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• W2067575053 abstract "S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of β-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-κB and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo. S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of β-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-κB and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo. S-Nitrosylation of cellular proteins has emerged as the key reaction through which NO 2The abbreviations used are: NOnitric oxideSNOS-nitrosothiolGSNORS-Nitrosoglutathione reductaseGSNOS-nitrosoglutathioneADHalcohol dehydrogenasesGCsoluble guanylyl cyclaseDMEMDulbecco's modified Eagle's mediumICAMintercellular adhesion moleculeSNPsodium nitroprusside12-HDDA12-hydroxydodecanoic acidp-IκBphosphorylated IκBl-NAMEN (ω) nitro-l-arginine methyl ester. controls the function of a wide array of cellular proteins (1.Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1730) Google Scholar). Through S-nitrosylation, NO has been shown to regulate smooth muscle tone, apoptosis, and NF-κB activity (2.Benhar M. Stamler J.S. Nat. Cell Biol. 2005; 7: 645-646Crossref PubMed Scopus (94) Google Scholar, 3.Kelleher Z.T. Matsumoto A. Stamler J.S. Marshall H.E. J. Biol. Chem. 2007; 282: 30667-30672Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 4.Que L.G. Liu L. Yan Y. Whitehead G.S. Gavett S.H. Schwartz D.A. Stamler J.S. Science. 2005; 308: 1618-1621Crossref PubMed Scopus (254) Google Scholar, 5.Whalen E.J. Foster M.W. Matsumoto A. Ozawa K. Violin J.D. Que L.G. Nelson C.D. Benhar M. Keys J.R. Rockman H.A. Koch W.J. Daaka Y. Lefkowitz R.J. Stamler J.S. Cell. 2007; 129: 511-522Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Whereas S-nitrosylation mediates many NO effects inside the body, denitrosylation pathways inside the cells terminate these effects. Among the enzymes capable of denitrosylating SNOs (6.Benhar M. Forrester M.T. Hess D.T. Stamler J.S. Science. 2008; 320: 1050-1054Crossref PubMed Scopus (455) Google Scholar, 7.Johnson M.A. Macdonald T.L. Mannick J.B. Conaway M.R. Gaston B. J. Biol. Chem. 2001; 276: 39872-39878Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 8.Trujillo M. Alvarez M.N. Peluffo G. Freeman B.A. Radi R. J. Biol. Chem. 1998; 273: 7828-7834Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 9.Wink D.A. Cook J.A. Kim S.Y. Vodovotz Y. Pacelli R. Krishna M.C. Russo A. Mitchell J.B. Jourd'heuil D. Miles A.M. Grisham M.B. J. Biol. Chem. 1997; 272: 11147-11151Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), the ubiquitously expressed GSNOR has been shown to be operative in vivo (10.Liu L. Hausladen A. Zeng M. Que L. Heitman J. Stamler J.S. Nature. 2001; 410: 490-494Crossref PubMed Scopus (753) Google Scholar, 11.Liu L. Yan Y. Zeng M. Zhang J. Hanes M.A. Ahearn G. McMahon T.J. Dickfeld T. Marshall H.E. Que L.G. Stamler J.S. Cell. 2004; 116: 617-628Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). nitric oxide S-nitrosothiol S-Nitrosoglutathione reductase S-nitrosoglutathione alcohol dehydrogenase soluble guanylyl cyclase Dulbecco's modified Eagle's medium intercellular adhesion molecule sodium nitroprusside 12-hydroxydodecanoic acid phosphorylated IκB N (ω) nitro-l-arginine methyl ester. GSNOR, a member of the alcohol dehydrogenase family (12.Eklund H. Müller-Wille P. Horjales E. Futer O. Holmquist B. Vallee B.L. Höög J.O. Kaiser R. Jörnvall H. Eur. J. Biochem. 1990; 193: 303-310Crossref PubMed Scopus (102) Google Scholar), indirectly regulates SNOs inside the cells by reducing GSNO, a NO metabolite arising from the reaction of glutathione with reactive nitrogen species (11.Liu L. Yan Y. Zeng M. Zhang J. Hanes M.A. Ahearn G. McMahon T.J. Dickfeld T. Marshall H.E. Que L.G. Stamler J.S. Cell. 2004; 116: 617-628Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Due to its role in the turnover of nitrosylation of intracellular proteins, GSNOR has become an important target for developing agents that modulate NO bioactivity inside cells. The therapeutic potential of preventing the breakdown of SNOs via inhibition of GSNOR has been demonstrated in mice lacking GSNOR. GSNOR-deficient mice showed significantly low airway hyperresponsivity to allergen challenge and increased β-adrenergic receptor expression and function in lungs and heart (4.Que L.G. Liu L. Yan Y. Whitehead G.S. Gavett S.H. Schwartz D.A. Stamler J.S. Science. 