N-acetyl lysyltyrosylcysteine amide inhibits myeloperoxidase, a novel tripeptide inhibitor

Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity. We show KYC inhibits MPO-mediated hypochlorous acid (HOCl) formation and nitration/oxidation of LDL. Disulfide is the major product of MPO-mediated KYC oxidation. KYC (⩽4,000 μM) does not induce cytotoxicity in bovine aortic endothelial cells (BAECs). KYC inhibits HOCl generation by phorbol myristate acetate (PMA)-stimulated neutrophils and human promyelocytic leukemia (HL-60) cells but not superoxide generation by PMA-stimulated HL-60 cells. KYC inhibits MPO-mediated HOCl formation in BAEC culture and protects BAECs from MPO-induced injury. KYC inhibits MPO-mediated lipid peroxidation of LDL whereas tyrosine (Tyr) and tryptophan (Trp) enhance oxidation. KYC is unique as its isomers do not inhibit MPO activity, or are much less effective. Ultraviolet-visible spectral studies indicate KYC binds to the active site of MPO and reacts with compounds I and II. Docking studies show the Tyr of KYC rests just above the heme of MPO. Interestingly, KYC increases MPO-dependent H2O2 consumption. These data indicate KYC is a novel and specific inhibitor of MPO activity that is nontoxic to endothelial cell cultures. Accordingly, KYC may be useful for treating MPO-mediated vascular disease. Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity. We show KYC inhibits MPO-mediated hypochlorous acid (HOCl) formation and nitration/oxidation of LDL. Disulfide is the major product of MPO-mediated KYC oxidation. KYC (⩽4,000 μM) does not induce cytotoxicity in bovine aortic endothelial cells (BAECs). KYC inhibits HOCl generation by phorbol myristate acetate (PMA)-stimulated neutrophils and human promyelocytic leukemia (HL-60) cells but not superoxide generation by PMA-stimulated HL-60 cells. KYC inhibits MPO-mediated HOCl formation in BAEC culture and protects BAECs from MPO-induced injury. KYC inhibits MPO-mediated lipid peroxidation of LDL whereas tyrosine (Tyr) and tryptophan (Trp) enhance oxidation. KYC is unique as its isomers do not inhibit MPO activity, or are much less effective. Ultraviolet-visible spectral studies indicate KYC binds to the active site of MPO and reacts with compounds I and II. Docking studies show the Tyr of KYC rests just above the heme of MPO. Interestingly, KYC increases MPO-dependent H2O2 consumption. These data indicate KYC is a novel and specific inhibitor of MPO activity that is nontoxic to endothelial cell cultures. Accordingly, KYC may be useful for treating MPO-mediated vascular disease. Myeloperoxidase (MPO) is a heme peroxidase released from activated neutrophils, macrophages, and monocytes that plays important roles in host defense (1Winterbourn C.C. Vissers M.C. Kettle A.J. Myeloperoxidase.Curr. Opin. Hematol. 2000; 7: 53-58Crossref PubMed Scopus (278) Google Scholar, 2Arnhold J. Flemmig J. Human myeloperoxidase in innate and acquired immunity.Arch. Biochem. Biophys. 2010; 500: 92-106Crossref PubMed Scopus (201) Google Scholar, 3Klebanoff S.J. Myeloperoxidase: friend and foe.J. Leukoc. Biol. 2005; 77: 598-625Crossref PubMed Scopus (1726) Google Scholar). Ferric MPO reacts with hydrogen peroxide (H2O2) to form compound I, an oxy-ferryl-cation radical (P•Fe4+=O) intermediate. This intermediate can oxidize a wide variety of substrates to generate an equally wide variety of toxic oxidants and free radicals to kill invading bacteria. Compound I oxidizes (pseudo)halides [such as chloride (Cl−), bromide (Br−), and thiocyanate (SCN−)] via direct, two-electron reduction (halogenation cycle) to form corresponding (pseudo)hypohalous acids [such as hypochlorous acid (HOCl), hypobromous acid, and hypothiocynate]. MPO oxidizes organic substrates such as tyrosine (Tyr) and tryptophan (Trp) to form tyrosyl (Tyr•) and tryptophanyl (Trp•) radicals, respectively. MPO also oxidizes a wide variety of ionic species [nitrite (NO2−), ascorbate, and urate) via one-electron reduction (peroxidation cycle) to form free radicals [nitrogen dioxide radicals (•NO2), ascorbyl radicals, and urate radicals] (4Battistuzzi G. Bellei M. Bortolotti C.A. Sola M. Redox properties of heme peroxidases.Arch. Biochem. Biophys. 