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Mechanistic Studies on a Novel, Highly Potent Gold-Phosphole Inhibitor of Human Glutathione Reductase

2005; Elsevier BV; Volume: 280; Issue: 21 Linguagem: Inglês

10.1074/jbc.m412519200

ISSN

1083-351X

Autores

Marcel Deponte, Sabine Urig, L. David Arscott, Karin Fritz‐Wolf, Régis Réau, Christel Herold‐Mende, Saša Končarević, Markus Meyer, Elisabeth Davioud–Charvet, David P. Ballou, Charles H. Williams, Katja Becker,

Tópico(s)

Metal-Catalyzed Oxygenation Mechanisms

Resumo

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When ∼1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a Kd(app) for GSSG of 63 μm, and a k(obs)max of 106 s-1 at 4 °C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (Kd = 0.46 μm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range. The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When ∼1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a Kd(app) for GSSG of 63 μm, and a k(obs)max of 106 s-1 at 4 °C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (Kd = 0.46 μm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range. The antioxidant enzyme glutathione reductase (GR) 1The abbreviations used are: GR, glutathione reductase; (h)GR, (human) glutathione reductase; Eox, enzyme in oxidized state; EH2, enzyme in a two-electron reduced state; CTC charge-transfer complex; I0.5, inhibitor concentration for 50% inhibition; GoPI, gold-phosphole inhibitor; DMF, N,N-dimethylformamide; PPh3AuCl, chloro(triphenylphosphine)gold(I); SELDI-TOF MS, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; GSNO, S-nitrosoglutathione. 1The abbreviations used are: GR, glutathione reductase; (h)GR, (human) glutathione reductase; Eox, enzyme in oxidized state; EH2, enzyme in a two-electron reduced state; CTC charge-transfer complex; I0.5, inhibitor concentration for 50% inhibition; GoPI, gold-phosphole inhibitor; DMF, N,N-dimethylformamide; PPh3AuCl, chloro(triphenylphosphine)gold(I); SELDI-TOF MS, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; GSNO, S-nitrosoglutathione. plays an essential role in the cellular redox metabolism of most organisms, catalyzing the regeneration of reduced GSH (1Becker K. Rahlfs S. Nickel C. Schirmer R.H. Biol. Chem. 2003; 384: 551-566PubMed Google Scholar). For this reason the mechanism and structure of GRs from several organisms including man (2Nordhoff A. Bücheler U.S. Werner D. Schirmer R.H. Biochemistry. 1993; 32: 4060-4066Crossref PubMed Scopus (106) Google Scholar, 3Krauth-Siegel R.L. Arscott L.D. Schönleben-Janas A. Schirmer R.H. Williams Jr., C.H. Biochemistry. 1998; 37: 13968-13977Crossref PubMed Scopus (58) Google Scholar, 4Pai E.F. Schulz G.E. J. Biol. Chem. 1983; 258: 1752-1757Abstract Full Text PDF PubMed Google Scholar, 5Karplus P.A. Schulz G.E. J. Mol. Biol. 1989; 210: 163-180Crossref PubMed Scopus (289) Google Scholar, 6Savvides S.N. Scheiwein M. Böhme C.C. Arteel G.E. Karplus P.A. Becker K. Schirmer R.H. J. Biol. Chem. 2002; 277: 2779-2784Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), Escherichia coli (7Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar), and the malaria-causing parasite Plasmodium falciparum (6Savvides S.N. Scheiwein M. Böhme C.C. Arteel G.E. Karplus P.A. Becker K. Schirmer R.H. J. Biol. Chem. 2002; 277: 2779-2784Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 8Böhme C.C. Arscott L.D. Becker K. Schirmer R.H. Williams Jr., C.H. J. Biol. Chem. 2000; 275: 37317-37323Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9Sarma G.N. Savvides S.N. Becker K. Schirmer M. Schirmer R.H. Karplus P.A. J. Mol. Biol. 2003; 328: 893-907Crossref PubMed Scopus (117) Google Scholar), have been extensively studied. GSH is present in most eukaryotic cells at millimolar concentrations. It is involved in redox homeostasis, formation