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

Article abstract of DOI:10.1194/jlr.M038273

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 MPOdependent 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. Copyright

Full text of DOI:10.1194/jlr.M038273

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
N-acetyl lysyltyrosylcysteine amide inhibits
myeloperoxidase, a novel tripeptide inhibitor1
Hao Zhang,2,*,† Xigang Jing,*,† Yang Shi,*,†,§ Hao Xu,*,† Jianhai Du,*,† Tongju Guan,*,†
Dorothee Weihrauch,** Deron W. Jones,*,† Weiling Wang, ††,§§ David Gourlay,*,† Keith T. Oldham,*,†
,
Cheryl A. Hillery,***,††† and Kirkwood A. Pritchard, Jr.1 *,†
Department of Surgery, Division of Pediatric Surgery,* Department of Pediatrics, Division of Hematology/
Oncology,*** Department of Anesthesiology,** Blood Research Institute of Wisconsin,††† and Children’s
Research Institute,† Medical College of Wisconsin, Milwaukee, WI; Department of Geriatrics†† and Key
Laboratory of Cardiovascular Proteomics of Shandong Province,§§ Qilu Hospital of Shandong University,
Jinan, Shandong, China; and Patient Centered Research,§ Aurora Health Care, Milwaukee, WI
Abstract Myeloperoxidase (MPO) plays important roles in
disease by increasing oxidative and nitrosative stress and
oxidizing lipoproteins. Here we report N-acetyl lysyltyrosyl-
cysteine amide (KYC) is an effective inhibitor of MPO activ-
ity. 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 ␮M) 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 inhib-
its 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 non-
toxic to endothelial cell cultures. Accordingly, KYC may
be useful for treating MPO-mediated vascular disease.—
Zhang, H., X. Jing, Y. Shi, H. Xu, J. Du, T. Guan, D. Weihrauch,
D. W. Jones, W. Wang, D. Gourlay, K. T. Oldham, C. A. Hillery,
and K. A. Pritchard, Jr. N-acetyl lysyltyrosylcysteine amide
inhibits myeloperoxidase, a novel tripeptide inhibitor. J. Lipid
Res. 2013. 54: 3016–3029.
Myeloperoxidase (MPO) is a heme peroxidase released
from activated neutrophils, macrophages, and monocytes
that plays important roles in host defense (1–3). 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 gen-
erate an equally wide variety of toxic oxidants and free radi-
cals to kill invading bacteria. Compound I oxidizes (pseudo)
halides [such as chloride (Clꢀ), bromide (Brꢀ), and thio-
cyanate (SCNꢀ)] via direct, two-electron reduction (halo-
genation cycle) to form corresponding (pseudo)hypohalous
acids [such as hypochlorous acid (HOCl), hypobromous
acid, and hypothiocynate]. MPO oxidizes organic sub-
strates 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 diox-
ide radicals (•NO2), ascorbyl radicals, and urate radicals]
(4, 5). Although MPO is released as a means of killing
invading bacteria, activated immune cells have been re-
ported to release MPO even in the absence of infection,
which unfortunately induces vascular injury and damage
(2, 6, 7). Growing evidence supports the idea that MPO
Abbreviations: BAEC, bovine aortic endothelial cell; Cys, cysteine;
DiTyr, dityrosine; DPBS, Dulbecco’s phosphate-buffered saline; DTPA,
diethylene triamine pentaacetic acid; DTT, dithiothreitol; Em, emiss-
sion; Ex, excitation; GSH, glutathione; HBSS, Hank’s balanced salt
solution; HL-60, human promyelocytic leukemia cells; HOCl, hypochlo-
rous acid; KFC, N-acetyl lysylphenylalanylcysteine amide; KYC, N-acetyl
lysyltyrosylcysteine amide; KYS, N-acetyl lysyltyrosylserine amide; MDA,
malondialdehyde; MPO, myeloperoxidase; •NO2, nitrogen dioxide radi-
cal; NO2Tyr, nitrotyrosine; NOX, NADPH oxidase; O2•ꢀ, superoxide anion;
Phe, phenylalanine; PMA, phorbol myristate acetate; SCNꢀ, thiocyanate;
TMB, 3,3',5,5'- tetramethylbenzidine; Trp, tryptophan; Tyr, tyrosine
UV-Vis, ultraviolet-visible.
Supplementary key words lipid peroxidation • hypochlorous acid •
nitrogen dioxide • nitration • chlorination • low density lipoproteins •
apolipoprotein A1
This work was supported by American Heart Association Grant 11SDG5120015
(H.Z.); R01-HL-089779 (D.W.), R01-HL-102836 (K.A.P., C.A.H.), R21-
HL-102996 (K.A.P.), U54-HL-090503 (C.A.H.); and a generous gift from
Ms. Poblocki of Elm Grove, WI (K.A.P.).
1 See referenced companion article, J. Lipid Res. 2013, 54: 3009–3015.
2 To whom correspondence should be addressed.
Manuscript received 27 March 2013 and in revised form 18 July 2013.
e-mail: hzhang@mcw.edu (H.Z.); kpritch@mcw.edu (K.A.P.)
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of three figures.
Published, JLR Papers in Press, July 19, 2013
DOI 10.1194/jlr.M038273
Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.
3016
Journal of Lipid Research Volume 54, 2013
This article is available online at http://www.jlr.org

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
plays important roles in the pathogenesis of disease by
oxidants that oxidize large proteins to form nitrotyrosine
(NO2Tyr) and/or dityrosine (DiTyr) adducts (49). Here,
we explored the possibility of using a series of novel trip-
eptides containing both Tyr and cysteine (Cys) as MPO
inhibitors, whereby a Tyr• formed by MPO activity is scav-
enged by the thiol of the adjacent Cys (48, 50, 51). 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, pro-
tein nitration, and LDL oxidation. Further, KYC specifi-
cally inhibits MPO and induces little if any cytotoxicity,
making it highly effective for protecting cells from MPO-
induced injury.
increasing oxidative and nitrosative stress (6). Oxidative
stress induced by aberrant MPO activity has been observed
in inflammatory lung disease (8), rheumatoid arthritis
(9, 10), peripheral artery disease (11), cardiovascular disease
(7, 12), and diabetes (13, 14). Even basic science studies in
rats have shown that MPO directly correlates with severity
of myocardial infarction (15, 16). Recently, immunochem-
ical studies revealed that MPO is expressed in microglia,
astrocytes, and certain types of neurons, suggesting that
MPO could play an important role in neurodegenerative
disease (17), such as multiple sclerosis (18–20), Alzheim-
er’s (21, 22), and Parkinson’s disease (23). Interestingly,
MPO has even been implicated as a risk factor for some
forms of cancers (24, 25). Some of the earliest evidence
that MPO plays a role in cardiovascular disease comes
from studies showing that chlorotyrosine on LDL is in-
creased in human vascular lesions (26). More recently,
several groups have suggested that MPO oxidation of HDL
may also play a role in atherosclerosis (26–29). With such
growing evidence that MPO plays a causal role in a variety
of diseases, it seems important to develop an inhibitor
that can be used to prevent MPO-dependent oxidative
damage (30).
A variety of different approaches have been used to in-
hibit MPO-mediated cell injury (31): antioxidant scaveng-
ing of MPO oxidants/radicals; inhibiting H2O2 production
in vivo; and directly inhibiting MPO activity. Antioxidant
scavenging of MPO oxidants and free radicals turned out
to be an ineffective approach because the reaction between
MPO oxidants (i.e., HOCl and hypobromous acid) and an-
tioxidants was not fast enough to prevent tissue damage
(32–34). Inhibiting cell injury by MPO via blocking H2O2
production in vivo was also considered impractical because
multiple pathways exist for generating H2O2 and none of
the agents were able to block H2O2 from all sources (35).
Although suicide inhibitors (i.e., azides, hydrazides, and
hydroxamic acids) that irreversibly modify the iron-heme site
of MPO are highly effective for inhibiting enzyme activity in
vitro (31), they lack specificity and are inherently toxic, which
makes them undesirable as therapeutic agents (35). Several
indole derivatives have been used as reversible inhibitors of
MPO because they effectively compete with Clꢀ and SCNꢀ to
prevent compound I from generating HOCl and hypothio-
cynate (36, 37). However, during oxidation, these agents are
converted into radicals that are toxic and capable of increas-
ing oxidative stress in vivo (36, 38, 39). Phenolic compounds
have also been used to inhibit MPO because they compete
with the other substrates for compounds I and II (35, 40, 41).
However, MPO oxidization of phenolic compounds also re-
sults in the formation of toxic radicals that can increase oxi-
dative stress. For example, MPO has been shown to oxidize
several phenolic compounds into radicals that actually accel-
erate LDL oxidation (42–44). A significant amount of ef-
fort has gone into designing and testing agents that block
MPO activity. Recent reports show that 2-thioxanthine and
INV-315 inhibit MPO in vivo (45, 46).
MATERIALS AND METHODS
Materials
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 ana-
logs were either synthesized by the Blood Center of Wisconsin
(Milwaukee, WI) or Biomatik (Wilmington, DE). All other chem-
icals 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 ob-
tained and maintained as previously described (52). The Homo-
geneous Caspases Assay kit (catalog number 03005372001) was
from Roche (Indianapolis, IN). CellTiter 96® Aqueous One Solu-
tion Cell Proliferation Assay kit (catalog number G3580) and Mito-
chondrial ToxGlo™ Assay kit (catalog number G8000) were from
Promega (Madison, WI).
MPO-catalyzed HOCl production
MPO (20 nM) was incubated with H2O2 (50 ꢂM), NaCl (150 mM),
taurine (5 mM), and increasing concentrations of KYC in a phos-
phate buffer (100 mM, pH 7.4) containing diethylene triamine
pentaacetic acid (DTPA) (100 ꢂM) to prevent nonspecific diva-
lent 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
(53). Briefly, 400 ꢂl of reaction solution was mixed with 100 ꢂl of
2 mM TMB, 100 ꢂM potassium iodide (KI) containing 10% dim-
ethylformamide 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).
MPO-mediated LDL conjugated diene formation
Reaction mixtures contained LDL (0.15 mg/ml), NaNO2
(100 ꢂM), H2O2 (100 ꢂM), MPO (20 nM), and increasing con-
centrations of KYC or equimolar concentrations of various com-
pounds in a phosphate buffer (100 mM, pH 7.4) containing DTPA
(100 ꢂM). Rates of LDL conjugated diene formation were de-
termined by following changes in absorbance at 234 nm, the
absorption maximum for conjugated dienes, on a UV-Vis spec-
trophotometer (Agilent Model 8453) at room temperature.
MPO-mediated LDL malondialdehyde formation
It is well-known that MPO oxidizes the phenol side
chain of Tyr in small peptides (47, 48) and it generates
Reaction mixtures contained LDL (0.5 mg/ml), NaNO2 (50 ꢂM),
H2O2 (50 ꢂM), MPO (50 nM), and increasing concentrations of
Novel tripeptide inhibitors of myeloperoxidase activity
3017

