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M. M. Armstrong et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
stimulate proliferation in cancer cell lines through a specific
2. Materials and methods
2.1. Materials
G
a
i-coupled receptor.8–10 5-oxo-ETE also plays an important role
in the asthmatic inflammatory response,11 gastrointestinal
diseases12 and activation of peroxisome proliferator-activated
receptor
c (PPARc
) transcriptional activity.13 Another oxo-lipid
All commercial fatty acids (Sigma–Aldrich Chemical Company)
were re-purified using a Higgins HAIsil Semi-Preparative (5 mM,
250 Â 10 mm) C-18 column. Solution A was 99.9% MeOH and 0.1%
acetic acid; solution B was 99.9% H2O and 0.1% acetic acid. An
isocratic elution of 85% A: 15% B was used to purify all fatty acids,
which were stored at À80 °C for a maximum of 6 months. HPLC
grade solvents were used for both semi-preparative HPLC purifica-
tion and analytical HPLC analysis of LOX products. Large scale prod-
uct purification was achieved by using a C18HAIsil 250 Â 10 mm
which plays a role in the cell is 12-oxo-5,8,10,14-(Z,Z,E,Z)-eicosa-
tetraenoic acid (12-oxo-ETE). Powell et al.14,15 observed that
12-oxo-ETE had effects on cytosolic calcium levels at concentra-
tions of 10 l
M, but Naccache et al.16 reported calcium effects as
low as 10 nM. The third oxo-lipid generated from AA is 15-oxo-
5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE). For this
oxo-lipid, an esterified, 15-oxo-ETE phospholipid has been
detected in patients with cystic fibrosis (CF)13 and shown to acti-
vate transcriptional activity in PPARc 12
Activation of PPARc
.
semi-preparative column, whereas
a
C18HAIsil 250 Â 4.6 mm
expression in CF mice ameliorates disease severity, suggesting that
15-oxo-ETE might potentially act to lower inflammation in CF.13
Finally, there is an oxo-lipid generated from LA, 13-oxo-9,11-
(Z,E)-octadecadienoic acid (13-oxo-ODE), which was found to be
analytical column was used for product separation in tandem with
MS/MS analysis. Both columns were purchased from Higgins
Analytical (Mountain View, CA). All other chemicals were reagent
grade or better and were used without further purification.
an endogenous ligand to PPAR
13-Oxo-ODE mediated the activation of PPAR
c
in intestinal epithelial cells (IEC).
to reduce mucosal
2.2. Protein expression
c
damage and down-regulate inflammation in several mouse models
of intestinal colitis,17 implicating it as a possible therapeutic target
for the treatment of inflammatory bowel disease.17
All the LOX isozymes used in this publication were expressed
and purified as previously published (h5-LOX,28 h12-LOX,25
h15-LOX-125 and s15-LOX-1,26 h15-LOX-227 and r15-LOX29).
Chemically, the oxo-lipids, like 5-oxo-ETE, 15-oxo-ETE, 12-oxo-
ETE and 13-oxo-ODE, are unique in that they contain an a, b unsat-
2.3. General procedure for the synthesis of oxo-lipids
urated carbonyl that can readily react with nucleophiles, such as
proteins and glutathione (GSH), via Michael addition reaction,
resulting in covalent modifications. The reversible conjugation of
13-oxo-ODE by GSH18,19 has been shown to occur by both enzy-
matic and non-enzymatic pathways, with the conjugate being
exported from the cell via an energy-dependent process.20 Similar
synthetic molecules that form covalent linkages to their targets
have been considered as therapeutics, but have traditionally been
disfavored due to concerns for their off-target reactivity, either
through direct tissue damage or through haptenization of proteins,
which could elicit an immune response.21 However, as selectivity
and drug resistance remain a serious issue for reversible inhibitors,
a resurgence of interest in this class of therapeutics has emerged.
For example, Taunton and co-workers,22 developed a fluorometh-
ylketone-substituted ligand (fmk), which irreversibly inactivates
p90 ribosomal protein S6 kinase (RSK1/2) in human cells at nano-
molar concentrations by modifying an active site cysteine, without
inhibiting over 130 other kinases.22 A similar covalent inhibitor,
JNK-IN-8, was discovered as a specific, irreversible intracellular
inhibitor against the mitogen-activated kinase JNK.23 JNK-IN-8
inhibits phosphorylation of c-Jun, a direct substrate of JNK, by
covalent modification of a conserved cysteine residue in the
ATP-binding motif.23 Both of these studies argue against the widely
held view that electrophilic inhibitors are inherently nonselec-
tive22 and therefore it is possible that the oxo-lipids target non-
conserved, non-catalytic cysteines in many proteins in the cell,
such as lipoxygenase.
