CHAPTER SIX

Glutathione Transferases in the Bioactivation of Azathioprine Olof Modén*, Bengt Mannervik*,†,1

*Department of Chemistry-BMC, Uppsala University, Uppsala, Sweden † Department of Neurochemistry, Stockholm University, Stockholm, Sweden 1 Corresponding author: e-mail address: [email protected]

Contents 1. Preamble 2. Background 2.1 Azathioprine 2.2 Glutathione 2.3 Glutathione transferases 3. Polymorphisms 3.1 Genetic polymorphism of human GST A2-2 3.2 Phenotypic differences in expression of different GST genotypes 3.3 Thermal inactivation of allelic GST 2-2 variants 4. Azathioprine and Immunosuppression 4.1 Azathioprine and inflammatory bowel disease 4.2 Azathioprine metabolism 5. Adverse Effects of Azathioprine 5.1 Dose adjustment 5.2 Various adverse effects 5.3 Cancer 5.4 Selectivity 6. Polymorphisms in the Metabolic Pathways of Azathioprine 6.1 Clinical observations 6.2 GST polymorphism 6.3 TPMT polymorphism 6.4 ITPA polymorphism 6.5 Polymorphism in transporters 6.6 XO and AO polymorphisms 6.7 Rac1 polymorphism 6.8 Polymorphism summary 7. Structural Requirements for High GST Activity with Azathioprine 7.1 Chimeric GST variants obtained by DNA shuffling of homologous sequences 7.2 Stochastic chimeragenesis

Advances in Cancer Research, Volume 122 ISSN 0065-230X http://dx.doi.org/10.1016/B978-0-12-420117-0.00006-2

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7.3 Sequence analysis 7.4 Structure–activity relationships 7.5 Some H-site residues conserved among the active chimeric mutants 7.6 Other segments of interest 7.7 Structural interpretation 7.8 An example of an active GST lacking C-terminal amino acid 222 8. Rational Design of Chimeras 8.1 Strategy 8.2 Design of chimeric GSTs with flanking N- and C-terminal sequences from GST A2-2 8.3 Characterization of the designed chimeras 9. Saturation Mutagenesis of two H-Site Residues in the C-Terminal Region 9.1 Synthesis of mutant library 9.2 Screening and mutant characterization 10. Redesign of GST A2-2 for Enhanced Azathioprine Activity 10.1 Targeted H-site residues 10.2 Mutant library based on reduced codon sets 10.3 Kinetic characterization of mutants isolated 10.4 Comparison with other alpha class sequences 10.5 Structural comparisons 11. Concluding Remarks Acknowledgments References

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Abstract The prodrug azathioprine is primarily used for maintaining remission in inflammatory bowel disease, but approximately 30% of the patients suffer adverse side effects. The prodrug is activated by glutathione conjugation and release of 6-mercaptopurine, a reaction most efficiently catalyzed by glutathione transferase (GST) A2-2. Among five genotypes of GST A2-2, the variant A2*E has threefold–fourfold higher catalytic efficiency with azathioprine, suggesting that the expression of A2*E could boost 6-mercaptopurine release and adverse side effects in treated patients. Structure–activity studies of the GST A2-2 variants and homologous alpha class GSTs were made to delineate the determinants of high catalytic efficiency compared to other alpha class GSTs. Engineered chimeras identified GST peptide segments of importance, and replacing the corresponding regions in low-activity GSTs by these short segments produced chimeras with higher azathioprine activity. By contrast, H-site mutagenesis led to decreased azathioprine activity when active-site positions 208 and 213 in these favored segments were mutagenized. Alternative substitutions indicated that hydrophobic residues were favored. A pertinent question is whether variant A2*E represents the highest azathioprine activity achievable within the GST structural framework. This issue was addressed by mutagenesis of H-site residues assumed to interact with the substrate based on molecular modeling. The mutants with notably enhanced activities had small or polar residues in the mutated positions. The most active mutant L107G/L108D/F222H displayed a 70-fold enhanced catalytic efficiency with azathioprine. The determination of its

GSTs Activate Azathioprine

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structure by X-ray crystallography showed an expanded H-site, suggesting improved accommodation of the transition state for catalysis.

1. PREAMBLE Azathioprine was originally developed as an anticancer agent based on its biotransformation that releases the antimetabolite 6-mercaptopurine (6MP). However, both azathioprine and 6-MP have been used more widely as immunosuppressive 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 drugs that not only act faster than the thiopurines but also have more adverse effects (Barba et al., 2012), while the results for organ function and cancer incidence are similar in the longer perspective (Clayton, McDonald, Chapman, & Chadban, 2012). In spite of the widespread clinical use of azathioprine for half a century, the enzymology of the first step of the activation of azathioprine has remained obscure until recently (Eklund, Moberg, Bergquist, & Mannervik, 2006). In this chapter, the role of glutathione transferases (GSTs) will be placed in the context of azathioprine metabolism with possible implications for clinical side effects. Particular focus will be placed on human glutathione transferase A2-2 and molecular features that govern relationships between protein structure and catalytic activity with azathioprine.