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
KYC in a phosphate buffer (100 mM, pH 7.4) containing DTPA
Phorbol myristate acetate-induced HOCl formation by
HL-60 cells
(100 ꢂM). 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 (54, 55). 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 vol-
ume of N-methyl-2-phenylindole [13.4 mM in acetonitrile/meth-
anol (3:1)]. After centrifugation (13,000 g, 5 min), 330 ꢂl of the
supernatants were mixed with 57.5 ꢂl 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).
HL-60 cells were cultured in RPMI 1640 medium contain-
ing 10% FBS (passages 20–50). Cells were harvested by centrifu-
gation (1,000 rpm, 10 min) and washed twice with Dulbecco’s
phosphate-buffered saline (DPBS) with glucose. HL-60 cells (1.2 ×
107/ml) were resuspended in DPBS with glucose. The washed
HL-60 cells were either stimulated with phorbol myristate acetate
(PMA) (10 ꢂM) or not, and incubated with taurine (5 mM) and
increasing concentrations of KYC at 37°C for 30 min. Catalase
(2,000 units/ml) was added to halt the reaction. After centrifuga-
tion, the supernatants were analyzed using the TMB assay as out-
lined above.
PMA-induced HOCl formation by human neutrophils
Human neutrophils were isolated according to a previous re-
port (56). All protocols utilizing human neutrophils were approved
by the Medical College of Wisconsin Institutional Review Board.
The KYC inhibition of HOCl formation from PMA-stimulated
neutrophils was analyzed as described in (57). Briefly, neutrophils
(3 × 106 cells/ml) were mixed with different amounts of KYC in
Hank’s balanced salt solution (HBSS) containing MgCl2 (0.5 mM),
CaCl2 (1.26 mM), glucose (5.5 mM), and taurine (5 mM). The cells
were stimulated with PMA (100 ng/ml) and incubated at 37°C for
20 min. The reactions were stopped by catalase (2,000 units/ml).
After centrifugation, the supernatants were analyzed using the
TMB assay as previously described.
MPO-mediated LDL Trp oxidation
Reaction mixtures containing LDL (0.15 mg/ml), NaNO2
(100 ꢂM), H2O2 (100 ꢂM), MPO (20 nM), and increasing con-
centrations of KYC in a phosphate buffer (100 mM, pH 7.4) con-
taining DTPA (100 ꢂM) 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 deter-
mined by measuring changes in the intrinsic fluorescence of Trp
(Ex 294 nm/Em 345 nm) using a LC-50 fluorometer (Perkin Elmer,
Waltham, MA).
MPO-mediated nitration of LDL
PMA-stimulated HL-60 cell O2•؊ formation
LDL (0.5 mg/ml) was incubated with MPO (50 nM), H2O2
(50 ꢂM), NaNO2 (50 ꢂM), and increasing concentrations of KYC
in phosphate buffer (100 mM, pH 7.4) containing DTPA (100 ꢂM)
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 cen-
trifuged (12,000 g, 15 min). Aliquots of supernatants were ap-
plied 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).
HL-60 cells were harvested by centrifugation at 1,000 rpm for
10 min and washed twice with DPBS with glucose to remove cul-
ture medium. The HL-60 cells (1.2 × 107/ml) were resuspended
in DPBS with glucose. After stimulation with PMA (10 ꢂM), the
HL-60 cells were incubated with cytochrome c (40 ꢂM) with or
without superoxide dismutase (500 units/ml) at 37°C for 10 min
and then the HL-60 cells were removed by centrifugation. Cyto-
chrome c reduction in supernatants was measured at 550 nm
using a UV-Vis spectrophotometer (Agilent Model 8453).
MPO-mediated BAEC injury
HPLC analysis
BAECs (passages 6–8) were cultured in 24-well plates in DMEM
containing 10% FBS until 70–80% confluent. The cells were washed
with HBSS two times and then incubated with MPO (2.5 ꢂg/ml)
and H2O2 KYC at the concentrations indicated in HBSS (0.5 ml) at
37°C for 30 min. In the case of neutrophils as a source of both MPO
and H2O2, neutrophils (0.2 × 106 cells/0.5 ml) were added into 24-
well plates in the presence of different amounts of KYC in HBSS
with MgCl2, CaCl2, and glucose. The cells were stimulated with 2 ꢂM
PMA at 37°C for 30 min. The cultured cells were washed with HBSS
three times. Finally, BAECs were examined visually and images cap-
tured for permanent record as described (58) using a Nikon Eclipse
microscope (Model TS100) fitted with a Nikon Digital Sight DS-U2
camera and NIS-Elements F 3.0 imaging software. Images are repre-
sentative of three independent experiments.
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 ana-
lyzed on a C-18 column (2.2 × 150 mm) and eluted with an ace-
tonitrile gradient (5–30%, containing 0.1% trifluoroacetic acid)
for 25 min.
Cytotoxicity assays
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 concen-
trations 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 Pro-
liferation 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.
Effects of KYC on MPO UV-Vis spectra
MPO (1.4 ꢂM) was incubated with KYC (50 ꢂM) with or without
H2O2 (40 ꢂM) and NaCl (150 mM) at room temperature. The
changes in UV-Vis spectra were recorded as indicated. Compound II
was prepared by mixing MPO (1.4 ꢂM) with H2O2 (300 ꢂM) for 20 s.
Excess H2O2 was removed by addition of catalase (5 ꢂg/ml). The
reaction was immediately mixed with KYC (50 ꢂM) and the changes
in heme spectra were recorded. Experiments were also performed
in the presence of methionine (1 mM). The results show no differ-
ence in UV-Vis spectra in the presence or absence of methionine.
3018
Journal of Lipid Research Volume 54, 2013

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
Statistics
HOCl or taurine chloramine by KYC, we mixed KYC with
HOCl or preformed taurine chloramine and analyzed the
remaining HOCl or taurine chloramine by TMB/KI assay.
Briefly, KYC [6.25 ꢂM (n = 3) and 12.5 ꢂM KYC (n = 3)]
was incubated with either 50 ꢂM HOCl or 50 ꢂM taurine
chloramine under our assay conditions for 30 min, then
measurements of HOCl or taurine chloramine were per-
formed as described in Materials and Methods. Our data
show that one molecule of KYC scavenges 1.21 0.15 (n = 6)
HOCl molecules or 0.97 0.01 (n = 6) molecules of tau-
rine chloramine. No significant differences were noted
between the two KYC concentrations used (6.25 ꢂM and
12.5 ꢂM). These results suggest that KYC reduces HOCl
formation by inhibiting MPO activity, not just scavenging
HOCl or taurine chloramine. The other tripeptides scav-
enge HOCl production by (ف27–0%). These data indicate
that although KYC scavenges HOCl to the same extent
as other thiol peptides, significantly greater inhibition is
achieved when it is treated with MPO that cannot be ex-
plained as scavenging HOCl. KYC’s ability to inhibit MPO
is comparable to the ability of Trp to inhibit MPO (36).
Interestingly, when arginine, another positively charged
amino acid, is substituted for Lys, the ability of RYC to in-
hibit MPO-catalyzed HOCl production is markedly reduced
(KYC = ف75% vs. RYC = ف22% inhibition). These data in-
dicate that the charge, size, and hydrophobicity of the first
amino acid are all important properties for how tripep-
tides inhibit MPO activity.
Data are presented as mean SD unless stated otherwise and
analyzed with the Student’s t-test where appropriate using Prism
5.0 (Graph Pad, Inc.) for two group comparison.
RESULTS
Effects of tripeptides on MPO-catalyzed HOCl
production
To determine the extent to which tripeptides inhibit MPO
activity, we synthesized a series of six tripeptides contain-
ing Tyr and Cys (XYC) and studied their effects on MPO
HOCl generation. As shown in Fig. 1A, at 12.5 ꢂM, among
the tripeptides tested, KYC was the only tripeptide that re-
duced HOCl production by ف75%. To exclude the possi-
bility that such reduction is due to a direct scavenging of
To further assess the efficiency of KYC for inhibiting
MPO, we next determined dose-dependent effects of KYC
on MPO-catalyzed HOCl production. Figure 1B shows that
KYC dose-dependently inhibited HOCl production with
an IC50 of ف7 ꢂM. At 25 ꢂM, KYC completely inhibited
HOCl production (Fig. 1B).
To understand the importance of the phenol of Tyr and
the thiol of Cys for KYC inhibiting MPO, we also compared
the dose-dependent effects of KYC with two structural ana-
logs, KFC and KYS. Without a free thiol, KYS failed to in-
hibit MPO-catalyzed HOCl production (Fig. 1C). Although
KFC decreased the amount of HOCl detected by the tau-
rine/TMB assay, its ability to decrease HOCl was much less
than KYC (Fig. 1D). Where KYC (25 ꢂM) completely ab-
lated MPO-catalyzed HOCl production, KFC (25 ꢂM) re-
duced HOCl by only 35% (Fig. 1D). As both phenylalanine
(Phe) and Cys are considered poor substrates for MPO
and there is almost no direct oxidation by MPO, it is likely
that KFC’s mechanism of action has more to do with the
free thiol scavenging than actually entering the active site
of MPO and reacting compound I or II, as does KYC. While
Tyr and Cys are required for KYC to inhibit MPO, these data
suggest that Lys also plays an important role in orienting
the tripeptide for optimal inhibition.
Fig. 1. Effects of Tyr- and Cys-containing tripeptides on MPO-
catalyzed HOCl formation. MPO (20 nM) was incubated with H2O2
(50 ꢂM), NaCl (150 mM), taurine (5 mM), and various amounts
of tripeptides in phosphate buffer (100 mM, pH 7.4) containing
DTPA (100 ꢂM) for 30 min. Reactions were halted by addition of
catalase (2,000 units/ml). Taurine chloramine was quantified us-
ing the TMB assay. A: The effect of different tripeptides on the
MPO-mediated HOCl formation. All tripeptide concentrations
were at 12.5 ꢂM. B–D: The dose-dependent inhibition of MPO-
catalyzed HOCl formation by KYC (B), KYS (C), and KFC (D).
KYC inhibited HOCl formation with an IC50 of ف7 ꢂM. E: Com-
parison of the effects of equimolar concentrations (12.5 ꢂM) of
KYC isomers on MPO-catalyzed HOCl formation. F: Comparison
of the effect of equimolar concentrations (12.5 ꢂM) of KYC with
Tyr and Trp on MPO-catalyzed HOCl formation. All data (mean
SD, n = 3) are expressed as percent of control (in the absence of
tripeptides).
To investigate the effect of D-isomers and sequence iso-
mers of KYC on MPO inhibition, we compared the effects
of KYC, made with all L-amino acids, with the effects of
D-amino acids and sequence isomers of KYC on MPO-
catalyzed HOCl production. Figure 1E shows that replac-
ing an L-amino acid with a D-amino acid at any position or
even at all three positions in KYC dramatically decreased
Novel tripeptide inhibitors of myeloperoxidase activity
3019

