Evaluation of inhibitory actions of flavonols and related substances on lysophospholipase D activity of serum autotaxin by a convenient assay using a chromogenic substrate

Article abstract of DOI:10.1021/jf904155a

Overproduction of lysophosphatidic acid (LPA) by lysophospholipase D/autotaxin (lysoPLD/ATX) is postulated to be involved in the promotion of cancer and atherosclerosis. A lysoPLD inhibitor may be utilized to ameliorate the LPA-related pathological condit

Full text of DOI:10.1021/jf904155a

J. Agric. Food Chem. 2010, 58, 6053–6063 6053
DOI:10.1021/jf904155a
Evaluation of Inhibitory Actions of Flavonols and
Related Substances on Lysophospholipase D Activity
of Serum Autotaxin by a Convenient Assay Using a
Chromogenic Substrate
K
AORI
U
S
EDA, MASANORI
Y
OSHIHARA, MICHIYASU
N
AKAO, TAMOTSU
T
ANAKA
,
†
HIGEKI
S
ANO, KENJI
F
UKUZAWA
,
AND
A
KIRA OKUMURA
T
*
Institute of Health Biosciences, University of Tokushima Graduate School, 1-78-1 Shomachi, Tokushima
770-8505, Japan. † Present address: Faculty of Pharmaceutical Sciences, Yasuda Women’s University,
6-13-1 Andoucho, Asaminami-ku, Hiroshima 731-3167, Japan.
Overproduction of lysophosphatidic acid (LPA) by lysophospholipase D/autotaxin (lysoPLD/ATX) is
postulated to be involved in the promotion of cancer and atherosclerosis. A lysoPLD inhibitor may be
utilized to ameliorate the LPA-related pathological conditions. In this study, a new assay was
devised to quantify p-nitrophenol from hydrolysis of chromogenic substrate by serum lysoPLD
without tedious lipid extraction procedures. Flavonols, phenolic acids, free fatty acids, and
N-acyltyrosines inhibited lysoPLD activity in a micromolar range. They were classified into
competitive, noncompetitive, or mixed type inhibitors. The results show that the low hydrophobicity
of an inhibitor is a critical factor in its preference for the binding to a noncatalytic binding site over a
catalytic binding site. Considering its reported bioavailability and the low dependency of its inhibitory
activity on serum dilution, flavonol is likely to be a more effective lysoPLD inhibitor in human blood
circulation in vivo than the other inhibitors including LPA.
KEYWORDS: Autotaxin; lysophosphatidic acid; lysophospholipase D; flavonol; phenolic acid; free
fatty acid
INTRODUCTION
Several assays have been used to screen inhibitors of the
lysoPLD activity of ATX. An assay with radioactive LPC was
first used to determine lysoPLD activity in animal body
fluids (8, 18, 19) and recently was applied to screen inhibitors of
lysoPLD activity of ATX (20). Although the assay, which is
accompanied by a tedious lipid extraction procedure, needs great
care to avoid radioactive contamination throughout its perfor-
mance, it identified the known phosphodiesterase inhibitors
calmidazolium and vinpocetine, the protein kinase inhibitors
damnacanthal and hypericin, and the tyrosine protein kinase
inhibitors tyrphostin AG8 and AG213 as weak inhibitors of
lysoPLD. Enzyme-coupled assays for the determination of cho-
line (2, 3) and LPA (21) are also applicable to monitoring of
lysoPLD activity in animal biological fluids, but a possibility
remains that the candidate compounds inhibit the activities of
secondary enzymes such as choline oxidase, rather than the
primary enzyme, lysoPLD. Thus, the first aim of this study was
to devise a convenient and reliable assay to screen potential
inhibitors of the lysoPLD activity of ATX.
Lysophosphatidic acid (LPA) is an important intercellular
mediator that plays a physiological role in a large array of animal
cells through its specific G-protein-coupled receptors. This lyso-
phospholipid mediator is known to participate in several patho-
logical dysfunctions such as cancer metastasis and atherosclerosis.
In fact, plasma lysophospholipase D (lysoPLD), an LPA-produ-
cing enzyme, was originally identified as autotaxin (ATX), which
was first isolated as a tumor cell motility-stimulating protein in the
culture medium of human melanoma cells (1-3). The lysoPLD
activity of ATX has been detected in the following human body
fluids inaddition to plasmaandserum:urine(4), ascitic fluid (5,6),
peritoneal washing fluid (6), seminal fluid (7), follicular fluid (8),
and cerebrospinal fluid (9). Overexpression of ATX was found in
malignanttumorcellssuchasmelanoma(1), teratocarcinoma(10),
lung cancer cells (11), breast cancer cells (12), thyroid carcinoma
cells (13), prostate cancer cells (14), neuroblastoma (15), glioblas-
toma multiforme (16), and EBV-positive Hodgkin’s lymphoma (17).
Considered altogether, ATX, if expressed at an unusually high level,
exerts tumor-aggressive and angiogenetic effects. Thus, development
of inhibitors of ATX should be explored as potential candidates for
clinical use.
LPA and its analogues, cyclic phosphatidic acid (PA) and its
carba analogues, fatty alcohol phosphates as LPA analogues,
acyl thiophosphates as LPA analogues, β-keto and β-hydroxy
phosphonate analogues of LPA, R-substituted phosphonate
analogues of LPA, R- and β-substitutes phosphonate analogues
of LPA, sn-2-aminooxy analogue of LPA, Darmstoff ana-
logues, and phosphonothioate and fluoromethylene phosphonate
*Author to whom correspondence should be addressed (tele-
phone þ81 88 633 7248; fax þ81 88 633 9572; e-mail tokumura@
ph.tokushima-u.ac.jp).
© 2010 American Chemical Society
Published on Web 04/23/2010
pubs.acs.org/JAFC

6054 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Ueda et al.
analogues of cyclic PA, were reported to be inhibitors of lysoPLD
activity (22-24). However, little is known about the inhibitory
effects of nonphospholipidic natural substances on the plasma
lysoPLD activity of ATX. Thus, in the current study, we first
attempted to explore the inhibitory potentials of natural phenolic
antioxidants, including flavonols, on the plasma lysoPLD acti-
vity of ATX because flavonols have been shown to inhibit not
only nucleotide phosphodiesterases (25) but also lipid-metaboli-
zing enzymes such as lipoxygenases (26) and phospholipase
A2 (27). This novel effect of flavonols, if found, is expected
to strengthen their beneficial effects including anticancer acti-
vity and therapeutic potential for human health. Therefore, we
focused our attention onexamining whether flavonols and related
substances, which have a wide range of distribution in the plant
kingdom, inhibit the lysoPLD activity of ATX, a member of
the ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP)
family (28).
determined according to the method of Chalvardjian and Rudnicki (31).
