Triazolopyrimidines identified as reversible myeloperoxidase inhibitors

Article abstract of DOI:10.1039/c7md00268h

Myeloperoxidase, a mammalian peroxidase involved in the immune system as an anti-microbial first responder, can produce hypochlorous acid in response to invading pathogens. Myeloperoxidase has been implicated in several chronic pathological diseases due to the chronic production of hypochlorous acid, as well as other reactive radical species. A high throughput screen and triaging protocol was developed to identify a reversible inhibitor of myeloperoxidase toward the potential treatment of chronic diseases such as atherosclerosis. The identification and characterization of a reversible myeloperoxidase inhibitor, 7-(benzyloxy)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine is described.

Full text of DOI:10.1039/c7md00268h

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RESEARCH ARTICLE
Triazolopyrimidines identified as reversible
myeloperoxidase inhibitors†
Cite this: DOI: 10.1039/c7md00268h
*
*
Franck Duclos, ‡ Lynn M. Abell,‡ David G. Harden, Kristen Pike,
Kimberly Nowak, Gregory A. Locke, Gerald J. Duke, Xiaoqin Liu, Gayani Fernando,
Scott A. Shaw, Benjamin P. Vokits, Nicholas R. Wurtz, Andrew Viet,
Meriah N. Valente, Sylwia Stachura,
Paul Sleph, Javed A. Khan,
Ji Gao,
Ashok R. Dongre, Lei Zhao, Ruth R. Wexler,
David A. Gordon and Ellen K. Kick
Myeloperoxidase, a mammalian peroxidase involved in the immune system as an anti-microbial first re-
sponder, can produce hypochlorous acid in response to invading pathogens. Myeloperoxidase has been
implicated in several chronic pathological diseases due to the chronic production of hypochlorous acid, as
well as other reactive radical species. A high throughput screen and triaging protocol was developed to
identify a reversible inhibitor of myeloperoxidase toward the potential treatment of chronic diseases such
as atherosclerosis. The identification and characterization of a reversible myeloperoxidase inhibitor,
7-(benzyloxy)-3H-[1,2,3]triazoloij4,5-d]pyrimidin-5-amine is described.
Received 24th May 2017,
Accepted 5th October 2017
DOI: 10.1039/c7md00268h
rsc.li/medchemcomm
over, HOCl-modified HDL and LDL lipoproteins have been
detected in advanced human atherosclerotic lesions.10–12
Introduction
The myeloid hemoprotein myeloperoxidase (MPO) is a mam-
malian peroxidase that uniquely catalyzes the oxidation of
chloride ion in the presence of hydrogen peroxide (H2O2) to
generate hypochlorous acid (HOCl), a powerful oxidant. MPO
can also act as a classical peroxidase, producing a wide range
of free radicals and reactive oxidant species. MPO is synthe-
sized in promyelocytes and promonocytes in the bone marrow
and stored in the azurophilic granules of neutrophils where it
constitutes 2–5% of total cellular protein content, and is pres-
ent in lower abundance in subpopulations of monocytes and
macrophages. MPO plays an important role in the immune
system to help kill invading pathogens.1 Recent evidence sup-
ports a pathological role of MPO in various chronic inflamma-
tory conditions;2–6 therefore, MPO inhibitors may be useful to
treat inflammatory diseases including cardiovascular disease.
MPO has been implicated in cardiovascular disease in
human clinical studies and by mechanistic work linking dele-
terious MPO oxidation of high density lipoprotein (HDL) and
low density lipoprotein (LDL) to disease progression.3–7 MPO
is expressed in the shoulder regions and necrotic core of
human atherosclerotic lesions and active enzyme has been
isolated from autopsy specimens of human lesions.8,9 More-
After isolation from atherosclerotic lesions, the HDL apolipo-
protein, ApoA1, had chlorinated and nitrated tyrosine
residues.3,5,13–15 Chlorotyrosine modifications of apoA1 are
proposed to be specific biomarkers of MPO activity, and were
shown to be associated with impaired cholesterol acceptor
function.15,16 The lipid and protein content of LDL are also
targets for MPO oxidation, and LDL particles exposed to MPO
in vitro have impaired binding to the LDL-receptor. In the
atherosclerotic plaque context, oxidized LDL is most likely
taken up by macrophage scavenger receptors leading to
macrophage foam cell formation.17 Further evidence impli-
cating MPO in atherosclerosis pathophysiology comes from
the study of hMPO transgenic mice crossed with LDL-R KO
mice showing significantly larger aortic lesions than control
LDL-R KO mice.18 Thus, MPO appears to play a role in the
generation of oxidized, dysfunctional HDL and LDL, which
contribute to atherosclerosis plaque development.