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
the ability of the tripeptide to inhibit MPO-catalyzed HOCl
(trace b and c) shows that when KYC disulfide was incu-
bated with GSH, the KYC disulfide was completely reduced
to its KYC monomer (Fig. 3, trace b). More so, DTT com-
pletely reduced KYC disulfide to its KYC monomer within
5 min (Fig. 3, trace c). These data demonstrate that oxida-
tion of KYC results in the formation of simple disulfides
that are easily regenerated to its active monomeric form
with physiologically relevant concentrations of GSH.
production. Likewise, YKC and CYK failed to inhibit MPO-
catalyzed HOCl production to the same extent as KYC (all
L-amino acids) (Fig. 1E). Finally, we compared the effects
of KYC on MPO-catalyzed HOCl production to the effects
of free Tyr and Trp (Fig. 1F). Consistent with another
report (36), Trp was an effective inhibitor of MPO-cata-
lyzed HOCl production. In contrast, Tyr alone had little, if
any, effect on MPO-catalyzed HOCl production, as has been
reported (36). The lack of effect of Tyr on MPO activity is
also in agreement with data showing that KYS, which also
contains a single Tyr, is not an effective inhibitor of MPO-
catalyzed HOCl production. These data indicate that KYC’s
sequence is unique and that steric conformation and amino
acid sequence order are important structural requirements
for KYC to inhibit MPO.
KYC specifically inhibits MPO activity from HL-60 cells
and human neutrophils
HL-60 cells were stimulated with PMA to induce the re-
lease of MPO and treated with KYC to determine its effects
on HOCl production. Without PMA stimulation, HL-60
cells produced little, if any, HOCl (Fig. 4A, inset). However,
after PMA stimulation, HL-60 cells generated high levels
of HOCl (Fig. 4A, inset). KYC dose-dependently inhibited
HOCl production by PMA-stimulated HL-60 cells with an
IC50 ف7 ꢂM (Fig. 4A). MPO-mediated HOCl formation
MPO-catalyzed KYC oxidation product analysis
To determine how MPO oxidizes KYC, we analyzed reac-
tion products by HPLC. MPO/H2O2 systems oxidized KYC
essentially to a single product that eluted around 7.6 min
(Fig. 2A, trace b). Although KYC can be oxidized by H2O2
directly, incubations with H2O2 alone yielded very little of
the 7.6 min product (Fig. 2A, trace a), which has the same
retention time as authentic KYC disulfide (Fig. 2A, trace e).
Monitoring the eluate from the HPLC with a fluorescent
detector (Ex = 290 nm/Em = 410 nm), the fluorescent
characteristic of a DiTyr showed no significant peak for-
mation (Fig. 2B, trace a and b). This lack of fluorescence
rules out DiTyr as a major product of oxidation. When KYS
was oxidized with the MPO/H2O2 system, several products
were observed to elute between 7 and 10 min (Fig. 2C, trace
a and b). The major peak in this trace has a fluorescent pro-
file that is characteristic of DiTyr (Fig. 2D, trace b), suggest-
ing that, unlike KYC, Tyr in KYS was oxidized by MPO to
form Tyr•, which in turn forms DiTyr. Incubation of KFC,
another KYC analog, with the MPO/H2O2 system did not
yield MPO-dependent oxidation products (Fig. 2E, F),
although small amounts of disulfides could be observed,
which is likely a result of slow oxidation of the thiols by
H2O2. Such data clearly indicate that, although MPO is
able to oxidize Tyr in both KYC and KYS directly, oxida-
tion of the Tyr in KYS forms DiTyr products, whereas the
Cys in KYC rapidly scavenges Tyr• radical leading to the
formation of a disulfide instead of DiTyr. Analysis of prod-
ucts from the MPO/H2O2/NO2ꢀ system (Fig. 2A, trace c)
or the MPO/H2O2/Clꢀ system (Fig. 2A, trace d) shows
that KYC disulfide is the major oxidation product of these
systems. These data suggest that regardless of the condi-
tions under which MPO oxidizes KYC, in the presence of
•ꢀ
requires H2O2 that is derived from O2 generated by
NADPH oxidase (NOX). To determine whether KYC or
•ꢀ
KYC disulfide inhibited NOX O2 generation in HL-60
cells, we quantified O2•ꢀ production using the cytochrome
c assay. This is important because, if KYC inhibited NOX
activity, it would decrease H2O2 formation making it appear
as if KYC inhibited MPO. Neither KYC nor KYC disulfide
•ꢀ
had any significant effect on O2 production in PMA-
stimulated HL-60 cells (Fig. 4B). HPLC analysis of the oxi-
dation products revealed that KYC disulfide was the major
product from PMA-stimulated HL-60 cells (Fig. 4C). Taken
together, these data suggest that KYC inhibited MPO
but not NOX activity, and that KYC disulfide is the major
oxidation product when activated HL-60 cells are incubated
with KYC. Figure 4D shows that KYC inhibits neutrophil-
mediated HOCl generation. Adding KYC to the PMA-
stimulated neutrophils dose-dependently inhibits MPO
mediated HOCl formation.
Cytotoxicity of KYC on BAECs
As a first step toward determining whether KYC is suit-
able for in vivo treatments, we incubated BAEC cultures
with increasing KYC concentrations (4,000 ꢂM). After
24 h, we analyzed the impact of KYC exposure on cell via-
bility, apoptosis, necrosis, and mitochondria function. Sup-
plementary Fig. IA shows the effects of KYC on BAEC
viability. No increases in cell death were induced by KYC
even at 4,000 ꢂM. To assess the impact of KYC on BAEC
apoptosis, we used the Homogeneous Caspases Assay kit
from Roche to analyze changes in the activity of multiple
capases in BAEC cultures because this kit is able to detect
multiple activated caspases (caspases 2, 3, 6, 7, 8, 9, and 10).
Caspase activity was essentially unaltered in BAEC cultures
incubated with increasing KYC concentrations (supple-
mentary Fig. IB). These data indicate that KYC does not
induce apoptosis. Membrane integrity studies showed that
KYC had no effects on protease activity, an index of necro-
sis, in BAEC cultures after 24 h (supplementary Fig. IC).
Finally, cellular ATP, an index of mitochondrial function,
was unaltered in BAEC cultures incubated with KYC
ꢀ
NO2 or Clꢀ, the major oxidation product is KYC disul-
fide, not DiTyr. Such findings clearly indicate that MPO
directly oxidizes the Tyr in the tripeptides. This can only
be accomplished if KYC enters the active site of MPO to
generate a Tyr• radical that is subsequently detoxified by
the free thiol of Cys with high efficiency.
To confirm that KYC oxidation by MPO yields a simple
disulfide, we reduced the product using simple thiols such
as glutathione (GSH) and dithiothreitol (DTT). Figure 3
3020
Journal of Lipid Research Volume 54, 2013

