A structure-based mechanism of cisplatin resistance mediated by glutathione transferase P1-1.

Cisplatin [cis-diamminedichloroplatinum(II) (cis-DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of glutathione transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis-DDP-binding protein. Our results show that cis-DDP is not a substrate for the glutathione (GSH) transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.

Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine.

Glutathione transferases (GSTs) form a family of detoxication enzymes instrumental in the inactivation and elimination of electrophilic mutagenic and carcinogenic compounds. The Pi class GST P1-1 is present in most tissues and is commonly overexpressed in neoplastic cells. GST P1-1 in the dog, Canis lupus familiaris, has merits as a marker for tumors and as a target for enzyme-activated prodrugs. We produced the canine enzyme CluGST P1-1 by heterologous bacterial expression and verified its cross-reactivity with antihuman-GST P1-1 antibodies. The catalytic activity with alternative substrates of biological significance was determined, and the most active substrate found was benzyl isothiocyanate. Among established GST inhibitors, Cibacron Blue showed positive cooperativity with an IC50 value of 43 nM. Dog GST P1-1 catalyzes activation of the prodrug Telcyta, but the activity is significantly lower than that of the human homolog.

The Relationship of Glutathione-S-Transferase and Multi-Drug Resistance-Related Protein 1 in Nitric Oxide (NO) Transport and Storage.

Nitric oxide is a diatomic gas that has traditionally been viewed, particularly in the context of chemical fields, as a toxic, pungent gas that is the product of ammonia oxidation. However, nitric oxide has been associated with many biological roles including cell signaling, macrophage cytotoxicity, and vasodilation. More recently, a model for nitric oxide trafficking has been proposed where nitric oxide is regulated in the form of dinitrosyl-dithiol-iron-complexes, which are much less toxic and have a significantly greater half-life than free nitric oxide. Our laboratory has previously examined this hypothesis in tumor cells and has demonstrated that dinitrosyl-dithiol-iron-complexes are transported and stored by multi-drug resistance-related protein 1 and glutathione-S-transferase P1. A crystal structure of a dinitrosyl-dithiol-iron complex with glutathione-S-transferase P1 has been solved that demonstrates that a tyrosine residue in glutathione-S-transferase P1 is responsible for binding dinitrosyl-dithiol-iron-complexes. Considering the roles of nitric oxide in vasodilation and many other processes, a physiological model of nitric oxide transport and storage would be valuable in understanding nitric oxide physiology and pathophysiology.

Differential responses of a pi-class glutathione S-transferase (CnGSTp) expression and antioxidant status between golden and brown noble scallops under pathogenic stress.

Glutathione S-transferases (GSTs) play important roles in immunity by protecting organisms against the damage of reactive oxygen species (ROS). In this study, a pi-class GST cDNA sequence was first cloned from noble scallop Chlamys nobilis (named CnGSTp). The full length cDNA of CnGSTp was 922 bp, encoding a cytosolic protein of 202 amino acids residues, with predicted molecular masses of 23.1 kDa. Then an acute Vibrio Parahaemolyticus challenge experiment was conducted by using the Golden and Brown noble scallops with different total carotenoids content (TCC), and CnGSTp expression level, TCC and ROS level was separately determined. The results showed that ROS and CnGSTp expression levels were significantly up-regulate under Vibrio Parahaemolyticus challenge than the control group (P < 0.05). The Golden scallops showed significantly higher CnGSTp expression level and lower ROS level in hemocytes than the Brown ones (P < 0.05). Moreover, there is a significantly positive correlation between TCC and ROS in the Golden scallops. The present results revealed that CnGSTp plays important roles in immune response and carotenoids play assistant roles in antioxidant defense system under pathogenic stress in the noble scallop.

A Covalent Inhibitor for Glutathione S-Transferase Pi (GSTP1-1 ) in Human Cells.

Glutathione S-transferase π (GSTP1-1 ) is overexpressed in many types of cancer and is involved in drug resistance. Therefore, GSTP1-1 is an important target in cancer therapy, and many GST inhibitors have been reported. We had previously developed an irreversible inhibitor, GS-ESF, as an effective GST inhibitor; however, its cellular permeability was too low for it to be used in inhibiting intracellular GST. We have now developed new irreversible inhibitors by introducing sulfonyl fluoride (SF) into chloronitrobenzene (CNB). The mechanism of action was revealed to be that CNBSF first reacts with glutathione (GSH) through an aromatic substitution in the cell, then the sulfonyl group on the GSH conjugate with CNBSF reacts with Tyr108 of GST to form a sulfonyl ester bond. Our new inhibitor irreversible inhibited GSTP1-1 both in vitro and in cellulo with a long duration of action.

Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine.

Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation.

Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.

Phosphorylation of Glutathione S-Transferase P1 (GSTP1) by Epidermal Growth Factor Receptor (EGFR) Promotes Formation of the GSTP1-c-Jun N-terminal kinase (JNK) Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells.

Under normal physiologic conditions, the glutathione S-transferase P1 (GSTP1) protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the mitogen-activated protein kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by epidermal growth factor receptor (EGFR) at tyrosine residues 3, 7, and 198. Here we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1, and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr-198 by EGFR and is associated with a >2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 cross-talk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs.

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.

Involvement of reactive persulfides in biological bismethylmercury sulfide formation.

Bismethylmercury sulfide (MeHg)2S has been found to be a detoxified metabolite of methylmercury (MeHg) that is produced by SH-SY5Y cells and in livers of rats exposed to MeHg. (MeHg)2S could be formed through the interactions between MeHg and sulfur species such as hydrogen sulfide (H2S or HS(-)), but the origin of its sulfur has not been fully identified. We herein examined the formation of (MeHg)2S through interactions between MeHg and persulfides, polysulfides, and protein preparations. Investigations using HPLC/atomic absorption spectrophotometry and EI-MS revealed that NaHS and Na2S4 react readily with MeHg to give (MeHg)2S, and similar results were found using GSH persulfide (GSSH) formed endogenously or generated enzymatically in vitro. (MeHg)2S was also formed by incubation of MeHg with liver and heart cytosolic fractions prepared from wild-type mice but not with those from mice lacking cystathionine γ-lyase (CSE) that catalyzes the formation of cysteine persulfide. Consistent with this, (MeHg)2S was detected in a variety of tissues taken from wild-type mice intraperitoneally injected with MeHg in vivo but not in those from MeHg-injected CSE knockout mice. By separating liver fractions by column chromatography, we found numerous proteins that contain persulfides: one of the proteins was identified as being glutathione S-transferase pi 1. These results indicate that the formation of (MeHg)2S can be attributed to interactions between MeHg and endogenous free persulfide species, as well as protein-bound cysteine persulfide.

PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells.

A novel biomarker for marine environmental pollution of pi-class glutathione S-transferase from Mytilus coruscus.

Glutathione S-transferases (GSTs) are the superfamily of phase II detoxification enzymes that play crucial roles in innate immunity. In this study, a pi-class GST homolog was identified from Mytilus coruscus (named as McGST1, KC525103). The full-length cDNA sequence of McGST1 was 621bp with a 5' untranslated region (UTR) of 70bp and a 3'-UTR of 201bp. The deduced amino acid sequence was 206 residues in length with theoretical pI/MW of 5.60/23.72kDa, containing the conserved G-site and diversiform H-site. BLASTn analysis and phylogenetic relationship strongly suggested that this cDNA sequence was a member of pi class GST family. The prediction of secondary structure displayed a preserved N-terminal and a C-terminal comprised with α-helixes. Quantitative real time RT-PCR showed that constitutive expression of McGST1 was occurred, with increasing order in mantle, muscle, gill, hemocyte, gonad and hepatopancreas. The stimulation of bacterial infection, heavy metals and 180CST could up-regulate McGST1 mRNA expression in hepatopancreas with time-dependent manners. The maximum expression appeared at 6h after pathogenic bacteria injected, with 10-fold in Vibrio alginolyticus and 16-fold in Vibrio harveyi higher than that of the control. The highest point of McGST1 mRNA appeared at different time for exposure to copper (10-fold at day 15), cadmium (9-fold at day10) and 180 CST (10-fold at day 15). These results suggested that McGST1 played a significant role in antioxidation and might potentially be used as indicators and biomarkers for detection of marine environmental pollution.

Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells.

The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS). More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1), heat shock 70 kDa protein 9 (HSPA9) and pyruvate kinase M2 (PKM2), were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL) suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

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.

Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics.

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.

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.

Glutathione S-transferase π complexes with and stimulates Na⁺,K⁺-ATPase.

Glutathione S-transferase (GST) was found to complex with the Na⁺,K⁺-ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α-subunit of the Na⁺,K⁺-ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺-ATPase. Co-immunoprecipitation experiments, using the anti-α-subunit antiserum to precipitate the GST-Na⁺,K⁺-ATPase complex and then using antibodies specific to an isoform of GST to identify the co-precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺-ATPase. GST stimulated the Na⁺,K⁺-ATPase activity up to 1.4-fold. The level of stimulation exhibited a saturable dose-response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺-ATPase activity was similar to that when untreated GST was added. When GST was treated with H2O2, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺-ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺-ATPase and stimulates the latter independent of its GST activity.

