Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation

Article abstract of DOI:10.1073/pnas.1001529107

Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC₅₀ ～ 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.

Full text of DOI:10.1073/pnas.1001529107

Boronic acid-based inhibitor of autotaxin reveals
rapid turnover of LPA in the circulation
Harald M. H. G. Albersa,b, Anping Dongc, Laurens A. van Meeterena, David A. Egand, Manjula Sunkarac,
Erica W. van Tilburga, Karianne Schuurmana, Olaf van Tellingene, Andrew J. Morrisc, Susan S. Smythc,
Wouter H. Moolenaara,f, and Huib Ovaaa,b,1
f
aDivision of Cell Biology, bNetherlands Proteomics Centre, Centre of Biomedical Genetics, dDivision of Molecular Carcinogenesis, and eDepartment of
c
Clinical Chemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; and Division of Cardiovascular Medicine, University of
Kentucky, Lexington, KY 40536
Communicated by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, February 16, 2010 (received for review October 21, 2009)
Autotaxin (ATX) is
a
secreted nucleotide pyrophosphatase/
have been explored as ATX inhibitors (23–26). However, such
lipid inhibitors have the inherent danger of inadvertently activat-
ing downstream LPA/S1P receptors, thereby inducing the oppo-
site of the intended effect. Furthermore, lipids offer relatively few
avenues for chemical diversification and usually have poor phar-
macokinetic properties. Nonlipid inhibitors of ATX have recently
been identified, but their potencies are low (27).
In this study we screened small-molecule libraries to search for
unique ATX inhibitors. We identified thiazolidinedione com-
pounds that selectively inhibit ATX activity and are readily amen-
able to further chemical diversification. We have optimized these
molecules by adopting an active-site-targeted strategy that has
proved successful for the development of the boronic acid-based
proteasome inhibitor bortezomib (28), which is in clinical use
(29). We show that a boronic acid-based inhibitor potently inhi-
bits ATX both in vitro and in vivo. When administered to mice,
our compound (HA130) induces a remarkably rapid fall in plasma
LPA levels, indicating that the turnover of circulating LPA is
much more dynamic than previously appreciated. We conclude
that boronic acid-based inhibitors hold promise as candidate
drugs to target the ATX-LPA axis in vivo.
phosphodiesterase that functions as a lysophospholipase D to
produce the lipid mediator lysophosphatidic acid (LPA), a mitogen,
chemoattractant, and survival factor for many cell types. The ATX-
LPA signaling axis has been implicated in angiogenesis, chronic
inflammation, fibrotic diseases and tumor progression, making this
system an attractive target for therapy. However, potent and selec-
tive nonlipid inhibitors of ATX are currently not available. By
screening a chemical library, we have identified thiazolidinediones
that selectively inhibit ATX-mediated LPA production both in vitro
and in vivo. Inhibitor potency was approximately 100-fold increased
(IC50 ∼ 30 nM) after the incorporation of a boronic acid moiety,
designed to target the active-site threonine (T210) in ATX. Intrave-
nous injection of this inhibitor into mice resulted in a surprisingly
rapid decrease in plasma LPA levels, indicating that turnover of
LPA in the circulation is much more dynamic than previously appre-
ciated. Thus, boronic acid-based small molecules hold promise as
candidate drugs to target ATX.
high-throughput screening ∣ lysophosphatidic acid ∣ lysophospholipase D ∣
small-molecule inhibitor ∣ phosphodiesterase
utotaxin (ATX or NPP2) is a secreted nucleotide pyropho-
Results
A
sphatase/phosphodiesterase (NPP) originally isolated as an
Discovery of Small-Molecule Inhibitors of ATX. The hydrolytic activity
of ATX originates from a single catalytic site at threonine
210 (T210) in the central phosphodiester domain (5) (Fig. 1).
To discover unique ATX inhibitors, we screened a collection
of ∼40; 000 drug-like small molecules using the hydrolysis of
bis(4-nitrophenyl) phosphate (bis-pNPP) by ATX as a readout.
Among the most potent hits, we selected a thiazolidinedione ser-
ies for optimization since the thiazolidinedione core is readily
amenable to chemical diversification (Fig. 2A). Inhibitor (A)
showed an IC50 value of 56 nM using 1 mM bis-pNPP as substrate.
For validation of A, we measured the inhibition of the ATX-
catalyzed release of choline from LPC. We established that
recombinant ATX has a Km value for LPC of 94 μM (Fig. S1).
