequences, 221
mutants were screened (Engler et al., 2009). Eight mutants had higher fitness
than 50 % of the best template and the best had a 4-fold improvement.
Among these eight mutants, two recombination segments out of nine were
conserved with seven or eight mutants having the same origin in those seg-
ments. Screening of 24 mutants in the second generation produced one
mutant with an additional 2-fold improvement. This mutant was still contain-
ing the conserved segments, also confirming that enhanced proteins can be

39
generated using multiple generations of recombinations that keep important
segments while varying other segments.

Comments on the alpha class GSTs included in the library

Three human alpha class GSTs, A1-1, A2-2 and A3-3, were used for the con-
struction of the library. In order to provide more variation to the library three
alpha class GSTs from other species were also incorporated. By including
enzymes with a lot of sequence variation and with both high and low activity
towards azathioprine, regions of importance for the function could be identi-
fied. Human A2-2 shares 95 % amino acid sequence identity with human
A1-1, 89 % with human A3-3 and human A5-5, 81 % with bovine A1-1, 73
% with rat A2-2 and rat A3-3, and 54 % with human A4-4. These two rat
GSTs are however not that similar, sharing only 68 % identity with each oth-
er.

Comments on the alpha class GSTs excluded from the library

Human A5-5
The gene encoding human A5-5 is probably not expressed in vivo. Due to the
high similarity to the H-site residues of human A2-2 and A1-1, one would
assume that if human A5-5 was expressed in vitro and assayed with aza-
thioprine it would show similar activity. Only P110L, F111I and I12A are
different from human A2-2, and P110S at least is tolerated in human A2-2
(Paper II), F111I was introduced into a mutant of hA2-2 with retention of
azathioprine activity, and I12A is present in human A1-1, which also has
activity with azathioprine. On the other hand, the G-site mutation R15S in
human A5-5 might be detrimental to the catalytic activity in general, consid-
ering the conservation of a basic residue in this position in alpha class se-
quences (Björnestedt et al., 1995).

Human A4-4
Human A4-4 had too low sequence identity with the other GSTs to be in-
cluded in the shuffling process. The genes encoding the alpha class GSTs are
located on chromosome 6, accompanied by several gene fragments. Both the
genes and the fragments are the results of more or less successful gene du-
plications. The order of the genes on the chromosome represents A2-2, A1-1,
A5-5, A3-3 and at last A4-4. A closer look at the fragments and the alpha
class GSTs in different species drew attention to the fragment between hu-
man A3-3 and A4-4. Its sequence was similar to rat A4-4 as well as a corres -
ponding gene in Macaca mulatta. Rat A4-4 and this GST in Macaca mulatta
share 76 % amino acid identity. They both share only 56−60 % amino acid
identity with human A1-1, A2-2, A3-3, A4-4 and A5-5, indicating that this

40
fragment represented a branch of the alpha class GSTs that was lost in hu-
mans.
In fact, a cDNA search for human A4-4 as a homologue to rat A4-4 failed
because of the low sequence identity (Mannervik et al., 2000). However,
when human A4-4 later was identified and expressed, its substrate specificity
profile was still similar to that of rat A4-4 (Hubatsch et al., 1998). This func-
tional similarity could partly be due to eight of twelve H-site residues being
identical between human A4-4 and rat A4-4, compared to for example only
three identical between human A4-4 and human A1-1. One example of re-
design of the H-site was the study where human A1-1 was mutated into an
enzyme with hA4-4 like substrate selectivity by mutating one position in re-
gion A, three positions in region B and the whole region C (Nilsson et al.,
2000).

Another example using DNA shuffling

In paper I, analysis of structure-activity relationships revealed segments with
H-site residues as primary determinants of the activity with azathioprine. But
the mutations introduced by DNA shuffling targets the whole sequence of
the enzyme except for the conserved parts. Another study using DNA shuff-
ling and a growth selection system found 13 amino acids mutated in an as-
partate aminotransferase with modified specificity (Yano et al., 1998). Six of
them were together responsible for a 20000-fold increase in catalytic effi-
ciency with one of the substrates while the other seven mutations contributed
cooperatively to an additional 3-fold enhancement. However, only one of the
six functionally important residues were located close enough for direct in-
teraction with the substrate. This demonstrated the complementary effects of
shuffling to active site redesign.

Suggested screening modifications

One attempt was made at testing azathioprine in a negative selection system.
E. coli with plasmids encoding different GST were grown in media or on
plates with azathioprine. The survival was not correlated to the activity of
the GSTs but could depend on several other factors, including expression
level of the GSTs but also viability during recombinant expression or pos-
sibly previous treatment of the bacterial colonies.
The specific activities with the fluorogenic substrate monochlorobimane
were determined for the wild type enzymes and eight mutants. There was no
correlation between the specific activities with azathioprine and mono-
chlorobimane (R2 = 0.02). The fluorogenic substrate can be used for initial
selection of colonies expressing functional GSTs (Eklund et al., 2002) but
biased substrate specificities would obviously follow.

41
 For estimations of specific activities already in the lysates, the enzyme
concentrations could have been determined using fusion tags or antibody de-
tection but this was never performed in this paper. One such example used
green fluorescent protein cloned downstream of the protein of interest in or-
der to determine the concentration of the fusion protein as well as distinguish
mutants with frame shifts (Denis-Quanquin et al., 2007).

Conclusion
In this project a library of chimeric enzymes was constructed by DNA shuff-
ling of five alpha class GSTs and a point mutation library. Comparison of the
sequences and their corresponding enzymatic activities with azathioprine in-
dicated that segments 1−2 in the N-terminus and 20−22 in the C-terminus
were important for this function. These segments contained several H-site
residues forming part of the substrate binding site. Both the segments and the
H-site residues will be further examined in papers II, III and IV. One issue
that will be addressed is whether these important segments of human A2-2
should be conserved or targeted when attempting to improve the catalytic ef-
ficiency with azathioprine by mutagenesis. Aside from the differences
among alpha class GSTs there is also variation present among individuals re-
garding single enzymes. The human A2-2 variant named A2*C was used in
paper I. The next chapter will be about the polymorphism of human GST
A2-2.

Polymorphism in human GST A2-2 (Paper II)

Introduction
The metabolism of azathioprine involves several steps of activation and mul-
tiple inactivation routes (Paper II: Figure 4). Genetic variation in TPMT can
have drastic effects on the outcome of clinical treatment of patients with the
drug. For double null-mutants the dose administered must be radically
lowered to avoid acute bone marrow degeneration (Roberts and Barclay,
2012). TPMT status can however only explain a small part of the adverse ef-
fects (Dong et al., 2010) and the results of polymorphism in other steps have
not been thoroughly examined. The first step involves glutathione conjuga-
tion by GSTs, in human mainly by A2-2, A1-1 and M1-1 (Eklund et al.,
2006). Different rates of activation could be related to adverse effects as seen
in a study monitoring the presence of M1-1 null-mutants (Stocco et al.,
2007). However, no articles have been published investigating the effects of
differences in phenotype for A2-2 and A1-1, despite their high activity with
azathioprine and high expression in several tissue types. In this paper, the

42
polymorphic variants of A2-2 were characterized with respect to catalytic ef-
ficiency with azathioprine and alternative substrates as well as thermostabil -
ity.
Table 2. (Paper II: Table 1). Amino acid variations in allelic variants of human GST
A2-2. Position marked "-" is not located close to the H-site.
Residue 110 112 196 210
H-site region B B - C
(according to Paper I)
GST A2*A Pro Ser Lys Glu
GST A2*B Pro Ser Lys Ala
GST A2*C Pro Thr Lys Glu
GST A2*D Pro Ser Asn Ala
GST A2*E Ser Ser Lys Glu

Kinetic characterization
Five genotypes of human GST A2-2 have been found, see Table 2. All but
A2*D have been confirmed in population studies. In paper II the five de-
scribed A2-2 genotypes were cloned, expressed and characterized. The
steady-state kinetics parameters are presented in Paper II: Table 4. Assuming
in vivo azathioprine concentrations below 10 μM (Odlind et al., 1986) and
KM values over 1 mM indicated that the activation rate under physiological
conditions was proportional to kcat/KM. Variant A2*E turned out to catalyze
this reaction 3−4-fold faster than the other variants of A2-2 and about 6-fold
faster than A1-1 and M1-1.
Structurally A2*E differs from the other variants by the P110S mutation.
The proline in position 110 is otherwise conserved in all human alpha class
GSTs. Serine in position 110 is present in one other alpha class GST, in
Monodelphis domestica. Positions 107, 108, 110 and 111 are all potential H-
site residues, located near the end of helix 4. Prolines can disrupt secondary
structure elements and mutations in this position could lead to reorientation
of the adjacent H-site residues.

Figure 5. (Paper II: Figure 1). Structures of Aza, NPTI, CMNI and CDNB. Arrows
indicate the positions of nucleophilic substitution by GSH. All compounds but
CDNB give the same imidazolyl-GSH conjugate.

43
Alternative substrates
The enzymes in paper I were also characterized with alternative substrates
(Figure 5). NPTI (Kurtovic et al., 2008) is an azathioprine analogue in which
the leaving group is 4-nitrothiophenol instead of 6-MP, facilitating colori-
metric screening. CMNI has the same substituted imidazole ring as aza-
thioprine and NPTI but only a chloride ion as leaving group. CMNI has been
tested as a radiosensitizer (Watts et al., 1980) with the primary purpose to
deplete cellular glutathione before radiation therapy (Sauer et al., 1988).
CDNB has a size similar to CMNI and is the standard substrate for measure-
ment of GST activity.
The catalytic efficiency with NPTI was about 3-fold higher for A2*E than
for the other variants, while it was lower with CMNI and CDNB (Paper II:
Table 4). Azathioprine, NPTI and CMNI all form the same glutathione con-
jugate. Since kcat was different for the three substrates it indicated that the re-
action rates were not limited by the release of this conjugate, at least not in
the cases with lower kcat.
Principal component analysis of the normalized catalytic efficiencies with
the four substrates was performed (Paper II: Figure 2). Principal component
1 accounted for 88 % of the total variability in the data set and confirmed
that variant A2*E was functionally different from the other four variants,
mainly due to its higher catalytic efficiency with azathioprine and NPTI as
well as lower catalytic efficiency with CDNB and CMNI. Principal compon-
ent 2 explained only 12 % of the variation in the data set, separating variants
A2*A-D depending on their relative catalytic efficiency with CMNI versus
CDNB.