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
Fig. 2. HPLC analysis of MPO-mediated KYC product formation. A: HPLC analysis of KYC products from
various MPO-mediated oxidation reactions detected at 280 nm (A280). KYC (440 ꢂM) was incubated with
H2O2 (100 ꢂM) in phosphate buffer (100 mM, pH 7.4) containing DTPA (100 ꢂM) at room temperature for
30 min and the reaction products analyzed by HPLC (trace a). Reaction conditions and incubation times
were the same as in trace a but with MPO included (50 nM) (trace b). Reaction conditions and incubation
times were the same as in trace b but with NaNO2 (0.5 mM) included (trace c). Reaction conditions and
incubation times were the same as in trace b but with NaCl (100 mM) included (trace d). HPLC trace of KYC
disulfide standard (trace e). B: HPLC analysis of KYC products from MPO/H2O2-mediated oxidation de-
tected by fluorescent detection at Ex = 290 nm (EX290) and Em = 410 nm (EM410). KYC/H2O2 (trace a) and
MPO/KYC/H2O2 (trace b). C: HPLC analysis of KYS products from MPO/H2O2-mediated oxidation reac-
tions detected at 280 nm (A280). KYS/H2O2 (trace a) and MPO/KYS/H2O2 (trace b). D: HPLC analysis of
KYS products from MPO/H2O2-mediated oxidation reactions detected by fluorescent detection at Ex = 290 nm
(EX290) and Em = 410 nm (EM410). KYS/H2O2 (trace a) and MPO/KYS/H2O2 (trace b). E: HPLC analysis of
KFC products from MPO/H2O2-mediated oxidation reactions detected at 220 nm (A220). KFC/H2O2 (trace a)
and MPO/KFC/H2O2 (trace b). F: HPLC analysis of KFC products from MPO/H2O2-mediated oxidation
reactions detected by fluorescent detection at Ex = 290 nm (EX290) and Em = 410 nm (EM410). KFC/H2O2
(trace a) and MPO/KFC/H2O2 (trace b).
(supplementary Fig. ID). On the basis of data from these
studies, we conclude that KYC does not induce cell dam-
age even at concentrations up to 4,000 ꢂM. Compared
with the basal levels of caspase activity (in the group with-
out KYC treatment), BAECs treated with high KYC con-
centrations tended to decrease caspase activity, which may
indicate that KYC protects cells at high concentration through
its free thiol antioxidant properties. This may explain why
protease activity was also decreased in BAEC cultures incu-
bated with 4,000 ꢂM KYC.
KYC protects BAECs from MPO-induced injury
With data indicating that KYC is not toxic to BAEC cul-
tures, we next determined if KYC protects BAECs from
MPO-induced injury. BAECs in 96-well plates were treated
with 100 ꢂl of HBSS containing MPO (2.5 ꢂg/ml) and
H2O2 (50 ꢂM) for 30 min with or without KYC. Changes
in cell morphology were recorded as a direct measure of
injury as previously reported by others (58). Figure 5
shows that KYC dramatically increased BAEC viability
and survival. Incubation of BAECs in the MPO/H2O2/
Novel tripeptide inhibitors of myeloperoxidase activity
3021

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
Effects of KYC on MPO-mediated LDL lipid
peroxidation
MPO oxidation of LDL and HDL has been hypothe-
sized to play a causal role in the genesis of atheroscle-
rotic lesions. To determine if KYC inhibits LDL oxidation,
we incubated LDL in MPO/H2O2/NO2ꢀ reaction systems
containing increasing concentrations of KYC and mea-
sured changes in conjugated diene formation. The MPO/
ꢀ
H2O2/NO2 reaction system dramatically increased LDL
oxidation, confirming reports that MPO oxidizes lipids
•
by generating NO2 (59, 60). Adding KYC to the MPO/
H2O2/NO2ꢀ reaction system dose-dependently increased
the lag time and decreased the overall rate of LDL oxida-
tion. At 25 ꢂM, KYC completely suppressed LDL oxida-
tion (Fig. 6A). These data demonstrate that KYC is a
potent inhibitor of MPO-mediated LDL peroxidation.
Next, we compared the effects of KYC with those of KFC
and KYS (Fig. 6B). While KYC (25 ꢂM) completely ab-
lated LDL oxidation, KFC increased the lag phase and
delayed, but did not totally inhibit, LDL oxidation. On
the basis of these findings, we reasoned that limited ef-
fects of KFC on LDL oxidation were probably the result
of direct •NO2 scavenging via the thiol of KFC. KYS gave a
totally different inhibition profile. Instead of inhibiting
LDL oxidation, KYS actually accelerated LDL oxidation
(Fig. 6B). These data are consistent with the idea that the
Tyr enters the active site of MPO, becomes oxidized to
generate a Tyr• which then accelerates LDL oxidation.
Figure 6C compares the inhibitory effects of KYC with
those of Tyr, Trp, and GSH on LDL oxidation. In agree-
ment with a report by others (44), Tyr (dashed line) ac-
celerated and enhanced LDL oxidation. Although
findings by others (36) and our data (Fig. 1F) show that
Trp effectively inhibits MPO-catalyzed HOCl production,
adding Trp to the MPO/H2O2/NO2ꢀ reaction system did
not inhibit, but actually enhanced, LDL oxidation (Fig. 6C).
Even though initial rates of LDL oxidation in MPO/
H2O2/NO2ꢀ reaction systems were reduced by GSH (i.e.,
an increase in lag time), this delay was quickly lost with
what appears to be an increase in thiol oxidation. Regard-
less, for all practical purposes GSH was ineffective for in-
hibiting LDL oxidation compared with KYC.
Fig. 3. Thiol reduction of KYC disulfide. KYC (440 ꢂM) was mixed
with MPO (50 nM) and H2O2 (100 ꢂM) for 2 h (as in Fig. 2A, trace b)
and reaction products were analyzed by HPLC (trace a). An aliquot
(50 ꢂl) of the reaction mixture in trace a was mixed with 10 mM
GSH (50 ꢂl) for 120 min and the resultant products analyzed by
HPLC (trace b). An aliquot (50 ꢂl) of the reaction mixture in trace
a was mixed with 50 ꢂl of DTT (0.5 mM) for 5 min at room tem-
perature and the products analyzed by HPLC (trace c).
Clꢀ alone caused severe cell damage as demonstrated
by dramatic changes in cell morphology (Fig. 5A). KYC
dose-dependently increased protection of BAEC cultures
from MPO-induced injury (Fig. 5A). At 50 ꢂM KYC,
BAEC cultures had almost the same morphology as cul-
tures that were not exposed to MPO (control). KYC also
protected BAEC from even higher concentrations of
H2O2 (100 ꢂM) and MPO (5 ꢂg/ml) (Fig. 5B, C). This
study shows that KYC is fully capable of protecting BAECs
from the cytotoxic effects of MPO. Incubation of PMA-
stimulated neutrophils with BAECs also induced BAEC
damage (Fig. 5D), as did incubation with MPO/H2O2
(Fig. 5A). KYC protected BAECs from cell injury from
MPO released from PMA-stimulated neutrophils as well
(Fig. 5D).
To examine these observations from a different per-
spective, we determined if KYC can inhibit MPO HOCl
formation even in the presence of BAECs. Supplemen-
tary Fig. IIA shows that KYC dose-dependently inhibits
HOCl formation by MPO (2.5 ꢂg/ml) and H2O2 (50 ꢂM)
in the presence of BAECs. KYC inhibits HOCl formation
even when MPO and H2O2 are increased (5 ꢂg/ml and
100 ꢂM, respectively) (supplementary Fig. IIB). PMA-
stimulated neutrophils generate HOCl when incubated
with BAEC cultures (supplementary Fig. IIC). KYC inhib-
its HOCl formation by PMA-activated neutrophils in the
presence of BAECs. To determine the effects of KYC
on cell viability we also performed the MTS assay under
these same conditions. As anticipated, KYC protected
BAECs in the presence of MPO/H2O2 (supplementary
Fig. IIIA, B) and PMA-stimulated neutrophils (supple-
mentary Fig. IIIC). These data confirm that KYC inhibits
MPO/neutrophil-dependent HOCl generation even in
the presence of BAECs, which begins to explain why KYC
can protect BAECs from the harmful effects of MPO
activity.
Additional insight into the mechanisms by which KYC
inhibited MPO-mediated LDL oxidation was gained by re-
peating the MPO/H2O2 oxidation studies in the absence
ꢀ
of NO2 . In these studies, we observed that Tyr (Fig. 6D,
dashed line) and Trp (Fig. 6D, dotted line) increased MPO-
mediated LDL oxidation, which agrees with the fact that
both Tyr• and Trp•, formed by MPO oxidation, are potent
oxidants that accelerate LDL oxidation. In contrast, KYC
and GSH did not increase LDL oxidation. GSH is reported
to be a poor substrate for MPO/H2O2 systems whose rate
of reaction is on the order of 10–100 Mꢀ1sꢀ1 (32). In addi-
tion, the glutathionyl radical that would be formed is a
poor oxidant. Thus, in the presence of GSH the MPO/
H2O2 system was unable to oxidize LDL. In contrast, the
Tyr in KYC can be rapidly oxidized to Tyr• by MPO. The
fact that the MPO/H2O2 system failed to increase LDL oxi-
dation in the presence of KYC is consistent with the fact
3022
Journal of Lipid Research Volume 54, 2013

