Selective inhibitors and tailored activity probes for lipoprotein- associated phospholipase A2

Article abstract of DOI:10.1016/j.bmcl.2012.11.061

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂ or PLA₂G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA₂ has also been implicate

Full text of DOI:10.1016/j.bmcl.2012.11.061

Bioorganic & Medicinal Chemistry Letters 23 (2013) 839–843
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Selective inhibitors and tailored activity probes for lipoprotein-associated
phospholipase A2
Joseph M. G. Nagano a,b, Ku-Lung Hsu a,b, Landon R. Whitby a,b, Micah J. Niphakis a,b, Anna E. Speers a,b
,
Steven J. Brown a,c, Timothy Spicer d, Virneliz Fernandez-Vega d, Jill Ferguson a,c, Peter Hodder d,e
,
Prabhavathi Srinivasan f, Tara D. Gonzalez f, Hugh Rosen a,c, Brian J. Bahnson f, Benjamin F. Cravatt a,b,
⇑
a The Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd. La Jolla, CA 92037, United States
b The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd. La Jolla, CA 92037, United States
c The Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, 10550 N. Torrey Pines Rd. La Jolla, CA 92037, United States
d Lead Identification Division, Molecular Screening Center, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States
e Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, United States
f Department of Chemistry & Biochemistry, University of Delaware, Newark, DE 19716, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipoprotein-associated phospholipase A2 (Lp-PLA2 or PLA2G7) binds to low-density lipoprotein (LDL) par-
ticles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA2 has also been impli-
cated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA2 have been
described, including darapladib, which is currently in phase 3 clinical development for the treatment
of atherosclerosis. The selectivity that darapladib and other Lp-PLA2 inhibitors display across the larger
serine hydrolase family has not, however, been reported. Here, we describe the use of both general
and tailored activity-based probes for profiling Lp-PLA2 and inhibitors of this enzyme in native biological
systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors
inactivate Lp-PLA2 in mouse tissues and human cell lines with high selectivity. Our findings thus identify
both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA2 function in biolog-
ical systems.
Received 25 October 2012
Accepted 14 November 2012
Available online 2 December 2012
Keywords:
Phospholipase
Inhibitor
Screening
Proteomics
Ó 2012 Elsevier Ltd. All rights reserved.
Lipoprotein-associated phospholipase A2 (Lp-PLA2 or PLA2G7) is
a secreted serine hydrolase that is found on low-density lipopro-
tein (LDL) particles and, in response to oxidation of LDL particles
(ox-LDL), is able to hydrolyze oxidized phosphatidylcholine into
lysophosphatidylcholine (LPC) and oxidized fatty acids.1 The re-
leased LPC and oxidized fatty acids are thought to promote inflam-
mation,2,3 thus designating Lp-PLA2 as a potential risk factor in the
development of atherosclerosis. This premise is also supported by a
positive correlation between Lp-PLA2 levels and the incidence of
coronary heart disease.4 Genetic studies have further shown that
a small percentage of the Asian population carries an inactivating
mutation of a conserved valine (V279F) in Lp-PLA2,5 and both po-
sitive and negative correlations between this mutation and cardio-
vascular disease have been reported.6–8 Based on the connection
between Lp-PLA2 and cardiovascular disease, as well as the genetic
evidence that inactivation of Lp-PLA2 may protect against coronary
heart disease, this enzyme is considered a potential biomarker and
therapeutic target for the treatment of atherosclerosis. Several
studies have also connected elevated levels of Lp-PLA2 expression
to the progression of prostate cancer.9–11 Interestingly, siRNA-in-
duced Lp-PLA2 inactivation in the VCaP human prostate cancer cell
line has been shown to induce apoptosis and sensitize the cells to
oxidative stress.9 Thus, Lp-PLA2 may also serve as a biomarker and
therapeutic target for prostate cancer.
Due to the interest in Lp-PLA2 as a therapeutic target, several
inhibitors of this enzyme have been reported. These include b-lac-
tams,12 amides of xanthurenic acid,13 and the compound darapla-
dib,14 which is in Phase III clinical trials for the treatment of
atherosclerosis. Darapladib has been shown to significantly
decrease plasma Lp-PLA2 activity and atherosclerotic plaque
formation in ApoE-deficient mice,15 in diabetic and hypercholes-
terolemic swine,16 and in human patients with coronary heart
disease.17,18
Despite the generation of several classes of Lp-PLA2 inhibitors,
including a compound in late-stage clinical development, we are
not aware of any detailed reports on the selectivity of these agents.
This question is especially pertinent for inhibitors of serine hydro-
lases, like Lp-PLA2, given the huge number of enzymes from this
class in humans.19 Lp-PLA2 also lacks precise assays that report
on its activity in complex biological systems. Measurements of
⇑
Corresponding author.
E-mail address: cravatt@scripps.edu (B.F. Cravatt).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.061