MPO exerts its biological activity predominantly via a pair
of catalytic pathways (Fig. 1). The chlorination pathway starts
by reaction of the FeIJIII) native enzyme with H2O2 to generate
compound I, which contains two oxidizing equivalents more
than the native enzyme (Fig. 1). Compound I has a high
redox potential with the capacity to oxidize chloride ions to
form HOCl and regenerate native enzyme. Alternatively in the
peroxidase pathway, compound I is reduced by two consecu-
tive one electron reduction steps via compound II intermedi-
ate back to the native enzyme. In these redox reactions a
plethora of substrates can be oxidized to their corresponding
Bristol-Myers Squibb Company, P.O. Box 5400, Princeton, New Jersey 08543-5400,
USA. E-mail: Franck.Duclos@Bms.com
† Electronic supplementary information (ESI) available: General synthetic
methods, key compound characterization, assay methods, and crystallographic
information are available in the ESI. See DOI: 10.1039/c7md00268h
‡ The first two authors contributed equally to this work.
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Fig. 1 MPO catalytic mechanism.
free radicals. Substrate inhibitors such as tryptophan benzyl
ester (1), indoles and nitroxides compete with chloride for ox-
idation by rapidly reacting with highly redox active com-
pound I to form compound II and generating a radical
intermediate.19–23 While compound II will persist with some
substrates in vitro, it is likely a physiological substrate like ty-
rosine, urate, or ascorbate in the in vivo milieu would recycle
compound II back to its native state. Irreversible inhibitors
such as 2-thioxanthines (2,3) and 2-thiouracil 4 act in two
steps by first generating a free radical intermediate from re-
action with compound I, and then the radical forms a cova-
lent linkage with the heme moiety resulting in permanent in-
activation of MPO (Fig. 2).2,24,25 There is the potential for
these free radical intermediates to be released from the active
site and then react with other proteins.26 There have been
limited reports of inhibitors such as the hydroxamic acid 5
that tightly bind at the MPO active site and function as
reversible inhibitors of the enzyme; however 5 was also
reported to be metabolized to nitroxide radicals thereby act-
ing by a mixed mechanism.27 While several irreversible inhib-
itors (2–4) have progressed to clinical trials, and AZD-3241 (3)
was reported in clinical trials for multiple systems atrophy,
the efforts described herein were focused on the search for
reversible MPO inhibitors to determine if potent inhibition
could be obtained (Fig. 2).
Fig. 2 Literature MPO inhibitors (1–5) and HTS hits (6, 7).
hibitors from reversible inhibitors, the reactions were carried
out with and without a 10 minute pre-incubation step.
Potent, irreversible inhibitors showed a strong shift toward
increased potency in the APF assay with pre-incubation com-
pared to no pre-incubation as demonstrated with AZD-5904
(Ex. 2, Table 1), which had a 6 fold increase in potency.
Whereas, many MPO substrates had a potency loss in the AR
assay compared to APF assay as shown with tryptophan ben-
zyl ester (Ex. 1, Table 1), fourteen compounds of interest
remained after this triaging process that inhibited both MPO-
mediated chlorination and peroxidation reactions. Further
mechanistic characterization was performed as described
below for hits 6 and 7.
Neither 6 nor 7 appear to be irreversible inhibitors based
on similar potency in the presence or absence of an enzyme-
inhibitor preincubation step in the APF assay (Table 1). In or-
der to confirm reversibility, pre-incubation jump dilution ex-
periments were conducted under varying conditions of pre-
incubation time, peroxide concentration and presence or ab-
sence of chloride, and the results clearly showed that inhibi-
tion by compound 6 was reversible compared to a known ir-
reversible inhibitor (Fig. SI1. In ESI†).