2005; 308: 1618-1621Crossref PubMed Scopus (254) Google Scholar, 5.Whalen E.J. Foster M.W. Matsumoto A. Ozawa K. Violin J.D. Que L.G. Nelson C.D. Benhar M. Keys J.R. Rockman H.A. Koch W.J. Daaka Y. Lefkowitz R.J. Stamler J.S. Cell. 2007; 129: 511-522Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). This suggested GSNOR inhibition to be beneficial for the treatment of lung and cardiovascular diseases. We report here novel inhibitors of GSNOR that exclude GSNO from its binding site and preferentially inhibit GSNOR among the alcohol dehydrogenase (ADH) isozymes. Using the new inhibitors, we demonstrate that GSNOR actively regulates S-nitrosylation of proteins stemming from nitric-oxide synthase activity. Among the key cellular processes regulated by GSNOR are the activation of the transcription factor NF-κB and soluble guanylyl cyclase (sGC). All of the chemicals used in the experiments were purchased from Sigma. RAW 264.7 and A549 cells, DMEM, and fetal bovine serum were purchased from American Tissue and Cell Culture. Recombinant human GSNOR, ADH1B (β2-ADH), ADH4 (π-ADH), and ADH7 (σ-ADH) were expressed in Escherichia coli and purified in the Indiana University School of Medicine Protein Expression Core. Compounds C1–C3 were purchased from ChemDiv Inc. The screening for GSNOR inhibitors was performed using a library of 60,000 compounds from ChemDiv Inc. in the Chemical Genomics Core facility at Indiana University. Screening was conducted in 384-well plates and involved incubating GSNOR with 12.5 μm compound, 1 mm each NAD+ and octanol in 0.1 m sodium glycine, pH 10. Enzyme activity was determined by measuring the rate of production of NADH spectrophotometrically at 340 nm. Inhibition of GSNOR was calculated from the ratio of enzyme activity in the presence of compounds to that in no compound controls performed on the same plate. Following their identification from the high throughput screening, the GSNOR-inhibitory properties of the initial hits were confirmed at the pH 10 using octanol as the substrate and at pH 7.5 using GSNO as the substrate (see legend of Table 1 for details of the assay).TABLE 1Structures of GSNOR inhibitors Inhibition of the ADH1B (β2-ADH), ADH4 (π-ADH), and ADH7 (σ-ADH) was evaluated by determining the inhibitory effect of GSNOR inhibitors on the rate of oxidation of ethanol by each of these ADH isozymes. The assay mixture contained a saturating amount of NAD+ (1–2 mm) and ethanol at its Km concentration for each of the respective enzyme. All assays were performed at 25 °C in 50 mm potassium phosphate, pH 7.5, containing 0.1 mm EDTA and involved determining the rate of formation of NADH spectrophotometrically at 340 nm. Specific assay conditions for each isozyme are as follows. (a) Studies with ADH1B involved adding 3.5 μg of the enzyme to the assay mixture containing 2 mm NAD+, 1 mm ethanol, and the inhibitor. (b) Studies with ADH7 involved adding 0.5 μg of enzyme to the assay mixture containing 2 mm NAD+, 30 mm ethanol, and the inhibitor. (c) Studies with ADH4 involved adding 19.5 μg of the enzyme to the assay mixture containing 1 mm NAD+, 35 mm ethanol and the inhibitor. (d) Studies with GSNOR involved adding 0.1 μg of the enzyme to an assay mixture containing 15 μm NADH, 5 μm GSNO, and the inhibitor. Inhibition experiments with the GSNOR inhibitors were conducted at 25 °C in 3 ml of 50 mm potassium phosphate (pH 7.5) containing 0.1 mm EDTA. Five different concentrations of GSNO or NADH were used when they were the varied substrates and maintained at 10 and 15 μm, respectively, when present as the nonvaried substrate. A minimum of three inhibitor concentrations were used, and the rate of NADH and GSNO consumption was