Combining activity and expression in different tissues

In a previous paper (Eklund et al., 2006) the rate of activation of aza-
thioprine was estimated for human liver cytoplasm. The enzymatic activation
dominated over the uncatalyzed reaction and the enzymes contributing the
most were GST A2-2, A1-1 and M1-1. A similar estimation was performed
using the GST isoenzyme concentrations from Rowe et al., 1997, combined
with the catalytic efficiencies of the allelic variants determined in paper II.
The sum of the apparent rate constants (min -1) was estimated to be 2.8−4.5
(liver), 2.4−3.6 (testis), 0.6−1.8 (pancreas), 0.6−1.1 (kidney), 0.4−0.7 (adren-
al glands), 0.2−0.3 (brain), 0.05−0.07 (lung), 0.04−0.07 (pituitary gland) and
0.03 (heart). The range of the rates in different tissues represented the vari -
ation among individuals, assuming the same concentration of GSTs but with
either A2*E (higher value) or another A2-2 variant (lower value). The GST
concentrations were determined as the mean of two samples from different
individuals.

44
 Due to the higher catalytic efficiency of A2*E there is a 1.5−2 fold differ -
ence in the sum of the apparent rate constants in most tissue types, except
pancreas with a 3-fold difference and heart with no A2-2 expression. The un-
catalyzed reaction contributes only about 1 % or less in the five tissue types
with the highest apparent rate constant. A M1-null homozygous mutant
would only influence the apparent rate constant by a 5−20 % decrease in
these tissue types. In the four other tissue types the apparent rate constant of
the GSTs plus the uncatalyzed reaction would be 1.5−3 fold lower in sub-
jects with the M1-null genotype.

Activity and variation of expression

However, from studying the expression of GSTs in specific tissues using a
larger sample of individuals it is clear that the inter-individual differences in
expression levels also are of a notable magnitude. As discussed in the intro-
duction the ratios between the minimum and maximum expression levels
was at the most 50-fold for A1-1 and 15-fold for A2-2. The sum of the ap -
parent rate constants could vary in that order of magnitude between individu-
als with very high and very low concentrations of azathioprine-active GSTs,
even if the GSTs were of the same genotype. One measurement with aza-
thioprine using human liver cytosol resulted in a specific activity of 4.3 ± 2.1
nmol/min/mg (N = 8, range 1.6−7.6 nmol/min/mg, von Bahr et al., 1980).

Bioavailability and activation

The time to maximum concentrations of azathioprine and 6-MP after oral ad-
ministration of azathioprine varies significantly among subjects (range
15−240 min, N = 8) and the difference between the maximum times also
varies up to 4 hours (Odlind et al., 1986). The different rates of activation
might be related to the distribution and toxicity of the metabolites. The es-
timated GST catalyzed activation of azathioprine in liver, pancreas and the
small intestine is potentially very high with large variation among individu-
als and might be one factor involved in effectiveness of treatment as well as
the commonness of adverse effects in these tissues.

Genotyping for activity and expression

Part of the difference in expression levels can be attributed to known poly-
morphisms, with 3-fold lower hepatic expression for A2*C (Ning et al.,
2004) and differences in expression levels for A1-1 and A2-2 depending on
an A1-1 polymorphism in the 5' non-coding region (Coles and Kadlubar,
2005). Genotyping of patients might thus aside from identifying the specific
activities of the GSTs with azathioprine also provide some hints about their

45
expression levels. Based on the allele frequencies mentioned in the introduc-
tion, the occurrence of A2*E homozygosity could be up to 1.7 % in some
populations and the A2*E heterozygosity up to 23 %. In the Portuguese
Caucasian population the occurrence of homozygous A2*E individuals with
higher specific activity towards azathioprine would be 0.6 %, while 32 %
would have A2*C homozygosity with lower hepatic expression.

Thermal inactivation
Thermostability of the five A2-2 variants was assessed with the addition of
physiological concentrations of glutathione at 2 mM. At a temperature of 37
°C the half-life of the thermal inactivation of variant A2*C was about two
weeks while the others were more stable. At 48 °C it was shown that A2*E
was more stable than the other four variants (Paper II: Figure 3). However,
the half-lives might be even shorter in vivo (García-Mata et al., 1998; Shen
et al., 1995) indicating that thermostability is not a major determinant of the
intracellular GST concentrations.

Conclusion
Five genotypes of human GST A2-2 were characterized and variant A2*E
turned out to have a different substrate profile than the other variants, with
3−4-fold higher catalytic efficiency with azathioprine. The in vitro results
can be considered in order to try to understand the consequences of clinical
azathioprine usage and to estimate the impact of different GST genotypes.
Considering the additional contribution of A1-1 and M1-1 to the bioactiva-
tion of azathioprine, the effect of having the A2*E genotype could result in
an up to 3-fold difference in apparent rate constant in some tissues. Com-
bined with differences in expression levels, the rate of activation varies sub-
stantially among individuals, possibly affecting the localization and toxicity
of azathioprine metabolites. Genotyping of GSTs involved in azathioprine
activation is recommended to further study the connections among GSTs,
azathioprine dose, metabolite levels, efficacy and adverse effects. Variant
A2*E showed that mutagenesis close to the H-site could increase the specific
activity with azathioprine. Paper I identified segments of human A2-2 of in-
terest for the specific activity with azathioprine and these segments will be in
focus in the next chapter.

46
Structural elements supporting high azathioprine
activity (Paper III)

Introduction
The prodrug azathioprine is activated mainly by human GSTs A2-2, A1-1
and M1-1, of which A2-2 has the highest specific activity of the human
GSTs. There is polymorphism affecting the activity as well as expression of
the GSTs and faster activation of the prodrug might lead to adverse effects in
treated patients. In Paper III structure-activity relationships were sought in
order to understand the higher catalytic efficiency of human A2-2 compared
to the other GSTs examined. By DNA shuffling of GST A2-2 together with
other alpha class GSTs in Paper I, the sequences were divided into 23 seg-
ments separated by recombination events (Paper I: Figure 4). Segments 1−2
in the C-terminus and 20−22 in the N-terminus were conserved among hu-
man A2-2 and the mutants with more than 20 % of the specific azathioprine
activity of A2-2.
The significance of these conserved segments was in Paper III investig-
ated by insertion of the C- and N-terminus from human A2-2 into the low-
active GSTs human A3-3 and rat A3-3. These short segments contained sev-
eral H-site residues and two of them, M208 and L213, are conserved among
human A2-2, the mutants with high activity and human A1-1, which displays
about 60 % of the specific activity of human A2-2. These two H-site residues
were targeted by saturation mutagenesis in order to examine their importance
for the activity of human A2-2 with azathioprine. Aside from azathioprine,
the enzymes were also characterized using three other substrates undergoing
similar chemical transformations.

Chimeric mutants

Cloning
In this paper as well as in paper I, human A2-2 refers to allelic variant C of
human GST A2-2. Two chimeric mutants were based on human A3-3 and rat
A3-3 with sequence from human A2-2 in the N- and C-terminus. These
mutants were produced by PCR using long primers covering the inserted
hA2-sequences, some bases identical to the templates as well as the restric-
tion sites outside of the coding sequence. Although the C-terminal segment
23, including H-site position F222, was not part of the conserved segments
in Paper I, it was included in the insertion in the chimeric mutants produced
in Paper III because of its location between segments 20−22 and the restric-

47
tion site. The amino acid sequences of human A3-3 and rat A3-3 differ in 25
and 60 positions respectively from human A2-2. The inserted C- and N-ter-
minal sequences introduced 7 and 15 mutations respectively into the chimer-
ic mutants based on human A3-3 and rat A3-3 (Paper III: Figure 3).

Characterization
The chimeric mutants were folded and stable GSTs and their specific activit-
ies towards azathioprine were determined to 0.19 and 0.44 μmol·min-1·mg-1,
about 10-fold higher than that of their respective templates human A3-3 and
rat A3-3, and almost up to the level of human A2-2, 0.67 μmol·min-1·mg-1
(Paper III: Figure 3).
The chimeric mutants were also examined using three other substrates in-
troduced in Paper II, CDNB, CMNI and NPTI (Paper III: Figure 1b). The
least difference among the five enzymes was found with CDNB, the general
GST substrate, with only small changes upon insertion of the flanking se-
quences from human A2-2. Human A2-2 has more than 10-fold higher spe-
cific activity with CMNI than human A3-3 and rat A3-3, similarly to the
situation with azathioprine, but the chimeric mutants only displayed up to
1/7 of the specific activity of human A2-2 with CMNI. With NPTI the chi-
meric mutants had 100- and 15-fold higher specific activities than the tem-
plates human A3-3 and rat A3-3. The chimeric mutant with human A3-3 in
the central portion even had 10-fold higher specific activity than human A2-
2, with a value of 660 μmol·min-1·mg-1.
Because of the high reaction rates with some of the enzymes and sub-
strates, significant dilutions of the enzyme stock solutions were made before
measurement in the cuvette and the spectrophotometer. To avoid noticeable
absorption of enzymes to the test tube walls, the tubes were incubated over
night with 2.5 % bovine serum albumin before making the diluted enzyme
solutions (Sundberg et al., 1995; Fedulova and Mannervik, 2011).

Alternative approach
Multivariate methods like partial least squares regression could have been
used to estimate how much each segment in the shuffled library in Paper I
contributed to the activity of the chimeric enzymes and possibly to predict
the sequences of improved chimeras. However, many more mutants would
have had to be sequenced and characterized and the enzymes with low activ-
ity would have had to be characterized more accurately in order to get useful
quantitative data. Besides, the greater the number of positions mutated, the
less additivity is to be expected in terms of activity or function (Lunzer et
al., 2010), and using crossterms to account for epistatic interactions would
require even more data to avoid overfitting. Choosing to focus only on the
segments that were conserved among the active mutants proved to be useful
in Paper III in identifying the features of the primary structure of importance
for the azathioprine activity of human GST A2-2.

48
Saturation mutagenesis

Cloning
The M208X/L213X mutant library based on human A2-2 was prepared us-
ing inverted polymerase chain reaction. Full-length linear plasmids were
generated using adjacent 5'-phosphorylated primers pointing in opposite dir-
ections. The PCR product was purified after agarose gel electrophoresis and
its blunt ends ligated before transformation into E. coli. The use of degener-
ate primers with NNS codons in two positions led to a library of 400 pos-
sible mutants with amino acids in both positions as well as some mutants
with early stop codons.

Screening
Lysates from 1880 bacterial colonies were screened with azathioprine and
the library coverage was estimated by Monte Carlo simulation to be 93 ± 1
% or 374 ± 5 unique mutants without stop codons in the mutated positions
(Paper III: Figure 5e−f). 1504 of the lysates were also screened with CDNB
and 752 of them with NPTI and CMNI as well. Different volumes of lysates
were used in the separate papers indicating that direct comparisons are not
straightforward. In this paper replicates were made but with less lysate in
each compared to Paper I. By measuring with different substrates the ratio of
activity values with different substrates corresponds to the ratio of specific
activities and it is independent of expression level. During screening, only
three lysates had higher activity with any of the substrates, with values of
120−150 % of the human A2-2 CDNB activity (Paper III: Figure 5a−d).