Stopped-flow experiments were conducted with MPO and
inhibitors 6 and 7 to determine whether the compounds were
truly inhibitors of MPO or behaving as slow substrates. The
absorption spectrum of both native enzyme and compound I
is characterized by a Soret band at 430 nm, while compound
II and III absorb at 452 and 630 nm, respectively.28 Com-
pound I was pre-formed by the incubation of MPO with a
slight excess of H2O2 followed by rapid mixing with a solu-
tion of the MPO inhibitor plus peroxide. The amount of
peroxide used in these experiments was purposely limiting to
determine if the enzyme would return to its resting state
upon consumption of this limiting substrate. As expected
slow substrates such as tryptophan benzyl ester (1), showed
Results
The goal of this project was to identify a stable reversible
MPO inhibitor that would bind to the active site of the native
enzyme and block its function without generating intermedi-
ate radicals. A screening tree based on this strategy was
designed and tested on a 124 K compound collection (Fig. 3)
for proof-of-concept purposes. The first screening assay was
conducted with aminophenyl fluorescein (APF) to detect inhi-
bition of MPO chlorination. After retests, 10% of the screen-
ing collection were identified as “active hits” at 10 μM. Two
counter screens were employed to eliminate ∼50% of hits
that were HOCl scavengers and redox active leaving 6150 “ac-
tive hits”. After structure based clustering, 1000 hits were
tested in dose response mode in the APF assay, and in an
MPO peroxidase inhibition assay with amplex red (AR) detec-
tion and no chloride. To differentiate potent, irreversible in-
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Fig. 3 HTS testing and triaging operations.
Table 1 In vitro MPO inhibitor profile
Human MPO
APF assay
w pre-inc
IC50 μM
Human MPO
APF assay
w/o pre-inc
IC50 μM
Human MPO
AR assay
w pre-inc
IC50 μM
Mouse MPO
APF assay
w pre-inc
IC50 μM
Neutrophil HOCl
production
IC50 μM
TPO
IC50 μM
LPO
IC50 μM
Ex. #
EPX IC50 μM
1
2
6
7
0.2 0.05
0.009 0.001
0.11 0.046*
0.36 0.11*
0.11 0.026
0.056 0.013
0.084 0.033*
0.32 0.070*
>13
0.012 0.002
0.11 0.034a
0.84 0.32a
3.9 (n = 1)
>30
6.0 1.6*
1 (n = 1)
5 (n = 1)
1700 (n = 1)
>30
13 1.0
0.015 0.007*
0.16 0.063*
0.081 (n = 1)
0.23 (n = 1)
a
n > 15. All other values were tested between 3 and 14 replicates unless otherwise indicated, and standard deviation is reported. Procedures
are reported in ESI.
an initial rapid increase in the 452 nm band for MPO along
with a concomitant decrease in the 430 nm band and a small
increase in absorbance at 630 nm (Fig. 4A and D).23 At longer
incubation times, the 452 nm band decreased in intensity,
and the 430 nm band increased indicating the reversion of
the enzyme to its resting state after consumption of the avail-
able peroxide. Compound 7 showed a similar profile indicat-
ing it was a substrate for MPO (Fig. 4C and F); however, 7
had a prolonged plateau for the 450 nm peak compared with
1 indicating that 7 reacts more slowly with MPO contributing
to its potency in the biochemical assays. In contrast, as
shown in Fig. 4B and E, rapid mixing of compound 6 with
MPO showed no change in the intensity of the 430 nm Soret
band over 200 s, indicating no reaction occurred with the en-
zyme. The combination of the stopped-flow experiments and
the reversibility experiments allowed us to identify reversible,
unreactive inhibitors of MPO.
tively filling the active site (Fig. 5). The triazolopyrimidine
has three hydrogen bonds with the active site residues Gln-
91, His-95 and Arg-239, effectively matching the polar amino
acid residues in the active site. In addition the pendant
amine interacts with a heme carboxylic acid. Further stabiliz-
ing the complex, the phenyl group has hydrophobic interac-
tions with the Arg-239 methylenes. The orientation of the
benzyl group is almost orthogonal to the benzyl in the
reported structure of 5, showing different hydrophobic inter-
actions provide affinity for 6.27
Selectivity was assessed to ensure the inhibitors were not
general heme binders. Compounds 6 and 7 were >10-fold se-
lective compared to lactoperoxidase (LPO) and thyroid peroxi-
dase (TPO) while potent inhibition of eosinophil peroxidase
(EPX) was measured (Table 1). The compounds were also
tested for inhibition of cytochrome P450 enzymes (CYP).