Compound A inhibited ATX with an IC50 value of 2.5 μM using
40 μM LPC as a substrate (Fig. 3A). However, it should be noted
that A has a 35% residual ATX activity (Fig. 3B). Inhibition of
ATX-mediated LPA production was confirmed by measuring
autocrine motility factor from melanoma cells (1). ATX, a
∼120 kDa glycoprotein, is unique amongst the NPPs in that it
functions as a lysophospholipase D (lysoPLD) that converts ex-
tracellular lysophosphatidylcholine (LPC) into the lipid mediator
lysophosphatidic acid (LPA; mono-acyl-sn-glycero-3-phosphate)
(2–5). LPA acts on specific G protein-coupled receptors and
thereby stimulates the migration, proliferation, and survival of
many cell types (6 and 7) (Fig. 1). ATX is produced by various
tissues and is the major LPA-producing enzyme in the circulation.
Newly produced LPA is subject to degradation by membrane-
bound lipid phosphate phosphatases (LPPs) (8 and 9). However,
little is known about the dynamic regulation of steady-state LPA
levels in vivo.
ATX is essential for vascular development (10 and 11) and is
found overexpressed in various human cancers (12). Forced over-
expression of ATX or individual LPA receptors promotes tumor
progression in mouse models (13–16), while LPA receptor
deficiency protects from colon carcinogenesis (17). In addition
to its role in cancer, ATX-LPA signaling has been implicated
in lymphocyte homing and (chronic) inflammation (18), fibrotic
diseases (19 and 20), and thrombosis (21). Therefore, the ATX-
LPA axis qualifies as an attractive target for therapies.
Potent and selective ATX inhibitors are now needed as a start-
ing point for the development of targeted anti-ATX/LPA therapy.
Direct targeting of LPA receptors seems to be a less attractive
strategy, since LPA acts on multiple receptors that show overlap-
ping activities (2 and 6). Since the initial finding that ATX is sub-
ject to product inhibition by LPA and sphingosine 1-phosphate
(S1P) (22), various synthetic phospho- and phosphonate lipids
Author contributions: H.M.H.G.A., L.A.v.M., D.A.E., O.v.T., A.J.M., S.S.S., W.H.M., and H.O.
designed research; H.M.H.G.A., A.D., L.A.v.M., D.A.E., M.S., E.W.v.T., K.S., O.v.T., and H.O.
performed research; H.M.H.G.A., L.A.v.M., D.A.E., E.W.v.T., A.J.M., S.S.S., and H.O.
contributed new reagents/analytic tools; H.M.H.G.A., A.D., L.A.v.M., D.A.E., M.S.,
E.W.v.T., K.S., O.v.T., A.J.M., S.S.S., W.H.M., and H.O. analyzed data; and H.M.H.G.A.,
W.H.M., and H.O. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: h.ovaa@nki.nl.
This article contains supporting information online at www.pnas.org/cgi/content/full/
1001529107/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1001529107
PNAS ∣ April 20, 2010 ∣ vol. 107 ∣ no. 16 ∣ 7257–7262

Fig. 1. The ATX-LPA receptor signaling axis. Secreted ATX hydrolyzes extracellular LPC into LPA, a reaction catalyzed by active-site residue T210. LPA signals
through multiple G protein-coupled receptors to stimulate the proliferation, migration, and survival of many cell types. LPA is degraded to monoacylglycerol
(MAG) by lipid phosphate phosphatases (LPPs), which are membrane-bound ecto-enzymes.
the conversion of 14C-LPC to 14C-LPA using thin-layer chroma-
phosphate ester acceptor site in ATX and that the T210 oxygen
nucleophile could be targeted by a boronic acid moiety. Boronic
acid is known for its high affinity for hard oxygen nucleophiles
over soft nucleophiles, such as sulfur which is found in many
phosphate ester hydrolytic enzymes. This approach has an
important precedent in the proteasome inhibitor and anticancer
drug bortezomib (Velcade), which is a peptidyl boronic acid that
targets the threonine oxygen nucleophile in the proteasome active
site through its boronic acid moiety (28 and 30). We adopted a si-
milar approach to target the T210 oxygen nucleophile in ATX.
Replacing the carboxylic acid in HA51 by a boronic acid
yielded compound HA130, that resulted in a ∼100-fold increase
in potency compared to screening hit A (IC50 ¼ 28 nM) (Fig. 3A).