Other reagents were of analytical grade.
Assay of LysoPLD Activity. Test substances for assay of lysoPLD
activity were dissolved in the following vehicles: saline for 16:0-LPA
(∼3.4 mM), 18:1-LPA (∼3.4 mM), alkyl 16:0-LPA, and N-palmitoyletha-
nolaminephosphoric acid (∼0.17 mM); double-distilled water for 16:0-
LPA (∼1.2 mM), caffeine,
L
-(-)-phenylalanine, 4-(4-hydroxyphenyl)-2-
butanone, 18:1-FA, 18:2-FA, 18:3-FA, and 20:4-FA (∼100 mM); diluted
sodium hydroxide solution for
-tyrosine (∼100 mM); diluted hydro-
chloric acid for
-3-(3,4-dihydroxyphenyl)alanine (∼100 mM); and
L
L
DMSO for 20:5-FA (∼10 mM), isorhamnetin-3-O-glucoside (∼30 mM),
quercetagetin (∼60 mM), and all other substances tested (∼100 mM). The
final concentration of DMSO was below 1% in all assay mixtures.
In a standard assay, 50 μL of diluted FBS (5% in saline; final, 0.83%) in
each well of a 96-well microplate (Thermo Fisher Scientific, Waltham,
MA) was mixed with 172 μL of saline. Then, the mixture was incubated
with 3 μL of test solution in DMSO or other vehicle and 75 μL of the
substrate solution (LPN; final, 75 μM) in 20 mM Tris-HCl buffer (pH 8.0)
for up to 4 h at 37 °C. The absorbance at 405 nm was measured
immediately on the microplate reader at 0 h and then every 20 min, 30
min, or 1 h for 4 h. The differences in absorbance with and without the
substrate measured at different time intervals were calculated as the
increase of absorbance due to the production of p-nitrophenol from
LPN: Δ absorbance = absorbance (þ LPN) - absorbance (- LPN).
On the basis of the graphs plotted with the increase in absorbance versus
incubation time, the initial rate was calculated from the slope of the
straight line extrapolated from the graph. A control solution containing no
test substance was also tested similarly, and the inhibitory rate (percent)
was calculated.
MATERIALS AND METHODS
Materials. Lysophosphatidyl-p-nitrophenol [LPN, 2-linoleoyl (18:2)]
was kindly given by Dr. T. Kishimoto (Alfresa Pharma, Osaka, Japan).
1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphate sodium salt (16:0-LPA),
1-oleoyl-2-hydroxy-sn-glycero-3-phosphate sodium salt (18:1-LPA), and
1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine (alkyl 16:0-LPC) were
purchased from Avanti Polar Lipids (Alabaster, AL). Phospholipase D
from Streptomyces chromofuscus, daidzein, chrysin, p-coumaric acid, ferulic
acid (trans-4-hydroxy-3-methoxycinnamic acid), sinapic acid, chlorogenic
acid, 3-(4-hydroxyphenyl)propionic acid, 4-hydroxyphenylacetic acid,
Synthesis of N-Acyltyrosine (NAT). Synthesis of NAT was based on
the Schotten-Baumann reaction (32) as described below. Tyrosine
(2.4-16.6 mmol) was dissolved in 4 N NaOH (3-4.5 equiv), and acyl
chloride (1-4 equiv) was added in five portions with shaking to the
solution (<0 °C). After 1-2 h, the reaction mixture was treated with
concentrated HCl and then extracted with ethyl acetate. The extract was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (Cosmosil
75C18-OPN, H2O/acetonitrile 3:1 for 3:0-NAT and D-3:0-NAT, 2:1 for
4:0-NAT and 5:0-NAT, and 1:1 for 5:0-NAT), preparative TLC
(chloroform/methanol 9:1 for D-3:0-NAT, 4:0-NAT, and 5:0-NAT),
recrystallization (methanol/ethyl acetate/n-hexane for 10:0-NAT, 12:0-
NAT, and 14:0-NAT; methanol/diethyl ether for 14:0-NAT; methanol for
14:0-NAT and 16:0-NAT), or washing with n-hexane, diethyl ether, ethyl
acetate, and methanol to afford NAT (in 0.1-30% yield).
In the cases of 10:0-NAT, 12:0-NAT, 14:0-NAT, 16:0-NAT, and
D-16:0-NAT, N,O-diacylated derivatives were obtained as the major
product in the reaction described above. Therefore, 4 N NaOH (4 equiv
of diacyltyrosine) was added to the residue dissolved in tetrahydrofuran
(15 mL for 10:0-NAT, 5 mL for 12:0-NAT) or dispersed in methanol
(7 mL) for 14:0-NAT and/or tetrahydrofuran (42 mL for 14:0-NAT, 40
mL for 16:0-NAT, and 20 mL for D-16:0-NAT) at room temperature.
After 0.5-0.8 h, the reaction mixture was treated with concentrated HCl
and then extracted with ethyl acetate. The extract was dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The subsequent
purification afforded the desired NATs.
N-(4-hydroxyphenyl)glycine, N-acetyl-
(4-hydroxyphenoxy)propionate, 3-(4-hydroxyphenyl)-1-propanol, N-acetyl-
-tyrosine (2:0-NAT), and essentially fatty acid-free bovine serum albumin
L
-phenylalanine, methyl (R)-(þ)-2-
L
(BSA) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Calcium
chloride, 28% ammonia solution, 2,6-di-tert-butyl-p-cresol (BHT), gallic
acid,
L-tyrosine,
L-(-)-phenylalanine, L-3-(3,4-dihydroxyphenyl)alanine, n-
butyric acid (4:0-FA), n-hexanoic acid (6:0-FA), n-heptanoic acid (7:0-FA),
n-decanoic acid (10:0-FA), lauric acid (12:0-FA), myristic acid (14:0-FA),
propionyl chloride, n-butyryl chloride, and n-valeryl chloride were purchased
from Nacalai Tesque (Kyoto, Japan). 4-Dimethylaminopyridine, rutin,
caffeine monohydrate, apigenin, kaempferol, formic acid (1:0-FA), palmitic
acid (16:0-FA), and dimethyl sulfoxide (DMSO) were purchased from Wako
Pure Chemicals Industries (Osaka, Japan). Morin, quercetin dehydrate,
isoquercitrin, 3-tert-butyl-4-hydroxyanisole (BHA), n-propyl gallate, 3-phe-
nylpropionic acid, acetic acid (2:0-FA), n-octanoic acid (8:0-FA), n-decanoyl
chloride, lauroyl chloride, myristoyl chloride, palmitoyl chloride, and metha-
nol were purchased from Kanto Chemical Corp. (Tokyo, Japan). Filter-
sterilized fetal bovine serum (FBS) was purchased from Equitech-Bio
(Kerrville, TX). O-Phosphorylethanolamine and stearic acid (18:0-FA) were
purchased from Tokyo Chemical Industry (Tokyo, Japan). Palmitic anhy-
dride was purchased from Bachem (Bubendolf, Switzerland). Quercetagetin,
robinetin, isorhamnetin, isorhamnetin-3-O-glucoside, and tamarixetin were
purchased from Extrasynthese (Genay Cedex, France). Myricetin was
purchased from LKT Laboratories (St. Paul, MN). Fisetin, 4-(4-hydro-
xyphenyl)-2-butanone, and 8-hydroxyoctanoic acid (ωOH-8:0-FA) were
purchased from Acros Organics (Geel, Belgium). (þ)-Catechin hydrate
was purchased from Spectrum Chemical Manufacturing Corp. (Gardena,
CA). (-)-Epicatechin was purchased from Nagara Science (Gifu, Japan).
