Human Glutathione Transferase P1-1 Research Articles

Arylcoumarin and novel biscoumarin derivatives as potent inhibitors of human glutathione S-transferase

A series of arylcoumarin derivatives and two novel biscoumarin derivatives were investigated for their human recombinant glutathione S-transferase P1-1 (GSTP1-1) enzyme inhibitory activities for the first time. 4-(3,4-Dihydroxyphenyl)-6,7-dihydroxycoumarin (compound 24) was observed to be the most active coumarin derivative (IC50: 0.14 µM). The inhibition was found to be time-dependent and irreversible. Hypothetical binding modes of the ten most active compounds were calculated by molecular docking. Ligand efficiency indices (LEI) were estimated to better understand the binding performance of the coumarin derivatives. Extensive structure-activity relationship studies showed that hydroxy substitution on both the coumarin and the aryl ring enhanced the biological activity and the position of hydroxy group on the coumarin ring is critical for the binding pose and the activity. Top three ligands were subjected to molecular dynamics simulations and MM/PBSA for further investigation. Binding mode of compound 24 suggested that its high inhibitory activity might be attributed to its position between Tyr7 and the cofactor, glutathione (GS-DNB). Exhibiting favorable druglikeness profiles and pharmacokinetics based on ADME studies, compound 5 and 24 can be considered as potential drug leads in future studies for further development. Communicated by Ramaswamy H. Sarma

Mechanistic insights into the inhibition of human placental glutathione S-transferase P1-1 by abscisic and gibberellic acids: An integrated experimental and computational study.

The interactions of the classic phytohormones gibberellic acid (gibberellin A3 , GA3 ) and abscisic acid (dormin, ABA), which antagonistically regulate several developmental processes and stress responses in higher plants, with human placental glutathione S-transferase P1-1 (hpGSTP1-1), an enzyme that plays a role in endo- or xenobiotic detoxification and regulation of cell survival and apoptosis, were investigated. The inhibitory potencies of ABA and GA3 against hpGSTP1, as well as the types of inhibition and the kinetic parameters, were determined by making use of both enzyme kinetic graphs and SPSS nonlinear regression models. The structural basis for the interaction between hpGSTP1-1 and phytohormones was predicted with the aid of molecular docking simulations. The IC50 values of ABA and GA3 were 5.3 and 5.0 mM, respectively. Both phytohormones inhibited hpGSTP1-1 in competitive manner with respect to the cosubstrates GSH and CDNB. When ABA was the inhibitor at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v , Vm , Km , and Ki values were statistically estimated to be 205 ± 16 μmol/min-mg protein, 1.32 ± 0.18 mM, 1.95 ± 0.25 mM and 175 ± 6 μmol/min-mg protein, 0.85 ± 0.06 mM, 1.85 ± 0.16 mM, respectively. On the other hand, the kinetic parameters Vm , Km , and Ki obtained with GA3 at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v were found to be 303 ± 14 μmol/min-mg protein, 1.77 ± 0.13 mM, 3.38 ± 0.26 mM and 249 ± 7 μmol/min-mg protein, 1.43 ± 0.07 mM, 2.89 ± 0.19 mM, respectively. Both phytohormones had the potential to engage in hydrogen-bonding and electrostatic interactions with the key residues that line the G- and H-sites of the enzyme's catalytic center. Inhibitory actions of ABA/GA3 on hpGSTP1-1 may guide medicinal chemists through the structure-based design of novel antineoplastic agents. It should be noted, however, that the same interactions may also render fetuses vulnerable to the potentially toxic effects of xenobiotics and noxious endobiotics.

Monocarbonyl Curcumin Analogues as Potent Inhibitors against Human Glutathione Transferase P1-1.