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
that the Tyr• was rapidly scavenged by the thiol of Cys via
intramolecular electron transfer.
MDA is another biomarker of lipid peroxidation. To
determine the effects of KYC on MPO-dependent MDA
formation in LDL, we incubated LDL (0.5 mg/ml) in a
MPO/H2O2/NO2ꢀ reaction system (50 nM, 50 ꢂM, 50 ꢂM,
respectively) and measured changes in MDA formation.
Increasing concentrations of KYC dose-dependently inhib-
ited MPO-mediated MDA formation in LDL (Fig. 7E), just
as it did conjugated diene formation (Fig. 7A).
The mechanism of KYC inhibition
To probe the mechanism for KYC inhibiting MPO activ-
ity, first, we analyzed the changes in MPO spectra (61) after
incubation with KYC and H2O2. Native MPO has absorbance
peaks at 429 and 575 nm. Incubating MPO with KYC in-
duced a red shift in the 429 nm peak and formed a new
peak around 627 nm. The 575 nm peak was also slightly
decreased. Inclusion of catalase (500 U/0.5 ml) in the reac-
tion did not eliminate the peak at 627 nm (data not shown).
This result indicates that KYC directly binds to the active site
of MPO and interacts with the iron-heme center (Fig. 7A,
panel a). Incubation of KYC/MPO with H2O2 decreased
absorbance at 429 nm and 575 nm, indicating that H2O2
activated MPO, thereby decreasing native MPO. At the
same time a major increase in absorbance at 456 nm and
an increase in a broad band around 627 nm were observed,
indicating that KYC increases the accumulation of com-
pound II (Fig. 7A, panel b). Taken together, these spectra
clearly indicate that KYC enters the active site of MPO and
binds close to the iron-heme site. Experiments were also
performed in the presence of methionine (1 mM). The
results show no difference in the presence or absence of
methionine (data not shown). In this way, KYC is able to
rapidly react with compound I to form compound II. We
repeated the same experiment in the presence of 150 mM
NaCl (Fig. 7A, panels c and d). The results show the same
trend as the reaction without NaCl, although the changes
were smaller, suggesting that KYC might compete against
Clꢀ for the active site. Finally, we determined if KYC reacts
with compound II directly. MPO was incubated with an
excess amount of H2O2 to convert native MPO into com-
pound II. After removing excess H2O2 with catalase, add-
ing KYC to the reaction mixture caused a blue shift of the
456 nm band and an increased absorbance at 575 nm, in-
dicating that KYC reduced compound II to native MPO. At
the same time, a new narrow band at 627 nm formed,
which was similar to what was observed when KYC was in-
cubated with MPO (Fig. 7A, panel a). Taken together these
data suggest that KYC reacts with both compound I and
compound II; however, the rate for KYC reacting with com-
pound I is faster than it is for compound II.
Fig. 4. KYC specifically inhibits MPO in HL-60 cells and neutro-
phils. A: KYC inhibited HOCl formation. HL-60 cells (1.2 × 107/
ml) were incubated with PMA and taurine as described in Materials
and Methods. The reaction was stopped by catalase. After centrifu-
gation, taurine chloramine was quantified by the KI/TMB assay.
The inset panel shows that PMA is required to stimulate HL-60 cell
HOCl production. Panel A shows that KYC dose dependently in-
hibits HL-60 cell HOCl production. B: Effects of KYC and KYC dis-
ulfide on HL-60 cells O2•ꢀ production. O2•ꢀ formation was analyzed
by the superoxide dismutase-inhibitable cytochrome c reduction
assay. HL-60 cells were incubated with cytochrome c (40 ꢂM) with
or without superoxide dismutase at 37°C for 10 min. The reaction
was stopped by addition of catalase. After centrifugation, cyto-
chrome c reduction was determined at 550 nm using a UV-Vis
spectrophotometer. Results are expressed as percent of control,
the data represents mean SD (n = 3). C: Effects of PMA on HL-60
cell-induced KYC product formation. HL-60 cells were stimulated
with PMA (37°C for 30 min) and then incubated with KYC as above
in panel (A). KYC oxidation products were analyzed by HPLC. In-
cubation with PMA-stimulated HL-60 cells (trace a). Incubation
with nonstimulated HL-60 cells (trace b). KYC disulfide standard
(trace c). D: Shows that KYC dose dependently inhibits HOCl
production by PMA-stimulated neutrophils.
Novel tripeptide inhibitors of myeloperoxidase activity
3023

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
Fig. 5. The protection of BAECs by KYC from
MPO-induced injury. BAECs (passages 6–8) were
cultured in 24-well plates with DMEM and 10% FBS
until 70–80% confluent and then treated with MPO,
H2O2, and KYC as described in Materials and Meth-
ods. Images are representative of three independent
studies. A: KYC concentration-dependent protection
against MPO-induced BAEC injuries. B: BAECs incu-
bated with 2.5 ꢂg MPO and 100 ꢂM H2O2 with or
without 25 ꢂM KYC for 30 min. C: BAECs incubated
with 5 ꢂg MPO and 50 ꢂM H2O2 with or without
25 ꢂM KYC for 30 min. D: KYC concentration-
dependent protection against PMA-stimulated neu-
trophil-induced BAEC injury.
Previous studies have shown that most MPO inhibitors
disrupt MPO catalytic cycles. For example, irreversible in-
hibitors such as azide, hydrazide, etc., are known to modify
the heme to destroy the catalytic function of MPO, while
indole compounds react with compound I but not com-
pound II. All these inhibition mechanisms decrease MPO-
dependent H2O2 consumption. Here we propose that KYC
competes against MPO substrates by rapidly reacting with
both compound I and compound II, which is a mecha-
nism that is different from the mechanisms of other MPO
inhibitors. To determine if this mechanism increases H2O2
consumption as Tyr (62), we incubated KYC with MPO
and H2O2 in the presence of chloride and measured H2O2
consumption (63). Our results show that incubation of
MPO with H2O2 and Clꢀ induced H2O2 consumption and
that addition of KYC dose-dependently increased H2O2
consumption (Fig. 7B). As KYC inhibits HOCl formation,
this increase in H2O2 consumption once again suggests
that KYC directly reacts with both compound I and com-
pound II.
Additional evidence supporting the idea that KYC enters
the active site of MPO and reacts with compounds I and II
comes from docking studies. We simulated the binding of
KYC in the active site of MPO using a published X-ray crys-
tallographic structure of MPO (PDB ID: 3ZS1). Figure 7C
shows that KYC (red) fits in the active site without clashing
with MPO. This simulation shows that KYC can be oriented
in such a way that the phenolic group of Tyr is above the
iron-heme of MPO (Fig. 7C, blue molecule) with the shortest
distance of 2.9 Å, which is similar to that which was re-
ported for docking indole derivatives (64). Both Cys and
Tyr of KYC point toward the opening of the pocket of the
active site of MPO in such a way that the amino group of
the Lys side chain in KYC interacts with the carboxyl group
of Glu116 of MPO (Fig. 7C, green lines) and might form a
salt bridge (distance 3.2 Å). Thus, the Lys helps orient
KYC in the active site for optimal inhibition. This result
supports our MPO spectra studies showing that KYC binds
into the active site of MPO and directly interacts with the
iron-heme site.
KYC inhibits MPO-dependent lipoprotein oxidation,
nitration, and chlorination
MPO activity increases Trp oxidation, Tyr nitration, and
chlorination in a variety of proteins (66, 67). To determine
the effect of KYC on MPO-dependent Trp oxidation in LDL,
we incubated LDL with MPO/H2O2/NaNO2 for 30 min at
room temperature and then measured Trp oxidation in LDL
via changes in Trp intrinsic fluorescence. MPO induced
significant decreases (15%, P < 0.001, t-test) in Trp intrin-
sic fluorescence in LDL (Fig. 8A), confirming that Trp in
LDL was oxidized. Adding KYC to the reaction system pro-
tected Trp residues in LDL from being oxidized. Figure
8B shows that KYC dose-dependently reduced NO2Tyr for-
mation in LDL induced by the MPO/H2O2/NaNO2 reac-
tion system. Compared with KYC at 0 ꢂM, KYC at 6.25 ꢂM
inhibited NO2Tyr formation by 50% and essentially ablated
NO2Tyr formation in LDL when added at 25 ꢂM (Fig. 8B).
3024
Journal of Lipid Research Volume 54, 2013

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
Fig. 6. Effects of KYC on MPO-mediated LDL oxidation. LDL oxidation was induced by MPO (20 nM),
H2O2 (100 ꢂM), and NaNO2 (100 ꢂM) in phosphate buffer (100 mM, pH 7.4) containing DTPA (100 ꢂM)
at room temperature. A: KYC dose-dependently inhibits LDL lipid oxidation induced by MPO/H2O2/
NaNO2. Line graphs showing time- and concentration-dependent oxidation of LDL. KYC decreases MPO-
mediated oxidation as measured by absorbance of conjugated dienes at 234 nm. B: Effects of 25 ꢂM KYC,
KYS, and KFC on MPO/H2O2/NaNO2-mediated LDL conjugated diene formation. C: Effects of KYC, GSH,
Tyr, and Trp (25 ꢂM) on MPO/H2O2/NaNO2-mediated LDL conjugated diene formation. D: Effects of KYC,
GSH, Tyr, and Trp (25 ꢂM) on MPO/H2O2-induced LDL conjugated diene formation. Incubation condi-
tions were the same as above in (B) except without NaNO2 as outlined in Materials and Methods. E: MPO-
mediated LDL MDA formation determined by N-methyl-2-phenylindole assay at 586 nm. The data represent
three independent experiments.
These results indicate that KYC is an effective inhibitor of
MPO-dependent protein nitration. We also examined the
ability of KYC to inhibit MPO-mediated thiocyanate-induced
LDL peroxidation. Incubation of LDL with MPO, H2O2,
and NaSCN at 37°C for 2 h induced MDA formation. KYC
dose-dependently inhibited MPO-NaSCN-mediated LDL
oxidation (Fig. 8C). These data indicate that KYC is an ef-
fective inhibitor of MPO-dependent Trp oxidation and
Tyr nitration in lipoproteins. In addition, KYC reduces
MPO/ꢀSCN-induced lipid peroxidation in LDL.
concentrations as high as 4,000 ꢂM over 24 h. KYC pro-
tects BAEC cultures from MPO-induced injury and death
at 25–50 ꢂM, which is several orders of magnitude less
than used for cytotoxicity studies. Such differences indi-
cate that KYC may have a wide therapeutic window for
treating MPO-dependent vascular inflammation.
Several studies (36, 42) show that phenol and/or in-
dole-like compounds are capable of competing with ha-
lides for compound I and/or compound II of MPO to
prevent conversion of the halides into toxic hypohalous
acids. Even though such agents may out-compete halides,
their reaction with compounds I and II of MPO results in
the formation of toxic phenolic and indole radicals (36).
To develop a new class of MPO inhibitors with minimal
toxicity, we synthesized a series of tripeptides that con-
tained both Tyr and Cys. Here, we used Tyr, a natural
substrate of MPO, to react with MPO oxidation interme-
diates. As anticipated, the reaction of Tyr with activated
MPO resulted in the generation of a toxic Tyr•. However,
in the environment of the tripeptide, KYC, Tyr• is scav-
enged by Cys before it has a chance to leave the active site
of MPO and oxidize other cellular targets. Support for
DISCUSSION
This study shows that KYC is a novel tripeptide that in-
hibits MPO-dependent HOCl production, LDL lipid per-
oxidation, protein nitration, and Trp oxidation. KYC
specifically inhibits the activity of MPO that is released
from PMA-activated HL-60 cells but not NOX activity that
•ꢀ
is essential for HL-60 cells to generate O2 that can dis-
mutate to H2O2 to activate MPO. KYC does not induce
cytotoxicity in BAEC cultures even when incubated at
Novel tripeptide inhibitors of myeloperoxidase activity
3025

