Glutathione Transferases: Emerging Multidisciplinary

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Sep 1, 2009 - Abstract: Cytosolic glutathione transferases (GSTs) are a diverse family ... the conjugation of the tripeptide glutathione (GSH) to an electrophilic.
Recent Patents on Biotechnology 2009, 3, 211-223

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Glutathione Transferases: Emerging Multidisciplinary Tools in Red and Green Biotechnology Evangelia G. Chronopoulou and Nikolaos E. Labrou* Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855-Athens, Greece Received: July 23, 2009; Accepted: August 20, 2009; Revised: September 1, 2009

Abstract: Cytosolic glutathione transferases (GSTs) are a diverse family of enzymes involved in a wide range of biological processes, many of which involve the conjugation of the tripeptide glutathione (GSH) to an electrophilic substrate. Detailed studies of GSTs are justified because of the considerable interest of these enzymes in medicine, agriculture and analytical biotechnology. For example, in medicine, GSTs are explored as molecular targets for the design of new anticancer drugs as a plausible means to sensitize drug-resistant tumors that overexpress GSTs. In agriculture, GSTs are exploited in the development of transgenic plants with increased resistance to biotic and abiotic stresses. Recently, selected isoenzymes of GSTs have found successful applications in the development of enzyme biosensors for the direct monitoring of environmental pollutants, such as herbicides and insecticides. This review article summarizes recent representative patents related to GSTs and their applications in biotechnology.

Keywords: Glutathione transferase, CDNB, 1-chloro-2,4-dinitrobenzene, G-site, glutathione binding site, GSH, glutathione, GST, glutathione transferase, H-site, hydrophobic binding site. INTRODUCTION Glutathione transferases (GSTs, EC. 2.5.1.18, formerly known as glutathione S-transferases) are multifunctional Phase II detoxification enzymes which protect cellular macromolecules from being attacked by reactive electrophiles [13]. GSTs catalyze Fig. (1) the nucleophilic addition of the sulfur atom of glutathione (-L-Glu-L-Cys-Gly; GSH) to the electrophilic groups of a large variety of hydrophobic molecules (products of metabolism or xenobiotics), thereby increasing their solubility and aiding their excretion from the cell [1-6]. GSTs exhibit wide substrate specificity toward electrophilic molecules including organic halides, epoxides, arene oxides, - and -unsaturated carbonyls, organic nitrate esters, and organic thiocyanates [1-5]. Importantly, although many of these reactions are catalyzed by several different GSTs, each isoform exhibits its own substrate selectivity [6,7]. GSTs not only catalyze the conjugation of GSH to electrophilic compounds but they also have more functions. For example, some members are involved in the biosynthesis of prostaglandins, in GSH-dependent isomerization reactions (e.g. in GSH-dependent isomerization of maleylacetoacetate to fumarylacetoacetate, in double-bond isomerizations of delta(5)-androstene-3,17-dione and delta(5)-pregnene-3,20dione), catalyze the reduction of toxic organic hydroperoxides, providing protection against oxidative stress [710]. Typical GST-catalyzed reactions are shown in Fig. (1). In addition, in view of their ligand binding properties, GSTs are implicated in the intracellular transport and storage *Address correspondence to this author at the Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 - Athens, Greece; Tel/Fax: +30-210-5294308; E-mail: [email protected] 1872-2083/09 $100.00+.00

of a broad range of structurally diverse hydrophobic ligands, such as haem, bilirubin, hormones, flavonoids, fatty acids and xenobiotics [9,10]. STRUCTURE AND FUNCTION OF CYTOSOLIC GSTs Cytosolic GSTs are divided in multigene families in mammalian, insects, plants, bacteria and fungi [2,10,11] (Table 1). Each one of these families has been subdivided into classes based on a variety of criteria, including amino acid and nucleotide sequence, tertiary and quaternary structural properties [1, 2]. Among GSTs members of each class, there is amino acid sequence identity more than 40%, while between different classes proteins have less than 25% sequence identity [7]. GSTs are composed of two subunits Fig. (2) and are either homodimers, of a single gene product, or heterodimers encoded by different genes. Each subunit is composed of 200-250 amino acid residues with typical molecular masses ranging from 20–28 kDa [2,10,12-14]. The heterodimeric forms are of considerable interest because they seem to be induced under conditions of hormone action and stress [13]. Analysis of the GST gene family in the Human Genome Organization database showed 21 putatively functional genes [15]. Upon closer examination, however, GST-kappa 1 (GSTK1), prostaglandin E synthase (PTGES) and three microsomal GSTs (MGST1, MGST2, MGST3) were determined as encoding membrane-bound enzymes having GST-like activity, but these genes are not evolutionarily related to the GST gene family [15]. Microsomal GSTs are structurally distinct from the cytosolic as they homo- and heterotrimerize rather than dimerize to form a single active site [3]. Microsomal GSTs play a key role in the endogenous © 2009 Bentham Science Publishers Ltd.