of deoxyribonucleotides, and detoxification of peroxides, 2-oxoaldehydes, and xenobiotics. Cells that are exposed to oxidative stress as well as rapidly proliferating cells particularly depend on the regeneration of GSH. Thus, selective, highly potent inhibitors of GR (10Becker K. Herold-Mende C. Park J.J. Lowe G. Schirmer R.H. J. Med. Chem. 2001; 44: 2784-2792Crossref PubMed Scopus (129) Google Scholar, 11Davioud-Charvet E. Delarue S. Biot C. Schwobel B. Boehme C.C. Mussigbrodt A. Maes L. Sergheraert C. Grellier P. Schirmer R.H. Becker K. J. Med. Chem. 2001; 44: 4268-4276Crossref PubMed Scopus (127) Google Scholar, 12Becker K. Gui M. Schirmer R.H. Eur. J. Biochem. 1995; 234: 472-480Crossref PubMed Scopus (78) Google Scholar, 13Becker K. Schirmer R.H. Methods Enzymol. 1995; 251: 173-188Crossref PubMed Scopus (52) Google Scholar, 14Karplus P.A. Krauth-Siegel R.L. Schirmer R.H. Schulz G.E. Eur. J. Biochem. 1988; 171: 193-198Crossref PubMed Scopus (71) Google Scholar) are promising lead compounds for the development of novel anti-tumor or antiparasitic drugs (1Becker K. Rahlfs S. Nickel C. Schirmer R.H. Biol. Chem. 2003; 384: 551-566PubMed Google Scholar, 8Böhme C.C. Arscott L.D. Becker K. Schirmer R.H. Williams Jr., C.H. J. Biol. Chem. 2000; 275: 37317-37323Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9Sarma G.N. Savvides S.N. Becker K. Schirmer M. Schirmer R.H. Karplus P.A. J. Mol. Biol. 2003; 328: 893-907Crossref PubMed Scopus (117) Google Scholar, 10Becker K. Herold-Mende C. Park J.J. Lowe G. Schirmer R.H. J. Med. Chem. 2001; 44: 2784-2792Crossref PubMed Scopus (129) Google Scholar, 11Davioud-Charvet E. Delarue S. Biot C. Schwobel B. Boehme C.C. Mussigbrodt A. Maes L. Sergheraert C. Grellier P. Schirmer R.H. Becker K. J. Med. Chem. 2001; 44: 4268-4276Crossref PubMed Scopus (127) Google Scholar, 12Becker K. Gui M. Schirmer R.H. Eur. J. Biochem. 1995; 234: 472-480Crossref PubMed Scopus (78) Google Scholar, 13Becker K. Schirmer R.H. Methods Enzymol. 1995; 251: 173-188Crossref PubMed Scopus (52) Google Scholar, 14Karplus P.A. Krauth-Siegel R.L. Schirmer R.H. Schulz G.E. Eur. J. Biochem. 1988; 171: 193-198Crossref PubMed Scopus (71) Google Scholar, 15Irmler A. Bechthold E. Davioud-Charvet E. Hofman V. Réau R. Gromer S. Schirmer R.H. Becker K. Chapman S. Perham R. Scrutton N. Flavins and Flavoproteins2002. Agency for Scientific Publications, Berlin2002: 803-815Google Scholar). As a member of the pyridine nucleotide-disulfide oxidoreductase family of homodimeric flavoenzymes, each subunit of GR contains one FAD, two substrate binding sites, and, in oxidized GR(Eox), an intramolecular disulfide. During the reductive half-reaction electrons are transferred rapidly from NADPH on the re side of the flavin to the cystine disulfide on the si side of the flavin. In the oxidative half-reaction the final electron acceptor GSSG is reduced to 2 GSH, regenerating the disulfide at the active site of GR. Under physiological, reducing conditions GR(EH2) presumably occurs as the principal species of the enzyme in vivo (8Böhme C.C. Arscott L.D. Becker K. Schirmer R.H. Williams Jr., C.H. J. Biol. Chem. 2000; 275: 37317-37323Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The flow of electrons during a catalytic cycle can be monitored by specific spectral changes, using the flavin as an inherent redox indicator. The reductive half-reaction is characterized by a loss of absorbance at 460 nm coupled to a slight blue shift of the local maximum. In addition, the absorbance at 540 nm increases because of the formation of a thiolate-FAD charge-transfer complex (CTC), formed between the flavin and the proximal or charge-transfer thiolate of Cys63 in hGR. Cys58 in hGR provides the interchange or distal thiol that forms a mixed disulfide with glutathione as an intermediate. Reoxidation of the enzyme, which is coupled to the formation of GSH during the oxidative half-reaction, reverses the spectral changes (3Krauth-Siegel R.L. Arscott L.D. Schönleben-Janas A. Schirmer R.H. Williams Jr., C.H. Biochemistry. 