Furthermore, HA130 abolished the residual ATX activity ob-
served with compounds A and HA51. Kinetic analysis revealed
that HA130 is a mixed-type inhibitor, producing a reduction in
Vmax and an increase in Km (Fig. 3D). Thus, inhibition of
ATX by HA130 results from a combination of a decreased
turnover number and decreased affinity for its substrate. Washout
of HA130 and the other compounds fully restored ATX activity,
indicative of reversible inhibition (Fig. 3C).
tography (Fig. 2B).
Boronic Acid-Based Optimization. Having identified compound A as
a unique ATX inhibitor, we set out to improve its potency. Synth-
esis of A required the aldehyde building block 1 (Fig. S2). For this
purpose, vanillin was O-alkylated with methyl-4-(bromomethyl)
benzoate, using potassium hydroxide as a base, to afford the
desired methyl benzoate. Benzoic acid 1 was obtained after
hydrolysis of methyl benzoate. 2,4-Thiazolidinedione was N-alky-
lated with 4-fluorobenzyl bromide to yield monosubstituted thia-
zolane-2,4-dione 2. Knoevenagel condensation of 2 with benzoic
acid 1 yielded Z-isomer A (Fig. S3). This synthetic route allowed
the synthesis and isolation of more than 100 derivatives of A in a
short time frame.
All derivatives were tested in the ATX-mediated choline
release assay. Fig. 3A shows the IC50 values of the three most
important molecules in the optimization process. Omitting the
methoxy group and replacing the carboxylic acid to the meta
position (HA51) resulted in a 2.5-fold increase in potency,
concomitant with a significant drop in residual ATX activity, from
35% to 7% (Fig. 3A, B). Lineweaver-Burk analysis revealed that
A and HA51 act as competitive inhibitors (Fig. 3D), suggesting
that they bind at or close to the catalytic threonine resi-
due (T210).
Selective Inhibition of ATX. Since boronic acids can target the
proteasome active site, we examined whether HA130 may affect
proteasome activity. HA130 did not affect the chymotryptic,
caspase, and tryptic activities of the proteasome (Fig. S4B). Con-
versely, bortezomib did not affect ATX activity. We next tested
We next sought to target active-site residue T210. We reasoned
that the acid moiety of A and HA51 may strongly bind to the
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Albers et al.

A
HA130 Decreases Circulating LPA Levels in Mice. To investigate how
ATX inhibition affects circulating plasma LPA levels, we adminis-
tered HA130 (1 nmol g−1) or vehicle as a single bolus injection
into the jugular vein of anaesthetized mice. As a nonvehicle
control we used compound HA51. Levels of HA130 or LPA in
plasma samples rapidly isolated from venous blood were moni-
tored before and after dosing. As shown in Fig. 5A, intravenous
administration of vehicle or HA51 (Fig. S5) had little or no effect
on plasma LPA levels. Following administration of HA130
(t ¼ 10 min), plasma levels of the inhibitor rose rapidly to a
concentration approaching 0.35 μM. This was accompanied by
a parallel decrease in plasma LPA levels (3.8-fold) which returned
slowly towards baseline as plasma levels of HA130 declined.
Fig. 5B shows summarized data from replicate experiments in
which mice were dosed with vehicle or HA130 while plasma
LPA levels were measured before, 2 and 10 min post dosing
(see also Fig. S5). Administration of HA130 produced statistically
significant decreases in plasma LPA levels compared to baseline,
vehicle, and HA51. The mean decrease in plasma LPA levels was
48% of the baseline control at 2 min post administration of
HA130. Plasma HA130 levels correlated well (R2 ¼ 0.751) with
plasma LPA levels (Fig. 5C). Taken together, these findings
suggest that continual production of LPA by ATX-catalyzed
hydrolysis of circulating LPC is required to sustain plasma levels
of LPA at their observed steady-state level. To confirm this, we
injected C17-LPA and found that the levels of this unnatural
LPA analog were elevated at the earliest measurable time point,
but then decayed rapidly following pseudo first-order kinetics
with a half-life of approximately 3 min. (Fig. 5D).