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B

A (1)

(2)

(3)

(4)

(5)

Fig. (1). A: Glutathione conjugation to a generic xenobiotic (X) via GST results in the formation of a glutathione-S conjugate. B: Typical GST-catalyzed reactions. (1): nucleophilic aromatic substitution with 1-chloro-2,4-dinitrobenzene, (2): Michael-type addition reaction with ethacrynic acid, (3): nucleophilic addition to epoxide, (4): cis-trans double bond isomerization of delta(5)-androstene-3,17-dione, (5): hydroperoxide reduction with cumene hydroperoxide.

GSTs in Biotechnology

Table 1.

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Classes and Biological Function of Cytosolic GSTs

Organism

Class

Function

Active Site Residue

Mammalian

Alphaa

Isomerase activities, drug metabolism, peroxidase activity, detoxification.

Tyrosine

Drug metabolism.

Tyrosine

Pi

Drug metabolism.

Tyrosine

Theta

Prevention of hepatocarcinogenesis, metabolism of industrial compounds.

Serine

Zeta

Catalyze the metabolism of a-haloacids.

Serine

Omega

Oxidative stress.

Cysteine

Sigma

Prostaglandin synthesis.

Tyrosine

Catabolism of organic compounds.

Cysteine

Mu

a

a

Bacteria

Beta

a

(other GSTs are likely to be present) Chi

Unknown

a

Plants

Phi

Tau

Detoxification function, against oxidative stress, signaling function, noncatalytic bindingof flavonoids, participation in intermediary metabolism.

a

Theta

Serine Serine

Zeta

Serine

Lambda

a

Cysteine

Glutathione-dependent dehydroascorbate reductases (DHAR)

Cysteine

Alpha

Unknown

Mu

Unknown

Fungi

Gamma Insects

Delta

a

Unknown Probably detoxification of environmental xenobiotics.

Serine

Detoxification of insecticides, peroxidase activity, oxidative stress.

Serine

Theta

Unknown

Serine

Sigma

Probably against by products of oxidative stress. May have a role in muscle function.

Tyrosine

Zeta

Tyrosine degradation pathway.

Serine

Omega

Unclear (probably against oxidative stress).

Cysteine

Epsilon

a

Serine

a

organism-specific classes

metabolism of leukotrienes and prostaglandins [3]. Therefore, the complete human GST gene family comprises 16 genes in six subfamilies: alpha (GSTA), mu (GSTM), omega (GSTO), pi (GSTP), theta (GSTT) and zeta (GSTZ) (Fig. (3)). Although these classes have evolved from a single common ancestor, their substrate specificity and diversity have been reshaped by gene duplication, genetic recombination, and an accumulation of mutations [7,14]. In other organism additional soluble GST classes have been reported. For example, in insects: delta and epsilon [5,16]; in plants: phi, tau, lambda, dehydroascorbate reductase [10,17]; and in bacteria: beta [18] and chi [19]. The cytosolic GSTs are related to glutaredoxin, containing a thioredoxin domain with a unique  topology [20-22]. In glutaredoxins, the catalytic residues are

two cysteines in a Cys-Pro-Tyr-Cys motif. These cysteines form a disulfide bond when oxidized and a mixed disulfide bond between GSH and the first cysteine during reduction. The human omega and bacterial beta GSTs also form mixed disulfide bonds with GSH [22]. Each subunit of cytosolic GSTs has two domains (Fig. (2)), an / domain that includes 1-3 helices and a large helical domain comprised of helices 4-9 [7,9,14]. Furthermore, in each subunit there are four beta sheets to produce two distinct domains, the N- and C- terminal domains. The interface between the two subunits can be hydrophobic or hydrophilic, and interactions between residues in both subunits are essential for dimer stability [9, 21]. Each subunit have two distinct binding sites: the G-site and the H-