Mutant characterization
Sequencing and purification of selected variants confirmed that the wild-type
human A2-2 had the highest activity with azathioprine (Paper III: Table 1).
For CDNB, CMNI and NPTI there were mutants with about the same specif-
ic activities as human A2-2, even though fewer lysates were screened with
these substrates. The general trend confirmed by principal component ana-
lysis was that the mutations led to decreased catalytic activities with all four
tested substrates, even though some mutants had distinctly different substrate
profiles compared to the template, A2-2 (Paper III: Figure 6). The loading
vectors of the biplot showed that azathioprine and the chromogenic analog
NPTI did not have the highest covariation, highlighting the difficulty of
screening using analog substrates during directed evolution of enzyme prop-
erties.
In human GST A2-2, the α9 helix acts as a lid and forms one side of the
H-site. L213 is located in the α9 helix with M208 in the hinge segments pre-
ceding the helix and depending on the flexibility and dynamics they might
interact with each other. The mutants with about half or more of the aza-

49
thioprine activity retained had mainly hydrophobic residues in these posi-
tions, especially in position 208 (Paper III: Figure 7). One exception was the
M208D/L213R mutant, with 40 % of the wild-type specific activity towards
azathioprine, and possibly its mutated residues were stabilized by their op-
posite charges. A truncated variant with valine in position 208 and a stop
codon in 213 still had 31 % of the specific activity of human A2-2 with
CDNB, indicating that the α9 helix is not necessary for general catalytic
activity but that removal of the helix might be more detrimental for the cata -
lysis with some substrates.

Conclusion
Two chimeric GSTs were rationally designed by insertion of the N- and C-
terminal sequences from human A2-2 into two alpha class GSTs with low
specific azathioprine activities, human A3-3 and rat A3-3. The chimeric
mutants displayed a 10-fold improvement in specific activity towards aza-
thioprine, almost up to the level of human A2-2.
The H-site positions M208/L213 were conserved among the wild-types
and mutants that in Paper I displayed high azathioprine activity. Screening to
an estimated 93 % coverage of a saturation mutagenesis library targeting
these two positions revealed mutants which all had decreased specific activ-
ities towards azathioprine, while activities towards the other substrates were
slightly better tolerated. Analysis of the mutants with higher azathioprine
activity indicated that hydrophobic amino acid residues were preferred, espe-
cially in position 208 but also in position 213.
Analysis of the point mutants and the chimeras confirmed that screening
with alternative substrates during directed evolution can provide a conveni-
ent way of faster finding functional enzymes but not necessarily with the de -
sirable properties regarding substrate specificity.
The structure-activity relationships showed that segments 1−2 and 20−22
from human GST A2-2, that were conserved among the mutants with high
activity, could sustain high azathioprine activity within the context of the ex-
amined alpha class sequences, while the conserved H-site residues within
these segments did not tolerate mutations with retention of the activity to-
wards azathioprine. Mutational strategies attempting to improve the catalytic
efficiency with this substrate could preferably instead focus on the variable
segments among the mutants with high activity. Mutagenesis in the variable
segments 8 and 23 of human GST A2-2, will be further examined in paper
IV.

50
Redesign of GST A2-2 for enhanced azathioprine
activity (Paper IV)

Introduction
The preceding papers have discussed mutagenesis of human GST A2-2, in-
cluding mutations throughout the whole enzyme as well as in the active site.
However, the catalytic efficiency with azathioprine was found to be lower
than with many other substrates. If the GSTs could be redesigned to have a
catalytic efficiency with azathioprine much higher than that of the endogen-
ous enzymes, they could find use in ADEPT. In this paper molecular docking
was performed in order to suggest which H-site amino acids were most
likely to influence catalysis. Three positions were chosen for mutagenesis
and a mutant library was created using reduced sets of codons. Variant A2*E
was used as template due to its higher catalytic efficiency with azathioprine
compared to the other A2-2 variants (Paper II). The library was screened for
azathioprine activity and interesting hits were characterized. The structure of
the most active mutant was determined by X-ray crystallography.

Library design

Choice of mutations
The family shuffling in paper I did not generate any alpha class GSTs with
enhanced activity towards azathioprine. Paper IV instead focused on mutat-
ing active site residues that were more likely to influence the catalysis and
not only to amino acids already present in the alpha class GSTs in these posi-
tions. Based on molecular docking of azathioprine into a model of human
A2*E the H-site residues L107, L108, F111 and F222 were assumed to be
able to interact directly with the substrate (Paper IV: Figure S1A). In particu-
lar amino acids in positions 107, 108 and 222 were thought to possibly con-
tribute to the transition state stabilization by interacting with the nitro group
of the imidazole ring, the thiolate of the glutathione or the upcoming negat-
ive charge on the sulfur in the 6-mercaptopurine leaving group (Paper IV:
Figure 1). One library was constructed targeting positions 107, 108 and 222
simultaneously. Positions F111 and L213 were later mutated independently
without finding any enzyme with improved activity towards azathioprine.

Reduced codon sets

The three-position library was produced using reduced codon sets in the tar-
geted positions in order to obtain a high quality in terms of even frequency
of the different amino acids. It also reduced the size of the library to facilit -

51
ate screening of a larger proportion of the possible amino acid combinations.
The degenerate codon set YHC (coding for His, Leu, Phe, Pro, Ser and Tyr)
was used for position 222, since an F222A mutation in a human A1-1 mutant
had detrimental effects on catalysis indicating that large amino acids might
be needed in this position (Nilsson et al., 2002). NDT (coding for amino
acids Arg, Asn, Asp, Cys, Gly, His, Ile, Leu, Phe, Ser, Tyr and Val) was used
for positions 107 and 108, giving a wider range of different types of amino
acids, all with only one codon per amino acid. In total, the library contained
864 possible amino acid combinations.

Cloning
The library was prepared using the dual-tube megaprimer approach (Tseng
et al., 2008). By first amplifying the region between the mutated sites (cor-
responding to amino acids 107/108 and 222), the purified PCR product was
then used as primers during the amplification of the whole plasmid. After
DpnI digestion of the template plasmids and transformation into E. coli, se-
quencing of randomly sampled colonies confirmed adequate codon variabil-
ity and no overrepresentation of template sequences.

Screening
Lysates from 1901 bacterial colonies were screened for activity with aza-
thioprine and the coverage of the library was estimated to be 89 ± 1 %. The
distribution of activities was skewed toward higher values and 44 % of the
lysates had activities significantly higher than the positive control A2*E (Pa-
per IV: Figure 2).

Kinetic characterization
Mutants with high activity towards azathioprine were sequenced and purified
(Paper IV: Table S2 and Paper IV: Table 1). Mutant G107/D108/H222
(named "GDH") had the highest specific activity towards azathioprine, 128
μmol mg-1 min-1. The catalytic efficiency kcat/KM was 292 mM-1 s-1, 70 times
larger than A2*E, with 11-fold higher kcat and 6-fold lower KM (Paper IV:
Table 2). The pH-profiles of the examined GSTs show a specific activity
maximum around 7 (Fig IV_4b). This indicated that lowering of the pH from
7.4 to 7.0 during screening did not drastically modify the activity values ob -
tained from the screening.

Comparison to other alpha class sequences

The GSTs with highest activity in the screening had mainly hydrophilic or
small amino acids in the three positions mutated (Table 3). These positions
are primarily occupied by large hydrophobic amino acids in the alpha class

52
GSTs. P222 is present in four of 30 alpha class GSTs while G107 and D108
are rare with only one occurrence of G107, according to the sequences ex-
amined in paper I.
Table 3. Amino acids represented in the mutated positions of the GSTs with highest
activity in the screening with azathioprine. Note that mutants with C107 had higher
expression and lower specific activity relative to the other mutants examined. Some
alpha class GSTs end before amino acid 222 and the absence is marked as "-".
Amino acid position 107 108 222
H-site region according to Paper I B B C
Present in the most active GHSVC DGSN HPS
Absent in the most active DFILNRY CFHILRVY FLY
Present frequently in alpha class LIMAG LMQHYVIGS F-PIML

Structural interpretation

Comparison of A1-1, A2*C and GDH

In human A2*C and the A2*E-derived GDH mutant, F111 is pointing in to
the middle of the H-site making it less deep compared to the H-site of human
A1-1 (compare Paper I: Figure 8b and Paper III: Figure 2). On the other
hand, the GDH mutant lacks the larger hydrophobic residues L107/L108 that
in A2*C and A1-1 form one wall of the H-site. Instead G107 is small and
D108 is pointing in another direction making the H-site wide in the mutant
compared to A2*C (Paper IV: Figure 5). Besides, H222 in mutant GDH is a
bit shorter and a bit further away from the H-site compared to F222 in A2*C
and A1-1, making that side of the active site pocket slightly larger as well.
Probably azathioprine and in particular the transition state can fit more easily
into the wider pocket of the GDH mutant. This is in agreement with the se-
quences of the mutants that had high activity with azathioprine, which had
mainly smaller amino acid residues in positions 107, 108 and 222 compared
to the residues available in the mutagenesis of those positions (Table 3).

Comparison of A2*C and A2*E

It is also possible that the difference between A2*C and A2*E,
P110S/T112S, is influencing the orientation of the D108 side chain in the
GDH mutant. The T112S mutation only modified the kinetics with aza-
thioprine marginally, while P110S enhanced the catalytic efficiency 3−4-fold
(Paper II: Table 4). Without determining a structure of A2*E it is hard to
draw conclusions about the structural effects of the P110S mutation. Even in
A2*C and the GDH mutant, E104 and F111 are in almost the same locations,
but possibly small changes in positions and orientations of L107, L108 and
F111 induced by the P110S mutation are responsible for the modified cata-
lytic properties.

53
Three mutated positions
The structurally visible effect of the GDH mutation on the template enzyme
was the widening of the H-site while the mutated amino acids did not neces -
sarily interact directly with the substrate. However, docking results may also
depend on the flexibility of the model. In the structure determination by X-
ray crystallography the electron density was not well-defined neither for aza-
thioprine nor for the substituted imidazole group of the corresponding gluta-
thione conjugate. This suggests that there is not a single fixed binding con-
formation of the substrate and with flexibility the mutated positions could
possibly interact with the substrate. One of the purified mutants with very
high specific activity with azathioprine, V107/G108/P222, lacked hydrophil-
ic groups in the three mutated positions, indicating that it was not the inser -
tion of new polar or charged catalytic groups that caused the rate enhance-
ment. This experiment provided one example of the difficulty in rational
design and the benefits of combining it with stochastic mutagenesis. Even
though the result was satisfying in terms of rate enhancement the reason was
not as anticipated before the experiment. Detailed mechanistic understanding
requires further investigation.