Due to the fact that oxo-lipids have interesting biological
properties, that they are potential covalent modifiers and that they
have similar structures to LOX substrates, we hypothesized that
oxo-lipids could potentially inhibit LOX isozymes at concentrations
that are biologically relevant. This hypothesis is reinforced by the
fact that certain LOX isozymes have non-catalytic cysteines in their
active sites,24 which could serve as nucleophiles to oxo-lipids. In
the current work, we present inhibitory data of a variety of oxo-
lipids (5-oxo-ETE, 15-oxo-ETE, 12-oxo-ETE, and 13-oxo-ODE)
against LOX isozymes (h5-LOX, h15-LOX-1, human platelet
12-lipoxygenase (h12-LOX), human epithelial 15-lipoxygenase-2
(h15-LOX-2), soybean 15-lipoxygenase-1 (s15-LOX-1), and rabbit
reticulocyte 15-LOX (r15-LOX)) and demonstrate that certain
oxo-lipids are LOX inhibitors.
The synthesis of all oxo-lipids consists of two steps, the first
step is enzymatic while the second step is synthetic. In the synthe-
sis of 13-oxo-ODE, s15-LOX-1 is reacted with linoleic acid (LA) in
100 mL of 100 mM Borate (pH 9.2) generating 13-HpODE.
15-Oxo-ETE is generated by reaction between h15-LOX-2 and
40
generating 15-HpETE. 12-Oxo-ETE is generated by reaction
between h12-LOX and 40 M AA in 100 mL 25 mM HEPES (pH
8.0), generating 12-HpETE. 5-Oxo-ETE is generated by reaction
between 5-LOX and 40 M AA in 100 mL 25 mM HEPES (pH 7.3),
lM arachidonic acid (AA) in 100 mL of 25 mM HEPES (pH 7.5),
l
l
0.3 mM CaCl2, 0.1 mM EDTA, 0.2 mM ATP, generating 5-HpETE.
The reactions are quenched with 1–2% acetic acid and extracted
using dichloromethane (DCM). The formation of 13-HpODE, 15-
HpETE, 12-HpETE and 5-HpETE are monitored at 234 nm with a
Perkin Elmer Lambda 40 UV/vis spectrophotometer. The second
step is an overnight synthetic reaction in which the hydroperoxy
products are reacted with acetic anhydride and pyridine at 4 °C
in a 1:1 ratio to generate 13-oxo-ODE, 15-oxo-ETE, 12-oxo-ETE
and 5-oxo-ETE, respectively. The reactions are quenched with cold
Milli-Q water for 2 h. The oxo-lipids are purified via high perfor-
mance liquid chromatography (HPLC) using a Higgins HAIsiL
Semi-Preparative C-18 column. Solution A was 99.9% ACN and
0.1% acetic acid; solution B was 99.9% H2O and 0.1% acetic acid.
An isocratic elution of 55% A: 45% B was used to purify each oxo-
compound. The retention times for each oxo-lipid at 280 nm are
as follows: 13-oxo-ODE (30 min), 15-oxo-ETE (33 min), 12-oxo-
ETE (90 min) and 5-oxo-ETE (70 min). Analytical analysis was per-
formed by liquid chromatography–mass spectrometry (LC–MS/
MS). Solution A was 99.9% H2O and 0.1% formic acid; solution B
was 99.9% ACN and 0.1% formic acid. Oxo-lipids were injected onto
a Phenomenex Synergi (4
attached to Thermo LTQ LC–MS/MS. The elution protocol
consisted of 200 L/min, with an isocratic mobile phase of 45%
lM, 150 mm  4.6 mm) C-18 column
a
l
solution A and 55% solution B. Negative ion MS/MS was utilized
(collision energy of 35 eV) to determine the fragmentation patterns
of all the oxo-lipids. 13-Oxo-ODE, parent m/z = 293, fragments m/
z = 113, 249, 293; 15-oxo-ETE, parent m/z = 317, fragments
m/z = 113, 273, 299; 12-oxo-ETE, parent m/z = 317, fragments m/
Armstrong, Michelle M.