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A

B

C

Fig. (2). A. Subunit structure of tau class GmGSTU4-4 from soy (for details see [9]). The G- and H-site and the N- and C-terminal are labeled. Bound S-(p-nitrobenzyl)-glutathione (active-site inhibitor) is shown in a stick representation. B: Ribbon diagram of the dimeric GmGSTU4-4 structure. The 2-fold axis relating the dimer subunits is perpendicular to the plane of the page. Bound ligand (GSH) is shown in a stick representation. C: A molecular surface representation of GmGSTU4-4 dimer. Bound GSH is shown in stick representation. The Figures were produced using PyMol (DeLano Scientific).

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Fig. (3). Ribbon diagram representation of the common chain fold of the GST superfamily. GSTs of classes: alpha; theta, mu, omega, zeta, sigma, tau, phi, and pi are depicted. Images in this figure were drawn using the program PyMol (DeLano Scientific).

site for GSH and xenobiotic substrate binding, respectively Fig. (2). The G-site is mainly composed of amino acids in the N-terminal, including the active site residue. The H-site is mainly found in the C-terminal and determines the substrate specificity for the hydrophobic substrate [2, 7, 9, 14, 23-25].

roascorbate reductase), which contain several members [24, 25]. For example, 25 soybean (Glycine max), 42 maize (Zea mays) 59 rice (Oryza sativa), and 54 Arabidopsis (Arabidopsis thaliana) isoenzymes, have been identified so far. Some of them have been characterized in detail [9, 2529] and patented [30-32].

The fundamental basis for all GSTs catalytic activities, is the capacity of these enzymes to lower the pKa of the sulfydryl group of reduced GSH from 9.0, in aqueous solution, to about 6.5, when GSH is bound in the active site [26-28]. In the alpha, mu, pi, and sigma classes the active site residue is a Tyr (Table 1). In the delta, epsilon, theta, phi, tau and zeta GSTs, the active site residue is a Ser, and in omega and beta class GSTs, it is a Cys [22]. Several crystal structures have shown that the active site residue is in hydrogen bond with the sulfur atom of GSH and is located at a position that allows it to stabilize the thiolate anion of GSH and enhance its nucleophilicity [9, 26, 29].

Plant GSTs have been intensively studied for their involvement in herbicide detoxification (e.g. triazines, thiocarbamates, chloroacetanilides, diphenylethers, and aryloxyphenoxypropionates) and several GSTs are known to be responsible for herbicide tolerance in crops and resistance in weeds [6, 9, 13, 17, 25, 29]. The first GST reported to be involved in herbicide metabolism (atrazine) was isolated from maize [33]. Herbicide tolerance is based primarily on the differential ability of plant species to detoxify a herbicide, with the formation of a herbicide-GSH conjugate in the resistant but not in the susceptible species. The plantspecific phi and tau GSTs are primarily responsible for herbicide detoxification, showing class specificity in substrate preference [29, 34, 35]. For example, phi enzymes (GSTFs) are highly active toward chloroacetanilide and thiocarbamate herbicides [36], whereas the tau enzymes (GSTUs) are efficient in detoxifying diphenylethers and aryloxyphenoxypropionates [9, 29, 35].

DEVELOPMENT OF HERBICIDE AND STRESSTOLERANT PLANTS The family of plant GSTs is composed of eight classes (phi, tau, theta, zeta, lambda, glutathione-dependent dehyd-