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J. M. G. Nagano et al. / Bioorg. Med. Chem. Lett. 23 (2013) 839–843
Lp-PLA2 activity are typically performed using either the artificial
fluorescent substrate 1-decanoyl-2-(4-nitrophenylglutaryl)phos-
phatidylcholine (DNPG)20 or radioactive substrate [3H]-PAF,21 nei-
ther of which can readily distinguish Lp-PLA2 from several other
PAF hydrolases that exist in mammalian proteomes.22,23
With these considerations in mind, we set out to develop and
apply activity-based protein profiling (ABPP) methods24,25 for char-
acterizing Lp-PLA2 and its inhibitors, including: (1) performing a
high-throughput screen for novel classes of inhibitors, (2) identify-
ing tailored activity-based probes for detecting Lp-PLA2 activity in
cell and tissue proteomes, and (3) evaluating established and new-
ly discovered Lp-PLA2 inhibitors for target engagement and selec-
tivity in human cells. The high-throughput screen was performed
using purified human Lp-PLA2 protein26,27 and a recently described
fluorescence polarization-ABPP (FluoPol-ABPP) platform28 in col-
laboration with the Molecular Libraries Screening Center Network
at The Scripps Research Institute. FluoPol-ABPP assays showed
time-dependent increases in fluorescence polarization of Lp-PLA2
labeled with the serine hydrolase-directed activity-based probe
fluorophosphonate-rhodamine (FP-Rh), while catalytically inactive
enzyme, in which the serine nucleophile S273 was mutated to ala-
nine (Lp-PLA2SA), and no-enzyme control samples showed no in-
crease in fluorescence polarization (Fig. 1). From a library of
326,141 compounds screened at 3.39 lM (PubChem AID:
463082), we identified 4934 compounds (1.5%) that reduced the
fluorescence polarization (>24%) of Lp-PLA2 labeling by the FP-Rh
probe. A confirmation screen (AID 463230) was performed on
2500 compounds at the same concentration in triplicate and
1675 (71.6%) of the compounds were confirmed as active. From
these results, we chose 153 compounds based on their high
(>60%) inhibition of Lp-PLA2, selectivity for Lp-PLA2 compared to
other screened enzymes and bioassays listed in PubChem, lack of
undesired functional groups, and medicinal chemistry potential.
We rescreened these 153 compounds versus recombinant mouse
Lp-PLA2 by gel-based competitive ABPP.29 Briefly, lysates from
HEK-293T cells transfected with a mouse Lp-PLA2 cDNA were incu-
Figure 2. Structures of lead Lp-PLA2 inhibitors WWL153, P3, P9, and of the
optimized inhibitor JMN4.
bated with each test compound (5
lM) for 30 min at 37 °C and
then reacted with FP-Rh (1 M, 30 min, 25 °C). Proteomic reactions
l
were run on SDS–PAGE and visualized by in-gel fluorescence scan-
ning. Twelve compounds were confirmed to inhibit Lp-PLA2 by
>75% at 5 lM, and eight of these compounds were carbamates,
Figure 3. In vitro potency and selectivity profiles of Lp-PLA2 inhibitors WWL153,
P9, and JMN4. Evaluation of Lp-PLA2 inhibitors by competitive ABPP with the FP-Rh
probe and (A) recombinant mouse (mLp-PLA2) and (B) human (hLp-PLA2) Lp-PLA2
enzymes at indicated concentrations. (C) Evaluation of the selectivity of Lp-PLA2
inhibitors at indicated concentrations by competitive ABPP in the mouse brain
membrane proteome. Fluorescent gels are shown in gray scale.
Figure 1. Lp-PLA2 FluoPol-ABPP assay. A 10 nM sample of purified human Lp-PLA2,
catalytically inactive S273A mutant (Lp-PLA2 SA), or no enzyme was incubated with
75 nM FP-Rh at room temperature. Fluorescence polarization was measured as a
function of time. Z0 = 0.82 at t = 15 min.