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
Fig. 7. The mechanism of KYC inhibition of MPO. A: Effects of KYC on MPO UV-Vis spectra. a: MPO (1.4 ꢂM)
was incubated with KYC (50 ꢂM) at room temperature. The changes in UV-Vis spectra were recorded at dif-
ferent times as indicated. b: The same reaction as in (a), except H2O2 (40 ꢂM) was added. c: The same reac-
tion as in (a), except 150 mM NaCl was added. d: The same reaction as in (b), except 150 mM NaCl was
added. e: MPO (1.4 ꢂM) was mixed with H2O2 (300 ꢂM) for 20 sec. The reaction was stopped by addition of
catalase. Immediately after recording the compound II spectrum, the reaction was mixed with KYC (50 ꢂM)
and the changes of heme spectra were recorded. B: H2O2 consumption of MPO. MPO (2 nM) was incubated
with H2O2 (50 ꢂM) and NaCl (100 mM) with different amounts of KYC in phosphate buffer (100 mM, pH
7.4) containing DTPA (100 ꢂM) at room temperature. H2O2 consumption rates were determined using the
Fox assay in triplicate. C: Docking of KYC in the active site of MPO (eHit v9.1, SimBioSysTM Inc., Toronto,
Canada). a: shown KYC on the top of heme of MPO active center and b: shown Tyr of KYC is almost parallel
to heme of MPO active center. The blue molecule is the heme of MPO, the red molecule is KYC, and the
green line is the side chain of Glu116.
intramolecular electron transfer playing a role in the
mechanisms by which KYC detoxifies MPO activity comes
from data showing that MPO-dependent KYC oxidation
forms exclusively a disulfide, not DiTyr, because DiTyr is
only formed when one Tyr• condenses with another Tyr•.
These data, including data showing that the most effi-
cient tripeptide inhibitor is KYC made with all L-amino
acids, indicate that KYC is unique in its ability to inhibit
MPO activity.
Previous studies (65, 66) show that thiols are effective
scavengers of MPO oxidation products, such as HOCl and
•NO2. Accordingly, it is possible that the thiol of KYC may
have scavenged these same oxidants in our assays. How-
ever, several lines of evidence indicate that the Tyr of KYC
3026
Journal of Lipid Research Volume 54, 2013

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
inhibitors of MPO-catalyzed HOCl production, including
the all D-isomer, Ac-(d)K(d)Y(d)C-amide. Such results in-
dicate that KYC possesses a specific structure that makes it
uniquely suited for inhibiting MPO activity that goes well
beyond the fact that KYC contains Tyr and Cys. Finally,
MPO heme spectral studies clearly show that KYC binds to
the active site of MPO because KYC alone is sufficient to
induce changes of iron-heme spectra of native MPO. Al-
though the nature of such spectral change is not known, it
may be caused by either the direct interaction between KYC
and heme, or somehow KYC is able to promote compound
III formation after entering the reactive site, as the new
spectrum is similar to those of MPO compound III. The
spectra data also show that KYC reacts with both com-
pound I and compound II. The results from spectra analy-
ses are consistent with the docking studies showing that
KYC fits nicely into the active site and interacts with iron-
heme of MPO. Another finding that is consistent with our
proposed mechanism is that KYC increases H2O2 con-
sumption by MPO.
As our primary objective was to develop an effective in-
hibitor of MPO activity that was not toxic, it was encourag-
ing to see that BAECs could be incubated in KYC at high
concentrations (4,000 ꢂM) without any significant impact
on BAEC viability, apoptosis, necrosis, or mitochondrial func-
tion. This is important because even though other agents
are reported to effectively inhibit MPO activity in vitro,
their toxic side effects reduce their usefulness in vivo. For
example, large doses of melatonin have been used to im-
prove vasodilatation in hypertensive rats (68). However, the
melatonin treatments also increased the number of apop-
totic cells in the vessel wall of the hypertensive rats (68).
Likewise, when cholesterol-fed rabbits were treated with
4-aminobenzoic acid hydrazide, which is highly effective
for inhibiting MPO activity in vitro, the treatments actually
increased intimal hyperplasia by over 43% (69). Recent
reports indicate that 2-thioxanthine and INV-315 may be
effective for inhibiting MPO in vivo (45, 46). In the case of
KYC, a detoxification mechanism was purposely incor-
porated into the inhibitor to prevent the secondary oxida-
tion that may be induced by oxidation of the inhibitor
itself by MPO. Another unique property of KYC is that it
actually increases MPO-dependent H2O2 consumption,
which should decrease oxidative injury by decreasing H2O2
at sites of inflammation.
Fig. 8. The effects of KYC on MPO-mediated lipoprotein oxida-
tion, nitration, and chlorination. A: Inhibition of LDL Trp oxida-
tion by KYC. LDL (0.15 mg/ml), NaNO2 (100 ꢂM), H2O2 (100 ꢂM),
MPO (20 nM), and increasing concentrations of KYC in a phos-
phate buffer (100 mM, pH 7.4) containing DTPA (100 ꢂM) were
incubated at room temperature for 30 min. The oxidation of Trp
in LDL was determined by measuring the changes of Trp fluores-
cence (Ex = 294 nm and Em = 345 nm). B: The effect of KYC on
MPO-mediated Tyr nitration of LDL. LDL (0.5 mg/ml) was incu-
bated with MPO (50 nM), H2O2 (50 ꢂM), NaNO2 (50 ꢂM), and
increasing concentrations of KYC in phosphate buffer (100 mM,
pH 7.4) containing DTPA (100 ꢂM) at 37°C for 4 h. Reaction was
stopped by addition of catalase (2,000 units/ml). The formation of
NO2Tyr was assessed by dot blot analysis performed in triplicate for
each condition. C: Effect of KYC on MPO-mediated lipid peroxida-
tion of LDL induced by H2O2 and NaSCN. LDL (0.5 mg/ml) was
incubated with MPO (50 nM), H2O2 (50 ꢂM), NaSCN (250 ꢂM),
and increasing concentrations of KYC in phosphate buffer (100 mM,
pH 7.4) containing DTPA (100 ꢂM) at 37°C for 2 h. Reaction was
stopped by catalase (2,000 units/ml). The lipid peroxidation was
assessed by MDA assay performed in triplicate.
In addition to increasing specificity and avoiding toxic-
ity, hydrophobicity is another important characteristic of
MPO inhibitors that has impact on in vivo effectiveness.
Although 4-aminobenzoic acid hydrazide is an effective in-
hibitor of MPO in vitro, it is rapidly taken up by LDL because
it is also highly soluble in lipids, effectively reducing its ac-
cess to MPO which is usually in an aqueous environment
(70). With respect to KYC, Lys, a positively charged amino
acid, increases the tripeptide’s hydrophilic properties,
thus preventing its partitioning into the lipid domain. This
enhances KYC’s retention in hydrophilic environments in
vivo (i.e., bloodstream, matrix, and subendothelial spaces)
where MPO is known to induce tissue damage. Our data
show that KYC effectively inhibits MPO-mediated LDL lipid
directly reacts with MPO compounds I and II as the domi-
nant mechanism by which it inhibits MPO activity. First, our
data show that Tyr is required for a tripeptide to effectively
inhibit MPO activity. KYC is oxidized by the simplest MPO/
ꢀ
H2O2 reaction system (i.e., no Clꢀ and NO2 ). Moreover,
the MPO/H2O2 reaction system oxidizes KYS but not KFC.
These data indicate that in the tripeptides, Tyr, not Cys, is
the favored target of activated MPO in tripeptides that
contain both Tyr and Cys. Although KFC decreases HOCl,
the reaction between HOCl and amino acids (67) suggests
that the decrease in HOCl is a result of the thiol in KFC
rather than the Lys or Phe which are not effective HOCl
scavengers under the conditions of the assay. Second,
all 11 analogs were consistently less effective than KYC as
Novel tripeptide inhibitors of myeloperoxidase activity
3027