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The use of genetic engineering has made possible the enhancement of certain traits and the transfer of metabolic capabilities from one plant to another. GSTs, due to their ability to detoxify herbicides, and toxic organic peroxides are considered as candidates for the development of herbicide and stress tolerant transgenic crops. Several successful approaches have been reported for the generation of transgenic plants overexpressing GST’s isoenzymes [32, 3747]. For example, Roxas et al. showed enhanced seed germination and seedling growth under stressful condition by expressing the isoenzyme ZmGST27 from maize [39,40]. In these transgenic plants, increased GSH-dependent peroxide scavenging and alterations in glutathione and ascorbate metabolism lead to reduced oxidative damage achieved by these stressful conditions. In another study transgenic tobacco plants expressing a chimeric rat GST gene [46] or an engineered form of ZmGSTU1 from maize [37] demonstrated resistance to herbicides which are normally inhibitory to plants not expressing the engineered constructs. More recently, Karavageli et al., have developed transgenic tobacco plants expressing the GST I gene from maize [41] Fig. (4). The transgenic plants showed substantially higher tolerance to alachlor compared to non-transgenic plants in terms of root, leaves and vigorous development. In another example, transgenic cotton (Gossypium hirsutum L.) lines expressing the tobacco GST Nt107 isoenzyme were evaluated for tolerance to chilling, salinity, herbicides, antioxidant enzyme activity, antioxidant compound levels, and lipid peroxidation [42]. Although transgenic seedlings exhibited ten-fold and five-fold higher GST activity under normal and saltstress conditions, respectively, germinating seedlings did not show improved tolerance to salinity, chilling conditions, or herbicides. These results show that expression of Nt107 in cotton does not provide adequate protection against oxidative stress and suggests that

Chronopoulou and Labrou

the endogenous antioxidant system in cotton may be disrupted by the expression of the tobacco GST. On the other hand a GST gene (GST-cr1) from cotton was introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation [43]. Transgenic tobacco plants overexpressing GST-cr1 were normal in growth compared with control plants, exhibited much higher levels of GST and GPx activities. These transgenic plants showed an enhanced resistance to oxidative stress induced by methyl viologen. Toshikazu et al., have reported that expression of Suaeda salsa GST in transgenic rice (Oryza sativa) resulted in a different level of abiotic stress (salt, paraquat and chilling) resistance [47]. In another example, the PjGST gene from Prosopis juhflora has been explored for producing abiotic stress (salt and/or drought stress) tolerant transgenic rice and tobacco plants [48]. DEVELOPMENT OF BIOSENSORS In the last two decades there has been an intense research interest in the field of enzyme-based bioassays and biosensors, as the need for cheap, fast and easy assessment of the composition of a sample has significantly increased [49-51]. Biosensors can provide real-time qualitative and quantitative information with minimum sample preparation and data of superior accuracy and specificity compared to traditional wet chemistry and instrumental methods. Moreover they have the advantage of being environmentally benign and safer for the user [23] and allow the simultaneous measurement of several samples [52]. The ability of GSTs to catalyze GSH-conjugation reactions has been explored for the development of three enzyme biosensors for the determination of herbicides. For example, the isoenzyme GST I from maize was used in its immobilized form for the development of fiber-optic portable

Fig. (4). The influence of herbicide alachlor in transgenic and non-transformed tobacco plants after 20 days. On the left is a transgenic plant in Murashige and Skoog (MS) medium where the concentration of the herbicide is 15 mg/L and on the right is a non-transformed tobacco plant where the concentration of the herbicide is 15 mg/L (for details see [41]).

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biosensor for the determination of commonly used pesticide atrazine [53]. In this study calibration curve was obtained for atrazine, with analytically useful concentration range 2.52– 125 M. The sensor detection limit was 0.84 M. The reproducibility of atrazine sensing was in the order of ±3– 5%. In another example, the isoenzyme GmGSTU4-4 from soy was used in the development of biosensor for the determination of chloroacetanilide herbicide alachlor [49]. GmGSTU4-4 catalyses the conjugation of GSH with alachlor with concomitant release protons (HCl), the concentration of which is proportional to the amount of conjugated alachlor. The optical signal, as a result of the reaction, was measured at 600 nm using bromocresol green as a pH indicator. More recently, the GST isoenzyme AaGSTE2-2 from the mosquito Aedes aegypti was employed to produce a highly specific enzyme assay for the determination of DDT [dichlorodiphenyl-trichloroethane1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane][50]. Detection was based on the pH change occurring in an appropriate buffer system during the AaGSTE2-2-catalysed dehydrochlorination reaction. pH change was monitored potentiometrically or colorimetrically in the presence of a pH indicator. The theoretical limit of detection of the assay was determined 3.8 μg/mL and the linear range of quantification 11.5-250 g/mL. Enzyme-based bioassays are relay on the inhibition of the enzymes by xenobiotics which block substrate turnover and indirectly allow the detection of the xenobiotic [23, 51, 54]. While these systems are sensitive, their specificity is limited due to interference by homologues or irrelevant non-target molecules, which can also act as inhibitors. A lot of effort is required to discover or tailor enzymes to recognize target xenobiotic molecules with high specificity. Detection and