IC50 (nM)
Code
A
O
O
N
S
56
O
O
COOH
F
O
O
N
S
N
B
C
111
161
N
O
O
O
S
N
O
COOH
O
B
LPA
LPC
Discussion
ATX is a secreted lysoPLD and the primary determinant of
bioactive LPA in the circulation. Mouse model studies have re-
vealed an important role for the ATX-LPA receptor axis in
tumor progression as well as in other pathologies, ranging from
inflammation to fibrosis (2, 12–21). Accordingly, pharmacologi-
cal inhibition of ATX is an attractive way to interfere with LPA
signaling for therapeutic benefit. Most ATX inhibitors described
so far are LPA analogs, whose designs are based on the initial
observation that LPA can act as a feedback inhibitor in vitro
and that LPA binds to ATX with much higher apparent affinity
than the substrate LPC does (22). However, we found that
synthetic LPA analogs and related lysophospholipids, such as
the S1P analog FTY720-phosphate (26), are rather poor ATX
inhibitors in the presence of high LPC concentrations.
increasing concentration of
A
Fig. 2. ATX inhibitors discovered by high-throughput screening and
validation of compound A. (A) IC50 values based on bis-pNPP (1 mM) hydro-
lysis. (B) TLC analysis of 14C-LPC to 14C-LPA conversion at different concentra-
tions of A (range: 0–30 μM). For details SI Text.
our inhibitors for selectivity against recombinant NPP1, which is
the closest relative of ATX, alkaline phosphatase (AP) and a
broad-spectrum phosphodiesterase (PDE). None of these
enzymes were affected by the ATX inhibitors at doses up to
10 μM (Fig. S4A). Furthermore, cell viability was not compro-
mised by HA130 (Fig. S4C).
In this study we have identified and optimized a class of
thiazolidinedione compounds as potent and selective inhibitors
of ATX. Our chemical optimization strategy was based on target-
ing the catalytic T210 residue in ATX by introducing a boronic
acid moiety. Boronic acid has previously been shown to be instru-
mental in the anticancer drug bortezomib, which targets the
threonine oxygen nucleophile in the active site of the proteasome
(30). Strikingly, replacing the carboxylic acid in HA51 by a
boronic acid increased the potency for ATX inhibition by two or-
ders of magnitude compared to the screening hit A. The boronic
acid HA130 has an IC50 value of 28 nM using LPC (40 μM) as a
substrate and did not affect either NPP1 or proteasome activity.
Injection of HA130 into mice resulted in a rapid fall in circulating
LPA levels, which is in keeping with the rapid degradation of
intravenous administered C17-LPA observed in these animals.
This result indicates that maintenance of steady-state LPA levels
in plasma involves a highly dynamic balance between its
ATX-mediated synthesis and its degradation by LPPs. Consistent
with this, LPP1-deficient mice show a significantly reduced rate of
[32P]LPA degradation in the bloodstream (32). Thus, ATX and
LPPs are key determinants of LPA turnover in vivo and their ac-
tivity balance sets the steady-state level of LPA in the circulation
and, most likely, in the interstitial space.
Inhibition of ATX-Driven Melanoma Cell Migration. ATX was origin-
ally identified as an autocrine motility factor for human A2058
melanoma cells (1). We examined the ATX-mediated chemotac-
tic migration of A2058 cells using Boyden chamber assays. ATX
hydrolyzes exogenously added LPC into LPA, a potent chemoat-
tractant for A2058 cells. As shown in Fig. 4A, ATX inhibitors A,
HA51, and HA130 inhibited ATX-mediated cell migration with
increasing potencies. None of the inhibitors affected LPA-
induced cell migration, indicating that they do not act on LPA
receptor signaling pathways.
Inhibition of Plasma ATX Activity. ATX is identical to plasma ly-
soPLD (3 and 4) and responsible for virtually all LPA-producing
activity in plasma and serum (31). Inhibitors were tested for
their ability to inhibit ATX/lysoPLD activity in human plasma
ex vivo. As shown in Fig. 4B, all three inhibitors were found
to inhibit plasma ATX activity with the expected ranking order
of potency. Inhibition of plasma ATX activity was long lasting
(24 h), indicating that HA130 is metabolically stable in plasma
(Table S1).
Albers et al.