Whereas 6 inhibited CYP3A4 with weak potency (IC50 = 14
μM), and had no inhibition against six other CYP enzymes, 7
demonstrated potent inhibition (IC50 < 0.1 μM) of CYPs 2B6
and 2C9.
Crystallization of 6 with MPO provided additional evi-
dence of native MPO enzyme inhibition with the inhibitor
bound directly above the heme in a stacked fashion effec-
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Fig. 4 A–C: Multiwavelength stopped-flow scans over 50 ms after mixing 1 uM MPO, 5 uM peroxide and 80 uM compound 1 (4A), 40 uM com-
pound 6 (4B), and 7 (4C). D–F: Single wavelength traces from separate stopped-flow experiments showing the change in 430 nm and 450 nm
wavelengths over 200 s after mixing for compound 1 (4D), 6 (4E), and 7 (4F).
The biological significance of the mechanistic and selec-
tivity profiles exhibited by these compounds was studied in
the following set of experiments. Inhibitors 2, 6 and 7 were
tested in a dose-response for inhibition of HOCl production
using human neutrophils isolated from healthy donor blood.
Neutrophils were incubated with phorbol myristate acetate
(PMA) to trigger neutrophil phagolysosome and NADPH oxi-
dase complex assembly leading to MPO release from granules
and H2O2 production. Formation of HOCl was quantified by
luminol oxidation and resulting chemiluminescence was
measured in kinetic mode. The irreversible inhibitor 2 and
reversible inhibitor 6 demonstrated similar potency in hu-
man neutrophils, whereas the substrate inhibitor 7 was much
less potent (Table 1). These results confirmed that in the cell
milieu the availability of physiological substrate allowed
recycling of the enzyme, and both reversible and irreversible
inhibitors could efficiently block the enzyme activity.
The ability of 6 to inhibit MPO mediated oxidation of a
known physiological substrate ApoA1 was assessed by moni-
toring the formation of ApoA1 chlorotyrosine 192 (Y192Cl,
Fig. 6) and oxidation of tryptophan 50 (W50ox) using a sensi-
tive LC-MS/MS approach. In parallel, a cell-based cholesterol
efflux assay using ApoA1 as the acceptor was carried out with
MPO treated ApoA1 and varied concentrations of 6. As previ-
ously shown, MPO-mediated oxidation of ApoA1 impaired
in vitro cholesterol efflux from macrophages.29–31 After treat-
ment with inhibitor 6, an inverse correlation between the
levels of chlorinated Y192Cl and oxidized W50ox ApoA1 resi-
dues, and the acceptor capability of ApoA1 to exchange cho-
lesterol from THP-1 macrophages was observed with an EC50
value of 10 μM (Fig. 6 and ESI†). These observations support
the concept of using an MPO reversible inhibitor to prevent
MPO-mediated oxidation of ApoA1, and the impairment of
ABCA1 cholesterol efflux pathway to counteract foam cell for-
mation in the artery wall.
Fig. 5 Co-crystal structure of 6 with MPO. The carbons of compound
6 are colored in yellow and the carbons for heme are colored in green.
Light chain residues of MPO are shown in cyan and heavy chain in
magenta. Oxygens are colored red and nitrogens are blue. Hydrogen
bonds are indicated with dashed lines.
The potential of 6 to inhibit MPO in mice was assessed
using an acute neutrophil degranulation model with phorbol
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No luminescence signal was detected from MPO KO plasma
after PMA injection indicating the formation of oxidized
luminol was dependent on MPO production of HOCl (Fig.
SI3, ESI†).33 In wild type mice, PMA injection led to a rapid
formation of luminol-dependent chemiluminescence that
was inhibited ∼50 and 70% by pre-treatment with 6 dosed at
30 and 100 mpk, respectively (Fig. 7). These doses provided
plasma concentrations of 180 and 460 μM at 20 minutes,
which is 36 and 92 fold higher than the potency in the hu-
man in vitro neutrophil PMA assay (Table 1), likely due in
part to plasma protein binding (87% bound in mouse). The
potency to inhibit mouse and human MPO in the APF assay
was similar (Table 1). While a full-dose response in mice was
not determined to correlate in vitro and in vivo potency, this
result confirmed that an MPO reversible inhibitor can block
the enzymatic production of HOCl in vivo.