1998; 37: 13968-13977Crossref PubMed Scopus (58) Google Scholar, 7Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar, 8Böhme C.C. Arscott L.D. Becker K. Schirmer R.H. Williams Jr., C.H. J. Biol. Chem. 2000; 275: 37317-37323Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Phospholes are phosphacyclopentadienes that have very limited aromatic character (for an example, see the central unit of the compound shown in Fig. 1) and a nucleophilic phosphorus atom, which makes them good reagents for chemical modifications, for example, with thiols (16Hay C. Hissler M. Fischmeister C. Rault-Berthelot J. Toupet L. Nyulaszi L. Réau R. Chem. Eur. J. 2001; 7: 4222-4236Crossref PubMed Scopus (246) Google Scholar). Thus, they are interesting candidates for inhibitor studies on disulfide reductases such as thioredoxin reductase and GR because they exhibit inhibitor concentrations for 50% inhibition (I0.5) in the lower micromolar range (15Irmler A. Bechthold E. Davioud-Charvet E. Hofman V. Réau R. Gromer S. Schirmer R.H. Becker K. Chapman S. Perham R. Scrutton N. Flavins and Flavoproteins2002. Agency for Scientific Publications, Berlin2002: 803-815Google Scholar). Some palladium-containing phospholes even possess I0.5 values in the nanomolar range for thioredoxin reductase (15Irmler A. Bechthold E. Davioud-Charvet E. Hofman V. Réau R. Gromer S. Schirmer R.H. Becker K. Chapman S. Perham R. Scrutton N. Flavins and Flavoproteins2002. Agency for Scientific Publications, Berlin2002: 803-815Google Scholar). Several organic gold compounds, which have high affinity for sulfur- and selenium-containing ligands (17Shaw III, C.F. Chem. Rev. 1999; 99: 2589-2600Crossref PubMed Scopus (847) Google Scholar), e.g. proteins with activated cysteine or selenocysteine residue(s), are also potential agents for medical applications. Here, we describe a novel, highly potent gold-phosphole inhibitor (GoPI) of hGR. Both the mode of action of the inhibitor on hGR as well as the oxidative half-reaction of hGR during catalysis are studied in detail. We propose a mechanism for the reaction of GoPI with hGR based on studies using mass spectrometry, titrations, stopped-flow kinetics, and steady-state kinetics. The potential of GoPI as a lead compound for the development of novel anti-tumor drugs was confirmed by first experiments with glioblastoma cells cultured in vitro. Materials—Recombinant hGR was produced as described (2Nordhoff A. Bücheler U.S. Werner D. Schirmer R.H. Biochemistry. 1993; 32: 4060-4066Crossref PubMed Scopus (106) Google Scholar). The protein concentration of purified hGR was determined using ϵ463 nm(λmax) = 11.3 mm-1 cm-1 for Eox (FAD-containing subunit, see Ref. 3Krauth-Siegel R.L. Arscott L.D. Schönleben-Janas A. Schirmer R.H. Williams Jr., C.H. Biochemistry. 1998; 37: 13968-13977Crossref PubMed Scopus (58) Google Scholar). The ratio A274 nm/A463 nm of purified enzyme was 6.9. The specific enzymatic activity for hGR was measured as described (3Krauth-Siegel R.L. Arscott L.D. Schönleben-Janas A. Schirmer R.H. Williams Jr., C.H. Biochemistry. 1998; 37: 13968-13977Crossref PubMed Scopus (58) Google Scholar). All titrations, steady-state, and rapid reaction experiments were performed in GR buffer containing 47 mm potassium phosphate, 200 mm KCl, and 1 mm EDTA, pH 6.9, as described previously (3Krauth-Siegel R.L. Arscott L.D. Schönleben-Janas A. Schirmer R.H. Williams Jr., C.H. Biochemistry. 