trans-Resveratrol, luteolin, caffeic acid, eicosapentaenoic acid (20:5-FA), and
docosahexaenoic acid (22:6-FA) were purchased from Cayman Chemical
Co. (Ann Arbor, MI). Genistein was purchased from Enzo Life Science
International Inc. (Plymouth Meeting, PA). 3-(3,4-Dihydroxyphenyl)pro-
pionic acid was purchased from Alfa Aesar (Ward Hill, MA). Sodium oleate
(18:1-FA), sodium R-linoleate (18:2-FA), sodium linolenate (18:3-FA), and
sodium arachidonate (20:4-FA) were purchased from Nu-Chek Prep, Inc.
(Elysian, MN).
(S)-3-(4-Hydroxyphenyl)-2-propionamidopropanoic acid [3:0-NAT]:
29
colorless oil, [R]D þ46.1 (c 0.41, CH3OH); 1H NMR (400 MHz, CD3OD)
δ 1.04 (t, J = 7.6 Hz, 3H), 2.17 (q, J = 7.6 Hz, 2H), 2.86 (dd, J = 13.9, 9.0
Hz, 2H), 3.10 (dd, J = 13.9, 5.1 Hz, 1H), 4.57-4.65 (m, 1H), 6.66-6.75
(m, 2H), 6.98-7.08 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 10.3, 29.8,
37.6, 55.1, 116.1, 129.0, 131.2, 157.1, 175.0, 176.9. ESIMS calcd for
C12H15NO4Na, MW 260.0899; found, m/z 260.0896 (Mþ þ Na).
(R)-3-(4-Hydroxyphenyl)-2-propionamidopropanoic acid [D-3:0-NAT]:
colorless oil, [R]D29 -45.9 (c 1.04, CH3OH). ESIMS calcd for C12H15NO4-
Na, MW 260.0899; found, m/z 260.0907 (Mþ þ Na).
(S)-2-Butyramido-3-(4-hydroxyphenyl)propanoic acid [4:0-NAT]:
brown oil, [R]D þ33.7 (c 1.30, CH3OH); 1H NMR (400 MHz, CD3OD)
29
Alkyl 16:0-LPA was prepared from alkyl 16:0-LPC using phospholi-
pase D from S. chromofuscus, as previously reported (29). N-Palmitoy-
lethanolaminephosphoric acid was prepared according to the modi-
fied method described by Sugiura et al. (30). Lipid-phosphorus was
δ 0.84 (t, J = 7.6 Hz, 3H), 1.54 (sext, J = 7.6 Hz, 2H), 2.13 (t, J = 7.6 Hz,
2H), 2.83 (dd, J = 14.2, 9.3 Hz, 1H), 3.10 (dd, J = 14.2, 4.9 Hz, 1H),
4.56-4.63 (m, 1H), 6.64-6.73 (m, 2H), 6.97-7.09 (m, 2H); 13C NMR
(75 MHz, CD3OD) δ 13.8, 20.1, 37.4, 38.5, 55.0, 116.1, 128.8, 131.1, 156.8,

Article
J. Agric. Food Chem., Vol. 58, No. 10, 2010 6055
175.0, 176.0. ESIMS calcd for C13H17NO4Na, MW 274.1055; found, m/z
274.1059 (Mþ þ Na).
(S)-3-(4-Hydroxyphenyl)-2-pentanamidopropanoic acid [5:0-NAT]:
30
brown oil, [R]D þ27.8 (c 0.89, CH3OH); 1H NMR (400 MHz, CD3OD)
δ 0.86 (t, J = 7.3 Hz, 3H), 1.15-1.27 (m, 2H), 1.42-1.52 (m, 2H), 2.15 (t,
J = 7.3 Hz, 2H), 2.82 (dd, J = 13.9, 9.5 Hz, 1H), 3.12 (dd, J = 13.9, 4.9
Hz, 1H), 4.55-4.64 (m, 1H), 6.64-6.72 (m, 2H), 6.97-7.07 (m, 2H); 13C
NMR (75 MHz, CD3OD) δ 14.1, 23.1, 28.9, 36.5, 37.5, 55.0, 116.1, 129.0,
131.1, 157.1, 175.0, 176.1. ESIMS calcd for C14H19NO4Na, MW 288.1212;
found, m/z 288.1205 (Mþ þ Na).
(S)-2-Decanamido-3-(4-hydroxyphenyl)propanoic acid [10:0-NAT]:
23
colorless powder, [R]D þ24.4 (c 1.00, CH3OH); 1H NMR (400 MHz,
CD3OD) δ 0.89 (t, J = 6.8 Hz, 3H), 1.14-1.37 (m, 12H), 1.44-1.55 (m,
2H), 2.15 (t, J = 7.6 Hz, 2H), 2.83 (dd, J = 13.9, 9.3 Hz, 1H), 3.10 (dd, J =
13.9, 4.9 Hz, 1H), 4.56-4.65 (m, 1H), 6.61-6.74 (m, 2H), 6.95-7.09 (m,
2H); 13C NMR (75 MHz, CD3OD) δ 14.4, 23.7, 26.9, 30.1, 30.41, 30.45,
30.5, 33.0, 36.8, 37.6, 55.1, 116.1, 129.1, 131.2, 157.3, 175.0, 176.1. ESIMS
calcd for C19H29NO4Na MW 358.1994, found m/z 358.1997 (Mþ þ Na).
(S)-2-Dodecanamido-3-(4-hydroxyphenyl)propanoic acid [12:0-NAT]:
colorless needle, [R]D þ21.0 (c 1.00, CH3OH); 1H NMR (400 MHz,
22
CD3OD) δ 0.89 (t, J = 6.8 Hz, 3H), 1.10-1.39 (m, 16H), 1.42-1.58 (m,
2H), 2.15 (t, J = 7.6 Hz, 2H), 2.84 (dd, J = 13.9, 9.3 Hz, 1H), 3.10 (dd, J =
13.9, 4.9 Hz, 1H), 4.55-4.66 (m, 1H), 6.63-6.76 (m, 2H), 6.97-7.09 (m,
2H); 13C NMR (75 MHz, CD3OD) δ 14.4, 23.7, 26.9, 30.1, 30.5, 30.6,
30.74, 30.75, 33.1, 36.8, 37.7, 55.1, 116.2, 129.1, 131.2, 157.3, 175.0,
176.2. ESIMS calcd for C21H33NO4Na MW 386.2307, found m/z
386.2337 (Mþ þ Na).