2010; 500: 21-36Crossref PubMed Scopus (172) Google Scholar, 5Arnhold J. Furtmuller P.G. Obinger C. Redox properties of myeloperoxidase.Redox Rep. 2003; 8: 179-186Crossref PubMed Scopus (67) Google Scholar). 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In this way the ability of Tyr• to leave the active site and oxidize LDL and/or induce cytotoxicity is essentially eliminated. Our studies show that N-acetyl lysyltyrosylcysteine amide (KYC) inhibits MPO-dependent HOCl generation, protein nitration, and LDL oxidation. Further, KYC specifically inhibits MPO and induces little if any cytotoxicity, making it highly effective for protecting cells from MPO-induced injury. MPO and LDL were from Lee Biosolutions (St. Louis, MO). Catalase, superoxide dismutase, and rabbit anti-NO2Tyr polyclonal antibody were from EMD (Gibbstown, NJ). MPO antibody was from Calbiochem (Cambridge, MA). KYC and other tripeptide analogs were either synthesized by the Blood Center of Wisconsin (Milwaukee, WI) or Biomatik (Wilmington, DE). All other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO). Purity (>98%) and authenticity of the tripeptides were confirmed by HPLC analysis and mass spectrometry. Human promyelocytic leukemia (HL-60) cells were from American Type Culture Collection (ATCC) (Manassas, VA). Bovine aortic endothelial cells (BAECs) were obtained and maintained as previously described (52Ou Z. Ou J. Ackerman A.W. Oldham K.T. Pritchard Jr, K.A. L-4F, an apolipoprotein A-1 mimetic, restores nitric oxide and superoxide anion balance in low-density lipoprotein-treated endothelial cells.Circulation. 2003; 107: 1520-1524Crossref PubMed Scopus (90) Google Scholar). The Homogeneous Caspases Assay kit (catalog number 03005372001) was from Roche (Indianapolis, IN). CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (catalog number G3580) and Mitochondrial ToxGlo™ Assay kit (catalog number G8000) were from Promega (Madison, WI). MPO (20 nM) was incubated with H2O2 (50 μM), NaCl (150 mM), taurine (5 mM), and increasing concentrations of KYC in a phosphate buffer (100 mM, pH 7.4) containing diethylene triamine pentaacetic acid (DTPA) (100 μM) to prevent nonspecific divalent metal cation oxidation for 30 min. Reactions were halted by addition of catalase (2,000 units/ml). Taurine chloramine was quantified using the 3,3',5,5'- tetramethylbenzidine (TMB) assay (53Dypbukt J.M. Bishop C. Brooks W.M. Thong B. Eriksson H. Kettle A.J. A sensitive and selective assay for chloramine production by myeloperoxidase.Free Radic. Biol. Med. 2005; 39: 1468-1477Crossref PubMed Scopus (125) Google Scholar). Briefly, 400 μl of reaction solution was mixed with 100 μl of 2 mM TMB, 100 μM potassium iodide (KI) containing 10% dimethylformamide in 400 mM acetate buffer (pH 5.4). After 5 min, absorbance (650 nm) was recorded on a ultraviolet-visible (UV-Vis) spectrophotometer (Agilent Model 8453). Reaction mixtures contained LDL (0.15 mg/ml), NaNO2 (100 μM), H2O2 (100 μM), MPO (20 nM), and increasing concentrations of KYC or equimolar concentrations of various compounds in a phosphate buffer (100 mM, pH 7.4) containing DTPA (100 μM). Rates of LDL conjugated diene formation were determined by following changes in absorbance at 234 nm, the absorption maximum for conjugated dienes, on a UV-Vis spectrophotometer (Agilent Model 8453) at room temperature. Reaction mixtures contained LDL (0.5 mg/ml), NaNO2 (50 μM), H2O2 (50 μM), MPO (50 nM), and increasing concentrations of KYC in a phosphate buffer (100 mM, pH 7.4) containing DTPA (100 μM). After incubation at 37°C for 4 h, the reactions were stopped by addition of catalase (2,000 units/ml). The formation of malondialdehyde (MDA) was determined according to published procedures (54Erdelmeier I. Gerard-Monnier D. Yadan J.C. Chaudiere J. Reactions of N-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Mechanistic aspects of the colorimetric assay of lipid peroxidation.Chem. Res. Toxicol. 