2. BACKGROUND 2.1. Azathioprine The thiopurine drugs 6-MP and 6-thioguanine were developed in the 1950s by George Hitchings and Gertrude B. Elion (Giner-Sorolla, 1988). Hitchings had theorized that antagonists of DNA bases might be able to inhibit the growth of rapidly dividing cells, and Elion screened several purine derivatives for the inhibition of purine utilization (Elion, 1989). 6-MP turned out to be active against tumors in animal models and acute leukemia in children. Azathioprine is a 1-methyl-4-nitroimidazole-5-yl derivative of 6-MP (Fig. 6.1), which was designed as a prodrug intended to release 6-MP slowly and preferably by a tumor-specific enzyme. For childhood leukemia, the chemotherapeutic index of azathioprine and 6-MP was similar, while for a carcinoma model in mice, azathioprine was similarly active but less toxic.

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Azathioprine CH3

N

6-MP

N SH NO2

S

N

N

N

N

N N

N H

N H

Figure 6.1 Structural formulas of azathioprine and 6-mercaptopurine (6-MP).

Robert Schwartz proposed that since the immunoblastic lymphocytes formed during an immune response were similar to leukemic lymphocytes, the purine analogs might inhibit the immune response (Schwartz & Dameshek, 1959). 6-MP given to rabbits simultaneously with antigen administration was indeed effective. Calne and coworkers (1962) proved that kidney transplantation in dogs was possible with immunosuppression by 6-MP, and later, studies showed that azathioprine was superior for preventing rejection. Since its introduction, the clinical use of azathioprine has been dominated by its ability to suppress the immune system. In the early 1960s, kidney transplantations in humans became successful by the administration of azathioprine and antiinflammatory corticosteroids. The pharmacological action of azathioprine is based on the release of 6-MP effected by the chemical elimination of the imidazole moiety. Glutathione is the most abundant low-molecular-mass thiol in the cell ( Josephy & Mannervik, 2006), and the thiolysis of azathioprine by glutathione has been established as the pivotal biotransformation in crude liver preparations (Kaplowitz, 1976). Although the original work suggested the involvement of GST activity, it was not until studies involving purified enzymes were performed that the overwhelming contribution of the enzymatic reaction was established (Eklund et al., 2006).

2.2. Glutathione The tripeptide glutathione (Fig. 6.2) is synthesized intracellularly, while the degradation of glutathione conjugates is initiated extracellularly by g-glutamyl transferase on the cell membrane (Ballatori, Hammond, Cunningham, Krance, & Marchan, 2005). The concentration of reduced glutathione has been estimated at about 1–2 mM in human plasma and about 1 mM in whole blood (Andersson, Lindgren, Arnadottir,

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GSTs Activate Azathioprine

SH O



CH2

O N H

O NH3+

O H N

O–

O

Figure 6.2 Glutathione (g-L-glutamyl-L-cysteinylglycine or g-L-Glu-L-Cys-Gly).

Prytz, & Hultberg, 1999) while estimated to be in the range 0.5–10 mM inside mammalian cells (Meister & Anderson, 1983). The thiol group of glutathione is a nucleophile and the ionized thiolate is an even more reactive chemical group that can attack electrophilic centers in other molecules. In addition to conjugation reactions, glutathione can serve as an antioxidant and reduce peroxides and other reactive oxidative species.

2.3. Glutathione transferases 2.3.1 Function GSTs are enzymes that catalyze the reaction of glutathione with electrophiles of both endogenous and xenobiotic origins. The main biological roles of GSTs encompass detoxification and protection against oxidative stress. By conjugating glutathione with toxic electrophilic substrates, the resulting molecules generally become less reactive and more soluble, thus facilitating their excretion from cells and the organism. The GSTs are promiscuous in their substrate acceptance, and collectively, they can deal with various xenobiotics or metabolic by-products that otherwise could be harmful and damage DNA and other cell constituents ( Josephy & Mannervik, 2006). It is noteworthy that certain GSTs are upregulated in long-lived strains of nematodes, fruit flies, and mice (McElwee et al., 2007). The protective effects of GSTs against xenobiotics are also exemplified by herbicide resistance in plants (Dixon, Lapthorn, & Edwards, 2002), insecticide resistance in flies (Low et al., 2007), and drug resistance in tumors (Morgan et al., 1998). There are four families of GSTs: cytosolic, mitochondrial, microsomal/membrane-associated, and fosfomycin/ glyoxalase (Pearson, 2005). In humans, there are 17 different cytosolic GSTs, divided into 7 classes (alpha, mu, omega, pi, sigma, theta, and zeta) based on sequence similarity; the genes of the different classes are located on seven distinct chromosomes (Mannervik, Board, Hayes, Listowsky, & Pearson, 2005).