Design and synthesis of versatile GSTP1-specific fluorogenic substrates for the highly sensitive detection of GSTP1 activity in living cells.

Pi-class glutathione S-transferase (GSTP1) is a detoxification enzyme that is highly expressed in various types of cancer cells and is a promising target for cancer imaging and therapy. Ps-TAc, an acetylated derivative of the GSTP1-specific fluorogenic substrate Ps-TG, is attracting attention as an effective GSTP1 fluorescent probe, and has been successfully used to visualize intracellular GSTP1 activity. Ps-TAc is a prodrug type fluorescent probe in which the phenolic hydroxyl group of Ps-TG is acetylated and thus is susceptible to nonspecific hydrolysis, potentially compromising its ability to detect GSTP1 activity. Here, we describe the development of a highly selective fluorogenic GSTP1 substrate that is membrane permeable and does not involve esterification and show its application to live-cell imaging and FACS analysis. We designed and synthesized several compounds with benzylsulfone substituents instead of the mesyl group of Ps-TG and tested their fluorescence activation by GSTP1 catalysis in vitro and in cellulo. Of the test compounds, Ps-TG3 was the most suitable for the visualization of intracellular GSTP1 activity because the signal from living cells increased significantly when MK-571, an inhibitor of multidrug resistance proteins (MRPs), was simultaneously loaded. The results obtained by co-loading Ps-TG3 and MK571 into GSTP1-nonexpressing cells suggest that Ps-TG3 can be a substrate for MRPs. The usefulness of Ps-TG3 was demonstrated by fluorescence imaging of several cancer cell cultures and FACS analysis of lymphoma cells. The results presented here suggest that Ps-TG3, in combination with MK571, is useful for visualizing and detecting intracellular GSTP1 activity in cancer cells that highly express GSTP1.

Inhibition Analysis and High-Resolution Crystal Structure of Mus musculus Glutathione Transferase P1-1.

Multidrug resistance is a significant barrier that makes anticancer therapies less effective. Glutathione transferases (GSTs) are involved in multidrug resistance mechanisms and play a significant part in the metabolism of alkylating anticancer drugs. The purpose of this study was to screen and select a lead compound with high inhibitory potency against the isoenzyme GSTP1-1 from Mus musculus (MmGSTP1-1). The lead compound was selected following the screening of a library of currently approved and registered pesticides that belong to different chemical classes. The results showed that the fungicide iprodione [3-(3,5-dichlorophenyl)-2,4-dioxo-N-propan-2-ylimidazolidine-1-carboxamide] exhibited the highest inhibition potency (ΙC50 = 11.3 ± 0.5 µΜ) towards MmGSTP1-1. Kinetics analysis revealed that iprodione functions as a mixed-type inhibitor towards glutathione (GSH) and non-competitive inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). X-ray crystallography was used to determine the crystal structure of MmGSTP1-1 at 1.28 Å resolution as a complex with S-(p-nitrobenzyl)glutathione (Nb-GSH). The crystal structure was used to map the ligand-binding site of MmGSTP1-1 and to provide structural data of the interaction of the enzyme with iprodione using molecular docking. The results of this study shed light on the inhibition mechanism of MmGSTP1-1 and provide a new compound as a potential lead structure for future drug/inhibitor development.

Surface plasmon resonance (SPR) studies on the interactions of carotenoids and their binding proteins.

The xanthophyll carotenoids lutein and zeaxanthin constitute the major carotenoids of the macular pigment in the human retina where they are thought to act in part to prevent light induced oxidative damage associated with age-related macular degeneration (AMD). The highly selective uptake of these pigments is mediated by specific carotenoid-binding proteins (GSTP1 and StARD3) recently identified in our laboratory. Carotenoids are hydrophobic in nature, so we first systematically optimized carotenoid preparations that are nano-dispersed in aqueous buffers, and then we used a new-generation surface plasmon resonance (SPR) protocol called FastStep™, which is significantly faster than conventional SPR assays. We have explored carotenoid-binding interactions of five proteins: human serum albumin (HSA), ß-lactoglobulin (LG), steroidogenic acute regulatory domain proteins (StARD1, StARD3) and glutathione S- transferase Pi isoform (GSTP1). HSA and LG showed relatively weak interaction with carotenoids (K(D)>1 µM). GSTP1 evidenced high affinity and specificity towards zeaxanthin and meso-zeaxanthin with K(D) values 0.14±0.02 µM and 0.17±0.02 µM, respectively. StARD3 expressed a relative high specificity towards lutein with a K(D) value of 0.59±0.03 µM, whereas StARD1 exhibited a relatively low selectivity and affinity (K(D)>1 µM) towards the various carotenoids tested.