1998; 37: 13968-13977Crossref PubMed Scopus (58) Google Scholar, 7Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar, 8Böhme C.C. Arscott L.D. Becker K. Schirmer R.H. Williams Jr., C.H. J. Biol. Chem. 2000; 275: 37317-37323Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). NADPH and GSSG were dissolved in GR buffer, and their concentrations were determined spectrophotometrically or in enzymatic assays, respectively. Solutions of GoPI (see below) in N,N-dimethylformamide (DMF) were freshly prepared before each experiment. GoPI Synthesis—The synthesis of [1-phenyl-2,5-di(2-pyridyl)-phosphole}AuCl] was carried out in analogy to the synthesis of [{1-phenyl-2,5-di(2-thienyl)phosphole}AuCl] (18Fave C. Cho T.Y. Hissler M. Chen C.W. Luh T.Y. Wu C.C. Réau R. J. Am. Chem. Soc. 2003; 125: 9254-9255Crossref PubMed Scopus (187) Google Scholar, 19Fave C. Hissler M. Kárpáti T. Rault-Berthelot J. Deborde V. Toupet L. Nyulászi L. Réau R. J. Am. Chem. Soc. 2004; 126: 6058-6063Crossref PubMed Scopus (88) Google Scholar) and [{1-phenyl-2,5-di(2-pyridyl)phosphole}W(CO)5] (20Hay C. Le Vilain D. Deborde V. Toupet L. Réau R. Chem. Commun. 1999; : 345-355Crossref Scopus (72) Google Scholar). The reactant 1-phenyl-2,5-di(2-pyridyl)phosphole was synthesized as described previously (16Hay C. Hissler M. Fischmeister C. Rault-Berthelot J. Toupet L. Nyulaszi L. Réau R. Chem. Eur. J. 2001; 7: 4222-4236Crossref PubMed Scopus (246) Google Scholar). Au-Cl(tetrahydrothiophene) (26 mg, 81 μmol) was added to a solution of 10 ml of CH2Cl2 containing 30 mg (81 μmol) of 1-phenyl-2,5-di(2-pyridyl)phosphole. This mixture was stirred for 3 h at room temperature. Then all the volatile materials were removed in vacuo. The precipitate was washed with pentane (4 × 10 ml) and the gold-phosphole complex was obtained as an air-stable green-yellow solid (34 mg, 70% yield). Structure and purity of GoPI (600.4 g/mol) were confirmed using high resolution mass spectrometry (HR-MS), as well as 1H, 13C[1H], and 31P[1H] NMR spectroscopy. Selected spectroscopic data: 13C[1H] NMR (CDCl3; 75.46 MHz): δ22.7 (s, = CCH2CH2), 30.1 (s, = CCH2), 122.8 (s, C5 pyridyl), 124.5 (d, J(P,C) = 5.5 Hz, C3 pyridyl), 129.3 (d, J(P,C) = 12.4 Hz, Cm), 132.2 (s, Cp), 134.5 (d, J(P,C) = 14.0 Hz, Co) 137.2 (s, C4 pyridyl), 149.4 (s, C6 pyridyl), 152.2 (d, J(P,C) = 13.9 Hz, C2 pyridyl), 153.6 (m, Cβ), Cα and Cipso not observed. 31P[1H] NMR (CDCl3, 121.5 MHz): δ +39.9 (s). High resolution mass spectrometry (mNBA, FAB): (m/z) 601.0859 [M + H]+. C24H22PClN2Au was calculated as 601.0875. C24H22PCIN2AuCalculated:C47.98H3.52Found:C48.10H3.62(Eq. 1) UV-visual (CH2Cl2) λmax (nm) ϵ (m-1 cm-1): 271 (7850) and 383 (8300). Emission (CH2Cl2) λem (nm) was 495. Steady-state Kinetics and Inhibition Studies—Steady-state kinetics were monitored spectrophotometrically at 25 °C using a Hitachi U-2001 or a Beckmann DU 650 UV-visual spectrophotometer. All experiments were performed in the presence of the same concentration of DMF without inhibitor as a control. The type of inhibition was characterized by analyzing Km values for GSSG in the presence of GoPI or PPh3AuCl, and for NADPH in the presence of GoPI. All reactions were started by the addition of NADPH unless otherwise described. To determine the half-maximal inhibition of hGR by GoPI in comparison with other disulfide reductase inhibitors (I0.5), 1.4 or 2.8 nm hGR was incubated for 10 min with 100 μm NADPH and 0.5–10 nm GoPI or 10–100 nm PPh3AuCl, respectively. Then, 100 μm GSSG was added, and the residual activity of hGR was measured. Alternatively, hGR was incubated with GoPI for 10 min in the absence of NADPH, followed by addition of NADPH and GSSG. Time-dependent inactivation of hGR by GoPI was analyzed under two sets of conditions: first, as described by Kitz and Wilson (21Kitz R. Wilson I.B. J. Biol. Chem. 1962; 237: 3245-3249Abstract Full Text PDF PubMed Google Scholar), was to preincubate 0.1 μm NADPH-reduced hGR with 1–3-fold excess GoPI at 4 °C. Higher concentrations of GoPI lead to complete