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quantification of the xenobiotics can thus be based on inhibition measurements of reaction rates using spectrophotometric or ion-selective potentiometric methods [23, 51, 54]. For example, a colorimetric GST assay was developed for the quantification of pyrethroid insecticides, using iodometric titration [54]. However, the assay was technically demanding and of moderate accuracy, as it was based on the detection of the non conjugated substrate GSH, only a small fraction of which is utilized in the enzymatic reaction. In another example, an optical biosensor was developed for fast and sensitive detection of captan (up to 2 ppm) in water supplies. Captan is known as potential carcinogen and harmful chemical to water ecosystem [52]. More recently, the mutant Gln53Ala of maize GST I which exhibits 9.2-fold higher inhibition potency for the insecticide malathion Fig. (5), compared to the wild-type enzyme, was used for the development of potensiometric biosensor for the determination of malathion [23]. The assay explores the ability of malathion to promote inhibition of the GST-catalysing 1chloro-2,4-dinitrobenzene (CDNB)/GSH conjugation reaction. The sensing scheme is based on the pH change occurring in a low buffer system by the GST reaction, which is measured potentiometrically using a pH electrode Fig. (6). Calibration curves were obtained for malathion, with useful concentration ranges 0-20 μ. The method’s reproducibility was in the order of ±3–5% and malathion recoveries were 96.7±2.8%. Another interesting approach was resently developed by Hasegawa et al., (2007) for acrylamide detection in starchy foods [55]. Acrylamide is considered as a dangerous for human health substance and a rapid and inexpensive way for

Fig. (5). The predicted mode of interaction of the mutant Gln53Ala of maize GST I with malathion. Molecular surfaces around the binding site encompassing the bound inhibitor malathion were shown. Bound malathion is shown in a stick representation (for detals see [23]).

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Fig. (6). A: The reaction of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH, catalyzed by GSTs. B: Schematic diagram of a GST-electrode. The sensing bioactive material is an immobilized enzyme layer. The enzyme is immobilized in front of pH-electrode.

its detection is essential. The gst-4 gene from C. elegans was selected and constructed a gst::gfp fusion gene, which was used to transform C. elegans into a biosensor for acrylamide. This biosensor detects acrylamide as a GFP-expression signal in a dose- and time-dependent manner. In absence of acrylamide there is not emission of GFP signal while is emitted a very strong GFP signal from the whole body with 500 mg/L of acrylamide [55]. APPLICATIONS IN MEDICINE Diagnostics A strong aspect of GSTs in medicine is that cytosolic GSTs can play an important role in diagnosis and monitoring of clinic course of cancer. For example, GSTP1-1 levels of patients with gastric cancer [56] and gliomas [57] increased as the tumor progressed. Moreover, studies have shown that GSTP1-1 may be a useful immunohistological marker for many cancers since its expression was increased in precancerous lesions and in various malignant tumor tissues [58]. Recently was developed a simple method for assaying hepato-neoplastic lesion or pre-neoplastic lesion using an antibody recognizing placental GST and co-evaluating the expression level of the gene of 2-macroglobulin [59]. Epidemiological studies that investigate the association between genetics polymorphisms of GSTs and specific diseases such as bladder cancer risk have been carried out