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7259

A
B
C
O
100
75
50
25
0
A
HA51
HA130
N
S
R2
O
F
R1
R1
R2
Code
A
IC50 (nM)
RA(%)
O
COOH
-10 -9
-8
-7
-6
-5
-4
OMe
2500
35
log[inhibitor]
COOH
O
100
75
50
25
0
no wash-out
wash-out
H
H
HA51
1070
28
7
0
HO
B
OH
O
HA130
A
HA51
HA130
D
A
HA51
HA130
6
4
2
15
10
5
0 µM
0 µM
0 nM
15
10
5
0.75 µM
1.5 µM
3 µM
50 nM
0.75 µM
1.5 µM
3 µM
100 nM
200 nM
400 nM
6 µM
-0.0075
-0.0025
0.0025
0.0075
-0.0075
-0.0025
0.0025
0.0075
-0.0075
-0.0025
0.0025
0.0075
1/[S] (µM)
1/[S] (µM)
1/[S] (µM)
Fig. 3. Analysis of ATX inhibition by compounds A, HA51, and boronic acid-based HA130, as measured by choline release from LPC (40 μM). (A) IC50 values and
residual ATX activity for the three inhibitors tested. (B) Dose-inhibition curves for the compounds shown in (A). (C) Wash-out experiments showing that
ATX inhibition is reversible. (D) Lineweaver-Burk plot analysis of ATX inhibition, showing competitive inhibition by compounds A and HA51, and mixed-type
inhibition by HA130. For details SI Text.
Several questions concerning circulating ATX remain to be an-
A
swered, including its tissue origin and metabolic fate, although
recent evidence indicates that ATX is rapidly cleared from the
circulation by liver sinusoidal endothelial cells (33). Circulating
ATX and LPA do not, of course, reflect the levels in intercellular
spaces, since ATX is produced locally by many different cell types
while the LPC substrate level in interstitial fluids is much lower
than that in plasma. Another key question concerns how ATX
activity is regulated under (patho)physiological conditions.
Interestingly, ATX binds to activated lymphocytes and platelets
in an integrin-dependent manner (18 and 21), which could lead to
altered catalytic activity and serve as a mechanism for localized
LPA production at sites of inflammation and injury.
100
33 nM
330 nM
control
75
50
25
0
B
A
100
75
50
25
0
HA51
HA130
In conclusion, we have used a boronic acid-based inhibitor to
demonstrate that ATX is a valid target for manipulating LPA
levels in vivo. Further development of boronic acid inhibitors
of ATX holds promise for therapeutic use in ATX/LPA-
dependent pathologies, including chronic inflammation, tumor
progression, and fibrotic diseases.
-10 -9
-8
-7
-6
-5
-4
log[inhibitor]
Methods
ATX/lysoPLD Activity Assay. ATX/lysoPLD activity was measured by choline
release from LPC (18∶1) (80 μM) in 96-well plates. Inhibitors (5 μM) in DMSO
were added to recombinant ATX (40 nM) in Tris-HCl buffer (pH 7.4) at 37 °C.
Fig. 4. Effect of inhibitors on ATX-induced cell migration and on plasma
ATX/lysoPLD activity. (A) ATX (1.2 nM), LPC (1 μM), and BSA (1 mg mL−1) were
added to the lower chambers of 48-wells Boyden chambers and the transwell
migration of A2058 melanoma cells was assayed after 4 h in the presence or
absence of ATX inhibitors. None of the compounds inhibited LPA-induced cell
migration (LPA added at 0.3 μM). (B) Inhibition of endogenous ATX/lysoPLD
activity in human plasma ex vivo, as measured by choline release from LPC.
Inhibition was maintained for at least 24 h (Table S1).
After
sulfonic acid) (ABTS) (2 mM) and horseradish peroxidase (10 Unit mL−1) were
added to 50 μL of the reaction mixture. Choline oxidase (50 μL, 10 Unit mL−1
3
h
of incubation, 50 μL 2,20-azino-bis(3-ethylbenzothiazoline-6-
)
was added for the colorimetric reaction. Absorbance was measured at
405 nm and data were analyzed using Graphpad Prism software.
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Albers et al.