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Figure 6.3 Structure of human GST A2-2 in complex with glutathione determined by X-ray crystallography (PDB entry 2WJU). View along the twofold symmetry axis with the two subunits to the left and to the right, each binding a glutathione molecule (rendered as CPK structure) in the G-site.

2.3.2 Structure The cytosolic GSTs are dimeric proteins with one catalytic center in each subunit, located near the interface between the subunits (Fig. 6.3). The G-site binds and activates glutathione, while the H-site accommodates the hydrophobic second substrate (Mannervik, Guthenberg, Jakobson, & Warholm, 1978). The amino acids forming the H-site are located in three separate regions of the primary structure. Each subunit consists of an N-terminal thioredoxin-like domain with secondary structure bababba and an all-helical C-terminal domain. The alpha class has five homodimeric members in humans, GSTs A1-1 to A5-5, and the genes of the subunits are located in a cluster on chromosome 6. Most alpha class genes encode GSTs with 222 amino acids and each subunit has a molecular mass of around 26 kDa. In addition to homodimers, the subunits can also form heterodimers, for example, GST A1-2 (Mannervik et al., 2005). Among 14 human GSTs investigated, only three showed relatively high activity with azathioprine: GSTs A1-1, A2-2, and M1-1 (Eklund et al., 2006). GST M1-1 is polymorphic with a null allele and approximately half the human population is lacking this enzyme (Warholm, Guthenberg, Mannervik, & von Bahr, 1981). This chapter will focus primarily on the homodimeric GST A2-2, which has the highest catalytic efficiency of all GSTs.

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Glutathione

Azathioprine CH3

N

6-MP

N

CH3

SH



G S H+

GS-imidazole

S

+

NO2

N

N

N

N

N N

N H

N H

+

N S

N NO2

G

Figure 6.4 The reaction of glutathione with azathioprine. The electrophilic carbon of azathioprine is attacked by the sulfur of the glutathionylate, forming the glutathione–imidazole conjugate and 6-mercaptopurine (6-MP).

2.3.3 Mechanism of catalysis Glutathione bound to an alpha class GST is first activated by deprotonation of its thiol group. The glutamyl a-carboxylate group of the glutathione receives the proton, while active-site water assists as a bridge (Dourado, Fernandes, Mannervik, & Ramos, 2008). The thiolate formed is stabilized by hydrogen bonding to a conserved tyrosine (Tyr9) and thus ready for nucleophilic attack on the electrophilic second substrate. The reactions catalyzed by GSTs can be substitutions, additions, or isomerizations, depending on the enzyme and the nature of the second substrate ( Josephy & Mannervik, 2006). The reaction with azathioprine is an aromatic substitution reaction (Fig. 6.4). 2.3.4 Tissue distribution of human GSTs Human GSTs A1-1 and A2-2 are expressed at high levels in the liver, small intestine, testis, kidney, adrenal gland, and pancreas and at lower levels in other tissues (Table 6.1). GST A3-3 is expressed in steroidogenic tissues ( Johansson & Mannervik, 2001; Larsson, Mannervik, & Raffalli-Mathieu, 2011), and GST A4-4 is expressed in a wide range of tissues, whereas no expression of A5-5 has been noted. Variable concentrations of GSTs have been reported for alpha class GSTs in several tissues (Table 6.2). The ratios between the minimum and the maximum expression levels were at the most 50-fold for GST A1-1 and 15-fold for GST A2-2 (Coles & Kadlubar, 2005). 2.3.5 Biomarker applications The presence of alpha class GSTs in blood plasma is low in healthy individuals (median 2.6 mg l1, range 0.2–20.4 mg l1, and N ¼ 350; Mulder et al., 1999). Alpha class GSTs can therefore be used as a sensitive marker of liver disease

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Table 6.1 Tissue expression and specific activities with azathioprine of human GSTs Specific activity GST Small intestine Erythrocytes Liver (mmol mg1 min1)

A1-1

+



+

0.24

A2-2

+



+

0.53

A3-3







0.01

A4-4

+



+

0.01

M1-1

(+)



(+)

M2-2







0.05

M3-3

+



+

Glutathione transferases in the bioactivation of azathioprine.

The prodrug azathioprine is primarily used for maintaining remission in inflammatory bowel disease, but approximately 30% of the patients suffer adver...
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