over the last years [60]. It has been found that the polymorphisms of human GST genes are implicated in response to cancer therapy [61-67]. The subtypes and the variants of GSTs may become in the future additional factors to consider for the best individuated course of therapy [61,62] For example, the allozyme GSTP1 105Val has been reported to be associated with longer survival of patients who receive combination chemotherapy for advanced colorectal cancer [63]. In recent patents high-throughput PCR assays for characterizing a subject's genetic makeup are described. Specifically, these assays permit the rapid and accurate characterization of a subject's inherited alleles of the polymorphic GST genes GSTM1, GSTM3, GSTP1, and GSTT1 [64-68]. GSTs are not only related with cancer. The isoenzymes from pi and alpha classes are also highly expressed in liver and kidney diseases [69]. Plasma levels of GST-pi have been found to be elevated in chronic hepatitis, chronic cholestatic diseases, primary biliary cirrhosis and transplant rejection. Hence, these data suggest that GST measurements in plasma might be useful in reflecting hepatic status [58]. The Omega class GSTs (GSTO) have a unique range of enzymatic activities compared with other GSTs. For example, the isoenzyme GSTO1-1 exhibits dehydroascorbate reductase and thioltransferase activities and also catalyzes the reduction of monomethylarsonate, an intermediate in the

GSTs in Biotechnology

pathway of arsenic biotransformation [21,70]. Other diverse actions of human GSTO1-1 include modulation of ryanodine receptors and interaction with cytokine release inhibitory drugs. In addition, GSTO1 has been linked to the age at onset of both Alzheimer’s and Parkinson’s diseases [70]. In this case, the role of GSTO enzymes in the reduction of dehydroascorbate in the brain may be the basis of their genetic linkage to age at onset of Alzheimer’s and Parkinson’s disease [70,71]. In addition, several polymorphisms have been identified in the coding regions of the human GSTO genes. A method for the determination a GSTO2 sequence variant as well as the sequences of GSTO2 allozymes have been patented by Weinshilboum, et al. [72]. Drug and Pro-Drug Design It is well established that GSTs are involved in an efficient detoxification of several chemotherapeutics and therefore are considered potentially crucial in regulating the susceptibility to cancer [73]. Several studies have shown that GSTs are expressed at higher levels in drug-resistance cancer cell lines compared with parental cell lines and normal tissues [74-80]. The expression of GSTs, especially of the pi class, has been related to the expression of the multidrug resistance (MDR) related P-glycoprotein [79]. The pi and mu classes of GSTs play a regulatory role in the mitogenactivated protein (MAP) kinase pathway that participates in cellular survival and death signals via protein-protein interactions with c-Jun N-terminal kinase 1 (JNK1) and ASK1 (apoptosis signal-regulating kinase). It is plausible that GSTs serve two distinct roles in the development of drug resistance via direct detoxification as well as acting as an inhibitor of the MAP kinase pathway [73]. Hence, the therapeutic strategies that can be developed may involve: (a) the design of GST inhibitors to act as modulatory agents in cases where anticancer agents are detoxified by GSTs, (b) the design of compounds that can inhibit the protein-protein interactions of GSTs with stress kinases and (c) the exploitation of elevated expression of GST in tumors, particularly GSTP1-1, through design of GST-activated prodrugs [73]. In the past, modulation by inhibition of GST has been attempted as a means to improve response to cancer drugs [74]. In concept, the drug was envisioned as a plausible means to sensitize drug-resistant tumours that overexpress GSTs [74,75]. For example, derivatives of the heterocyclic compound 7-nitro-benzofurazan or 7-nitro-2,1,3-benzoxadiazole, are explored as agents having a strong inhibitory activity towards members of the GST family [76]. In addition, the inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX) is cytotoxic toward P-glycoproteinoverexpressing tumor cell lines [77], and recently has been reported that this inhibitor overcomes the multidrug resistance (MDR)-associated protein 1-mediated MDR in small cell lung cancer [78,81]. In another approach a highaffinity RNA aptamer with high specificity and affinity to GSTs was developed [82]. In another patent a method of simultaneously treating both cancer and the multidrug resistance phenotype via inhibition of a GST was developed [83].