Figure 1. Time course of production of p-nitrophenol from LPN incubated
in diluted FBS and its inhibition by ferulic acid. Diluted FBS (final, 0.83%)
was incubated with LPN (final, 75 μM) at 37 °C for 4 h in the presence or
absence of ferulic acid (final, 0.1 mM). The absorbance at 405 nm was
measured every 30 min or 1 h for 4 h. Values are means of duplicate
determinations.
(S)-3-(4-Hydroxyphenyl)-2-tetradecanamidopropanoic acid [14:0-NAT]:
white solid, [R]D þ24.3 (c 0.14, CH3OH); 1H NMR (400 MHz, DMSO-
21
d6) δ 0.85 (t, J = 7.1 Hz, 3H), 1.07-1.32 (m, 20H), 1.34-1.45 (m, 2H),
2.03 (t, J = 7.3 Hz, 2H), 2.71 (dd, J = 13.9, 9.8 Hz, 1H), 2.91 (dd, J =
13.7, 4.6 Hz, 1H), 4.27-4.38 (m, 1H), 6.58-6.70 (m, 2H), 6.94-7.06 (m,
2H), 8.01 (d, J = 8.3 Hz, 1H), 9.17 (s, 1H), 12.53 (br s, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 14.0, 22.3, 25.3, 28.7, 28.9, 29.0, 29.1, 29.20, 29.23,
29.25, 31.5, 35.2, 36.2, 38.9, 53.7, 114.9, 127.7, 129.9, 155.8, 172.1, 173.2.
ESIMS calcd for C23H37NO4Na MW 414.2620, found m/z 414.2601
(Mþ þ Na).
areas of 18:2-LPA to 17:0-LPA in LC-MS/MS. Analytical conditions were
described previously (34). For analysis of LPN, lipids in the assay solution
(100 μL) were extracted according to the method of Bligh and Dyer (33).
LPNs having an sn-1- or sn-2-linoleoyl group were analyzed by multiple
reaction monitoring in a negative ion mode under the same conditions for
LPA analysis by LC-MS-MS (34) except for setting Q1 and Q3 as [M - H]-
at m/z 554 and [linoleate]- at m/z 279, respectively.
(S)-3-(4-Hydroxyphenyl)-2-palmitamidopropanoic acid [16:0-NAT]:
21
white solid, [R]D þ18.8 (c 0.13, CH3OH); 1H NMR (400 MHz, DMSO-
d6) δ 0.85 (t, J = 7.1 Hz, 3H), 1.05-1.43 (m, 26H), 2.03 (t, J = 7.3 Hz, 2H),
2.66-2.75 (m, 1H), 2.87-2.94 (m, 1H), 4.27-4.36 (m, 1H), 6.60-6.67 (m,
2H), 6.96-7.23 (m, 2H), 8.01 (d, J = 8.1 Hz, 1H), 9.17 (s, 1H), 12.32 (br s,
1H); 13C NMR (75 MHz, DMSO-d6) δ 14.1, 22.4, 25.5, 28.9, 29.1, 29.2,
29.31, 29.38, 29.4, 31.6, 35.3, 36.3, 53.9, 115.2, 128.0, 130.2, 156.1, 172.7,
173.6. ESIMS calcd for C25H41NO4Na, MW 442.2933; found, m/z 442.2900
(Mþ þ Na).
RESULTS
Screening of Inhibitory Substances by a New Assay with a
Chromogenic Substrate. We chose various antioxidants, including
polyphenols, as the first screening target for development of
lysoPLD inhibitors because polyphenols have attracted increas-
ing attention as a dietary supplement that exerts beneficial effects
on human health due to its antioxidant activity and enzyme-
inhibitory activity [lipoxygenase (26), phospholipase A2 (27),
myeloperoxidase (35)]. Second, phenolic acids were examined
as potential inhibitors of serum lysoPLD because their structures
correspond to a partial structure of the flavonol skeleton. Because
phenolic acids are known to be present at higher concentrations
than flavonols in the blood after routine oral intake, their
physiological inhibition of plasma lysoPLD is expected to be
more significant. Third, we tested the lysoPLD-inhibiting activ-
ities of FAs with an aliphatic hydrocarbon chain as a series of
homologuesthat structurallyrelatetoa group ofphenolicacids to
assess the necessity of a benzene ring in the structures of a
phenolic acid for its inhibitory action on lysoPLD activity. Last,
we synthesized NATs with different acyl chains as structural
analogues of phenolic acids and tested their inhibitory effects on
lysoPLD activity. To test potential inhibitory effects of these
compounds, we needed a convenient and reliable assay for
lysoPLD activity. In the present assay system, we used LPN as
an artificial chromogenic substrate. LysoPLD activity in FBS
hydrolyzed LPN to LPA and p-nitrophenol (Figure 1), the
(R)-3-(4-Hydroxyphenyl)-2-palmitamidopropanoic acid [D-16:0-NAT]:
white solid, [R]D19 -12.7 (c 0.11, CH3OH). ESIMS calcd for C25H41NO4-
Na, MW 442.2933; found, m/z 442.2971 (Mþ þ Na).
Kinetic Analysis of Inhibition of Serum LysoPLD Activity. In our
standard assay of lysoPLD activity in FBS, the final concentration of the
substrate LPN was 0.075 mM. To analyze inhibition of the lysoPLD
activity kinetically, we measured the initial rate for 4-h of incubation at
37 °C at different final concentrations of the substrate LPN (0.025, 0.0375,
0.075, 0.1, 0.15, and 0.3 mM). On the basis of the graphs plotted with the
reciprocal of the initial rate versus that of the concentration of the
substrate, a straight line was extrapolated from the graph. The inhibitors
used for making Lineweaver-Burk plot were as follows: robinetin, 18:1-
LPA, and 12:0-FA at 0.1 mM, and 2:0-FA, 6:0-FA, 2:0-NAT, 5:0-NAT,
12:0-NAT at 0.3 mM as representatives of flavonols, LPAs, fatty acids,
and NATs, respectively.