The isoenzyme of human glutathione transferase P1-1 (hGSTP1-1) is involved in multi-drug resistance (MDR) mechanisms in numerous cancer cell lines. In the present study, the inhibition potency of two curcuminoids and eleven monocarbonyl curcumin analogues against hGSTP1-1 was investigated. Demethoxycurcumin (Curcumin II) and three of the monocarbonyl curcumin analogues exhibited the highest inhibitory activity towards hGSTP1-1 with IC50 values ranging between 5.45 ± 1.08 and 37.72 ± 1.02 μM. Kinetic inhibition studies of the most potent inhibitors demonstrated that they function as non-competitive/mixed-type inhibitors. These compounds were also evaluated for their toxicity against the prostate cancer cells DU-145. Interestingly, the strongest hGSTP1-1 inhibitor, (DM96), exhibited the highest cytotoxicity with an IC50 of 8.60 ± 1.07 μΜ, while the IC50 values of the rest of the compounds ranged between 44.59-48.52 μΜ. Structural analysis employing molecular docking, molecular dynamics (MD) simulations, and binding-free-energy calculations was performed to study the four most potent curcumin analogues as hGSTP1-1 inhibitors. According to the obtained computational results, DM96 exhibited the lowest binding free energy, which is in agreement with the experimental data. All studied curcumin analogues were found to form hydrophobic interactions with the residue Gln52, as well as hydrogen bonds with the nearby residues Gln65 and Asn67. Additional hydrophobic interactions with the residues Phe9 and Val36 as well as π-π stacking interaction with Phe9 contributed to the superior inhibitory activity of DM96. The van der Waals component through shape complementarity was found to play the most important role in DM96-inhibitory activity. Overall, our results revealed that the monocarbonyl curcumin derivative DM96 acts as a strong hGSTP1-1 inhibitor, exerts high prostate cancer cell cytotoxicity, and may, therefore, be exploited for the suppression and chemosensitization of cancer cells. This study provides new insights into the development of safe and effective GST-targeted cancer chemosensitizers.

Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione: Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions

Unsymmetrical bisacridines (UAs) are a novel potent class of antitumor-active therapeutics. A significant route of phase II drug metabolism is conjugation with glutathione (GSH), which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes. The aim of this work was to investigate the GSH-mediated metabolic pathway of a representative UA, C-2028. GSH-supplemented incubations of C-2028 with rat, but not with human, liver cytosol led to the formation of a single GSH-related metabolite. Interestingly, it was also revealed with rat liver microsomes. Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450 (CYP450) inhibitor 1-aminobenzotriazole. Therefore, the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate. In turn, incubations of C-2028 and GSH with human recombinant glutathione S-transferase (GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form, respectively. These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule. Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction. Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry. Mechanisms for their formation were proposed. The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important. This may affect the patient's drug clearance due to GST activity, loss of GSH, or the interactions with GSH-conjugated drugs. Moreover, GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

Assessment of the inhibitory activity of the pyrethroid pesticide deltamethrin against human placental glutathione transferase P1-1: A combined kinetic and docking study

Deltamethrin (DEL), which is a synthetic pyrethroid insecticide, has been used successfully all over the world to treat mosquito nets for the control of malaria. Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the conjugation of reduced glutathione (GSH) to a variety of xenobiotics and are normally recognized as detoxification enzymes. Here, we used a colorimetric assay based on the human placental GSTP1-1 (hpGSTP1-1)-catalyzed reaction between GSH and the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) as well as molecular docking to investigate the mechanistic and structural aspects of hpGSTP1-1 inhibition by DEL. We show that DEL is a potent, noncompetitive inhibitor of hpGSTP1-1 with an IC50 value of 6.1 μM and Ki values of 5.61 ± 0.32 μM and 7.96 ± 0.97 μM at fixed [CDNB]–varied [GSH] and fixed [GSH]–varied [CDNB], respectively. DEL appears to be accommodated well in an eccentric cavity located at the interface of the hpGSTP1-1 homodimer, presumably causing conformational changes to the enzyme’s substrate-binding sites such that the enzyme is no longer able to transform GSH and CDNB effectively. Correspondingly, considerable maternal exposure to and subsequent accumulation of DEL may interfere with the proper development of the vulnerable fetus, possibly increasing the risk of developing congenital defects.

Abstract 5257: Upregulation of glutathione S-transferase P1 by green tea polyphenols: A novel transcriptional target of p53 tumor suppressor gene