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In a resent invention the isoenzyme GSTA3-3 was targeted for the treatment of cancer and other diseases responsive to inhibition of steroid hormone production [84]. GSTA3-3 was found to efficiently catalyze obligatory double-bond isomerizations of delta(5)-androstene-3,17dione Fig. (1) and delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The invention relates to the use of inhibitors of GSTA3-3 for production of a drug for the treatment of steroid hormone dependent diseases, such cancer [84]. Pro-drug development using the platform of GST has recently produced a number of lead compounds [85-89]. GSTs, as it has been discussed, are frequently overexpressed in neoplastic tissues, with the GSTP1-1 isoform most commonly overexpressed in cancers resistant to drugs. Therefore GSTP1-1 proved to be an attractive molecular target for prodrug activation. For example, an interesting prodrug, illustrated in Fig. (7), that is activated by GSTP1-1 is TLK286 (Telcyta), [74]. This drug is activated by Tyr7 in the active site of human GSTP1-1, through a -elimination reaction. Following activation, the drug is cleavage into a phosphorodiamidate, which in turn spontaneously forms aziridinium species, the actual alkylating moieties, and a GSH analogue. In another approach Findlay et al., [85] have designed O2-arylated diazeniumdiolates derivatives, which are NOreleasing and GST-activated prodrugs Fig. (8) [86]. The general mechanism of action of such prodrugs involves nucleophilic aromatic substitution by GSH, generating the nitric oxide-releasing diazeniumdiolate ion. Due to the involvement of NO in diverse physiological processes, sitedirected delivery of therapeutic nitric oxide is essential to avoid any undesirable side-effects. Encouraging results in both cell and animal models of cancer have suggested that this prodrug exhibits improved cytotoxic selectivity toward cancer cells, most likely due to the high levels of GSTs in these cells. GSTs, in addition to GSH-conjugating activity, exhibit sulphonamidase activity and catalyze the GSH-mediated hydrolysis of sulphonamide bond [87,88]. Such reactions are of interest as potential tumour-directed pro-drug activation strategies. Axarli et al., (2009), [89] reported the design and synthesis of novel chimaeric sulphonamide-derivatives able to be activated by the human GSTA1-1 (hGSTA1-1). These derivatives bear a peptidyl-moiety (analogues of bombesin peptide) as molecular recognition element for targeting the drug selectively to tumour cells. Bombesin peptide is recognized by the cancer-specific bombesin receptor. The released S-alkyl-GSH, after hGSTA1-1-mediated cleavage of the sulphonamide bond, provides a strong inhibitor against GSTs, Fig. (9). CURRENT & FUTURE DEVELOPMENTS GSTs are a highly diverse family of enzymes with functions ranging from detoxification to biosynthesis and catalyze the conjugation of nonpolar compounds that contain an electrophilic carbon, nitrogen or sulfur atom to GSH, contributing to the metabolism of drugs, pesticides and other

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Fig. (7). Structure of TLK286 prodrug and its activation by hGSTP1-1. Tyr 7 is the catalytic residue of hGSTP1-1.

Fig. (8). Structure of an NO-releasing GST-activated pro-drug, and its reaction with GSH.

Fig. (9). Sulphonamide cleavage by hGSTA1-1. R: is the structure of bombesin peptide (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-GlyHis-Leu-Met-NH2).

xenobiotics. The wide catalytic and structural diversity of GSTs constitute an effective recourse for successful applications. In recent years, significant advances have been made

in the understanding of GSTs structure and function. Research on the properties of new isoenzymes of plant, mammalian or microbial origin continues to grow. Future

GSTs in Biotechnology

genomics and proteomics will provide more insight in the presence and activity of the GSTs in cell and about natural functions and their modulation under stress. The examples provided in this article highlight the applications of GSTs in plant biotechnology, medicine and analytical biotechnology. The potential economic, social and health benefits that may be derived from these applications are very significant and therefore the area will be growing at a constant rate. ACKNOWLEDGEMENT

Recent Patents on Biotechnology 2009, Vol. 3, No. 3 [13]

[14] [15] [16]

[17]

This work was co-funded by the European UnionEuropean Social Fund & National Resources. [18]

CONFLICT OF INTEREST The authors declare no conflict of interest. [19]

ABBREVIATIONS CDNB

=

1-Chloro-2,4-dinitrobenzene

G-site

=

Glutathione binding site

GSH

=

Glutathione

GST

=

Glutathione transferase

H-site

=

Hydrophobic binding site

MAP

=

Mitogen-activated protein

MDR

=

Multidrug resistance

[20]

[21]

[22]

[23]

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