Other positions involved

If the mutated residues did not provide new catalytic groups, perhaps the
already existing residues are more involved in the transition state stabiliza-
tion. Y9 is already known to stabilize the deprotonated sulfur on the activ-
ated glutathione (Dourado et al., 2008). Maybe R15 nearby could hydrogen
bond to the upcoming negative charge on the sulfur of the purine leaving
group or possibly to the nitro group of the imidazole ring. Position 15 is a
conserved basic residue in alpha class GSTs (Björnestedt et al., 1995) and its
charge is of importance for the activation of glutathione (Dourado et al.,
2010). F111 could possibly stabilize the purine ring of azathioprine depend-
ing on the conformation of the substrate and the transition state. Perhaps re-
placements to larger or polar amino acids in other H-site positions could also
lead to new interactions with the substrate while removal of bulky residues
might position the substrate or interacting amino acids even more favorably.
The three mutated positions were quite tolerant to substitutions and further
mutagenesis could allow some flexibility there.

Mutational strategies
Several mutational strategies were used in papers I−IV that all generated lib-
raries with folded and active GSTs. Paper IV approached the question wheth-
er it was possible to improve the catalytic efficiency with azathioprine. In
summary there was no improvement by shuffling with other alpha class
GSTs displaying low activity (Paper I), a 3-fold improvement by examining

54
the naturally occurring polymorphism in human A2-2 (Paper II), and an ad-
ditional 70-fold improvement in catalytic efficiency by simultaneously tar-
geting three H-site positions (Paper IV). These three positions were not con-
served in the azathioprine active GSTs in paper I while mutagenesis of two
H-site positions that were conserved in the GSTs with higher activity to-
wards azathioprine did not lead to improvement (Paper III). Mutants with
very high azathioprine activity in the three-position-library had small or po-
lar residues that were rare in alpha class sequences in these positions and that
could be one reason why the shuffling of alpha class sequences did not
identify mutants with higher specific activity than human A2-2 towards aza-
thioprine.

Conclusion
Human GST A2*E was targeted for mutagenesis in order to see if higher
activity with the prodrug azathioprine could be achieved. Molecular docking
of the substrate into a modeled structure of the template suggested for muta-
genesis three positions, in which new amino acids might influence the cata-
lysis. A library was constructed using reduced sets of codons and contained
864 possible unique mutants. 1901 bacterial lysates was screened for aza-
thioprine activity giving an estimated 89 ± 1 % coverage of the library. 44 %
of the examined lysates had activity values significantly higher than the tem-
plate. In general the mutants with the highest activity had small or polar
residues in the mutated positions. The best mutant, L107G/L108D/F222H
had a 70-fold improved catalytic efficiency with the targeted substrate. De-
termination of the structure of the mutant by X-ray crystallography showed a
wider H-site, suggesting that that the transition state could be accommodated
in a mode better suited for catalysis. A GST with improved catalytic effi-
ciency towards azathioprine might find use as a selectable marker in tempor-
al gene therapy or in ADEPT, especially for subjects with slow endogenous
azathioprine activation as discussed in Paper II.

Summary

Structure-function relationships of human GST A2-2

The prodrug azathioprine is metabolized along several pathways and the first
step is activated in human by GSTs A2-2, A1-1 and M1-1. To investigate the
faster azathioprine activation of human A2-2 compared to other GSTs, DNA
shuffling was performed using human GST A2-2 as template together with
four other alpha class GSTs from different species as well as a point muta-

55
tion library (Paper I). Five of the 23 recombined segments, located in the N-
and C-terminus, were conserved among mutants with more than 20 % of the
human A2-2 specific azathioprine activity.
The importance of these conserved segments was further examined by in-
sertion of the N- and C-terminal sequences from human A2-2 into the low-
active GSTs human A3-3 and rat A3-3 (Paper III). The resulting chimeric en-
zymes displayed a 10-fold enhanced specific activity towards azathioprine,
almost up to the level of human A2-2. The segments contained five H-site
residues and two of them, M208/L213, were conserved among the active
mutants as well as human A1-1, with 60 % of the human A2-2 activity.
These two conserved positions were targeted by saturation mutagenesis with
NNS codons (Paper III). After screening of approximately 93 % of the 400
enzyme combinations, all mutants examined had decreased activity towards
azathioprine, with a preference among the most active mutants for hydro-
phobic residues in the mutated positions, especially position 208.
The structure-activity relationships showed that the analyzed segments
could sustain azathioprine activity in the context of these alpha class se-
quences and that mutagenesis in the corresponding H-site residues was not
tolerated regarding the azathioprine activity. This suggested that mutagenesis
leading to higher rates of azathioprine activation instead should be focused
to the segments that were variable among the chimeric GSTs with higher
specific activity towards azathioprine.

Polymorphism of human GST A2-2

About 30 % of the patients undergoing azathioprine therapy experience ad-
verse effects, including bone marrow suppression, gastrointestinal disturb-
ance, hepatotoxicity and pancreatitis. Genotyping of the gene encoding the
inactivation enzyme TPMT identifies patients with TPMT deficiency and at
high risk for severe myelosuppression, but only a minor proportion of the
cases of adverse effects can be predicted. The differences in the rate of aza-
thioprine activation might affect metabolite concentrations and distributions.
Polymorphism in the azathioprine-activating GST M1-1 have been indicated
to be related to differences in the occurrence of adverse effects.
Five genotypes of human GST A2-2 were characterized and variant A2*E
turned out to have a 3−4-fold higher catalytic efficiency with azathioprine
(Paper II). Variant A2*E showed that the P110S mutation, adjacent to the H-
site positions in the central portion of the GST could increase the specific
activity with azathioprine, possibly by changing the orientation of the H-site
residues. For a homozygous A2*E carrier, the difference could, depending
on the expression levels of the relevant GSTs, result in an up to 3-fold differ-
ence in apparent rate constant in some tissues. Genotyping of GSTs involved
in azathioprine activation is recommended to further study the connections

56
among GSTs, azathioprine dose, metabolite levels, efficacy and adverse ef-
fects.

Characterization using alternative substrates

The catalytic efficiency with the chromogenic substrate NPTI was about 3-
fold higher for A2*E than for the other variants of human A2-2, while it was
lower with the radiosensitizer CMNI and the general GST substrate CDNB
(Paper II). Azathioprine, NPTI and CMNI all form the same glutathione con-
jugate and the difference in kcat indicates that the reaction rates of these en-
zymes are not limited by the release of this conjugate, at least not for the re-
actions with lower kcat.
In the principal component analysis, azathioprine and its surrogate sub-
strate NPTI showed more covariation than the other substrates when examin-
ing the polymorphic A2-2 variants (Paper II) but not when examining the
M208/L213 saturation library of A2-2 (Paper III). The specific activities
with the fluorogenic substrate monochlorobimane were not correlated with
the specific activities towards azathioprine in the alpha class chimeric GSTs.
Analysis of the point mutants and the chimeras confirmed that screening
with alternative substrates during directed evolution can provide a conveni-
ent way of faster finding functional enzymes but not necessarily with the de -
sirable properties regarding substrate specificity.

Redesign of human GST A2-2 for enchanced azathioprine

activity
Human A2-2 was further targeted by mutagenesis to see if the catalytic effi-
ciency with azathioprine could be improved. Molecular docking of the sub-
strate into a model of the variant A2*E suggested positions L107/L108/F222
for mutagenesis. Amino acids in these positions might directly interact with
the substrate and possibly affect the catalysis. A library with reduced codons,
NDT and YHC, was screened for azathioprine activity to an estimated 89 %
coverage. 44 % of the examined lysates had significantly higher activity val-
ues during the screening and the best mutant, L107G/L108D/F222H, dis-
played a 70-fold increased catalytic efficiency with azathioprine compared to
A2*E, a more than 200-fold improvement compared to the original starting
point A2*C.
The mutated positions that led to increased catalytic efficiency with aza-
thioprine, P110S/T112S (Paper II) and L107G/L108D//F222H (Paper IV),
were all part of the variable segments among the mutants with specific aza-
thioprine activity more than 20 % of that of A2*C (Paper I). The most active
mutants had small or polar residues in positions 107, 108 and 222 and de-
termination of the structure of the best mutant by X-ray crystallography

57
showed a wider H-site than A2*C (Paper IV), suggesting that that the trans-
ition state could be accommodated in a mode better suited for catalysis. A
GST with improved catalytic efficiency towards azathioprine might find use
in ADEPT, for example targeting the HER2 receptor on tumor cells.

58
Summary in Swedish

Mutationsanalys och förbättring av alfaklass-

glutationtransferaser med avseende på aktivitet med
proläkemedlet azatioprin

Bakgrund

Modifiering av enzymer
Enzymer utgör cellens maskineri och katalyserar biokemiska reaktioner
under fysiologiska betingelser. Därmed guidas cellens metabolism till att
genomföra användbara reaktioner. I naturen utvecklas generna som kodar för
enzymerna genom att variation skapas i form av duplikationer,
rekombinationer, punktmutationer, insättningar och borttagningar.
Organismer som överlever och reproducerar sig för vidare generna för de
enzymer som var bäst anpassade för sin situation. I ett laboratorium kan man
härma den evolutionära processen och skapa bibliotek av enzymvarianter,
mutanter, på olika sätt. DNA som kodar för de olika varianterna sätts in i
bakterier som odlas i näringslösning och som producerar enzymerna, vars
funktion sedan kan mätas direkt eller efter att ha renats fram.
För att sålla ut enzymer med de önskvärda egenskaperna bör man helst
mäta mutanternas aktivitet med det tilltänkta substratet. Använder man andra
liknande substrat är det inte säkert att man utvecklar den egenskap som man
har hoppats på. Andra faktorer som kan påverka utsållningen är till exempel
om enzymbiblioteket har många mutanter med ofullständig sekvens eller om
många exemplar av ursprungssekvensen finns kvar i omuterad form. Det kan
också vara lämpligt att använda sig av degenererade primrar med bara ett
kodon för varje aminosyra, vilket leder till en jämnare fördelning av
mutanternas variation i biblioteket. I den här avhandlingen har olika metoder
för att modifiera enzymer testats.
Enzymer har tekniska tillämpningar inom industri, medicin, diagnostik
och molekylärbiologi. Ett exempel på en medicinsk tillämpning är att koppla
ihop läkemedelsaktiverande enzym med en ligand som binder till receptorer

59
på oönskade celler, till exempel i en tumör, för att på så sätt få en lokal
aktivering av läkemedlet.