Analysis of LPA by LC-MS/MS. Fifty microliters of 5% FBS in
saline (final, 0.83%) as the enzyme source, 75 μL of 18:2-LPC and LPN in
20 mMTris-HClbuffer, pH 8.0 (final, 75 μM), as the substrate, and 3 μL of
the test solution in a vehicle such as DMSO or vehicle alone were mixed,
and the mixture was incubated for 4-6 h at 37 °C. After incubation with
LPA, lipids in the assay solution (100 μL) were extracted according to the
modified method of Bligh and Dyer (33) after the addition of 100 pmol of
17:0-LPA as an internal standard. 18:2-LPA in the lipid extract from the
acidified aqueous layer was quantified by obtaining the ratio of the peak

6056 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Ueda et al.
Figure 2. Inhibitory effects of robinetin and 16:0-NAT on lysoPLD activity
toward exogenous 18:2-LPC, which is a physiologically more appropriate
substrate than LPN. Five percent FBS was incubated with a solution
of 18:2-LPC (final, 75 μM) in the presence and absence of robinetin or
16:0-NAT (final, 0.1 mM) at 37 °C for 6 h. After incubation, the incubation
mixture was acidified, and lipids were extracted from the incubation mixture
according to the method of Bligh and Dyer. 18:2-LPA was quantified by
LC-MS/MS, as shown under Materials and Methods. Values are means (
SD of triplicate determinations. The results were compared by using an
analysis of variance followed by Student’s unpaired t test. The asterisks
show statistical significance (/, P < 0.05).
generation of which could be monitored by increased absorbance
at around 405 nm. Thus, lysoPLD activity is continuously
quantifiedasp-nitrophenolproductionfor various periods. Using
this assay, we tested the inhibitory activity of several compounds
in a 96-well microplate, allowing the assay to be a time- and
reagent-saving screening for lysoPLD inhibitors. We used FBS as
an enzyme source, because we were able to purchase a large
quantity in a single lot. In our standard assay, FBS was finally
diluted to 0.83%. As shown in Figure 1, an almost linear increase
in absorbance due to the production of p-nitrophenol was
observed with time after incubation of diluted FBS with LPN
in the presence and absence of ferulic acid (final, 0.1 mM). Thus,
we set the final concentration of the substrate LPN as 75 μM,
providing the absorbance change in a proper range. The maximal
conversion of LPN to p-nitrophenol produced an absorbance
increase of 0.1-0.2 at 405 nm for up to 4 h in diluted FBS, and an
absorbance increase of 0.3-0.6 in undiluted FBS. The incubation
time was up to 4 h. By LC-MS/MS, we found that percentages of
peak area of LPN having an sn-2-lionoleoyl group, a faster
migrating isomer than the corresponding sn-1 isomer on the LC
column, were 49.0 ( 0.29, 21.9 ( 3.32 and 22.9 ( 6.82 (triplicate
determinations) in the lipid extracts from the assay solution
incubated with 75 μM LPN for 0, 2, and 4 h, respectively. This
result showed the rapid equilibration between the sn-1 and sn-2
isomers (about 23:77) under our assay conditions, suggesting that
serum lysoPLD acted on LPN with the constant isomer ratio
during most of the assay time. Thus, we calculated the initial rate
from the slope of a straight line on the graphs plotted with the
increase of absorbance obtained versus the incubation time. By
analyzing the results in a Lineweaver-Burk plot, we obtained
a Km value of 15.1 ( 1.3 μM (n = 21) for LPN as a substrate
for the lysoPLD activity of ATX in FBS, which is about one-
seventh of that for 16:0-LPC (2). However, it is about 5 times
higher than the reported fluorogenic substrate for lysoPLD/
ATX (36).
Figure 3. Comparisons of the concentration dependence of inhibition by
polyphenols, FAs, and NATs on substances on lysoPLD. The final
concentration of the substrate was 75 μM. Due to the low solubility of
robinetinand16:0-NAT, their resultsat1mMwerenotobtained. Valuesare
means of duplicate determinations. N.D. = not determined.
of 18:2-LPA from exogenously added 18:2-LPC, a physiological
substrate for plasma lysoPLD in the presence and absence of
robinetin or 16:0-NAT as an inhibitor of lysoPLD, confirming
that the test substances really inhibited the lysoPLD activity
toward the physiological substrate LPC (Figure 2). Next, we
tested effects of 16:0-NAT and robinetin on the equipotent
lysoPLD activity of recombinant human ATX with that of
diluted FBS in our standard assay with 75 μM LPN and found
that 39.6 and 49.6% inhibitions were attained by 16:0-NAT and
robinetin at 0.1 mM, respectively. This result suggests that the
coexistence of components from diluted FBS in our assay system
caused no serious interference over the lysoPLD activity ATX.
Considering these results together, we concluded that the method
using LPN is convenient and suitable for the screening of
lysoPLD inhibitors.
The curves of concentration-dependent inhibition of lysoPLD
activity by the eight test substances were different (Figure 3). The
concentration dependences for robinetin, 12:0-FA, and 16:0-
NAT were higher than those for caffeic acid, 6:0-FA, and 2:0-
NAT. The former were found to function as competitive inhibi-
tors, except for robinetin, whereas the latter were found to work
mainly as noncompetitive inhibitors, except for the mixed type
inhibition for 6:0-FA, which will be described later. LPA data are
not shown because LPAs were hard to dissolve at final concen-
trations >0.3 mM.
As noted above, the artificial substrate LPN was used in our
assay. Thus, it was necessary to confirm whether the inhibitory
effect of the hydrolysis of artificial substrate by lysoPLD activity
corresponds to that of the hydrolysis of physiological substrate
(LPC). Using LC-MS/MS, we directly measured the production

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J. Agric. Food Chem., Vol. 58, No. 10, 2010 6057
Figure 4. Lineweaver-Burk plot of inhibition by LPA, FAs, NATs, and robinetin on lysoPLD activity of diluted FBS. LysoPLD activity in diluted FBS (final,
0.83%) was calculated from the initial rate measured in 4 h of incubation at 37 °C. Concentrations of the substrate LPN were 0.025, 0.0375, 0.05, 0.075, 0.1,
0.15, and 0.3 mM. Concentrations of the inhibitor were 0.1 mM for robinetin, 18:1-LPA (A-1), and 12:0-FA (A-2) and 0.3 mM for 2:0-FA (C-1), 6:0-FA (B-2),
2:0-NAT (C-2), 5:0-NAT (B-3), and 12:0-NAT (A-3). Open and solid circles show values measured in the presence and absence of the inhibitor, respectively.
Values are means of triplicate determinations. The mean and SD of Km/Vmax for the controls were 433 ( 86 (n = 8).