J. M. G. Nagano et al. / Bioorg. Med. Chem. Lett. 23 (2013) 839–843
841
Figure 4. In vitro potency and selectivity profiles of darapladib and JMN4 in mouse brain membrane and PC3 prostate cancer cells. (A) Inhibition and labeling of recombinant
mouse Lp-PLA2 with HT-01. Inhibition was measured by competitive ABPP with the FP-Rh probe and direct detection of labeling by HT-01, which contains a BODIPY
fluorophore. (B) Evaluation of darapladib and JMN4 inhibitory activity with mouse brain membranes probed with (B) HT-01 and (C) FP-Rh. (D) Evaluation of darapladib and
JMN4 inhibitory activity in PC3 cell proteomes probed with (D) HT-01 and (E) FP-Rh.
two of which contained a biphenyl moiety (P3 and P9, Fig. 2). A
previous study30 from our lab identified a structurally distinct car-
bamate WWL153 (Fig. 2) as a lead inhibitor for Lp-PLA2 that dis-
played good potency (IC50 = 290 nM), but sub-optimal selectivity
(see below). We therefore combined the 2-methyl-4-piperazino-
quinoline portion of WWL153 with the biphenyl moiety of P3
and P9 to create the compound JMN4 (Fig. 2).
Additional competitive gel-based ABPP assays revealed that
JMN4 exhibited superior potency for inhibiting recombinant
mouse (IC50 = 90 nM) and human (IC50 = 5.9 nM) Lp-PLA2 com-
pared to either WWL153 (IC50 = 290 nM for mouse Lp-PLA2;
IC50 = 250 nM for human Lp-PLA2) or P9 (IC50 = 470 nM for mouse
Lp-PLA2; IC50 = 100 nM for human Lp-PLA2) (Fig. 3A, B). Competi-
tive ABPP experiments in the mouse brain proteome also revealed
that JMN4 cross-reacted with fewer serine hydrolases than
WWL153 and P9, both which inhibited FAAH at high concentra-
tions (Fig. 3C). WWL153 also inhibited MAGL and ABHD6
(Fig. 3C). These data, taken together, designated JMN4 as a potent
and selective inhibitor of mouse and human Lp-PLA2.
It is important to note that, although Lp-PLA2 is expressed in the
mouse brain,30 it is not visualized by gel-based ABPP using FP-Rh,
presumably due to the presence of more abundant, co-migrating
serine hydrolases. We reasoned that a more selective activity-
based probe could facilitate the detection of endogenous Lp-PLA2
in complex proteomes such as mouse brain. Recently, we reported
a 1,2,3-triazole urea activity-based probe termed HT-01 and its use
to visualize the low-abundance serine hydrolase DAGLb in cell and
tissue proteomes.31 Examination of the HT-01 labeling profile of
mouse brain membrane proteome revealed the presence of an
additional tetrahydrolipstatin (THL)-sensitive serine hydrolase
activity that exhibited a molecular weight (ꢀ55 kDa doublet) con-
sistent with glycosylated Lp-PLA2. We had previously shown that
mouse Lp-PLA2 was inhibited by THL32 and various 1,2,3-triazole
ureas31 and found here that this enzyme was also inhibited by