Abstract Green tea polyphenols (GTPs) and its major constituent (-)-epigallocatechin-3-gallate (EGCG) reactivate epigenetically silenced genes in cancer cells. Glutathione S-transferase P1 (GSTP1), an enzyme involved in detoxification process, is frequently inactivated in prostate cancer due to epigenetic modifications. The mechanisms underlying the re-expression of GSTP1 expression by GTP/EGCG in prostate cancer is currently not well understood. Earlier we demonstrated the ability of GTP/EGCG to increase p53 transcriptional activity through suppression of class I histone deacetylases (Carcinogenesis 33(2):377-84, 2012). Here we report that the GSTP1 gene is an unrecognized downstream transcriptional target of the tumor suppressor p53, and that GTP/EGCG has ability to increase GSTP1 expression mediated through p53. Treatment of human prostate cancer LNCaP cells with 5μg/ml GTP and 20μM EGCG, activate p53 through acetylation at the Lys373 and Lys382 residues along with consequent increase in GSTP1 protein expression in time-dependent manner. To study the possible interaction between GSTP1 and p53, LNCaP cells stably-transfected with short hairpin-RNA against p53 (LNCaPshp53) and control vector (LNCaPshV) for generation of p53 knockout cells. GTP/EGCG treatment induced p53 activation and acetylation at p53 Lys373 and Lys382 specifically in LNCaPshV cells in time-dependent manner, but not in LNCaPshp53. This increase in p53 acetylation led to activation of GSTP1 in LNCaPshV cells both at protein and message levels. GTP/EGCG-mediated increase in GSTP1 expression is caused by increase binding of p53 to its consensus binding site on the intron 4 of GSTP1 gene as analyzed by electrophoretic mobility shift binding assay. Furthermore, this association was validated by ChIP assay where GTP/EGCG treatment led to an increase in the amount of acetylated p53-Lys373 associated within the intron site of GSTP1, compared to untreated controls. Taken together, our findings indicate the ability of p53 to transcriptionally activate the human GSTP1 gene, which defines a novel mechanism of protection of the genome by green tea polyphenols. Citation Format: Vijay S. Thakur, Gauri Deb, Mark W. Jackson, Sanjay Gupta. Upregulation of glutathione S-transferase P1 by green tea polyphenols: A novel transcriptional target of p53 tumor suppressor gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5257.

Glutathione analogues as substrates or inhibitors that discriminate between allozymes of the MDR-involved human glutathione transferase P1-1.

Glutathione (GSH) structure-guided tripeptide analogues were designed and synthesized by solid phase technology, purified (≥95%) by RP and/or GF column chromatography, to identify those that, compared with GSH, exhibited similar or higher binding and catalytic efficiency toward the MDR-involved human GSTP1-1 isoenzyme, and could discriminate between the allozymic expression products of the polymorphic human GSTP1 gene locus, designated as hGSTP1*A (Ile(104) /Ala(113) ), hGSTP1*B (Val(104) /Ala(113) ), and hGSTP1*C (Val(104) /Val(113) ). The analogues bear single amino acid alterations as well as alterations in more than one position. Some analogues showed remarkable allozyme selectivity, binding catalytically to A (I, II, IV, XII), to C (V and XVI), to A and C (III, VII, XIV) or to all three allozymes (XV). A heterocyclic substituent at positions 1 or 2 of GSH favors inhibition of A, whereas a small hydrophobic/hydrophilic amide substituent at position 2 (Cys) favors inhibition of B and C. Heterocyclic substituents at position 1, only, produce catalytic analogues for A, whereas less bulky and more flexible hydrophobic/hydrophilic substituents, at positions 1 or 3, lead to effective substrates with C. When such substituents were introduced simultaneously at positions 1 and 3, the analogues produced have no catalytic potential but showed appreciable inhibitory effects, instead, with all allozymes. It is anticipated that when GSH analogues with selective inhibitory or catalytic binding, were conjugated to allozyme-selective inhibitors of hGSTP1-1, the derived leads would be useful for the designing of novel chimeric inhibitors against the MDR-involved hGSTP1-1 allozymes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 330-344, 2016.

Nitrobenzoxadiazole-based GSTP1-1 inhibitors containing the full peptidyl moiety of (pseudo)glutathione

Context: The inhibition of glutathione S-transferase P1-1 (GSTP1-1) is a sound strategy to overcome drug resistance in oncology practice.Objective: The nitrobenzoxadiazolyl (NBD) S-conjugate of glutathione and the corresponding γ-oxa-glutamyl isostere (compounds 1 and 5, respectively) have been disclosed as GST inhibitors. The rationale of their design is discussed in juxtaposition to non-peptide NBD thioethers.Materials and methods: Synthesis of derivatives 1 and 5 and in vitro evaluation on human GSTP1-1 and M2-2 are reported.Results: Conjugates 1 and 5 were found to be low micromolar inhibitors of both isoforms. Furthermore, they display a threefold reduction in selectivity for GSTM2-2 over the P1-1 isozyme in comparison with the potent non-peptide inhibitor nitrobenzoxadiazolyl-thiohexanol (NBDHEX).Discussion and conclusions: Spectroscopic data are congruent with the formation of a stable sigma-complex between GSH and the inhibitors in the protein active site. Conjugate 5 is suitable for in vivo modulation of GST activity in cancer treatment.

8-Methoxypsoralen is a competitive inhibitor of glutathione S-transferase P1-1.

The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy.

Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator

Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.

Synthesis and Biological Evaluation of 2‐Substituted‐5‐(4‐nitrophenylsulfonamido)benzoxazoles as Human GST P1‐1 Inhibitors, and Description of the Binding Site Features

Glutathione-S-transferases (GSTs) are enzymes involved in cellular detoxification by catalyzing the nucleophilic attack of glutathione (GSH) on the electrophilic center of numerous of toxic compounds and xenobiotics, including chemotherapeutic drugs. Human GST P1-1, which is known as the most prevalent isoform of the mammalian cytosolic GSTs, is overexpressed in many cancers and contributes to multidrug resistance by directly conjugating to chemotherapeutics. It is suggested that this resistance is related to the high expression of GST P1-1 in cancers, thereby contributing to resistance to chemotherapy. In addition, GSTs exhibit sulfonamidase activity, thereby catalyzing the GSH-mediated hydrolysis of sulfonamide bonds. Such reactions are of interest as potential tumor-directed prodrug activation strategies. Herein we report the design and synthesis of some novel sulfonamide-containing benzoxazoles, which are able to inhibit human GST P1-1. Among the tested compounds, 2-(4-chlorobenzyl)-5-(4-nitrophenylsulfonamido)benzoxazole (5 f) was found as the most active hGST P1-1 inhibitor, with an IC50 value of 10.2 μM, showing potency similar to that of the reference drug ethacrynic acid. Molecular docking studies performed with CDocker revealed that the newly synthesized 2-substituted-5-(4-nitrophenylsulfonamido)benzoxazoles act as catalytic inhibitors of hGST P1-1 by binding to the H-site and generating conjugates with GSH to form S-(4-nitrophenyl)GSH (GS-BN complex) via nucleophilic aromatic substitution reaction. The 4-nitrobenzenesulfonamido moiety at position 5 of the benzoxazole ring is essential for binding to the H-site and for the formation of the GST-mediated GSH conjugate.

Expression of human glutathione S-transferase A1, P1 and T1 in Escherichia coli

To construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli). Human GSTA1, GSTP1 and GSTT1 gene whole length cDNAs were amplified by RT-PCR and then subcloned into pET-28a(+) vectors. The proteins were expressed in E. coli BL21(DE3). After purified by Ni2+ affinity chromatography, the enzymatic activities of GSTs were measured with 1-chloro-2,4 -dinitrobenzene (CDNB) as substrate. The correct GSTA1, GSTP1 and GSTT1 genes were cloned. And soluble GSTA1, GSTT1, GSTP1 proteins were expressed in E.coli. After purification, GSTA1, GSTT1 and GSTP1 showed good enzymatic activities, which were 17.55, 0.02, 18.75 μmol·min-1·mg-1, respectively. The expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully.

S-Nitrosation Destabilizes Glutathione Transferase P1-1

Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.

FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1

ObjectiveGlutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. MethodsWe tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. ResultsWe identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. Conclusion and practical implicationsIn their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. We used transient kinetic methods to determine a minimal mechanism for spontaneous S-nitrosoglutathione (GSNO)-mediated transnitrosation of human glutathione transferase (GST) P1-1, a major detoxification enzyme and key regulator of cell proliferation. Cys(47) of GSTP1-1 is S-nitrosated in two steps, with the chemical step limited by a pre-equilibrium between the open and closed conformations of helix α2 at the active site. Cys(101), in contrast, is S-nitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. Despite the presence of a GSNO binding site at the active site of GSTP1-1, isothermal titration calorimetry as well as nitrosation experiments using S-nitrosocysteine demonstrate that GSNO binding does not precede S-nitrosation of GSTP1-1. Kinetics experiments using the cellular reductant glutathione show that Cys(101)-NO is substantially more resistant to denitrosation than Cys(47)-NO, suggesting a potential role for Cys(101) in long term nitric oxide storage or transfer. These results constitute the first report of the molecular mechanism of spontaneous protein transnitrosation, providing insight into the post-translational control of GSTP1-1 as well as the process of protein transnitrosation in general.