Kinetic Analysis of Inhibitions on LysoPLD Activity. We per-
formed kinetic analysis of the inhibition of the lysoPLD activity,
using a Lineweaver-Burk plot. We expected LPA to inhibit the
activity of serum lysoPLD in a competitive manner, because LPA
is the product generated by lysoPLD. As shown by the Line-
weaver-Burk plot in Figure 4A-1, the intersection point of the
graphs with and without LPA was just on the y-axis. This
indicates that LPA behaves as a competitive inhibitor. On the
other hand, the modes of action by flavonols such as robinetin
were noncompetitive or mixed (Figure 4B-1). Interestingly, in the
case of FAs and NATs, the kinetic type of enzyme inhibition was
dependent on the length of the carbon chain of the fatty acid or
fatty acyl moiety. Liposoluble substances with longer carbon
chains (12:0-FA, 12:0-NAT) functioned mainly as competitive
inhibitors as well as LPAs, whereas those with shorter carbon
chains such as 6:0-FA and 5:0-NAT acted via a mixed inhibition.
Both 2:0-FA and 2:0-NAT were noncompetitive inhibitors.
Considering these results together, test substances could be
classified by differences in their affinities to the catalytic and
noncatalytic binding sites as a competitive, noncompetitive
inhibitor and a mixed type inhibitor. These results suggest that
lysoPLD has a catalytic binding site on which the substrate,
competitive inhibitor, and mixed type inhibitor act and that it has
at least one noncatalytic binding site on which the noncompetitive
and mixed type inhibitors act. Even short fatty acids such as 2:0-
FA were capable of inducing a conformational change of serum
lysoPLD by binding to this noncatalytic binding site. Kinetic
parameters of inhibition by test substances on hydrolysis of LPN
by lysoPLD are shown in Table 1.
Dilution Dependence of Inhibition of LysoPLD Activity by
Various Types of Substances. As shown in Figure 5, the increases
in absorbance that correspond to the production of p-nitrophenol
were observed with time after incubation of diluted and undiluted
FBS with LPN in the presence and absence of a test substance. In
the case of LPA used as an inhibitor, there was a large difference
between its inhibitory effects when incubated with diluted and
undiluted FBS. A significant inhibition of lysoPLD activity by
LPAs at 0.1 mM was observed on lysoPLD activity when 0.5%
FBS was used as the enzyme-containing solution (final, 0.83%).
However, no significant inhibition by LPAs was observed at this

6058 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Ueda et al.
Table 1. Comparison of Kinetic Parameters Obtained by Lineweaver-Burk
Plot Analysis of LPA-, FA-, NAT-, and Robinetin-Induced Inhibition of LysoPLD
Activity against LPNa
K
m (μM)
V
max (abs/h)
(þ) ratio of Vmax (()
test substance (-) (þ) ratio of Km (() (-)
18:1-LPA
12:0-FA
12:0-NAT
robinetin
6:0-FA
12 163
24 126
13 213
13.1
5.3
0.040 0.038
1.0
1.3
1.7
0.8
0.8
0.9
0.7
0.7
0.052 0.068
0.040 0.068
0.043 0.033
0.053 0.042
0.061 0.052
0.043 0.031
0.042 0.030
16.9
2.6
14
33
26
18
19
35
43
31
15
14
1.3
1.2
5:0-NAT
2:0-FA
2:0-NAT
0.8
0.7
a Kinetic parameters of inhibition by 18:1-LPA, 12:0-FA, 12:0-NAT, robinetin, 6:0-
FA, 5:0-NAT, 2:0-FA, and 2:0-NAT were obtained by Lineweaver-Burk plot analysis
(Figure 4). The upper, middle, and lower two or three substances in the table were
competitive, mixed, and noncompetitive inhibitors, respectively. LysoPLD activity in
0.83% FBS was calculated from the initial rate measured for 4 h of incubation at
37 °C. Both Km and Vmax were measured in the presence (þ) and absence (-) of
the inhibitor, respectively. Values are means of duplicate determinations of one to
four experiments.
concentration when undiluted FBS was used as the enzyme
solution. Potent product inhibition by LPA in a mainly compe-
titive manner was observed in a previous experiment in which
conditioned medium from ATX-transfected insect sf9 cells was
used as an enzyme source (37). The discrepancy between our
result with undiluted FBS and the previous result may be
explained by considering that higher concentrations of certain
proteins in undiluted FBS than in conditioned medium of sf9 cells
prevent the product inhibition by LPA in undiluted FBS. A
similar diluted FBS-dependent inhibition was observed for FAs,
whereas only a small inhibition of lysoPLD activity was observed
in undiluted FBS. On the other hand, NATs inhibited lysoPLD in
diluted and undiluted FBS to a similar extent. Among the test
compounds, flavonols were the most refractory to reduction of
their inhibitory actions when tested on undiluted FBS. These
results indicate that the bioavailability of LPAs and FAs might be
minimal in vivo. On the other hand, the flavonols and NATs
tested were less sensitive to adsorption by serum proteins and,
thus, may behave as more potent inhibitors in vivo than in the in
vitro assay system. Flavonols and related substances including
NATs may be more practical for development as a medical
prototype candidate drug than LPAs and FAs.
Figure 5. Different inhibitory potencies of LPAs (1), FAs (2), NATs (3),
and flavonols (4) on lysoPLD activity in diluted and undiluted FBS. (A, B)
Diluted (final, 0.83%) and undiluted (final, 16.7%) FBS was incubated with
LPN (final, 75 μM) at 37 °C for 4 h in the presence or absence of the
inhibitory substance (final, 0.1 mM). The absorbance at 405 nm was
measured every 20 min, 30 min, or 1 h for 4 h. Values are means of
duplicate determinations. (C) Inhibitory effects of 16:0-LPA, 20:4-FA, 12:0-
NAT, and myricetin on lysoPLD activity are compared as representative of
LPA, FA, NAT, and flavonol groups. White and black columns show
percentages of inhibition by the test substances measured with diluted
(final, 0.83%) and undiluted (final, 16.7%) FBS, respectively.
Structure-Activity Relationship of Inhibition on LysoPLD Activity by
Polyphenols. The structures of flavonols, phenolic acids, and
related substances tested in this study are shown in Table 2. Some
flavonols (kaempferol, tamarixetin), flavones (luteolin, apigenin,
chrysin), flavanols [(þ)-catechin, (-)-epicatechin], isoflavones
(daidzein, genistein), other polyphenols (resveratrol), caffeine,
isorhamnetin-3-O-glucoside was almost equipotent to its aglycone
isorhamnetin in inhibition of serum lysoPLD activity. These results
indicate the free hydroxyl group at the 3-position on the C ring
might be not essential to inhibit serum lysoPLD activity.
L-amino acids [tyrosine, phenylalanine, and 3-(3,4-dihydroxy-
The inhibitory effects of various phenolic acids on lysoPLD
activity were relatively lower than that of 16:0-LPA (Figure 6B). We
confirmed that caffeic acid as a representative phenolic acid func-
tioned as a noncompetitive inhibitor of the lysoPLD activity. In the
FA and NAT classes, the substances with a longer methylene chains
had stronger inhibitory effects on lysoPLD activity (Figure 6C,D).