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J. M. G. Nagano et al. / Bioorg. Med. Chem. Lett. 23 (2013) 839–843
A
B
Fig. 5. ABPP-SILAC analysis of inhibitor-treated PC3 cells. Heavy-labeled cells were treated in situ for 4 h with 100 nM darapladib (A) or JMN4 (B); light-labeled cells were
treated with DMSO. Data are reported as mean values SEM of all peptides quantified for each serine hydrolase. SILAC ratios were normalized to a control experiment where
both heavy and light cells were treated with DMSO.
HT-01 with an IC50 of 39 nM (Fig. 4A). Based on these results, we
tested both darapladib and JMN4 in gel-based competitive ABPP
experiments with HT-01 and found that these compounds blocked
HT-01 labeling of the 55 kDa mouse membrane enzyme, support-
ing its designation as endogenous Lp-PLA2 (Fig. 4B). JMN4 showed
could not be detected by the general serine hydrolase probe FP-
Rh in the PC3 cell proteome (Fig. 4E), presumably due to overlap-
ping signals from more abundant, co-migrating serine hydrolases.
These FP-Rh profiles did, however, provide evidence that darapla-
dib and JMN4 exhibited good selectivity for Lp-PLA2 in that other
detected serine hydrolase activities in the PC3 cell proteome were
not inhibited by these compounds (Fig. 4E).
lower potency against mouse brain Lp-PLA2 (IC50 ꢀ1
lM) com-
pared to recombinant mouse Lp-PLA2, which we speculate may
be due to the greater amount of total proteome needed in the assay
to detect endogenous Lp-PLA2 in mouse brain (1.0 mg brain pro-
tein/mL vs 0.25 mg transfected cell protein/mL). HT-01 also identi-
fied a ꢀ70 kDa protein that was inhibited by JMN4, but not
darapladib (Fig. 4B). This enzyme is likely the blood-derived serine
hydrolase carboxylesterase ES1, which has been a common off-tar-
get for many carbamate inhibitors.30 Finally, we performed a com-
petitive gel-based ABPP analysis with FP-Rh, which showed that
neither darapladib nor JMN4 cross-reacted with other serine
hydrolases detected in mouse brain at concentrations up to
We next asked whether darapladib and JMN4 could inhibit
human Lp-PLA2 in situ by treating PC3 cells with these com-
pounds and then analyzing the serine hydrolase activity profiles
by the liquid chromatography–mass spectrometry (LC–MS) plat-
form ABPP-SILAC.33–36 PC3 cells were cultured in isotopically
heavy or light media and treated with 100 nM darapladib or
JMN4 (heavy cells) or DMSO (light cells) for 4 h. Cells were har-
vested, lysed, and labeled with the activity-based probe FP-bio-
tin.29 Light and heavy fractions were mixed in a 1:1 ratio,
enriched with avidin-conjugated beads, digested on-bead with
trypsin and analyzed by liquid chromatography–tandem MS
(LC–MS/MS) using an LTQ-Velos Orbitrap instrument. SILAC ra-
tios for each peptide were generated using the CIMAGE soft-
ware37 to quantify light and heavy signals from the parent ion
(MS1) peaks. Protein identities were determined from product
ion peptide profiles (MS2) using the ProLuCID search algo-
rithm.38 We also performed a control experiment where both
heavy and light cells were treated with DMSO to control for
the effects of isotopic growth media on protein expression and
activity. This analysis showed that both darapladib (Fig. 5A)
and JMN4 (Fig. 5B) completely inhibited Lp-PLA2 in human pros-
tate cancer cells, while not affecting any of the other ꢀ40 serine
hydrolases detected in these cells.
10
lM (Fig. 4C).
The aforementioned studies indicated that JMN4 and darapla-
dib were selective inhibitors of mouse Lp-PLA2 and that HT-01
served as a tailored activity-based probe for detection of this en-
zyme in native proteomes. We next investigated the performance
of these inhibitors/probes in human proteomes. As previously
mentioned, human prostate cancer cells have been found to ex-
press Lp-PLA2.9–11 Consistent with these past studies, we detected
an ꢀ50 kDa protein in the PC3 human prostate cancer cell prote-
ome that exhibited darapladib- and JMN4-sensitive labeling by
the HT-01 probe (Fig. 4D). These features indicated that the
50 kDa protein was human Lp-PLA2. Interestingly, as we previously
observed in the mouse brain proteome (Fig. 4C), human Lp-PLA2

J. M. G. Nagano et al. / Bioorg. Med. Chem. Lett. 23 (2013) 839–843
843
10. Vainio, P.; Lehtinen, L.; Mirtti, T.; Hilvo, M.; Seppänen-Laakso, T.; Virtanen, J.;
In conclusion, we have shown that both darapladib and the
newly developed compound JMN4 are potent and selective inhibi-
tors of Lp-PLA2 in both mouse tissues and human cell lines, indicat-
ing that these compounds can be used as a structurally and
mechanistically39 distinct pair of probes to study Lp-PLA2 function
in biological systems. We also believe that the tailored probe HT-
01 should prove useful for profiling Lp-PLA2 activity in native pro-
teomes, which is otherwise difficult to detect by gel-based ABPP
assays using more general serine hydrolase-directed probes. We
speculate, for instance, that HT-01 could form the basis for an
ex vivo target engagement assay to confirm Lp-PLA2 inhibition in
human clinical studies, as has recently been performed for protea-
some inhibitors using FP-biotin.40
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Acknowledgments
We thank Mr. Pierre Baillargeon and Mrs. Lina DeLuca (Lead
Identification Division, TSRI Florida) for their assistance with com-
pound management. This work was supported by the National
Institutes of Health (DA033760, MH084512, HL084366), the Pros-
tate Cancer Foundation, the Achievement Rewards For College Sci-
entists (ARCS) Foundation (fellowship for J.M.G.N.) and the Skaggs
Institute for Chemical Biology.
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Supplementary data associated with this article can be found, in
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061.
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