Fig. 6 Inhibition of MPO mediated chlorination by 6 (blue) rescued
ApoA1 cholesterol efflux capacity (red).
Initial SAR focused on investigating the triazolopyrimidine
core to determine the critical interactions to maintain activity
(Fig. 8). Changes to the pendant amino group resulted in a
loss of potency as observed for the pyrimidine 8, pyrimidinol
9, and pyrimidine alkyl amine 10. Methylation of the triazole
at any position caused loss of MPO inhibition (11, 12). The
purine analogs (13–14) and indazole 15 had a loss of activity,
indicating the pKa of the triazolopyrimidine (pKa = 6.6) may
be important for the binding.
Interactions with the hydrophobic pocket were best
maintained with the benzyl ether lead, which had better ac-
tivity than the phenyl ether (16), or phenethyl ether (17,
Table 2). Small substituents on the pendant benzyl had simi-
lar potency in the APF assay, with small electron withdrawing
fluorine substitutions trending toward increased potency (18,
21, 25), and the methyl or methoxy substitutions showing a
modest reduction in potency (20, 23, 27, Table 2). Inhibitor 6
has been described in the literature as a reactive inhibitor of
Fig. 7 Inhibition of MPO-mediated formation of HOCl after dosing 6
at 30 and 100 mpk as measured by luminol chemiluminescence. *P <
0.05, vs. vehicle, t test.
myristate acetate (PMA) based on a similar method described
by Gross, S. et al.32 Initially the model was validated by com-
paring the production of HOCl-mediated chemiluminescence
in MPO KO versus wild type mice upon injection with PMA.
Fig. 8 SAR of the triazolopyrimidine core of lead 6.
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Table 2 Ether substitution and MPO inhibition
LDL
LPO
Low density lipoprotein
Lactoperoxidase
MGMT
MPO
PMA
Pre-inc
RT
Methyl guanine methyl transferase
Myeloperoxidase
Phorbol myristate acetate
Preincubation
Retest
SAR
TPO
w pre-inc
Structure activity relationship
Thyroid peroxidase
With preincubation
APF assay w pre-inc
Ex. #
n
R
IC50a μM
w/o pre-inc. Without preincubation
16
17
18
19
20
21
22
23
24
25
26
27
0
2
1
1
1
1
1
1
1
1
1
1
H
H
2-F
2-Cl
2-CH3
3-F
3-Cl
3-OMe
3-OCF2H
4-F
4-CF3
4-CH3
>13
0.63
0.078
0.18
0.47
0.040
0.20
0.49
0.20
0.047
0.14
0.51
Conflicts of interest
The authors declare no competing interests.
References
1 S. J. Klebanoff, Science, 1970, 169, 1095.
2 M. Ali, B. Pulli, G. Courties, B. Tricot, M. Sebas, Y. Iwamoto,
I. Hilgendorf, S. Schob, A. Dong, W. Zheng, A. Skoura, A.
Kalgukar, C. Cortes, R. Ruggeri, F. K. Swirski, M.
Nahrendorf, L. Buckbinder and J. W. Chen, JACC Basic
Transl. Sci., 2016, 1, 633.
3 Y. Huang, J. A. DiDonato, B. S. Levison, D. Schmitt, L. Li, Y.
Wu, J. Buffa, T. Kim, G. S. Gerstenecker, X. Gu, C. S.
Kadiyala, Z. Wang, M. K. Culley, J. E. Hazen, A. J. Didonato,
X. Fu, S. Z. Berisha, D. Peng, T. T. Nguyen, S. Liang, C. C.
Chuang, L. Cho, E. F. Plow, P. L. Fox, V. Gogonea, W. H.
Tang, J. S. Parks, E. A. Fisher, J. D. Smith and S. L. Hazen,
Nat. Med., 2014, 20, 193.
4 B. Ikitimur and B. Karadag, Future Cardiol., 2010, 6, 693.
5 B. Shao, S. Pennathur and J. W. Heinecke, J. Biol. Chem.,
2012, 287, 6375.
6 B. S. van der Veen, M. P. de Winther and P. Heeringa,
Antioxid. Redox Signaling, 2009, 11, 2899.