Glutationtransferas (GST)
GSTer katalyserar reaktionen mellan antioxidanten glutation och diverse
toxiska molekyler i cellen som annars skulle kunna bidra till oxidativ stress
eller skador på DNA. Genom att konjugera glutationet till de reaktiva
substraten blir de mindre reaktiva och mer lösliga och kan lättare utsöndras
från cellen och kroppen.
GSTerna är promiskuösa vad gäller substratacceptansen och kan
katalysera reaktionen med många olika substrat med olika hastigheter. I
människan finns det 17 olika cytosoliska GSTer och tillsammans utgör de en
arsenal av avgiftningsenzymer som alla är redo att ta hand om olika substrat.
Fem av dessa GSTer tillhör alfaklassen och framför allt A2-2 var i fokus i
denna avhandling. A2-2 uttrycks i hög nivå i levern, tunntarmen, testiklarna,
njurarna, binjurarna och bukspottkörteln.
De flesta alfaklass GSTerna är uppbyggda av två subenheter med 222
aminosyror var. Varje subenhet har ett G-säte som binder glutation och ett H-
säte som binder de hydrofoba substraten. Ett av substraten som A2-2 kan
katalysera reaktionen med är läkemedlet azatioprin. Varför A2-2 har så hög
specifik aktivitet med azatioprin jämfört med andra GSTer har undersökts i
denna avhandling.

Azatioprin (Imurel®)
Azatioprin är ett immundämpande läkemedel som används till exempel vid
inflammatoriska tarmsjukdomar som ulcerös kolit och Chrons sjukdom.
Azatioprin är ett proläkemedel som aktiveras i flera steg innan den aktiva
substansen bildas. Först aktiveras azatioprin genom konjugering med
glutation vilket frisläpper 6-merkaptopurin (6-MP). Det är en analog till
baserna i DNA och inkorporering i DNA leder till skador på DNA-strängarna
och celldöd. Metaboliterna kan även störa andra enzymer. Ungefär 30 % av
patienterna upplever bieffekter i form av skador på exempelvis benmärgen,
magtarmkanalen, levern och bukspottkörteln. Har man brist på ett av
enzymerna som inaktiverar 6-MP kan man vara extra känslig och behöva
lägre dos av azatioprin.
En stor del av bieffekterna kan dock inte förklaras av kända faktorer.
Aktiveringen av azatioprin skiljer sig åt mellan individer och hastigheten kan
påverka koncentrationen av metaboliter i senare steg, vilket också kan
påverka uppkomsten av bieffekter. Aktivitet och uttryck av olika GSTer i
olika vävnader borde alltså kunna påverka effekterna av behandling med
azatioprin. Aktiviteten hos olika varianter av det azatioprin-aktiverande GST
A2-2 har undersökts i den här avhandlingen. Om enzymet dessutom kunde
förbättras med avseende på aktiveringen av azatioprin skulle enzymet kunna

60
kopplas till en målsökande ligand för att få en mer kontrollerad lokalisering
av metaboliterna.

Avhandlingens artiklar

Struktur-funktionssamband för humana GST A2-2

Det humana GST A2-2 rekombinerades med fyra andra alfaklass-GSTer från
olika arter samt ett punktmutationsbibliotek (Artikel I). Av de 23
rekombinerade segmenten var det fem som hade behållits likadana inom
mutanterna med mer än 20 % av aktiviteten med humana A2-2. Dessa
segment undersöktes ytterligare genom att sätta in dem i GSTer med låg
aktivitet (Artikel III). I dessa mutanter var aktiveringen av azatioprin 10
gånger högre än för de låga GSTerna, nästan upp till nivån för humana A2-2.
Segmenten betydelse undersöktes ytterligare genom att mutera flera H-
sätespositioner. I det fall mutationerna förlades till de nämnda segmenten
blev aktiviteten med azatioprin bara lägre (Artikel III) medan när
mutationerna förlades till andra segment så hade mutantenzymerna både
högre och lägre aktivitet (Artiklar II och IV). De nämnda segmenten, som
låg i början och slutet av enzymets aminosyrasekvens, var alltså viktiga för
aktiviteten med azatioprin och tolererade inte att H-sätespositionerna
muterades annat än till hydrofoba aminosyror som i vissa fall bara gav lite
minskad aktivitet.

Polymorfi i humana GST A2-2

I den mänskliga befolkningen existerar fem kända genetiska varianter av A2-
2. Variant A2*E visade sig ha 3−4 gånger högre katalytisk effektivitet med
azatioprin (Artikel II). Mutationen P110S befinner sig intill H-sätet och har
förmodligen påverkat orienteringen av H-sätespositionerna. Aktiveringen i
kroppen påverkas även av uttrycksnivån av enzymet i olika vävnader.
Skillnaden i aktivering mellan olika individer kan ge olika koncentration och
fördelning av proläkemedlets aktiva metaboliter, och kan vara relaterat till
uppkomsten av bieffekter. Fortsatta studier rekommenderas, som skulle
kunna undersöka kopplingen för patienter mellan GST-polymorfi,
azatioprinaktivitet, metabolitfördelning och bieffekter.

Karaktärisering med alternativa substrat

GSTerna undersöktes förutom med azatioprin även med två substrat som ger
upphov till samma glutationkonjugat men andra frisläppande grupper.
Värdena på kcat var inte lika för de olika substraten (Artikel II), vilket antyder
att reaktionshastigheterna inte är begränsade av frisläppningen av det
gemensamma glutationkonjugatet, åtminstone inte för de reaktioner med
långsammare kcat.

61
 Aktiviteten med azatioprin och ett surrogatsubstrat samvarierade med
vissa mutanter men inte med andra (Artiklar II och III). Ingen korrelation
syntes mellan azatioprin och ett fluorogent GST-substrat. Analys av
mutanternas aktiviteter med andra substrat bekräftar att även om utsållning
kan ske snabbare med hjälp av andra substrat så ger det i första hand en
utsållning av enzym som klarar av att uttryckas och inte säkert en förbättring
av den substratselektivitet man är ute efter.

Förbättring av azatioprinaktiveringen hos humana GST A2-2

Datoriserad dockning av azatioprin mot en modellerad struktur av A2*E
föreslog tre positioner som vid mutagenes skulle kunna påverka katalysen
(Artikel IV). Ett mutantbibliotek skapades med tolv eller sex alternativa
aminosyror i varje muterad position och bara ett kodon per aminosyra. En
stor andel av mutanterna visade sig utföra förbättrad azatioprinaktivering och
den allra snabbaste, L107G/L108D/F222H hade 70 gånger högre katalytisk
effektivitet än A2*E. Dess struktur bestämdes med röntgenkristallografi och
dess H-säte verkade vara bredare än i A2*C, vilket tyder på att reaktionens
övergångstillstånd passar in i ett läge bättre lämpat för katalys.

Slutsats
Mutationsanalysen som utförts ökade förståelsen av azatioprinaktiveringen i
alfaklass-GSTer och belyste A2*E som en faktor bakom läkemedlets
bieffekter. En framgångsrikt förändrad GST, med 200 gånger högre
katalytisk effektivitet med azatioprin jämfört med ursprungsvarianten A2*C,
kan komma till användning inom målinriktad läkemedelsaktivering.

62
Acknowledgments

Bengt Mannervik, tack för att du tog emot mig som doktorand i din
forskargrupp. Det har varit inspirerande, givande och lärorikt. Du besitter
mycket kunskap och idéer och är en framgångsrik forskare. Tack också för
din och Anne-Charlottes gästfrihet och för de intressanta konferenser du har
ordnat med prominenta gäster. Hoppas att din forskning bär frukt i många år
framöver!
Tack till min biträdande handledare Mikael Widersten som har bidragit
med värdefulla synpunkter vid upprepade tillfällen. Tack också för att du lett
institutionens arbete de senaste åren.
Jag vill tacka mina medförfattare, framför allt Wei för samarbetet och
umgänget. Lycka till i fortsättningen!
Tack Birgitta för ditt grundläggande arbete med GST och azatioprin. Även
tack till Thomas för ditt projektarbete inom GST och din nyfikenhet och
förmåga att hitta lösningar.
Stort tack till Saga, Natalia, och Arna som har delat det lilla rummet med
mig. Tack för glädjen och humorn och för utbytet av erfarenheter och
upplevelser. Ni är bäst!
Tack till många kollegor som generöst delat med sig av sina färdigheter
eller diskuterat lösningar, bland andra Birgit, Gun, Françoise, Emilia, Ann,
Diana, Cissi och Nasim.
Tack Inger för allt fixande med papper.
Tack Lilian och Anne-Maj för att ni håller ordning i kurslabben. Tack
också Gunnar för bra samarbete under undervisningen. Sofia och Angelica,
ni är grymma labhandledare!
Tack till gamla och nya medlemmar i Bengts grupp för en trevlig
stämning i labbet, på gruppmöten eller vid andra sammankomster; Malena,
Abeer, Usama, Yaman, Nese, Emil, Fatima, Ronnie, Camilla, Andreas,
Daniel, Aslam och Marianne.
Tack även till övriga kollegor i Mickes, Helenas, Gunnars, Stellans och
Birgittas forskargrupper för ert bidrag till institutionens välbefinnande.
Jag vill också passa på att tacka Herbert & Karin Jacobssons Stiftelse och
Cancerfonden för finansiellt stöd.
Sist men inte minst vill jag tacka familjen, släkten och vännerna för ert
stöd. Ni betyder mycket för mig.

63
References

Afshar S., Asai T. & Morrison S.L. (2009) Humanized ADEPT comprised of an en-
gineered human purine nucleoside phosphorylase and a tumor targeting peptide
for treatment of cancer. Mol Cancer Ther. 8, 185-193
Al-Safi S., Tashtoush B. & Hassan M. (2003) Comparison of the effects of aza -
thioprine and its novel non-mercaptopurine analog on antibody response in rab-
bits. Pol J Pharmacol. 55, 239-243
Allorge D., Hamdan R., Broly F., Libersa C. & Colombel J.F. (2005) ITPA genotyp-
ing test does not improve detection of Crohn's disease patients at risk of aza-
thioprine/6-mercaptopurine induced myelosuppression. Gut. 54, 565
Andersson A., Lindgren A., Arnadottir M., Prytz H. & Hultberg B. (1999) Thiols as
a measure of plasma redox status in healthy subjects and in patients with renal or
liver failure. Clin Chem. 45, 1084-1086
Arenas M., Simpson G., Lewis C.M. et al. (2005) Genetic variation in the MTHFR
gene influences thiopurine methyltransferase activity. Clin Chem. 51, 2371-2374
Baert F., Noman M., Vermeire S. et al. (2003) Influence of immunogenicity on the
long-term efficacy of infliximab in Crohn's disease. N Engl J Med. 348, 601-608
Bagshawe K.D. (1987) Antibody directed enzymes revive anti-cancer prodrugs
concept. Br J Cancer. 56, 531-532
Ballatori N., Hammond C.L., Cunningham J.B., Krance S.M. & Marchan R. (2005)
Molecular mechanisms of reduced glutathione transport: role of the
MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins. Toxicol
Appl Pharmacol. 204, 238-255
Ban H., Andoh A., Imaeda H. et al. (2010) The multidrug-resistance protein 4 poly-
morphism is a new factor accounting for thiopurine sensitivity in Japanese pa-
tients with inflammatory bowel disease. J Gastroenterol. 45, 1014-1021
Barabino A., Torrente F., Ventura A., Cucchiara S., Castro M. & Barbera C. (2002)
Azathioprine in paediatric inflammatory bowel disease: an Italian multicentre
survey. Aliment Pharmacol Ther. 16, 1125-1130
Barba J., Rosell D., Rincon A. et al. (2012) New immunosuppressive therapies and
surgical complications after renal transplantation. Transplant Proc. 44, 1275-
1280
Beaugerie L., Brousse N., Bouvier A.M. et al. (2009) Lymphoproliferative disorders
in patients receiving thiopurines for inflammatory bowel disease: a prospective
observational cohort study. Lancet. 374, 1617-1625
Benkovic S. & Hammes-Schiffer S. (2003) A perspective on enzyme catalysis. Sci-
ence. 301, 1196-1202
Björnestedt R., Stenberg G., Widersten M. et al. (1995) Functional significance of
arginine 15 in the active site of human class alpha glutathione transferase A1-1.
J Mol Biol. 247, 765-773
Bourgine J., Garat A., Allorge D. et al. (2011) Evidence for a functional genetic
polymorphism of the Rho-GTPase Rac1. Implication in azathioprine response?
Pharmacogenet Genomics. 21, 313-324