B
**
A
HA130
vehicle
HA130
vehicle
*
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
1.50
1.25
1.00
0.75
0.50
0.25
0.00
LPA
HA130
pre-bleed 2 min
10 min
0
5
10
15
20
25
time (min)
time (min)
5
C
D
10
1
R2 = 0.751
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
0.5
0.25
0.125
0.0
0.1
0.2
0.3
0.4
0.5
0
10
20
time (min)
30
plasma HA130 (µM)
Fig. 5. Effects of HA130 on circulating LPA levels in mice. (A) Vehicle or HA130 was administered intravenously into an anaesthetized mouse and plasma levels
of the inhibitor or total LPA were determined at the indicated time points. Compound HA51 was used as a nonvehicle control (Fig. S5). (B) Plasma levels of LPA
were determined at baseline and at 2 and 10 min post dosing of vehicle or HA130. The data shown are means þ ∕ − SD (six mice treated with HA130; five mice
treated with vehicle alone). The difference between baseline LPA levels and LPA levels at 2 min in drug-treated mice are statistically significant (P ¼ 0.001) by
paired t-test. (C) Plasma levels of LPA and HA130 at 2 min post administration closely correlate (R2 ¼ 0.751). (D) C17-LPA (10 μL of a 10 mM solution in saline
containing 0.1% fatty acid-free BSA) was injected into the jugular vein of anaesthetized mice and plasma levels of C17-LPA were determined at different time
points. The inset shows a semilog plot used to calculate the half-life for clearance of C17-LPA from the circulation. Data points are means þ ∕ − SD (n ¼ 3).
Human Plasma ATX Activity. Plasma ATX activity was measured in 96-wells
plates using LPC (18∶1) as a substrate. Heparin-treated human plasma
(2 μL) was added to 38 μL Tris-HCl buffer (100 mM Tris-HCl, pH 9, 500 mM
NaCl, 5 mM MgCl2 and 0.05% Triton X-100). Subsequently, 0.8 μL inhibitor
in DMSO was added. Finally, 40 μL of 2 mM LPC (18∶1) in Tris-HCl buffer
was added to each well and the plate was incubated at 37 °C. The mixture
with DMSO alone was used as a control. Plasma without added LPC was taken
as control for endogenous LPA production. After 1.5 h of incubation, 150 μL
homovanillic acid (2 mM) and horseradish peroxidase (1.6 Unit mL−1) in
Tris-HCl (0.01% Triton X-100, 20 mM CaCl2 and 50 mM Tris-HCl, pH ¼ 7.4)
was added to 20 μL of the reaction mixture. Choline oxidase was added
Studies in Mice. For intravenous administration, HA130 and HA51 in DMSO
were diluted 10-fold into saline to give a drug concentration solution of
1 mM and a final DMSO concentration of 10%. This material was kept at
37 °C and bath sonicated for 30 s immediately prior to intravenous adminis-
tration. Male FVB mice were anaesthetized with isofluorane and dissected to
expose the jugular vein. HA130 (1 μL g−1, 1 mM) or vehicle were injected in-
travenously. HA51 served as a nonvehicle control. Whole blood was sampled
from the jugular vein and collected directly into anticoagulant, mixed,
centrifuged, and plasma transferred to glass tubes containing acidified
solvents for extraction of HA130 and LPA. All animal experiments conformed
to the recommendations of the “Guide for the Care and Use of Laboratory
Animals” (Department of Health, Education, and Welfare publication
number NIH 78-23, 1996) and were approved by the institutional Animal Care
and Use Committee.
(40 μL, 4 Unit mL−1
320∕450 nm.
)
and fluorescence was determined at λex∕λem
¼
ATX-Mediated Cell Migration. A2058 melanoma cell chemotaxis was assayed
using 48-well Boyden chambers. Fibronectin-coated polycarbonate
membranes (8 μM pores, NeuroProbe Inc.) were used to separate the upper
from the lower chamber. The lower chamber contained DMEM with BSA
(1 mg mL−1), ATX (1.2 nM) and LPC (1 μM). Cells (0.75 × 106 mL−1) were
loaded in the upper wells and the chamber was incubated at 37 °C for
4 h. Nonmigrated cells were removed from the membrane and migrated cells
were fixed and stained in Diff-Quick (Medion Diagnostics AG). The mem-
brane was mounted on a glass slide and migrated cells were quantified.
Additional assays and chemical synthesis: SI Text.
ACKNOWLEDGMENTS. We thank Anastassis Perrakis and Andreas Kremer for
helpful discussions. This work was supported by grants from the Netherlands
Organization for Scientific Research (NWO) (to H.O., W.H.M.), the Dutch Can-
cer Society (to W.H.M.), the Center for Biomedical Genetics (to W.H.M.), the
Netherlands Genomics Initiative (to H.O.), and the National Institutes of
Health (NIH; HL074219 and HL078663 (to S.S.S.)); and GM050388 (to A.J.M.).
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