7 D. Kutter, P. Devaquet, G. Vanderstocken, J. M. Paulus, V.
Marchal and A. Gothot, Acta Haematol., 2000, 104, 10.
8 A. Daugherty, J. L. Dunn, D. L. Rateri and J. W. Heinecke,
J. Clin. Invest., 1994, 94, 437.
9 S. Sugiyama, Y. Okada, G. K. Sukhova, R. Virmani, J. W.
Heinecke and P. Libby, Am. J. Pathol., 2001, 158, 879.
10 C. Delporte, K. Z. Boudjeltia, C. Noyon, P. G. Furtmuller, V.
Nuyens, M. C. Slomianny, P. Madhoun, J. M. Desmet, P.
Raynal, D. Dufour, C. N. Koyani, F. Reye, A. Rousseau, M.
Vanhaeverbeek, J. Ducobu, J. C. Michalski, J. Neve, L.
Vanhamme, C. Obinger, E. Malle and P. Van Antwerpen,
J. Lipid Res., 2014, 55, 747.
a
Assay was run n = 1 or 2 for each compound.
methyl guanine methyl transferase (MGMT),34 and subse-
quent publications will address optimization of MPO potency
and efforts to remove the reactivity with MGMT by optimiz-
ing the benzyl ether.
Conclusions
A triazolopyrimidine reversible inhibitor of MPO was identi-
fied using a carefully constructed HTS testing protocol. While
many substrate and irreversible inhibitors of MPO have been
reported, compound 6 is the first non-reactive MPO inhibitor
reported. The reversible, non-reactive nature of inhibitor 6
was confirmed using multiple types of mechanism studies.
The crystallographic data is consistent with this conclusion
and provides some indication of the important protein-
inhibitor contacts that are necessary for potency. Compound
6 can inhibit the in vitro chlorination of ApoA1, which is
thought to be deleterious toward atherosclerotic plaque for-
mation, and the neutrophil production of HOCl in an acute
mouse model. Further optimization of MPO inhibitory po-
tency and the selectivity for MPO over MGMT would be re-
quired for progression to a lead compound for chronic
in vivo studies.
Abbreviations
11 L. J. Hazell, L. Arnold, D. Flowers, G. Waeg, E. Malle and R.
Stocker, J. Clin. Invest., 1996, 97, 1535.
12 S. L. Hazen and J. W. Heinecke, J. Clin. Invest., 1997, 99,
2075.
ApoA1
APF
AR
Apolipoprotein A1
Aminophenyl fluorescein
Amplex red
CYP
EPX
HDL
HTS
Cytochrome P450
13 J. A. DiDonato, Y. Huang, K. S. Aulak, O. Even-Or, G.
Gerstenecker, V. Gogonea, Y. Wu, P. L. Fox, W. H. Tang,
E. F. Plow, J. D. Smith, E. A. Fisher and S. L. Hazen,
Circulation, 2013, 128, 1644.
Eosinophil peroxidase
High density lipoprotein
High throughput screening
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2017

View Article Online
MedChemComm
Research Article
14 S. Pennathur, C. Bergt, B. Shao, J. Byun, S. Y. Kassim, P.
Singh, P. S. Green, T. O. McDonald, J. Brunzell, A. Chait,
J. F. Oram, K. O'Brien, R. L. Geary and J. W. Heinecke,
J. Biol. Chem., 2004, 279, 42977.
15 B. Shao, C. Tang, A. Sinha, P. S. Mayer, G. D. Davenport, N.
Brot, M. N. Oda, X. Q. Zhao and J. W. Heinecke, Circ. Res.,
2014, 114, 1733.
16 L. Zheng, B. Nukuna, M. L. Brennan, M. Sun, M.
Goormastic, M. Settle, D. Schmitt, X. Fu, L. Thomson, P. L.
Fox, H. Ischiropoulos, J. D. Smith, M. Kinter and S. L.
Hazen, J. Clin. Invest., 2004, 114, 529.
17 E. A. Podrez, M. Febbraio, N. Sheibani, D. Schmitt, R. L.
Silverstein, D. P. Hajjar, P. A. Cohen, W. A. Frazier, H. F.
Hoff and S. L. Hazen, J. Clin. Invest., 2000, 105, 1095.
18 L. W. Castellani, J. J. Chang, X. Wang, A. J. Lusis and W. F.
Reynolds, J. Lipid Res., 2006, 47, 1366.