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
18. Nagra, R. M., B. Becher, W. W. Tourtellotte, J. P. Antel, D. Gold,
peroxidation and protein oxidation/nitration. On the ba-
sis of these observations, KYC may be a more suitable in-
hibitor of MPO in lipid-rich regions than agents that possess
a more hydrophobic character such as 4-aminobenzoic acid
hydrazide.
T. Paladino, R. A. Smith, J. R. Nelson, and W. F. Reynolds. 1997.
Immunohistochemical and genetic evidence of myeloperoxidase
involvement in multiple sclerosis. J. Neuroimmunol. 78: 97–107.
19. Gray, E., T. L. Thomas, S. Betmouni, N. Scolding, and S. Love.
2008. Elevated myeloperoxidase activity in white matter in multiple
sclerosis. Neurosci. Lett. 444: 195–198.
In conclusion, KYC, a novel tripeptide, is a specific po-
tent nontoxic inhibitor of MPO activity that may be suit-
able for in vivo studies. As KYC effectively inhibits MPO
HOCl generation and LDL oxidation, as well as protects
BAEC cultures from MPO-mediated injury, KYC may be
an effective inhibitor for investigating MPO-dependent
mechanisms mediating oxidative/nitrosative stress in a va-
riety of diseases. The principles explained here concern-
ing the biochemistry by which KYC inhibits MPO may lead
to the development of new therapeutic tools targeting
other oxidative enzymes involved in the pathogenesis of
vascular disease and inflammation.
20. Gray, E., T. L. Thomas, S. Betmouni, N. Scolding, and S. Love.
2008. Elevated activity and microglial expression of myeloperoxi-
dase in demyelinated cerebral cortex in multiple sclerosis. Brain
Pathol. 18: 86–95.
21. Green, P. S., A. J. Mendez, J. S. Jacob, J. R. Crowley, W. Growdon,
B. T. Hyman, and J. W. Heinecke. 2004. Neuronal expression of
myeloperoxidase is increased in Alzheimer’s disease. J. Neurochem.
90: 724–733.
22. Maki, R. A., V. A. Tyurin, R. C. Lyon, R. L. Hamilton, S. T. DeKosky,
V. E. Kagan, and W. F. Reynolds. 2009. Aberrant expression of my-
eloperoxidase in astrocytes promotes phospholipid oxidation and
memory deficits in a mouse model of Alzheimer disease. J. Biol.
Chem. 284: 3158–3169.
23. Choi, D. K., S. Pennathur, C. Perier, K. Tieu, P. Teismann, D. C.
Wu, V. Jackson-Lewis, M. Vila, J. P. Vonsattel, J. W. Heinecke, et
al. 2005. Ablation of the inflammatory enzyme myeloperoxidase
mitigates features of Parkinson’s disease in mice. J. Neurosci. 25:
6594–6600.
REFERENCES
24. Ahn, J., M. D. Gammon, R. M. Santella, M. M. Gaudet, J. A.
Britton, S. L. Teitelbaum, M. B. Terry, A. I. Neugut, P. D. Josephy,
and C. B. Ambrosone. 2004. Myeloperoxidase genotype, fruit and
vegetable consumption, and breast cancer risk. Cancer Res. 64:
7634–7639.
25. Taioli, E., S. Benhamou, C. Bouchardy, I. Cascorbi, N. Cajas-Salazar,
H. Dally, K. M. Fong, J. E. Larsen, L. Le Marchand, S. J. London,
et al. 2007. Myeloperoxidase G-463A polymorphism and lung can-
cer: a HuGE genetic susceptibility to environmental carcinogens
pooled analysis. Genet. Med. 9: 67–73.
26. Zheng, L., B. Nukuna, M. L. Brennan, M. Sun, M. Goormastic,
M. Settle, D. Schmitt, X. Fu, L. Thomson, P. L. Fox, et al. 2004.
Apolipoprotein A-I is a selective target for myeloperoxidase-cata-
lyzed oxidation and functional impairment in subjects with cardio-
vascular disease. J. Clin. Invest. 114: 529–541.
27. Panzenboeck, U., S. Raitmayer, H. Reicher, H. Lindner, O. Glatter,
E. Malle, and W. Sattler. 1997. Effects of reagent and enzymatically
generated hypochlorite on physicochemical and metabolic proper-
ties of high density lipoproteins. J. Biol. Chem. 272: 29711–29720.
28. Nicholls, S. J., L. Zheng, and S. L. Hazen. 2005. Formation of dys-
functional high-density lipoprotein by myeloperoxidase. Trends
Cardiovasc. Med. 15: 212–219.
29. Bergt, C., S. Pennathur, X. Fu, J. Byun, K. O’Brien, T. O. McDonald,
P. Singh, G. M. Anantharamaiah, A. Chait, J. Brunzell, et al. 2004.
The myeloperoxidase product hypochlorous acid oxidizes HDL in
the human artery wall and impairs ABCA1-dependent cholesterol
transport. Proc. Natl. Acad. Sci. USA. 101: 13032–13037.
30. Spickett, C. M. 2007. Chlorinated lipids and fatty acids: an emerg-
ing role in pathology. Pharmacol. Ther. 115: 400–409.
31. Malle, E., P. G. Furtmuller, W. Sattler, and C. Obinger. 2007.
Myeloperoxidase: a target for new drug development? Br. J.
Pharmacol. 152: 838–854.
32. Skaff, O., D. I. Pattison, and M. J. Davies. 2007. Kinetics of hypo-
bromous acid-mediated oxidation of lipid components and antioxi-
dants. Chem. Res. Toxicol. 20: 1980–1988.
33. Pattison, D. I., and M. J. Davies. 2004. Kinetic analysis of the reac-
tions of hypobromous acid with protein components: implications
for cellular damage and use of 3-bromotyrosine as a marker of oxi-
dative stress. Biochemistry. 43: 4799–4809.
1. Winterbourn, C. C., M. C. Vissers, and A. J. Kettle. 2000. Myeloperox-
idase. Curr. Opin. Hematol. 7: 53–58.
2. Arnhold, J., and J. Flemmig. 2010. Human myeloperoxidase in in-
nate and acquired immunity. Arch. Biochem. Biophys. 500: 92–106.
3. Klebanoff, S. J. 2005. Myeloperoxidase: friend and foe. J. Leukoc.
Biol. 77: 598–625.
4. Battistuzzi, G., M. Bellei, C. A. Bortolotti, and M. Sola. 2010. Redox
properties of heme peroxidases. Arch. Biochem. Biophys. 500: 21–36.
5. Arnhold, J., P. G. Furtmuller, and C. Obinger. 2003. Redox proper-
ties of myeloperoxidase. Redox Rep. 8: 179–186.
6. Hoy, A., B. Leininger-Muller, D. Kutter, G. Siest, and S. Visvikis.
2002. Growing significance of myeloperoxidase in non-infectious
diseases. Clin. Chem. Lab. Med. 40: 2–8.
7. Lau, D., and S. Baldus. 2006. Myeloperoxidase and its contributory
role in inflammatory vascular disease. Pharmacol. Ther. 111: 16–26.
8. Van Der Vliet, A., M. N. Nguyen, M. K. Shigenaga, J. P. Eiserich, G.
P. Marelich, and C. E. Cross. 2000. Myeloperoxidase and protein
oxidation in cystic fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol.
279: L537–L546.
9. Nurcombe, H. L., R. C. Bucknall, and S. W. Edwards. 1991.
Activation of the neutrophil myeloperoxidase-H2O2 system by syn-
ovial fluid isolated from patients with rheumatoid arthritis. Ann.
Rheum. Dis. 50: 237–242.
10. Schiller, J., J. Arnhold, K. Sonntag, and K. Arnold. 1996. NMR stud-
ies on human, pathologically changed synovial fluids: role of hy-
pochlorous acid. Magn. Reson. Med. 35: 848–853.
11. Ali, Z., P. Sarcia, T. H. Mosley, Jr., V. Kondragunta, and I. J. Kullo.
2009. Association of serum myeloperoxidase with the ankle-bra-
chial index and peripheral arterial disease. Vasc. Med. 14: 215–220.
12. Nicholls, S. J., and S. L. Hazen. 2005. Myeloperoxidase and cardio-
vascular disease. Arterioscler. Thromb. Vasc. Biol. 25: 1102–1111.
13. Heilman, K., M. Zilmer, K. Zilmer, M. Lintrop, P. Kampus, J. Kals,
and V. Tillmann. 2009. Arterial stiffness, carotid artery intima-me-
dia thickness and plasma myeloperoxidase level in children with
type 1 diabetes. Diabetes Res. Clin. Pract. 84: 168–173.
14. Wiersma, J. J., M. C. Meuwese, J. N. van Miert, A. Kastelein, J. G.
Tijssen, J. J. Piek, and M. D. Trip. 2008. Diabetes mellitus type 2 is
associated with higher levels of myeloperoxidase. Med. Sci. Monit.
14: CR406–CR410.
15. Gumina, R. J., J. el Schultz, Z. Yao, D. Kenny, D. C. Warltier, P. J.
Newman, and G. J. Gross. 1996. Antibody to platelet/endothelial
cell adhesion molecule-1 reduces myocardial infarct size in a rat
model of ischemia-reperfusion injury. Circulation. 94: 3327–3333.
16. Yunoki, K., T. Naruko, R. Komatsu, N. Shirai, M. Nakagawa,
K. Sugioka, Y. Ikura, K. F. Kusano, A. Itoh, K. Haze, et al. 2010.
Relation of elevated levels of plasma myeloperoxidase to impaired
myocardial microcirculation after reperfusion in patients with
acute myocardial infarction. Am. J. Cardiol. 105: 922–929.
17. Lefkowitz, D. L., and S. S. Lefkowitz. 2008. Microglia and myeloper-
oxidase: a deadly partnership in neurodegenerative disease. Free
Radic. Biol. Med. 45: 726–731.
34. Pattison, D. I., and M. J. Davies. 2006. Reactions of myeloperoxi-
dase-derived oxidants with biological substrates: gaining chemical
insight into human inflammatory diseases. Curr. Med. Chem. 13:
3271–3290.
35. Koelsch, M., R. Mallak, G. G. Graham, T. Kajer, M. K. Milligan,
L. Q. Nguyen, D. W. Newsham, J. S. Keh, A. J. Kettle, K. F. Scott,
et al. 2010. Acetaminophen (paracetamol) inhibits myeloper-
oxidase-catalyzed oxidant production and biological damage at
therapeutically achievable concentrations. Biochem. Pharmacol. 79:
1156–1164.
36. Kettle, A. J., and L. P. Candaeis. 2000. Oxidation of tryptophan
by redox intermediates of myeloperoxidase and inhibition of hy-
pochlorous acid production. Redox Rep. 5: 179–184.
3028
Journal of Lipid Research Volume 54, 2013