and very rapid inactivation of hGR, which prevented measurement. Aliquots of 5 μl were taken after 0.5–10 min and used for measuring the residual activity at 25 °C in a 500-μl assay mixture. Second, 1.7 nm NADPH-reduced hGR at 25 °C was incubated with inhibitor (1.7–5.0 nm). After various intervals (0.5–10 min) of incubation, the enzymatic activity was measured by adding 1 mm GSSG to the mixture and compared with that of an untreated control. Based on this second approach a second-order rate constant of the reaction between enzyme and inhibitor was determined from the equation, d[E]/dt = k[E][I] (22Davioud-Charvet E. McLeish M.J. Veine D.M. Giegel D. Arscott L.D. Andricopulo A.D. Becker K. Müller S. Schirmer R.H. Williams Jr., C.H. Kenyon G.L. Biochemistry. 2003; 42: 13319-13330Crossref PubMed Scopus (61) Google Scholar). Titration and Rapid Reaction Experiments—Titrations were carried out at 25 °C and stopped-flow measurements at 4 °C. To produce hGR(EH2) in the absence of NADP(H), NaBH4 was added at a ∼50-fold molar excess over hGR(Eox) (7Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar, 8Böhme C.C. Arscott L.D. Becker K. Schirmer R.H. Williams Jr., C.H. J. Biol. Chem. 2000; 275: 37317-37323Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). NaBH4 was dissolved in NaOH, pH 10 (where it is quite stable), prior to each experiment. Salt concentrations and pH of the GR buffer do not change significantly after addition of the NaBH4-containing solution (<4% v/v); excess NaBH4 is degraded rapidly at pH 6.9 (7Rietveld P. Arscott L.D. Berry A. Scrutton N.S. Deonarain M.P. Perham R.N. Williams Jr., C.H. Biochemistry. 1994; 33: 13888-13895Crossref PubMed Scopus (74) Google Scholar, 8Böhme C.C. Arscott L.D. Becker K. Schirmer R.H. Williams Jr., C.H. J. Biol. Chem. 2000; 275: 37317-37323Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 23Davis R.E. Swain C.G. J. Am. Chem. Soc. 1960; 82: 5949-5950Crossref Scopus (71) Google Scholar). Absorption spectra were recorded on a Varian Cary 3 or a Hewlett-Packard 8453 UV-visual spectrophotometer. Titration of Oxidized and Reduced hGR with GoPI—Oxidized enzyme was titrated (in duplicate) with 0–10 eq of GoPI under aerobic conditions (initial concentration of enzyme was 9.6 or 9.7 μm, the final concentration of DMF was 1.8 or 1.6%, v/v). For each experiment GR buffer was titrated with inhibitor as a reference. In a control experiment hGR was titrated with DMF without GoPI, and the spectra and specific enzymatic activity were not affected significantly up to a concentration of 2.7% (v/v). NaBH4-reduced hGR(EH2) was titrated with 0–1.1 eq of inhibitor under anaerobic conditions (initial concentration of enzyme was 16.6 μm, final concentration of DMF was 2.7%, v/v). At the end of the titration, the protein solution was washed repeatedly in a Centricon 30 (Millipore) and assayed for specific activity to test the reversibility of the inhibition. Reduction of hGR(Eox) after Preincubation with GoPI—Oxidized enzyme (8.2 μm) was incubated aerobically with 10 eq of inhibitor, followed by titration with 0–11.3 eq of NADPH (0–88 μm) under anaerobic conditions. At the end of the titration, the protein solution was washed repeatedly in Centricon 30 to attempt to remove the inhibitor. Alternatively, 16.3 μm hGR(Eox) was incubated with 1.25 eq of GoPI, followed by a reduction with 0–20 eq of NaBH4 under aerobic conditions. GR buffer containing only the inhibitor was treated identically and served as a spectral control. Reactivity of GoPI with Dithiothreitol, GSH, and Bovine Serum Albumin—To analyze spectral changes of GoPI in the presence of different thiols, solutions of 15–20 μm inhibitor in GR buffer were incubated for 10 min with 10 eq of GSH or dithiothreitol. In addition, GoPI was added to a solution containing 48 μm (2.4 eq) bovine serum