The inhibition of lysoPLD activity by substances functioning mainly
as a noncompetitive inhibitors was rather moderate, except for some
flavonols, whereas the substances that showed stronger inhibitory
potency functioned as competitive inhibitors.
phenyl)alanine] and phenolic antioxidants (propyl gallate,
BHT, BHA), methyl (R)-(þ)-2-(4-hydroxyphenoxy)propionate,
3-(4-hydroxyphenyl)-1-propanol, and 4-(4-hydroxyphenyl)-2-bu-
tanone were inactive. The potencies of lysoPLD inhibition of active
flavonols and phenolic acids relative to that of 16:0-LPA were
obtained and are shown in Figure 6A,B, respectively. The open and
shaded columns show results with the inhibitors at 0.1 and 0.3 mM,
respectively. The pattern of relative inhibitory potency obtained
at 0.3 mM was essentially similar to that at 0.1 mM. Among
group A, flavonols such as myricetin, robinetin, and quercetagetin
showed a stronger inhibitory effect on the lysoPLD activity than
16:0-LPA when assayed at 0.1 mM (Figure 6A). The inhi-
bitory potencies of isoquercitrin and rutin on serum lysoPLD
activity were roughly equal to its aglycone quercetin. Similarly,
DISCUSSION
To identify nonlipid inhibitors of lysoPLD activity of ATX
with satisfactory bioavailability, a virtual screening approach was

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J. Agric. Food Chem., Vol. 58, No. 10, 2010 6059
Table 2. Structural Features and Inhibitory Effects on LysoPLD Activity of Fiavonols, Phenolic Acids, and Their Related Substances Testeda
a The four compounds are abbreviated as follows: methyl (R)-(þ)-2-(4-hydroxyphenoxy)propionate (MHPP); 3-(4-hydroxypheny)-1-propano1 (HPP); 4-(4-hydroxypheny)-2-
butanone (HPB); and L-3-(3,4-dihydroxyphenyl)alanine (L-DOPA). ; shows substances providing <5% inhibition at the maximal concentration tested (0.3 or 1 mM).
developed based on the structural model of the ATX catalytic site
that was simulated by computational strategies such as database
screening guided by docked complexes of candidate structures
and a binary QSAR model based on known substrates and
inhibitors of ATX. Nine compounds were identified that inhibit
lysoPLD activity toward the synthetic substrate 1 μM FS-3 at an
IC50 value of <10 μM (22). A recent study with a high-
throughput screening test assessment found a potent inhibitor
of the lysoPLD activity of ATX purified from the culture medium
of adipocytes, which were effective at nanomolar concentra-
tions (38). In the current study, we found several substances
inhibited lysoPLD activity under our assay conditions, although
their potencies were not higher than the reported lysoPLD
inhibitors with an IC50 value at submicromolar concentra-
tions (38, 39). Owing to a 75 times higher concentration of
LPN than that of FS-3, however, the direct comparison of IC50
values of inhibitory substances obtained in this study with those
reported previously (22,39) may not be useful. The IC50 values of
several substances tested in the current study would be reduced
considerably when assayed with LPN at the low concentration
used for FS-3. Among flavonols tested in this study, myricetin,
robinetin, and quercetagetin were found to exert the most potent
inhibitory effect on lysoPLD activity in diluted FBS. As their
common structural feature, they have three hydroxyl groups
bound to the A or B ring, one double bond, and one carbonyl
group in the C ring in the flavonol skeleton, allowing these three
rings to exist on the same plane. Because flavanols such as
catechin that have three hydroxyl groups on the A or B ring
but no double bond and carbonyl group in the C ring failed to
inhibit the lysoPLD activity (Table 2), the coplanarity of the A, B,
and C rings is necessary to bind stereospecifically to the lysoPLD
enzyme, inhibiting its activity in a noncompetitive fashion.
Synthetic antioxidants such as BHA and BHT did not inhibit
lysoPLD activity in this study, indicating that the observed
inhibitory action of some flavonols was not due to their anti-
oxidant activity. Judging from the reported structure-activity
relationships of the metal-chelating activity of flavonols (40, 41)
that were distinct from that of the lysoPLD-inhibitory activity
found in this study, the inhibition of lysoPLD activity of ATX by
some flavonols is unlikely due to the flavonol-induced metal
chelation from ATX, a metalloenzyme (2,28). Deletion of the C-
30 hydroxyl group of the B ring of luteolin to form apigenin was
shown to abolish the inhibitory effects on phosphodiesterases 4
and 5 (25). By analogy with this paper, deletion of the C-30

6060 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Ueda et al.
Figure 6. Relativepotencies of inhibitory activity of different compounds on lysoPLD activity in 4 h of incubation of 0.83% FBS toward LPN to that of 16:0-LPA.
The final concentrations of the substrate (LPN) and the inhibitors were 75 μM and 0.1 or 0.3 mM, respectively. Values are means of duplicate determinations.
N.D. = not determined. The following abbreviations are used in this figure: HPPA, 3-(4-hydroxyphenyl)propionic acid; HPAA, 4-hydroxyphenylacetic acid;
NHPG, N-(4-hydroxyphenyl)glycine; 2:0-NAP, N-acetyl-L-phenylalanine; GA, gallic acid; PPA, 3-phenylpropionic acid; DPPA, 3-(3,4-dihydroxyphenyl)pro-
pionic acid.
hydroxyl of the B ring of quercetin to form kaempferol was found
to result in the loss of the inhibitory effect in this study. In
addition, methylation of the C-40 hydroxyl of the B ring of
quercetin to form tamarixetin eliminated the inhibitory effect,
indicating that an intact hydroxyl group is essential for the
inhibitory action, whereas no significant reduction of the inhibi-
tory effect due to methylation of the C-30 hydroxyl group of
quercetin to form isorhamnetin was observed. The lysoPLD-
inhibiting activity of morin with C-20- and C-40- but not C-30-
hydroxyl groups on the B ring was equipotent to that of quercetin
having C-30- and C-40-hydroxyl groups on the B ring.
Both LPA and FA, naturally occurring lipids, showed signi-
ficant inhibitory effects on serum lysoPLD activity when FBS
was diluted. However, we found that both acidic lipids failed to
inhibit lysoPLD in undiluted serum. This result means that the
inhibitory potency of LPA is masked by one or more components
in serum ex vivo and plasma in the blood circulation in vivo,
respectively. In fact, LPA accumulates at an almost constant rate
for 1-2 days in serum and plasma preparations from various
animal species during incubation at 25-37 °C, indicating no
potent inhibition of lysoPLD activity by LPA produced by this
enzymeduringexvivoincubationsofplasmaandserum(8,18,42).