determined spectrophotometrically at 340 nm. The data were fit to the competitive, noncompetitive, and uncompetitive inhibition models, and the model best fitting the data was chosen on the basis of F-statistics performed using the Graphpad Prism 4.0. Fluorescence studies were conducted in 50 mm potassium phosphate, pH 7.5, at room temperature using a Fluoromax-2 fluorescence spectrometer (Instruments S.A., Inc., Edison, NJ). The equilibrium dissociation constant of GSNOR inhibitors was determined by measuring the changes in the fluorescence of GSNOR-bound NADH (λex = 350 nm; λem = 455 nm) upon the addition of inhibitor. During the experiment, increasing amounts of inhibitor were added to a solution containing 2 μm GSNOR and 1.7 μm NADH. The decrease in fluorescence at 455 nm with each addition of inhibitor was plotted against the final concentration of inhibitor, and the data were fitted to Equation 1 using nonlinear regression to obtain the dissociation constant of the inhibitor for GSNOR-NADH complex, ΔF=ΔFLt+Et+KD−Lt+Et+KD−4LTET2ET(Eq. 1) In Equation 1, ΔF is the change in the fluorescence at 455 nm upon the addition of inhibitor. ΔFM is the maximum fluorescence change that was obtained from curve fitting. [ET] and [LT] are the concentrations of GSNOR and inhibitor, respectively. KD is the equilibrium dissociation constant for the formation of GSNOR·NADH·inhibitor complex. The data were fitted using the Graphpad Prism 4.0. RAW 264.7 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 200 units/ml penicillin, and 200 μg/ml streptomycin. The cells were incubated at 37 °C in an atmosphere containing 5% CO2 and 95% air. For the experiments, 1–2 × 106 cells were plated in 6-well plates with or without 33 μm compounds 16 h before the experiment. (Later experiments showed that pretreatment with compounds had no effect on the rate of accumulation of nitroso species inside the cells). On the day of the experiment, the medium was replaced with a fresh 3 ml of medium, and the cells were treated with compounds for a predetermined length of time. Following the incubation period, the cells were washed three times with phosphate-buffered saline and scraped off of the plate in 250 μl of lysis buffer (50 mm potassium phosphate, pH 7.0, containing 50 mm N-ethylmaleimide and 1 mm EDTA) and lysed by sonication using a microtip probe (three pulses of 30% duty cycle; 2 output control on a Branson Sonicator). The cell debris was pelleted by centrifugation (10 min at 16,000 × g), and the cell lysate was analyzed for protein concentration using the Bio-Rad dye-binding protein assay. The concentration of nitroso compounds in the cell lysate was determined using the triiodide-based chemiluminescence method (13.Samouilov A. Zweier J.L. Anal. Biochem. 1998; 258: 322-330Crossref PubMed Scopus (110) Google Scholar) using a Sievers 280 nitric oxide analyzer. Briefly, cell lysates were treated with 15% (v/v) of a sulfanilamide solution (5% (w/v) in 0.2 m HCl) and kept at room temperature for 5 min to remove nitrite. The triiodide mixture was prepared fresh every day as described earlier (14.Zhang Y. Hogg N. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7891-7896Crossref PubMed Scopus (157) Google Scholar) and kept at 60 °C in the reaction vessel. The concentration of nitroso species was derived from a standard curve generated using GSNO. RAW 264.7 cells were cultured in 100-mm dishes using DMEM containing 10% heat-inactivated serum and 100 units/ml penicillin, 100 μg/ml streptomycin. On the day of the experiment, the medium was replaced with a fresh medium containing C3 (33 μm) alone or a mixture of C3 and l-NAME (1.1 mm), and the cells were incubated at 37 °C for varied lengths of time. At indicated times, the cells were quenched, and the lysate was analyzed for S-nitrosothiol content by the biotin switch assay as described by Jaffrey et al. (15.Jaffrey S.R. Methods Enzymol. 