albumin, and incubated for 10 min. Rapid Reaction Kinetics—Rapid reaction kinetics of the oxidative half-reaction were measured under anaerobic conditions in the diode array mode in a Hi-Tech SF-61DX2 stopped-flow photometer (Hi-Tech Scientific, Salisbury, Wiltshire, United Kingdom). The reaction was initiated by mixing equal volumes of substrate and NaBH4-reduced enzyme (preincubated with or without GoPI) in the stopped-flow instrument: in a series of control experiments the first syringe was loaded with 28.1 μm hGR(EH2), and the second syringe was loaded with GR buffer containing 0, 30, 60, 120, or 600 μm GSSG. Afterward, 0.9 eq of GoPI was added to hGR(EH2) in the first syringe (0.86% DMF, v/v) and mixed again with 0, 30, 60, 120, or 600 μm GSSG. Finally, GoPI was added to the enzyme-containing solution at an excess over hGR (∼2.4 eq, ∼3% DMF, v/v) and mixed with 600 μm GSSG. The data from kinetic traces at selected wavelengths were fitted to single exponential functions using the software KinetAsyst3 (Hi-Tech Scientific). Molecular Modeling—GoPI was manually built with GaussView (Gaussian, Inc.). The geometry of the molecule was subsequently refined by molecular mechanics methods using the UFF force field. GoPI was manually fitted to the active site of hGR (5Karplus P.A. Schulz G.E. J. Mol. Biol. 1989; 210: 163-180Crossref PubMed Scopus (289) Google Scholar) with the program O (24Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar) and refined with the program CNS (25Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16949) Google Scholar). SELDI-TOF MS—Purified hGR was analyzed on NP20 Protein-Chip® Arrays (normal phase, hydrophilic properties; Ciphergen Biosystems, Inc.) to detect masses of untreated hGR(Eox) compared with enzyme after pre-reduction with NADPH and incubation with GoPI. Each sample was prepared in triplicate. GR (85 μm) was reduced with 50 eq of NADPH and then incubated with 1 or 10 eq of GoPI for 30 min at room temperature. The resulting solution was diluted 1:100 in double-distilled water and 1 μl of each sample (0.85 pmol) was applied directly to NP20 ProteinChip® Array spots, which were prewetted with 1 μl of double-distilled water. The array surface was allowed to dry for 30 min, and was washed twice with 5 μl of double-distilled water to remove residual salt. Saturated sinapinic acid solution (1 μl 50%) dissolved in 50% acetonitrile, 0.5% trifluoroacetic acid was added and air-dried twice. Subsequently, mass analysis of bound hGR was performed using the ProteinChip Reader (PBS IIc, Ciphergen Biosystems, Inc.). The ProteinChip Arrays were analyzed by averaging 100 laser shots collected in the positive ion mode. The accelerating potential was +20 kV and the extraction delay time was set to 1620 ns. Deflector settings were used to filter out peaks with m/z <8000. Calibration was performed with the following proteins: bovine β-lactoglobulin A (18,363 Da), horseradish peroxidase (43,240 Da), bovine serum albumin (66,433 Da), and chicken conalbumin (77,490 Da). Proliferation Assays—Cell cultures of glioblastoma cells NCH37, NCH82, and NCH89 were established in our laboratory (26Herold-Mende C. Steiner H.H. Andl T. Riede D. Buttler A. Reisser C. Fusenig N.E. Mueller M.M. Lab. Investig. 1999; 79: 1573-1582PubMed Google Scholar) and cultured routinely in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics at 37 °C, 5% CO2, and 95% air in a humidified incubator with medium changes twice a week. After reaching confluence, cells were harvested by a brief incubation with a trypsin/EDTA solution (Viralex, PAA, Linz, Austria) and seeded into a fresh 75-cm2 plastic tissue culture flask. Proliferation assays were performed as described earlier (26Herold-Mende C. Steiner H.H. Andl T. Riede D. Buttler A. Reisser C. Fusenig N.E. Mueller M.M. Lab. Investig. 