64
Breen D.P., Marinaki A.M., Arenas M. & Hayes P.C. (2005) Pharmacogenetic asso-
ciation with adverse drug reactions to azathioprine immunosuppressive therapy
following liver transplantation. Liver Transpl. 11, 826-833
Cara C.J., Pena A.S., Sans M. et al. (2004) Reviewing the mechanism of action of
thiopurine drugs: towards a new paradigm in clinical practice. Med Sci Monit.
10, RA247-254
Chalmers A.H., Knight P.R. & Atkinson M.R. (1967) Conversion of azathioprine
into mercaptopurine and mercaptoimidazole derivatives in vitro and during im-
munosuppressive therapy. Aust J Exp Biol Med Sci. 45, 681-691
Chen M.M., Snow C.D., Vizcarra C.L., Mayo S.L. & Arnold F.H. (2012) Comparis-
on of random mutagenesis and semi-rational designed libraries for improved
cytochrome P450 BM3-catalyzed hydroxylation of small alkanes. Protein Eng
Des Sel. 25, 171-178
Chouchana L., Narjoz C., Beaune P., Loriot M.A. & Roblin X. (2012) Review art-
icle: the benefits of pharmacogenetics for improving thiopurine therapy in in-
flammatory bowel disease. Aliment Pharmacol Ther. 35, 15-36
Clayton P.A., McDonald S.P., Chapman J.R. & Chadban S.J. (2012) Mycophenolate
versus azathioprine for kidney transplantation: a 15-year follow-up of a random-
ized trial. Transplantation. 94, 152-158
Coles B.F., Anderson K.E., Doerge D.R., Churchwell M.I., Lang N.P. & Kadlubar
F.F. (2000) Quantitative analysis of interindividual variation of glutathione S-
transferase expression in human pancreas and the ambiguity of correlating geno-
type with phenotype. Cancer Res. 60, 573-579
Coles B.F., Chen G., Kadlubar F.F. & Radominska-Pandya A. (2002) Interindividual
variation and organ-specific patterns of glutathione S-transferase alpha, mu, and
pi expression in gastrointestinal tract mucosa of normal individuals. Arch Bio-
chem Biophys. 403, 270-276
Coles B.F. & Kadlubar F.F. (2005) Human alpha class glutathione S-transferases: ge-
netic polymorphism, expression, and susceptibility to disease. Methods Enzymol.
401, 9-42
Connell W.R., Kamm M.A., Ritchie J.K. & Lennard-Jones J.E. (1993) Bone marrow
toxicity caused by azathioprine in inflammatory bowel disease: 27 years of ex-
perience. Gut. 34, 1081-1085
Crawford D.J., Maddocks J.L., Jones D.N. & Szawlowski P. (1996) Rational design
of novel immunosuppressive drugs: analogues of azathioprine lacking the 6-
mercaptopurine substituent retain or have enhanced immunosuppressive effects.
J Med Chem. 39, 2690-2695
DeLeve L.D., Wang X., Kuhlenkamp J.F. & Kaplowitz N. (1996) Toxicity of aza-
thioprine and monocrotaline in murine sinusoidal endothelial cells and hepato-
cytes: the role of glutathione and relevance to hepatic venoocclusive disease.
Hepatology. 23, 589-599
Denis-Quanquin S., Lamouroux L., Lougarre A. et al. (2007) Protein expression
from synthetic genes: selection of clones using GFP. J Biotechnol. 131, 223-230
Dixon D.P., Lapthorn A. & Edwards R. (2002) Plant glutathione transferases. Gen-
ome Biol. 3, reviews/3004.1-10
Dong X.W., Zheng Q., Zhu M.M., Tong J.L. & Ran Z.H. (2010) Thiopurine S-
methyltransferase polymorphisms and thiopurine toxicity in treatment of inflam-
matory bowel disease. World J Gastroenterol. 16, 3187-3195
Dourado D.F., Fernandes P.A., Mannervik B. & Ramos M.J. (2008) Glutathione
transferase: new model for glutathione activation. Chemistry. 14, 9591-9598

65
Dourado D.F., Fernandes P.A., Mannervik B. & Ramos M.J. (2010) Glutathione
transferase A1-1: catalytic importance of arginine 15. J Phys Chem B. 114,
1690-1697
Dubinsky M.C., Lamothe S., Yang H.Y. et al. (2000) Pharmacogenomics and meta-
bolite measurement for 6-mercaptopurine therapy in inflammatory bowel dis-
ease. Gastroenterology. 118, 705-713
Ehren J., Govindarajan S., Moron B., Minshull J. & Khosla C. (2008) Protein engin-
eering of improved prolyl endopeptidases for celiac sprue therapy. Protein En-
gineering Design & Selection. 21, 699-707
Eklund B.I., Edalat M., Stenberg G. & Mannervik B. (2002) Screening for recom-
binant glutathione transferases active with monochlorobimane. Anal Biochem.
309, 102-108
Eklund B.I., Moberg M., Bergquist J. & Mannervik B. (2006) Divergent activities of
human glutathione transferases in the bioactivation of azathioprine. Mol Phar-
macol. 70, 747-754
Elion G.B. (1989) The purine path to chemotherapy. Science. 244, 41-47
Endelman J.B., Silberg J.J., Wang Z.G. & Arnold F.H. (2004) Site-directed protein
recombination as a shortest-path problem. Protein Eng Des Sel. 17, 589-594
Engler C., Gruetzner R., Kandzia R. & Marillonnet S. (2009) Golden gate shuffling:
a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS
One. 4, e5553
Fahey R.C. & Sundquist A.R. (1991) Evolution of glutathione metabolism. Adv En-
zymol Relat Areas Mol Biol. 64, 1-53
Fedulova N. & Mannervik B. (2011) Experimental conditions affecting functional
comparison of highly active glutathione transferases. Anal Biochem. 413, 16-23
Fox R. (2005) Directed molecular evolution by machine learning and the influence
of nonlinear interactions. J Theor Biol. 234, 187-199
Fraser A.G., Orchard T.R. & Jewell D.P. (2002) The efficacy of azathioprine for the
treatment of inflammatory bowel disease: a 30 year review. Gut. 50, 485-489
French H., Dalzell A., Srinivasan R. & El-Matary W. (2011) Relapse Rate Following
Azathioprine Withdrawal in Maintaining Remission for Crohn's Disease: A
Meta-Analysis. Digestive Diseases and Sciences. 56, 1929-1936
Frey P.A. & Hegeman A.D. (2007) Enzymatic reaction mechanisms, Oxford Univer-
sity Press, Oxford
Frushicheva M. & Warshel A. (2012) Towards Quantitative Computer-Aided Studies
of Enzymatic Enantioselectivity: The Case of Candida antarctica Lipase A.
Chembiochem. 13, 215-223
García-Mata R., Conde R.D. & Sanllorenti P.M. (1998) Contribution of breakdown
in the regulation of mouse liver glutathione S-transferase subunits during protein
depletion and re-feeding. Biochim Biophys Acta. 1448, 46-50
Gardiner S.J., Gearry R.B., Begg E.J., Zhang M. & Barclay M.L. (2008) Thiopurine
dose in intermediate and normal metabolizers of thiopurine methyltransferase
may differ three-fold. Clin Gastroenterol Hepatol. 6, 654-660
Gearry R.B. & Barclay M.L. (2005) Azathioprine and 6-mercaptopurine pharmaco-
genetics and metabolite monitoring in inflammatory bowel disease. J Gastroen-
terol Hepatol. 20, 1149-1157
Gearry R.B., Roberts R.L., Barclay M.L. & Kennedy M.A. (2004) Lack of associ-
ation between the ITPA 94C>A polymorphism and adverse effects from aza-
thioprine. Pharmacogenetics. 14, 779-781
González-Lama Y., Bermejo F., López-Sanromán A. et al. (2011) Thiopurine
methyl-transferase activity and azathioprine metabolite concentrations do not