2005; 396: 105-118Crossref PubMed Scopus (29) Google Scholar) with modifications suggested by Wang et al. (16.Wang X. Kettenhofen N.J. Shiva S. Hogg N. Gladwin M.T. Free Radic. Biol. Med. 2008; 44: 1362-1372Crossref PubMed Scopus (92) Google Scholar) and Zhang et al. (17.Zhang Y. Keszler A. Broniowska K.A. Hogg N. Free Radic. Biol. Med. 2005; 38: 874-881Crossref PubMed Scopus (103) Google Scholar). Briefly, free sulfydryls in ∼200 μg of cell lysate were blocked with 20 mm S-methyl methanethiosulfonate in 1 ml of HEN buffer (250 mm HEPES, pH 7.7, containing 1 mm EDTA and 0.1 mm Neocupronine) containing 2% SDS at 50 °C for 20 min. Free S-methyl methanethiosulfonate was removed by gel filtration spin columns, and the blocked proteins were labeled with 1 mm (N-(6-(biotinamido)hexyl)-3′-(2′-pyridyldithio)-propionamide) (Pierce) in the presence or absence of 30 mm ascorbate and 2 μm CuCl for 3 h. Equal amounts of proteins were loaded in each lane, and the degree of biotinylation (and hence S-nitrosylation) determined using an anti-biotin antibody (Sigma). For the isolation of biotinylated proteins, equal amounts of biotinylated RAW cell extract were treated with streptavidin-agarose and incubated at 4 °C for 4 h. The resin was extensively washed with a washing buffer (25 mm HEPES, pH 7.7, containing 600 mm NaCl, 1 mm EDTA, and 1% β-octyl glucoside) before eluting them using SDS-PAGE sample buffer containing 500 mm β-mercaptoethanol at 40 °C for 2 h. The eluted proteins were separated using SDS-PAGE and blotted onto a polyvinylidene difluoride membrane. The blots were probed with IKKβ antibody (Cell Signaling Technology) and immunodetected by chemiluminescence. A549/NfκB-luc cells (Panomics; Quantitative Biology) harboring a luciferase reporter under the control of six consensus NF-κB binding motifs were propagated at 37 °C in the presence of 5% CO2 and in DMEM with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal bovine serum. During the experiments, 105 cells were plated with 2 ml of medium and 100 μg/ml hygromycin in 12-well plates. Cells were treated with C3 for 4 h followed by TNFα (50 ng/ml) treatment for 6 h. Luciferase activity was measured in cell lysates using a commercial kit (Promega, Madison, WI). The luciferase activity was measured using a manual luminometer, and the data were normalized to the total protein concentration determined using the BCA protein assay. A549 or RAW 264.7 cells were cultured in F-12k or DMEM containing 10% heat-inactivated serum, 100 units/ml penicillin, and 100 mg/ml streptomycin, respectively. A549 (2 × 105) and RAW 264.7 (2.5 × 105) were plated in 35-mm plates, or 50,000 A549 cells were plated in 24-well plates on the day before the experiment. On the day of the experiment, the medium was replaced with fresh medium plus or minus varied concentrations of C3 for 4 h. In some experiments, the cells were also treated with MG132 (40 μm final) for 1 h. Following the pretreatments, the cells were treated with TNFα (10 ng/ml) for 5 min for the analysis of phospho-IκBα, IκBα, phospho-IKKα/β, and IKKβ and for 6 h for analysis of intercellular adhesion molecule-1 (ICAM-1) before quenching them with SDS-PAGE sample buffer containing 50 mm NaF. Samples were vortexed, boiled for 5 min, centrifuged at 16,000 × g for 5 min, and loaded onto a 10% precast SDS- polyacrylamide Tris-HCl gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane. The blots were probed overnight with primary antibodies at 4 °C and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The signal was detected using a GE Healthcare ECL Plus chemiluminescence kit. Mice were anesthetized with diethyl ether. A thoracotomy was performed to expose thoracic and abdominal aorta. A 25-gauge syringe was inserted into the apex of the left ventricle and perfused free of blood with oxygenated Krebs Henseleit buffer. The right atrium was cut to provide an exit for blood. The aorta was removed and cleaned of fat and adventitia. The aorta was cut into 2-mm-long segments and mounted on a four-channel wire myograph (AD Instruments). Vessel rings were maintained in 10-ml organ baths with oxygenated PSS (95% O2 and 5% CO2) at 37 °C. Rings were allowed to equilibrate for 80 min with the