1999; 79: 1573-1582PubMed Google Scholar) using the BrdU Labeling and Detection Kit III (Roche Diagnostics, Mannheim, Germany). Cells were seeded in 8 replicas in 96-well plates into Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics, each well receiving 7 × 103 cells. After 24 h GoPI was added at different concentrations (1, 5, 10, and 20 μm). 48 h later, 5′-bromo-2′-deoxyuridine was added to the wells at a final concentration of 100 μm. The 5′-bromo-2′-deoxyuridine incorporation assay was processed according to the manufacturer's instructions. The mean value of the absorbance of the control samples containing no GoPI was defined as 100% proliferation rate. All values are means of at least two independent experiments (each comprising 8 replicas). Chemical Properties of GoPI—GoPI is a symmetric aromatic phosphole-gold complex (Fig. 1), which is stable at room temperature. The phosphorus atom behaves as a classical 2-electron donor toward the gold(I) atom (18Fave C. Cho T.Y. Hissler M. Chen C.W. Luh T.Y. Wu C.C. Réau R. J. Am. Chem. Soc. 2003; 125: 9254-9255Crossref PubMed Scopus (187) Google Scholar). The simplicity of the 13C NMR spectra recorded in solution is in favor of a symmetric structure. However, theoretical calculations and modeling of the structure predict a low energy barrier for the pyridyl rings to rotate around the C-C bonds connecting them to the phosphole ring (16Hay C. Hissler M. Fischmeister C. Rault-Berthelot J. Toupet L. Nyulaszi L. Réau R. Chem. Eur. J. 2001; 7: 4222-4236Crossref PubMed Scopus (246) Google Scholar). It is thus very likely that this dynamic process is rapid in solution at room temperature. Steady-state Kinetics—Inhibition of hGR(Eox) by GoPI is competitive for GSSG at high concentrations of inhibitor (Fig. 2): all straight lines in the Lineweaver-Burk plot intersect at the ordinate with almost identical Vmax values, and plots of [GSSG]/ν against [GoPI] lead to predominantly parallel lines when using data points for 0.01, 0.5, 1.0, and 2.0 μm GoPI. The Kic is 0.46 μm (for competitive inhibition of pyridine nucleotide-disulfide oxidoreductases, see for example Ref. 10Becker K. Herold-Mende C. Park J.J. Lowe G. Schirmer R.H. J. Med. Chem. 2001; 44: 2784-2792Crossref PubMed Scopus (129) Google Scholar), calculated using the equation Kic = Km[GoPI]/(Km ′ - Km) for 0.5, 1.0, and 2.0 μm GoPI. Km ′ is the apparent Michaelis constant in the presence of inhibitor obtained from the x axis intercepts of the Lineweaver-Burk plots. The Km for GSSG in the absence of inhibitor is 71 μm. Km for NADPH (4.4–5.6 μm) was not significantly changed in the presence of 0.65–2.5 μm inhibitor (data not shown). In contrast to the competition with GSSG that was observed at high inhibitor concentrations, only ∼1 nm GoPI was sufficient to inactivate 50% of 1.4 nm NADPH-reduced hGR, when hGR was preincubated for 10 min with GoPI. Preincubating oxidized hGR with inhibitor in the absence of NADPH for 10 min requires 6.2 nm inhibitor to achieve 50% inactivation. Time-dependent Inactivation—GoPI also inhibits hGR irreversibly in a time-dependent process. The inactivation of GoPI was monitored over a period of 10 min using two different preincubation protocols as described under "Experimental Procedures." In the first approach enzyme activity decreased with pseudo first-order kinetics (kapp = kinact[I]0/(Ki + [I]0), see also Refs. 21Kitz R. Wilson I.B. J. Biol. Chem. 1962; 237: 3245-3249Abstract Full Text PDF PubMed Google Scholar and 27Bisswanger H. Enzymkinetik. 3rd Ed. Wiley-VCH, Weinheim2000Crossref Google Scholar), and was dependent on the inhibitor concentration (data not shown). The maximal rate constant for inhibition in the presence of excess inhibitor (kinact) was ∼1.2 min-1, and t½ = ln2/kinact for enzyme

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