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2013/07/24/jlr.M038273.DC1
.html
37. Galijasevic, S., I. Abdulhamid, and H. M. Abu-Soud. 2008.
54. Erdelmeier, I., D. Gerard-Monnier, J. C. Yadan, and J. Chaudiere.
1998. Reactions of N-methyl-2-phenylindole with malondialdehyde
and 4-hydroxyalkenals. Mechanistic aspects of the colorimetric as-
say of lipid peroxidation. Chem. Res. Toxicol. 11: 1184–1194.
55. Gérard-Monnier, D., I. Erdelmeier, K. Régnard, N. Moze-Henry,
J. C. Yadan, and J. Chaudière. 1998. Reactions of 1-methyl-2-phe-
nylindole with malondialdehyde and 4-hydroxyalkenals. Analytical
applications to a colorimetric assay of lipid peroxidation. Chem. Res.
Toxicol. 11: 1176–1183.
56. Haynes, J., Jr., and B. Obiako. 2002. Activated polymorphonuclear
cells increase sickle red blood cell retention in lung: role of phos-
pholipids. Am. J. Physiol. Heart Circ. Physiol. 282: H122–H130.
57. Forbes, L. V., P. G. Furtmuller, I. Khalilova, R. Turner, C. Obinger,
and A. J. Kettle. 2012. Isoniazid as a substrate and inhibitor of my-
eloperoxidase: identification of amine adducts and the influence
of superoxide dismutase on their formation. Biochem. Pharmacol. 84:
949–960.
Melatonin is a potent inhibitor for myeloperoxidase. Biochemistry.
47: 2668–2677.
38. Ximenes, V. F., G. J. Maghzal, R. Turner, Y. Kato, C. C. Winterbourn,
and A. J. Kettle. 2009. Serotonin as a physiological substrate for my-
eloperoxidase and its superoxide-dependent oxidation to cytotoxic
tryptamine-4,5-dione. Biochem. J. 425: 285–293.
39. Ximenes, V. F., A. S. Pessoa, C. Z. Padovan, D. C. Abrantes, F. H.
Gomes, M. A. Maticoli, and M. L. de Menezes. 2009. Oxidation of
melatonin by AAPH-derived peroxyl radicals: evidence of a pro-
oxidant effect of melatonin. Biochim. Biophys. Acta. 1790: 787–792.
40. Kato, Y., A. Nagao, J. Terao, and T. Osawa. 2003. Inhibition of
myeloperoxidase-catalyzed tyrosylation by phenolic antioxidants in
vitro. Biosci. Biotechnol. Biochem. 67: 1136–1139.
41. Kettle, A. J., and C. C. Winterbourn. 1991. Mechanism of inhibition
of myeloperoxidase by anti-inflammatory drugs. Biochem. Pharmacol.
41: 1485–1492.
42. Heinecke, J. W. 2002. Tyrosyl radical production by myeloperoxi-
dase: a phagocyte pathway for lipid peroxidation and dityrosine
cross-linking of proteins. Toxicology. 177: 11–22.
43. Hermann, M., S. Kapiotis, R. Hofbauer, C. Seelos, I. Held, and B.
Gmeiner. 1999. Salicylate promotes myeloperoxidase-initiated LDL
oxidation: antagonization by its metabolite gentisic acid. Free Radic.
Biol. Med. 26: 1253–1260.
44. Savenkova, M. L., D. M. Mueller, and J. W. Heinecke. 1994. Tyrosyl
radical generated by myeloperoxidase is a physiological catalyst for
the initiation of lipid peroxidation in low density lipoprotein. J.
Biol. Chem. 269: 20394–20400.
45. Churg, A., C. V. Marshall, D. D. Sin, S. Bolton, S. Zhou, K. Thain, E.
B. Cadogan, J. Maltby, M. G. Soars, P. R. Mallinder, et al. 2012. Late
intervention with a myeloperoxidase inhibitor stops progression of
experimental chronic obstructive pulmonary disease. Am. J. Respir.
Crit. Care Med. 185: 34–43.
58. Pullar, J. M., C. C. Winterbourn, and M. C. Vissers. 1999. Loss
of GSH and thiol enzymes in endothelial cells exposed to sub-
lethal concentrations of hypochlorous acid. Am. J. Physiol. 277:
H1505–H1512.
59. Hazen, S. L., R. Zhang, Z. Shen, W. Wu, E. A. Podrez, J. C.
MacPherson, D. Schmitt, S. N. Mitra, C. Mukhopadhyay, Y. Chen,
et al. 1999. Formation of nitric oxide-derived oxidants by myeloper-
oxidase in monocytes: pathways for monocyte-mediated protein ni-
tration and lipid peroxidation in vivo. Circ. Res. 85: 950–958.
60. Byun, J., D. M. Mueller, J. S. Fabjan, and J. W. Heinecke. 1999.
Nitrogen dioxide radical generated by the myeloperoxidase-hy-
drogen peroxide-nitrite system promotes lipid peroxidation of low
density lipoprotein. FEBS Lett. 455: 243–246.
61. Meotti, F. C., G. N. Jameson, R. Turner, D. T. Harwood, S. Stockwell,
M. D. Rees, S. R. Thomas, and A. J. Kettle. 2011. Urate as a physi-
ological substrate for myeloperoxidase: implications for hyperuri-
cemia and inflammation. J. Biol. Chem. 286: 12901–12911.
62. Kettle, A. J., and C. C. Winterbourn. 2001. A kinetic analysis of the
catalase activity of myeloperoxidase. Biochemistry. 40: 10204–10212.
63. Jiang, Z. Y., A. C. Woollard, and S. P. Wolff. 1990. Hydrogen perox-
ide production during experimental protein glycation. FEBS Lett.
268: 69–71.
46. Liu, C., R. Desikan, Z. Ying, L. Gushchina, T. Kampfrath, J. Deiuliis,
A. Wang, X. Xu, J. Zhong, X. Rao, et al. 2012. Effects of a novel
pharmacologic inhibitor of myeloperoxidase in a mouse athero-
sclerosis model. PLoS ONE. 7: e50767.
47. Tien, M. 1999. Myeloperoxidase-catalyzed oxidation of tyrosine.
Arch. Biochem. Biophys. 367: 61–66.
48. Zhang, H., Y. Xu, J. Joseph, and B. Kalyanaraman. 2005.
Intramolecular electron transfer between tyrosyl radical and
cysteine residue inhibits tyrosine nitration and induces thiyl radi-
cal formation in model peptides treated with myeloperoxidase,
H2O2, and NO2-: EPR SPIN trapping studies. J. Biol. Chem. 280:
40684–40698.
49. Eiserich, J. P., M. Hristova, C. E. Cross, A. D. Jones, B. A. Freeman,
B. Halliwell, and A. van der Vliet. 1998. Formation of nitric oxide-
derived inflammatory oxidants by myeloperoxidase in neutrophils.
Nature. 391: 393–397.
50. Zhang, H., Y. Xu, J. Joseph, and B. Kalyanaraman. 2008. Influence
of intramolecular electron transfer mechanism in biological nitra-
tion, nitrosation, and oxidation of redox-sensitive amino acids.
Methods Enzymol. 440: 65–94.
51. Zhang,H.,J.Zielonka,A.Sikora,J.Joseph,Y.Xu,andB.Kalyanaraman.
2009. The effect of neighboring methionine residue on tyrosine
nitration and oxidation in peptides treated with MPO, H2O2, and
NO2(-) or peroxynitrite and bicarbonate: role of intramolecular
electron transfer mechanism? Arch. Biochem. Biophys. 484: 134–145.
52. Ou, Z., J. Ou, A. W. Ackerman, K. T. Oldham, and K. A. Pritchard,
Jr. 2003. L-4F, an apolipoprotein A-1 mimetic, restores nitric oxide
and superoxide anion balance in low-density lipoprotein-treated
endothelial cells. Circulation. 107: 1520–1524.
64. Hallingbäck, H. R., R. R. Gabdoulline, and R. C. Wade. 2006.
Comparison of the binding and reactivity of plant and mamma-
lian peroxidases to indole derivatives by computational docking.
Biochemistry. 45: 2940–2950.
65. Ford, E., M. N. Hughes, and P. Wardman. 2002. Kinetics of the
reactions of nitrogen dioxide with glutathione, cysteine, and uric
acid at physiological pH. Free Radic. Biol. Med. 32: 1314–1323.
66. Peskin, A. V., and C. C. Winterbourn. 2001. Kinetics of the reac-
tions of hypochlorous acid and amino acid chloramines with thiols,
methionine, and ascorbate. Free Radic. Biol. Med. 30: 572–579.
67. Pattison, D. I., and M. J. Davies. 2001. Absolute rate constants for
the reaction of hypochlorous acid with protein side chains and pep-
tide bonds. Chem. Res. Toxicol. 14: 1453–1464.
68. Rezzani, R., E. Porteri, C. De Ciuceis, F. Bonomini, L. F. Rodella,
S. Paiardi, G. E. Boari, C. Platto, A. Pilu, D. Avanzi, et al. 2010.
Effects of melatonin and Pycnogenol on small artery structure
and function in spontaneously hypertensive rats. Hypertension. 55:
1373–1380.
69. Békési, G., H. Heinle, R. Kakucs, T. Pázmány, D. Szombath, M.
Dinya, Z. Tulassay, J. Fehér, K. Rácz, B. Székács, et al. 2005. Effect
of inhibitors of myeloperoxidase on the development of aortic ath-
erosclerosis in an animal model. Exp. Gerontol. 40: 199–208.
70. Jerlich, A., G. Fritz, H. Kharrazi, M. Hammel, S. Tschabuschnig, O.
Glatter, and R. J. Schaur. 2000. Comparison of HOCl traps with my-
eloperoxidase inhibitors in prevention of low density lipoprotein
oxidation. Biochim. Biophys. Acta. 1481: 109–118.
53. Dypbukt, J. M., C. Bishop, W. M. Brooks, B. Thong, H. Eriksson, and
A. J. Kettle. 2005. A sensitive and selective assay for chloramine pro-
duction by myeloperoxidase. Free Radic. Biol. Med. 39: 1468–1477.
Novel tripeptide inhibitors of myeloperoxidase activity
3029