buffer in each organ bath changed every 20 min. One gram of pretension was placed on each aortic ring (appropriate starting tension for optimal vasomotor function, as determined in previous experiments). An eight-channel octal bridge (Powerlab) and data acquisition software (Chart version 5.2.2) were used to record all force measurements. After equilibration for 80 min, 1 μm phenylephrine was added to each ring for submaximal contraction. After stabilization, either C3 or sodium nitroprusside (SNP) was added to the rings, and the tone of the rings was determined. For the determination of SNP and C3 dose-response relationships, aortic rings were precontracted with 10−6m phenylephrine, and SNP or C3 was then added in increasing concentrations from 10−9 to 10−4m. The cytosolic fraction of RAW264.7 cells (20 μl) was mixed with 40 μl of reaction buffer (50 mm triethanolamine, pH 7.4, 0.1 mm EGTA, 0.1 mg/ml creatine kinase, 200 μm phosphocreatine, 100 μm cGMP) and, when necessary, supplied with 50 μm C3 and incubated for 10 min at room temperature. The samples were then supplied with 50 μm GSNO and incubated for an additional 10 min. At the end of incubation, 0.3 μg of purified sGC was added to the sample, which was transferred to 37 °C, and the cyclase reaction was initiated by 1 mm GTP/[α-32P]GTP. After 10 min, the reaction was stopped by 0.5 ml of 150 mm zinc acetate, followed by the addition of 0.5 ml of 200 mm sodium carbonate. The formed zinc carbonate pellet containing the bulk of unreacted GTP was precipitated by 10 min of centrifugation at 15,000 × g, and the supernatant was loaded on a 1-ml alumina oxide column equilibrated with 50 mm Tris, pH 7.5. cGMP was eluted from the column with 10 ml of 50 mm Tris, pH 7.5, collected, and quantified by a scintillation counter. A high throughput screening approach was used for identifying novel inhibitors of GSNOR. Screening for GSNOR inhibitors was performed in the presence of saturating and unsaturating concentrations of NAD+ and the alcohol substrate, octanol (18.Estonius M. Höög J.O. Danielsson O. Jörnvall H. Biochemistry. 1994; 33: 15080-15085Crossref PubMed Scopus (33) Google Scholar), to increase the probability of identifying compounds that bind only in the GSNO binding site of GSNOR. Three compounds (C1–C3), displaying high affinity and specificity for inhibiting GSNOR within the alcohol dehydrogenase family, were selected for further investigation (Tables 1 and S1). Each of the selected compounds obeyed Lipinski's rules (19.Lipinski C.A. Lombardo F. Dominy B.W. Feeney P.J. Adv. Drug Deliv. Rev. 2001; 46: 3-26Crossref PubMed Scopus (8492) Google Scholar) for druglike properties and had 2-order of magnitude lower IC50 than the present GSNOR inhibitor, dodecanoic acid (20.Sanghani P.C. Stone C.L. Ray B.D. Pindel E.V. Hurley T.D. Bosron W.F. Biochemistry. 2000; 39: 10720-10729Crossref PubMed Scopus (63) Google Scholar) (Table 1). Dead end kinetic inhibition and fluorescence binding studies were performed to determine the mechanism by which C1–C3 inhibited GSNOR. As evident from Table 2 and supplemental Fig. S1, the inhibitors exhibit noncompetitive or uncompetitive inhibition against varied concentrations of GSNO or NADH. This suggested that neither of the substrates were able to completely prevent the compounds from binding to GSNOR. The inability of both of the substrates to overcome the inhibition completely is also consistent with C1–C3 binding to multiple enzyme complexes in the kinetic pathway (shown in Scheme 1). Inhibition by binding to a site outside the active site in GSNOR was unlikely, since the type of inhibition by the compounds against NADH and GSNOR would have been similar. An additional dead-end inhibition study involving dodecanoic acid as inhibitor against varied GSNO was performed to determine the type of complexes that an inhibitor binding in the substrate binding site (20.Sanghani P.C. Stone C.L. Ray B.D. Pindel E.V. Hurley T.D. Bosron W.F. Biochemistry. 2000; 39: 10720-10729Crossref PubMed Scopus (63) Google Scholar, 21.Sanghani P.C. Robinson H. Bosron W.F. Hurley T.D. Biochemistry. 