1998; 11: 1184-1194Crossref PubMed Scopus (190) Google Scholar, 55Gérard-Monnier D. Erdelmeier I. Régnard K. Moze-Henry N. Yadan J.C. Chaudière J. Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation.Chem. Res. Toxicol. 1998; 11: 1176-1183Crossref PubMed Scopus (353) Google Scholar). Briefly, incubation mixtures (containing 25 mM butylated hydroxytoluene) were adjusted to pH 1.5 and incubated at 60°C for 80 min to hydrolyze the Schiff bases formed from MDA and protein. The samples were mixed with 3-fold volume of N-methyl-2-phenylindole [13.4 mM in acetonitrile/methanol (3:1)]. After centrifugation (13,000 g, 5 min), 330 μl of the supernatants were mixed with 57.5 μl of concentrated HCl and incubated at 45°C for another 60 min. Finally, after centrifugation (13,000 g, 5 min), total MDA in the samples was determined from the absorbance at 586 nm using a UV-Vis spectrophotometer (Agilent Model 8453). Reaction mixtures containing LDL (0.15 mg/ml), NaNO2 (100 μM), H2O2 (100 μM), MPO (20 nM), and increasing concentrations of KYC in a phosphate buffer (100 mM, pH 7.4) containing DTPA (100 μM) were incubated at room temperature for 30 min. Reactions were stopped by addition of catalase (2,000 units/ml) and the oxidation of Trp in LDL was determined by measuring changes in the intrinsic fluorescence of Trp (Ex 294 nm/Em 345 nm) using a LC-50 fluorometer (Perkin Elmer, Waltham, MA). LDL (0.5 mg/ml) was incubated with MPO (50 nM), H2O2 (50 μM), NaNO2 (50 μM), and increasing concentrations of KYC in phosphate buffer (100 mM, pH 7.4) containing DTPA (100 μM) at 37°C for 4 h. Reactions were stopped by addition of catalase (2,000 units/ml). Formation of NO2Tyr was assessed by dot blot analysis. Briefly, LDL solutions were mixed with 1% SDS and centrifuged (12,000 g, 15 min). Aliquots of supernatants were applied to a nitrocellulose membrane using a dot blot apparatus (Bio-Rad model Bio-Dot). The levels of NO2Tyr were visualized using a rabbit polyclonal anti-NO2Tyr antibody (EMD) and the ECL plus kit from Thermo-Pierce (Rockford, IL). KYC oxidation products were analyzed by reverse phase HPLC using a C-18 column (4.6 × 150 mm). The peptide and products were eluted using an acetonitrile gradient (5–10%, containing 0.1% trifluoroacetic acid) for 20 min. Elution was monitored at both 220 nm and 280 nm. N-acetyl lysyltyrosylserine amide (KYS) and N-acetyl lysylphenylalanylcysteine amide (KFC) were analyzed on a C-18 column (2.2 × 150 mm) and eluted with an acetonitrile gradient (5–30%, containing 0.1% trifluoroacetic acid) for 25 min. BAECs (passages 4–10) were seeded onto 96-well plates and cultured in MEM medium containing 10% FBS in a 5% CO2 and 100% humidity environment at 37°C. Increasing concentrations of KYC (0 to 4 mM, final concentration) were added to the culture medium and cells were incubated for another 24 h. The effects of KYC on cell viability were determined by the MTS assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit, Promega). Caspase activities for apoptosis in the treated BAECs were measured with the Homogeneous Caspases Assay kit from Roche. Necrosis and mitochondrial functions were analyzed by Mitochondrial ToxGlo™ Assay kit (Promega). All determinations were performed according to manufacturer's instructions. HL-60 cells were cultured in RPMI 1640 medium containing 10% FBS (passages 20–50). Cells were harvested by centrifugation (1,000 rpm, 10 min) and washed twice with Dulbecco's phosphate-buffered saline (DPBS) with glucose. HL-60 cells (1.2