66
 predict clinical outcome in thiopurine-treated inflammatory bowel disease pa-
tients. Aliment Pharmacol Ther. 34, 544-554
Goyal K., Jo Kim B., Kim J.D., Kim Y.K., Kitaoka M. & Hayashi K. (2002) En-
hancement of transglycosylation activity by construction of chimeras between
mesophilic and thermophilic beta-glucosidase. Arch Biochem Biophys. 407, 125-
134
Grabulovski D., Kaspar M. & Neri D. (2007) A novel, non-immunogenic Fyn SH3-
derived binding protein with tumor vascular targeting properties. J Biol Chem.
282, 3196-3204
Grahn E., Novotny M., Jakobsson E. et al. (2006) New crystal structures of human
glutathione transferase A1-1 shed light on glutathione binding and the conforma-
tion of the C-terminal helix. Acta Crystallogr D Biol Crystallogr. 62, 197-207
Gumulya Y. & Reetz M.T. (2011) Enhancing the thermal robustness of an enzyme
by directed evolution: least favorable starting points and inferior mutants can
map superior evolutionary pathways. Chembiochem. 12, 2502-2510
Gustafsson C., Govindarajan S. & Minshull J. (2003) Putting engineering back into
protein engineering: bioinformatic approaches to catalyst design. Current Opin-
ion in Biotechnology. 14, 366-370
Hansson L.O., Widersten M. & Mannervik B. (1997) Mechanism-based phage dis-
play selection of active-site mutants of human glutathione transferase A1-1 cata-
lyzing SNAr reactions. Biochemistry. 36, 11252-11260
Hiraga K. & Arnold F.H. (2003) General method for sequence-independent site-dir-
ected chimeragenesis. J Mol Biol. 330, 287-296
Holliger P. & Hudson P.J. (2005) Engineered antibody fragments and the rise of
single domains. Nat Biotechnol. 23, 1126-1136
Hosse R.J., Rothe A. & Power B.E. (2006) A new generation of protein display scaf-
folds for molecular recognition. Protein Sci. 15, 14-27
Hubatsch I., Ridderström M. & Mannervik B. (1998) Human glutathione transferase
A4-4: an alpha class enzyme with high catalytic efficiency in the conjugation of
4-hydroxynonenal and other genotoxic products of lipid peroxidation. Biochem
J. 330 ( Pt 1), 175-179
Hughes M.D., Nagel D.A., Santos A.F., Sutherland A.J. & Hine A.V. (2003) Remov-
ing the redundancy from randomised gene libraries. J Mol Biol. 331, 973-979
Johansson A.S. & Mannervik B. (2001) Human glutathione transferase A3-3, a
highly efficient catalyst of double-bond isomerization in the biosynthetic path-
way of steroid hormones. J Biol Chem. 276, 33061-33065
Jones A., Lamsa M., Frandsen T. et al. (2008) Directed evolution of a maltogenic al-
pha-amylase from Bacillus sp TS-25. Journal of Biotechnology. 134, 325-333
Josephy P.D. & Mannervik B. (2006) Molecular toxicology. 2nd edn. Oxford Uni-
versity Press, New York, Oxford
Karran P. (2006) Thiopurines, DNA damage, DNA repair and therapy-related cancer.
Br Med Bull. 79-80, 153-170
Kazlauskas R.J. & Bornscheuer U.T. (2009) Finding better protein engineering
strategies. Nat Chem Biol. 5, 526-529
Kennedy N.A., Asser T.L., Mountifield R.E., Doogue M.P., Andrews J.M. & Bamp-
ton P.A. (2013) Thiopurine metabolite measurement leads to changes in manage-
ment of inflammatory bowel disease. Intern Med J. 43, 278-286
Kim B., Singh S. & Hayashi K. (2006) Characteristics of chimeric enzymes con-
structed between Thermotoga maritima and Agrobacterium tumefaciens beta-
glucosidases: Role of C-terminal domain in catalytic activity. Enzyme and Mi-
crobial Technology. 38, 952-959

67
Kirk O., Borchert T.V. & Fuglsang C.C. (2002) Industrial enzyme applications. Curr
Opin Biotechnol. 13, 345-351
Knight D.M., Trinh H., Le J. et al. (1993) Construction and initial characterization
of a mouse-human chimeric anti-TNF antibody. Mol Immunol. 30, 1443-1453
Kudo M., Moteki T., Sasaki T. et al. (2008) Functional characterization of human
xanthine oxidase allelic variants. Pharmacogenet Genomics. 18, 243-251
Kurtovic S., Grehn L., Karlsson A., Hellman U. & Mannervik B. (2008) Glutathione
transferase activity with a novel substrate mimics the activation of the prodrug
azathioprine. Anal Biochem. 375, 339-344
Lakatos P.L. & Kiss L.S. (2011) Current status of thiopurine analogues in the treat-
ment in Crohn's disease. World J Gastroenterol. 17, 4372-4381
Larsson E., Mannervik B. & Raffalli-Mathieu F. (2011) Quantitative and selective
polymerase chain reaction analysis of highly similar human alpha-class gluta-
thione transferases. Anal Biochem. 412, 96-101
Lee A.U. & Farrell G.C. (2001) Mechanism of azathioprine-induced injury to hep-
atocytes: roles of glutathione depletion and mitochondrial injury. J Hepatol. 35,
756-764
Lees C.W., Maan A.K., Hansoti B., Satsangi J. & Arnott I.D. (2008) Tolerability and
safety of mercaptopurine in azathioprine-intolerant patients with inflammatory
bowel disease. Aliment Pharmacol Ther. 27, 220-227
Low W.Y., Ng H.L., Morton C.J., Parker M.W., Batterham P. & Robin C. (2007)
Molecular evolution of glutathione S-transferases in the genus Drosophila. Ge-
netics. 177, 1363-1375
Lowry P.W., Franklin C.L., Weaver A.L. et al. (2001) Measurement of thiopurine
methyltransferase activity and azathioprine metabolites in patients with inflam-
matory bowel disease. Gut. 49, 665-670
Lunzer M., Golding G.B. & Dean A.M. (2010) Pervasive cryptic epistasis in mo-
lecular evolution. PLoS Genet. 6, e1001162
Maddox J.S. & Soltani K. (2008) Risk of nonmelanoma skin cancer with aza-
thioprine use. Inflamm Bowel Dis. 14, 1425-1431
Mannervik B., Etahadieh M., Fernandez E. et al. (2000) Novel natural substrates and
novel isoenzymes of glutathione transferase. Clin Chem Enzymol Commun. 8,
189–202
Marinaki A.M., Duley J.A., Arenas M. et al. (2004) Mutation in the ITPA gene pre-
dicts intolerance to azathioprine. Nucleosides Nucleotides Nucleic Acids. 23,
1393-1397
McElwee J.J., Schuster E., Blanc E. et al. (2007) Evolutionary conservation of regu-
lated longevity assurance mechanisms. Genome Biol. 8, R132
Meister A. & Anderson M.E. (1983) Glutathione. Annu Rev Biochem. 52, 711-760
Meyer M.M., Hochrein L. & Arnold F.H. (2006) Structure-guided SCHEMA recom-
bination of distantly related beta-lactamases. Protein Eng Des Sel. 19, 563-570
Morgan A.S., Sanderson P.E., Borch R.F. et al. (1998) Tumor efficacy and bone mar-
row-sparing properties of TER286, a cytotoxin activated by glutathione S-trans-
ferase. Cancer Res. 58, 2568-2575
Morley K.L. & Kazlauskas R.J. (2005) Improving enzyme properties: when are
closer mutations better? Trends Biotechnol. 23, 231-237
Mulder T.P., Court D.A. & Peters W.H. (1999) Variability of glutathione S-trans-
ferase alpha in human liver and plasma. Clin Chem. 45, 355-359
Mulder T.P., Peters W.H., Court D.A. & Jansen J.B. (1996) Sandwich ELISA for
glutathione S-transferase Alpha 1-1: plasma concentrations in controls and in pa-
tients with gastrointestinal disorders. Clin Chem. 42, 416-419

68
Neylon C. (2004) Chemical and biochemical strategies for the randomization of pro-
tein encoding DNA sequences: library construction methods for directed evolu-
tion. Nucleic Acids Res. 32, 1448-1459
Nilsson L.O., Edalat M., Pettersson P.L. & Mannervik B. (2002) Aromatic residues
in the C-terminal region of glutathione transferase A1-1 influence rate-determin-
ing steps in the catalytic mechanism. Biochim Biophys Acta. 1598, 199-205
Nilsson L.O., Gustafsson A. & Mannervik B. (2000) Redesign of substrate-selectiv-
ity determining modules of glutathione transferase A1-1 installs high catalytic
efficiency with toxic alkenal products of lipid peroxidation. Proc Natl Acad Sci
U S A. 97, 9408-9412
Ning B., Wang C., Morel F. et al. (2004) Human glutathione S-transferase A2 poly-
morphisms: variant expression, distribution in prostate cancer cases/controls and
a novel form. Pharmacogenetics. 14, 35-44
O'Donovan P., Perrett C.M., Zhang X. et al. (2005) Azathioprine and UVA light gen-
erate mutagenic oxidative DNA damage. Science. 309, 1871-1874
Odlind B., Hartvig P., Lindström B., Lönnerholm G., Tufveson G. & Grefberg N.
(1986) Serum azathioprine and 6-mercaptopurine levels and immunosuppressive
activity after azathioprine in uremic patients. Int J Immunopharmacol. 8, 1-11
Orlova A., Magnusson M., Eriksson T.L. et al. (2006) Tumor imaging using a pico-
molar affinity HER2 binding affibody molecule. Cancer Res. 66, 4339-4348
Otey C.R., Landwehr M., Endelman J.B., Hiraga K., Bloom J.D. & Arnold F.H.
(2006) Structure-guided recombination creates an artificial family of cyto-
chromes P450. PLoS Biol. 4, e112
Parikh M.R. & Matsumura I. (2005) Site-saturation mutagenesis is more efficient
than DNA shuffling for the directed evolution of beta-fucosidase from beta-
galactosidase. J Mol Biol. 352, 621-628
Park H.S., Nam S.H., Lee J.K. et al. (2006) Design and evolution of new catalytic
activity with an existing protein scaffold. Science. 311, 535-538
Pearson D.C., May G.R., Fick G.H. & Sutherland L.R. (1995) Azathioprine and 6-
mercaptopurine in Crohn disease. A meta-analysis. Ann Intern Med. 123, 132-
142
Pearson W.R. (2005) Phylogenies of glutathione transferase families. Methods En-
zymol. 401, 186-204
Poppe D., Tiede I., Fritz G. et al. (2006) Azathioprine suppresses ezrin-radixin-
moesin-dependent T cell-APC conjugation through inhibition of Vav guanosine
exchange activity on Rac proteins. J Immunol. 176, 640-651
Porath J., Carlsson J., Olsson I. & Belfrage G. (1975) Metal chelate affinity chroma-
tography, a new approach to protein fractionation. Nature. 258, 598-599
Prefontaine E., Sutherland L.R., Macdonald J.K. & Cepoiu M. (2009) Azathioprine
or 6-mercaptopurine for maintenance of remission in Crohn's disease. Cochrane
Database Syst Rev, 1, CD000067
Reetz M.T., Kahakeaw D. & Lohmer R. (2008) Addressing the numbers problem in
directed evolution. Chembiochem. 9, 1797-1804
Reetz M.T., Prasad S., Carballeira J.D., Gumulya Y. & Bocola M. (2010) Iterative
saturation mutagenesis accelerates laboratory evolution of enzyme stereose-
lectivity: rigorous comparison with traditional methods. J Am Chem Soc. 132,
9144-9152
Reetz M., Bocola M., Carballeira J., Zha D. & Vogel A. (2005) Expanding the range
of substrate acceptance of enzymes: Combinatorial active-site saturation test.
Angewandte Chemie-International Edition. 44, 4192-4196