2002; 41: 10778-10786Crossref PubMed Scopus (64) Google Scholar) would form in the kinetic pathway of GSNO reduction. Dodecanoic acid was found to be a noncompetitive inhibitor against varied concentrations of GSNO, although it binds into the GSNO binding site (Table 2). The noncompetitive inhibition of GSNOR by dodecanoic acid can be explained by its binding to GSNOR at more than one place in the kinetic pathway, one where it competes with GSNO to bind to the enzyme (steps 1 and 2 in Scheme 1) and one where GSNO does not normally bind in the kinetic pathway (step 3 in Scheme 1). The noncompetitive inhibition of GSNOR by C1–C3 against varied GSNO can also be explained by their forming such E·NADH·I (EAI in Scheme 1) and E·NAD+·I (EQI in Scheme 1) complexes. The uncompetitive inhibition by C1 and C3 and almost uncompetitive inhibition by C2 (although inhibition by C2 statistically fits the noncompetitive mechanism better, the Kis is 5-fold higher than Kii and has high S.E. values) against varied NADH can be explained by the compounds binding to the E·NADH complex in the nearly ordered kinetic mechanism of GSNOR and the high affinity of NADH (KD = 0.05 μm) for GSNOR (20.Sanghani P.C. Stone C.L. Ray B.D. Pindel E.V. Hurley T.D. Bosron W.F. Biochemistry. 2000; 39: 10720-10729Crossref PubMed Scopus (63) Google Scholar). Both of these factors would make the contribution of E·I very small in the inhibition of the enzyme under the experimental conditions and make the inhibition uncompetitive. The mechanism by which C1–C3 inhibit GSNOR is similar to that by which the sulfoxide and amide compounds inhibit horse liver alcohol dehydrogenase (22.Chadha V.K. Leidal K.G. Plapp B.V. J. Med. Chem. 1983; 26: 916-922Crossref PubMed Scopus (27) Google Scholar). To summarize, the dead end kinetic inhibition studies are consistent with C1–C3 binding into the GSNO binding site and forming GSNOR·NADH·I (EAI in Scheme 1), GSNOR·NAD+·I (EQI), and GSNOR·I (EI) complexes.TABLE 2Mechanism of inhibition of GSNOR by GSNOR inhibitorsCompoundVaried substrateType of inhibitionKisKiiKDaKD is the equilibrium dissociation constant of the inhibitor for binding to the GSNOR·NADH complex, obtained by measuring the changes in the fluorescence of GSNOR-bound NADH with the addition of inhibitor (λex = 350 nm; λem = 455 nm). The dissociation constant was measured at 25 °C in 50 mm potassium phosphate, pH 7.5. Each KD value is an average of three independent experiments and is shown with the associated S.E.μmμmμmC1GSNONC1.7 ± 0.21.8 ± 0.11.3 ± 0.3NADHUC1.5 ± 0.1C2GSNONC1.9 ± 0.24.0 ± 0.36.5 ± 1.0NADHNC12 ± 52.5 ± 0.1C3GSNONC2.6 ± 0.31.6 ± 0.12.0 ± 0.1NADHUC1.7 ± 0.1Dodecanoic acidGSNONC280 ± 40190 ± 10a KD is the equilibrium dissociation constant of the inhibitor for binding to the GSNOR·NADH complex, obtained by measuring the changes in the fluorescence of GSNOR-bound NADH with the addition of inhibitor (λex = 350 nm; λem = 455 nm). The dissociation constant was measured at 25 °C in 50 mm potassium phosphate, pH 7.5. Each KD value is an average of three independent experiments and is shown with the associated S.E. Open table in a new tab Equilibrium binding studies were conducted to support the findings of the kinetic dead end inhibition studies. The addition of C3 to the GSNOR·NADH binary complex resulted in the quenching as well as blue shift in NADH emission (compare curves b and c in Fig. 1 A). This is consistent with the formation of the GSNOR·NADH·C3 ternary complex. A similar quenching of the fluorescence of the coenzyme dihydropyridine ring was observed when the amide inhibitors were added to the horse liver ADH·NADH complex (23.Sarma R.H. Woronick C.L. Biochemistry. 1972; 11: 170-179Crossref PubMed Scopus (20) Google Scholar). Fluorescence binding studies were also conducted in the presence of the alcohol substrate, 12-hydroxydodecanoic acid (12-HDDA), to determine if C3 was excluding the substrate from the GSNOR active site. The formation of GSNOR·NADH·12-hydroxydodecanoate abortive ternary complex was reported earlier (20.Sanghani P.C. Stone C.L. Ray B.D. Pindel E.V. Hurley T.D. Bosron W.F. Biochemistry. 