Engineering High Catalytic Efficiency of the Steroid Isomerase Activity of Human Glutathione S-transferase P1-1

Glutathione S-transferases (GSTs) are a family of multifunctional enzymes that catalyze the formation of conjugates between reduced glutathione (GSH) and a variety of carcinogenic, mutagenic, toxic and pharmacologically active compounds. 1,2 GSTs have also been shown to participate in other biological process. Certain GSTs can detoxify lipid and DNA hydroperoxide by their intrinsic peroxidase activity, while others can catalyze the isomerization of certain steroids. Mammalian cytosolic GSTs, which exist as homo- or heterodimers, are grouped into at least seven distinct classes alpha, mu, pi, sigma, theta, sigma, kappa and zeta - according to their physical, chemical, immunological and structural properties. 1,3,4 Human pi-class hGST P1-1 contains eight arginine residues. Among them, Arg13 is the only residue conserved in all the known Pi class GSTs and in most of the Alpha class GSTs. The highly conserved residues are expected to be important to the enzyme's structure and function. hGST P1-1 is particularly interesting as it can be used as a reliable preneoplastic or neoplastic marker because it is expressed at raised levels by a number of human tumors. 5,6 It has also

Hidden Allostery in Human Glutathione Transferase P1-1 Unveiled by Unnatural Amino Acid Substitutions and Inhibition Studies

Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis–Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kcat and 300-fold increased KMGSH values, displays an apparent Hill coefficient of 0.82±0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency kcat/KMGSH to near the wild-type value but still yields Hill coefficients of 0.61±0.08 and 0.86±0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99±0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.

Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1

The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene–glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1.

Identifying and Characterizing Binding Sites on the Irreversible Inhibition of Human Glutathione S‐Transferase P1‐1 by S‐Thiocarbamoylation

Human glutathione S-transferase P1-1 (hGST P1-1) is involved in cell detoxification processes through the conjugation of its natural substrate, reduced glutathione (GSH), with xenobiotics. GSTs are known to be overexpressed in tumors, and naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are effective cancer chemopreventive compounds. To identify and characterize the potential inhibitory mechanisms of GST P1-1 induced by isothiocyanate conjugates, we studied the binding of GST P1-1 and some cysteine mutants to the BITC-SG conjugate as well as to the synthetic S-(N-benzylcarbamoylmethyl)glutathione conjugate (BC-SG). We report here the inactivation of GST P1-1 through the covalent modification of two Cys47 residues per dimer and one Cys101. The evidence has been compiled by isothermal titration calorimetry (ITC) and electrospray ionization mass spectrometry (ESI-MS). ITC experiments suggest that the BITC-SG conjugate generates adducts with Cys47 and Cys101 at physiological temperatures through a corresponding kinetic process, in which the BITC moiety is covalently bound to these enzyme cysteines through an S-thiocarbamoylation reaction. ESI-MS analysis of the BITC-SG incubated enzymes indicates that although the Cys47 in each subunit is covalently attached to the BITC ligand moiety, only one of the Cys101 residues in the dimer is so attached. A plausible mechanism is given for the emergence of inactivation through the kinetic processes with both cysteines. Likewise, our molecular docking simulations suggest that steric hindrance is the reason why only one Cys101 per dimer is covalently modified by BITC-SG. No covalent inactivation of GST P1-1 with the BC-SG inhibitor has been observed. The affinities and inhibitory potencies for both conjugates are high and very similar, but slightly lower for BC-SG. Thus, we conclude that the presence of the sulfur atom from the isothiocyanate moiety in BITC-SG is crucial for its irreversible inhibition of GST P1-1.

Constitutive expression of an antioxidant enzyme, glutathione S-transferase P1, during differentiation of human intestinal Caco-2 cells

In this study, we investigated the molecular mechanism of the constitutive expression of an antioxidant enzyme, glutathione S-transferase (GST), during differentiation of human intestinal epithelial Caco-2 cells. We observed that the class π GST isozyme (GST P1) expression correlated with the increased expression of caudal-related homeodomain protein 2 (CDX2), a member of the mammalian homeobox family of transcription factors. In addition, transfection of Caco-2 cells with the human CDX2 cDNA resulted in enhanced expression of the GST P1 gene and protein. Chromatin immunoprecipitation assay demonstrated that CDX2 binds to the GST P1 promoter containing the putative consensus CDX-binding element, TTTAC, located at −247 upstream from the established site for transcription initiation. Using the dsDNA pull-down assay, it was revealed that CDX2 recognized and bound to the putative consensus CDX-binding element within the human GST P1 promoter region and that the amount of the CDX2 bound to the putative consensus CDX-binding element increased during Caco-2 cell differentiation. Furthermore, we demonstrated that CDX2 formed the transcriptional complex with Sp1 and bound to the putative consensus CDX-binding element within the human GST P1 promoter region. These data suggest that CDX2 binds to the human GST P1 promoter via complex formation with Sp1 and controls the constitutive expression of GST P1 during Caco-2 cell differentiation.