69
Reetz M., Wang L. & Bocola M. (2006) Directed evolution of enantioselective en -
zymes: Iterative cycles of CASTing for probing protein-sequence space. Ange-
wandte Chemie-International Edition. 45, 1236-1241
Roberts R.L. & Barclay M.L. (2012) Current relevance of pharmacogenetics in im-
munomodulation treatment for Crohn's disease. J Gastroenterol Hepatol. 27,
1546-1554
Roberts R. & Szostak J. (1997) RNA-peptide fusions for the in vitro selection of
peptides and proteins. Proceedings of the National Academy of Sciences of the
United States of America. 94, 12297-12302
Rowe J.D., Nieves E. & Listowsky I. (1997) Subunit diversity and tissue distribution
of human glutathione S-transferases: interpretations based on electrospray ioniz-
ation-MS and peptide sequence-specific antisera. Biochem J. 325 (Pt 2), 481-
486
Sauer H., Hantke U. & Wilmanns W. (1988) Azathioprine lymphocytotoxicity. Po-
tentially lethal damage by its imidazole derivatives. Arzneimittelforschung. 38,
820-824
Schwab M., Schäffeler E., Marx C. et al. (2002) Azathioprine therapy and adverse
drug reactions in patients with inflammatory bowel disease: impact of thiopurine
S-methyltransferase polymorphism. Pharmacogenetics. 12, 429-436
Scott J. & Smith G. (1990) Searching for peptide ligands with an epitope library.
Science. 249, 386-390
Senter P.D. & Springer C.J. (2001) Selective activation of anticancer prodrugs by
monoclonal antibody-enzyme conjugates. Adv Drug Deliv Rev. 53, 247-264
Shen H., Ranganathan S., Kuzmich S. & Tew K.D. (1995) Influence of ethacrynic
acid on glutathione S-transferase pi transcript and protein half-lives in human
colon cancer cells. Biochem Pharmacol. 50, 1233-1238
Shih D.Q., Nguyen M., Zheng L. et al. (2012) Split-dose administration of thiopur-
ine drugs: a novel and effective strategy for managing preferential 6-MMP meta-
bolism. Aliment Pharmacol Ther. 36, 449-458
Shipkova M., Franz J., Abe M., Klett C., Wieland E. & Andus T. (2011) Association
between adverse effects under azathioprine therapy and inosine triphosphate
pyrophosphatase activity in patients with chronic inflammatory bowel disease.
Ther Drug Monit. 33, 321-328
Siegel J.B., Zanghellini A., Lovick H.M. et al. (2010) Computational design of an
enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction. Science.
329, 309-313
Silva S.N., Azevedo A.P., Teixeira V., Pina J.E., Rueff J. & Gaspar J.F. (2009) The
role of GSTA2 polymorphisms and haplotypes in breast cancer susceptibility: a
case-control study in the Portuguese population. Oncol Rep. 22, 593-598
Smith M.A., Marinaki A.M., Arenas M. et al. (2009) Novel pharmacogenetic mark-
ers for treatment outcome in azathioprine-treated inflammatory bowel disease.
Aliment Pharmacol Ther. 30, 375-384
Sparrow M.P., Hande S.A., Friedman S., Cao D. & Hanauer S.B. (2007) Effect of al -
lopurinol on clinical outcomes in inflammatory bowel disease nonresponders to
azathioprine or 6-mercaptopurine. Clin Gastroenterol Hepatol. 5, 209-214
Stemmer W.P. (1994) Rapid evolution of a protein in vitro by DNA shuffling.
Nature. 370, 389-391
Stocco G., Martelossi S., Barabino A. et al. (2007) Glutathione-S-transferase geno-
types and the adverse effects of azathioprine in young patients with inflammat-
ory bowel disease. Inflamm Bowel Dis. 13, 57-64

70
Sundberg A.G., Nilsson R., Appelkvist E.L. & Dallner G. (1995) ELISA procedures
for the quantitation of glutathione transferases in the urine. Kidney Int. 48, 570-
575
Swann P.F., Waters T.R., Moulton D.C. et al. (1996) Role of postreplicative DNA
mismatch repair in the cytotoxic action of thioguanine. Science. 273, 1109-1111
Tawfik D. & Griffiths A. (1998) Man-made cell-like compartments for molecular
evolution. Nature Biotechnology. 16, 652-656
Tay B.S., Lilley R.M., Murray A.W. & Atkinson M.R. (1969) Inhibition of phos-
phoribosyl pyrophosphate amidotransferase from Ehrlich ascites-tumour cells by
thiopurine nucleotides. Biochem Pharmacol. 18, 936-938
Tetlow N. & Board P.G. (2004) Functional polymorphism of human glutathione
transferase A2. Pharmacogenetics. 14, 111-116
Tetlow N., Liu D. & Board P. (2001) Polymorphism of human Alpha class gluta-
thione transferases. Pharmacogenetics. 11, 609-617
Tiede I., Fritz G., Strand S. et al. (2003) CD28-dependent Rac1 activation is the mo-
lecular target of azathioprine in primary human CD4+ T lymphocytes. J Clin In-
vest. 111, 1133-1145
Tseng W.C., Lin J.W., Wei T.Y. & Fang T.Y. (2008) A novel megaprimed and ligase-
free, PCR-based, site-directed mutagenesis method. Anal Biochem. 375, 376-
378
Van den Brande J.M., Braat H., van den Brink G.R. et al. (2003) Infliximab but not
etanercept induces apoptosis in lamina propria T-lymphocytes from patients with
Crohn's disease. Gastroenterology. 124, 1774-1785
van Dieren J.M., van Vuuren A.J., Kusters J.G., Nieuwenhuis E.E., Kuipers E.J. &
van der Woude C.J. (2005) ITPA genotyping is not predictive for the develop-
ment of side effects in AZA treated inflammatory bowel disease patients. Gut.
54, 1664
van Egmond R., Chin P., Zhang M., Sies C.W. & Barclay M.L. (2012) High TPMT
enzyme activity does not explain drug resistance due to preferential 6-
methylmercaptopurine production in patients on thiopurine treatment. Aliment
Pharmacol Ther. 35, 1181-1189
Voigt C.A., Martinez C., Wang Z.G., Mayo S.L. & Arnold F.H. (2002) Protein build-
ing blocks preserved by recombination. Nat Struct Biol. 9, 553-558
von Ahsen N., Armstrong V.W., Behrens C. et al. (2005) Association of inosine tri-
phosphatase 94C>A and thiopurine S-methyltransferase deficiency with adverse
events and study drop-outs under azathioprine therapy in a prospective Crohn
disease study. Clin Chem. 51, 2282-2288
von Bahr C., Glaumann H., Gudas J. & Kaplowitz N. (1980) Inhibition of hepatic
metabolism of azathioprine by furosemide in human liver in vitro. Biochem
Pharmacol. 29, 1439-1441
Walter K.U., Vamvaca K. & Hilvert D. (2005) An active enzyme constructed from a
9-amino acid alphabet. J Biol Chem. 280, 37742-37746
Wang L. & Weinshilboum R. (2006) Thiopurine S-methyltransferase pharmacogen-
etics: insights, challenges and future directions. Oncogene. 25, 1629-1638
Warshel A. (2003) Computer simulations of enzyme catalysis: Methods, progress,
and insights. Annual Review of Biophysics and Biomolecular Structure. 32, 425-
443
Watts M.E., Hodgkiss R.J., Sehmi D.S. & Woodcock M. (1980) Rapid-mix studies
on the anomalous radiosensitization of mammalian cells by 5-chloro-1-methyl-
4-nitromidazole. Int J Radiat Biol Relat Stud Phys Chem Med. 38, 673-675

71
Weersma R.K., Peters F.T., Oostenbrug L.E. et al. (2004) Increased incidence of aza-
thioprine-induced pancreatitis in Crohn's disease compared with other diseases.
Aliment Pharmacol Ther. 20, 843-850
Weinshilboum R.M. & Sladek S.L. (1980) Mercaptopurine pharmacogenetics:
monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am
J Hum Genet. 32, 651-662
Whittington P.J., Piechocki M.P., Heng H.H. et al. (2008) DNA vaccination controls
Her-2+ tumors that are refractory to targeted therapies. Cancer Res. 68, 7502-
7511
Widersten M., Björnestedt R. & Mannervik B. (1994) Contribution of amino acid
residue 208 in the hydrophobic binding site to the catalytic mechanism of human
glutathione transferase A1-1. Biochemistry. 33, 11717-11723
Wielinga P.R., Reid G., Challa E.E. et al. (2002) Thiopurine metabolism and identi-
fication of the thiopurine metabolites transported by MRP4 and MRP5 overex-
pressed in human embryonic kidney cells. Mol Pharmacol. 62, 1321-1331
Wittrup K.D. & Verdine G.L. (2012a) Protein engineering for therapeutics, Part A.
Preface. Methods Enzymol. 502, xiii-xiv
Wittrup K.D. & Verdine G.L. (2012b) Protein engineering for therapeutics, part B.
Preface. Methods Enzymol. 503, xiii-xiv
Wong D.R., Derijks L.J., den Dulk M.O., Gemmeke E.H. & Hooymans P.M. (2007)
The role of xanthine oxidase in thiopurine metabolism: a case report. Ther Drug
Monit. 29, 845-848
Wong T.S., Wu N., Roccatano D., Zacharias M. & Schwaneberg U. (2005) Sensitive
assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic
compounds. J Biomol Screen. 10, 246-252
Wroblova K., Kolorz M., Batovsky M. et al. (2012) Gene polymorphisms involved
in manifestation of leucopenia, digestive intolerance, and pancreatitis in aza-
thioprine-treated patients. Dig Dis Sci. 57, 2394-2401
Xiong H., Xin H., Wu X., Li Q., Xiong L. & Yu A. (2010) Association between in -
osine triphosphate pyrophosphohydrolase deficiency and azathioprine-related
adverse drug reactions in the Chinese kidney transplant recipients. Fundamental
& Clinical Pharmacology. 24, 393-400
Yano T., Oue S. & Kagamiyama H. (1998) Directed evolution of an aspartate amino-
transferase with new substrate specificities. Proc Natl Acad Sci U S A. 95, 5511-
5515
Yates C.R., Krynetski E.Y., Loennechen T. et al. (1997) Molecular diagnosis of
thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and
mercaptopurine intolerance. Ann Intern Med. 126, 608-614
You L. & Arnold F. (1996) Directed evolution of subtilisin E in Bacillus subtilis to
enhance total activity in aqueous dimethylformamide. Protein Engineering. 9,
77-83
Zabala-Fernández W., Barreiro-de Acosta M., Echarri A. et al. (2011) A pharmaco-
genetics study of TPMT and ITPA genes detects a relationship with side effects
and clinical response in patients with inflammatory bowel disease receiving
Azathioprine. J Gastrointestin Liver Dis. 20, 247-253
Zielinski R., Lyakhov I., Hassan M. et al. (2011) HER2-affitoxin: a potent therapeut-
ic agent for the treatment of HER2-overexpressing tumors. Clin Cancer Res. 17,
5071-5081

72
Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 1050
Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science and

Technology, Uppsala University, is usually a summary of a
number of papers. A few copies of the complete dissertation
are kept at major Swedish research libraries, while the
summary alone is distributed internationally through
the series Digital Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Science and Technology.

ACTA
UNIVERSITATIS
UPSALIENSIS
Distribution: publications.uu.se UPPSALA
urn:nbn:se:uu:diva-167332 2013