2000; 39: 10720-10729Crossref PubMed Scopus (63) Google Scholar). Binding of 12-HDDA to the GSNOR·NADH complex resulted in an increase in the fluorescence of NADH, as shown in Fig. 1B (compare curves b and c). The addition of C3 to the mixture resulted in a spectrum (Fig. 1B, curve d) that was consistent with the displacement of 12-HDDA out of the active site and formation of the GSNOR·NADH·C3 ternary complex (curve d has higher fluorescence than that in Fig. 1A, curve c, because not all of 12-HDDA has been displaced by C3). These fluorescence binding experiments suggest that C3 excludes only the alcohol/aldehyde substrate from the binding site of GSNOR. C1 and C2 also exhibited similar effects on NADH fluorescence in the presence or absence of 12-HDDA, indicating that they too, like C3, only exclude the alcohol substrate from the GSNOR active site (data not shown). The fluorescence change observed upon the formation of the GSNOR·NADH·inhibitor complex was used to determine the equilibrium dissociation constant of the inhibitors for the GSNOR·NADH complex (Fig. 1C). The equilibrium dissociation constants of C1–C3 were in the low micromolar range (Table 2), suggesting that the affinity for the GSNOR·NADH complex was sufficient for the compounds to be effective inside the cells. C1 and C3 had a significantly higher affinity for the GSNOR·NADH complex than C2, as evident from their 3–5-fold lower equilibrium dissociation constants. The ability of the newly discovered inhibitors to inhibit GSNOR inside the cells was tested using a murine macrophage cell line (RAW 264.7 cells). RAW 264.7 cells were treated with inhibitors alone or in combination with GSNO. The accumulation of intracellular SNOs was determined as a function of time and inhibitor concentration (Fig. 2, A and B). Significantly higher amounts of SNOs accumulated in cells treated with both GSNO and the inhibitor than in those treated with GSNO alone (Fig. 2A). C1 and C3 appeared to be more effective than C2 in inhibiting GSNOR inside the cells, as evident from higher accumulation of SNOs (Fig. 2, A and B). These studies demonstrate that C1–C3 are able to inhibit intracellular GSNOR. The dependence of SNO accumulation on the concentration of compounds in RAW 264.7 cells (Fig. 2B) suggested that it was possible to regulate the extent of S-nitrosylation with the GSNOR inhibitors. An analysis of the molecular size of the nitrosylated species inside the treated cells showed that more than 95% of the nitrosylated species were greater than 5 kDa in size. Furthermore, 21–28% of the nitrosylated species in treated cells were resistant to mercury pretreatment, suggesting that N-nitrosothiolated proteins were also getting formed inside the cells. The effect of GSNOR inhibition on the nitrosylation of cellular proteins was also examined using the biotin switch assay technique developed by Jaffrey et al. (24.Jaffrey S.R. Erdjument-Bromage H. Ferris C.D. Tempst P. Snyder S.H. Nat. Cell Biol. 2001; 3: 193-197Crossref PubMed Scopus (1226) Google Scholar) with modifications by Wang et al. (16.Wang X. Kettenhofen N.J. Shiva S. Hogg N. Gladwin M.T. Free Radic. Biol. Med. 2008; 44: 1362-1372Crossref PubMed Scopus (92) Google Scholar). C3 increased the nitrosylation of cellular proteins in RAW 264.7 cells in a time-dependent manner (Fig. 2C). The effects of C3 on the nitrosylation of cellular proteins appeared to peak around 8 h before coming down. The accumulation of SNOs was less if cells were simultaneously treated with C3 and the nitric oxide synthase inhibitor, l-NAME (Fig. 2C). These data sug" @default.
• W2067575053 created "2016-06-24" @default.
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• W2067575053 date "2009-09-01" @default.
• W2067575053 modified "2023-09-27" @default.
• W2067575053 title "Kinetic and Cellular Characterization of Novel Inhibitors of S-Nitrosoglutathione Reductase" @default.
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