Y. Zhang, D. Bowles, D. W. Widlicka, H. Eng, T. Ryder, R.
Sharma, A. Wolford, C. Okerberg, K. Walters, T. S. Maurer,
Y. Zhang, P. D. Bonin, S. N. Spath, G. Xing, D. Hepworth, K.
Ahn and A. S. Kalgutkar, J. Med. Chem., 2015, 58, 8513.
25 A. K. Tiden, T. Sjogren, M. Svensson, A. Bernlind, R.
Senthilmohan, F. Auchere, H. Norman, P. O. Markgren, S.
Gustavsson, S. Schmidt, S. Lundquist, L. V. Forbes, N. J.
Magon, L. N. Paton, G. N. Jameson, H. Eriksson and A. J.
Kettle, J. Biol. Chem., 2011, 286, 37578.
26 J. Ward, S. N. Spath, B. Pabst, P. A. Carpino, R. B. Ruggeri,
G. Xing, A. E. Speers, B. F. Cravatt and K. Ahn, Biochemistry,
2013, 52, 9187.
27 L. V. Forbes, T. Sjogren, F. Auchere, D. W. Jenkins, B.
Thong, D. Laughton, P. Hemsley, G. Pairaudeau, R. Turner,
H. Eriksson, J. F. Unitt and A. J. Kettle, J. Biol. Chem.,
2013, 288, 36636.
19 M. Allegra, P. G. Furtmuller, G. Regelsberger, M. L. Turco-
Liveri, L. Tesoriere, M. Perretti, M. A. Livrea and C. Obinger,
Biochem. Biophys. Res. Commun., 2001, 282, 380.
28 P. G. Furtmuller, M. Zederbauer, W. Jantschko, J. Helm, M.
Bogner, C. Jakopitsch and C. Obinger, Arch. Biochem.
Biophys., 2006, 445, 199.
20 R. F. Queiroz, S. M. Vaz and O. Augusto, Biochem. J.,
2011, 439, 423.
29 C. Bergt, X. Fu, N. P. Huq, J. Kao and J. W. Heinecke, J. Biol.
Chem., 2004, 279, 7856.
21 M. D. Rees, S. E. Bottle, K. E. Fairfull-Smith, E. Malle, J. M.
Whitelock and M. J. Davies, Biochem. J., 2009, 421, 79.
22 J. Soubhye, I. Aldib, B. Elfving, M. Gelbcke, P. G. Furtmuller,
M. Podrecca, R. Conotte, J. M. Colet, A. Rousseau, F. Reye, A.
Sarakbi, M. Vanhaeverbeek, J. M. Kauffmann, C. Obinger, J.
Neve, M. Prevost, K. Zouaoui Boudjeltia, F. Dufrasne and P.
Van Antwerpen, J. Med. Chem., 2013, 56, 3943.
30 M. S. Borja, L. Zhao, B. Hammerson, C. Tang, R. Yang, N.
Carson, G. Fernando, X. Liu, M. S. Budamagunta, J. Genest,
G. C. Shearer, F. Duclos and M. N. Oda, PLoS One, 2013, 8,
e71541.
31 L. Zheng, M. Settle, G. Brubaker, D. Schmitt, S. L. Hazen,
J. D. Smith and M. Kinter, J. Biol. Chem., 2005, 280, 38.
32 S. Gross, S. T. Gammon, B. L. Moss, D. Rauch, J. Harding,
J. W. Heinecke, L. Ratner and D. Piwnica-Worms, Nat. Med.,
2009, 15, 455.
23 I. Sliskovic, I. Abdulhamid, M. Sharma and H. M. Abu-Soud,
Free Radical Biol. Med., 2009, 47, 1005.
24 R. B. Ruggeri, L. Buckbinder, S. W. Bagley, P. A. Carpino,
E. L. Conn, M. S. Dowling, D. P. Fernando, W. Jiao, D. W.
Kung, S. T. Orr, Y. Qi, B. N. Rocke, A. Smith, J. S. Warmus,
33 E. P. Brestel, Biochem. Biophys. Res. Commun., 1985, 126, 482.
34 M. Y. Chae, K. Swenn, S. Kanugula, M. E. Dolan, A. E. Pegg
and R. C. Moschel, J. Med. Chem., 1995, 38, 359.
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