Some plasma proteins including albumin may sequester LPA
away from its direct inhibitiory action toward lysoPLD activity of
ATX. However, this might not be the case for some biological
fluids, such as cerebrospinal fluid, that have a low protein
concentration. Thus, it is likely that LPA regulates its own
production by ATX/lysoPLD in certain local environments. On
the other hand, inhibition by some flavonols was still observed in
undiluted FBS, which reflects an in vivo pathological condition of
blood circulation. Flavonols are taken in as food constituents and
absorbed into the blood. In human blood circulation, flavonols
from the diet may be physiologically more important in inhibiting
plasma lysoPLD than endogenously circulating lipids such as
LPA and FA, which are largely sequestered by plasma proteins
including albumin. Interestingly, the inhibitory potencies of three

Article
J. Agric. Food Chem., Vol. 58, No. 10, 2010 6061
flavonol glycosides were almost equal to those of their aglycons in
inhibiting plasma lysoPLD activity, suggesting that the sugar
moiety of flavonol glycosides contributes to its inhibitory action
to only a small extent. Because flavonoids such as quercetin are
converted to glucuronidated or sulfated forms by intestinal and
hepatic enzymes, flavonoids exist mainly as conjugated forms in
the blood circulation. Our results showing essentially equipotent
inhibitoryactions by the glycosidesofquercetin and isorhamnetin
on serum lysoPLD activity to those of their aglycons suggest that
glucuronized flavonols in the blood circulation would be effective
in inhibiting plasma lysoPLD activity in vivo. In this sense, it
should be mentioned that the inhibitory effect of chemically
noncatalytic binding site. The dual inhibition also seems to be the
case of the lysoPLD substrate LPC. A recent study comparing the
inhibitory effect of LPCs with a distinct acyl chain length showed
that 14:0- and 16:0-LPCs more effectively reduced the hydrolysis
of FS-3, a fluorogenetic synthetic substrate for ATX (36), than
12:0-LPC did, whereas the choline production from exogenous
LPC by ATX was optimal for 12:0- and 14:0-LPC (45). They
hypothesized that 16:0-LPC binds to ATX more efficiently than
12:0-LPC, but that the former is not well positioned for enzymatic
hydrolysis. Thus, the substrate LPCs bound to both the catalytic
and noncatalytic binding sites with distinct affinities, the ratios of
which varied depending on its acyl chain length.
synthesized quercetin 3-
D
-O-glucuronide on myeloperoxidase
Several flavonols and phenolic acids were both found to inhibit
serum lysoPLD activity, mainly in a noncompetitive or mixed
inhibition manner. Interestingly, in the case of FAs and NATs,
the main mode of lysoPLD inhibition was dependent on the
length of the hydrocarbon chain of the free fatty acid or fatty acyl
moiety. The major mode of inhibition by more liposoluble
substances with longer carbon chains such as 12:0-FA and
12:0-NAT was competitive, whereas the mode of inhibition by
FAs and NATs with shorter carbon chains (2:0-FA and 2:0-
NAT) was noncompetitive. The mode of inhibition for the
analogues having a medium chain length (6:0-FA and 5:0-NAT)
was the mixed type. These results suggest that lysoPLD possesses
at least one noncatalytic binding site on which several low
molecular weight compounds bind toinhibit the lysoPLD enzyme
noncompetitively. The noncatalytic binding site seems to accept
smaller molecules, indicating a smaller entry cavity to the site, in
comparison with the catalytic binding site. On the other hand, the
test molecules with larger sizes correlate to the higher affinity to
the catalytic binding site, suggesting that the size of this entry
pocket is relatively large. Additionally, there were few differences
activity was reported to be comparable to that of the quercetin
aglycone (35). Although the blood concentration of flavonol
conjugates may not reach an effective concentration to inhibit
plasma lysoPLD significantly in vivo, an increase in its blood
concentration is expected when a larger amount of flavonols is
consumed as a dietary supplement. When our results are con-
sidered in combination with previous papers showing that most
reportedlysoPLDinhibitorshavepoorbioavailability(20,22,38),
the water-soluble flavonol glycosides or conjugates with better
bioavailability might be a prototype for developing a potential
medical drug and food supplement, respectively, based on the
inhibitory action on lysoPLD activity of ATX.
In this study, several phenolic acids were found to have a
moderate inhibitory effect on lysoPLD activity in diluted FBS. In
the case of phenolic acids, a free carboxylic group was necessary,
but unlike the case of flavonols, multiple hydroxyl groups on a
benzene ring were not a critical structural feature. The presence of
a free amino group in the structure of phenolic acids was not
effective in inhibiting plasma lysoPLD activity. Whereas the
major structural unit of phenolic acids corresponds to the partial
structural unit of flavonols, the mode of interaction of phenolic
acids with lysoPLD enzyme would be different from that of
flavonols.
The structure characterization and enzymatic properties of
ATX/lysoPLD have been well characterized, but there are few
reports on its three-dimensional structure. The information
available now is all indirect and involves simulation of data
obtained from crystallized bacterial NPPs, which have structural
homology with ATX/lysoPLD (43). In addition, the molecular
mechanism of its physiological regulation remains obscure. In
this study, we obtained information about the sizes of the
structural pockets of ATX/lysoPLD enzyme by systematically
comparing the inhibitory potency and effect of different test
substances on lysoPLD activity. Our results suggest the existence
of pockets in which some small-molecule compounds can be
sequestered, regulating the enzyme activity competitively and
noncompetitively. LPA, a product of lysoPLD, was reported to
behave as an inhibitor of lysoPLD itself in vitro, as well as
sphingosine 1-phosphate (37). The mixed inhibition mode, which
has a much larger contribution to competitive inhibition, was
suggested for the inhibitory effect of these lipid phosphates on
lysoPLD activity of recombinant ATX toward a fluorescence
energy transfer-based phosphodiesterase sensor, because the
intersection point of the graph with and without LPA deviated
slightly from the y-axis of the Lineweaver-Burk plot (37). Liu
et al. reported that 16:0-, 18:1-, 18:2-, and 20:4-LPA inhibited the
activity of lysoPLD purified from FBS in a competitive fashion
with Ki values of 1.4, 1.2, 0.21, and 0.55 μM, respectively (44).
We confirmed that the main mode of LPA action on lysoPLD is
competitive inhibition by the Lineweaver-Burk plot of
data obtained under our assay conditions, indicating a much
higher affinity of LPA to the catalytic binding site than to the
in inhibitory potencies on lysoPLD activity between L- and D-
forms of 3:0-NAT and 16:0-NAT. This poor stereospecificity for
inhibition of lysoPLD activity might mean high flexibility of the
catalytic and noncatalytic pockets, which can accept different
types of molecules, leading to conformational changes around the
catalytic site. In this sense, NATs and related substances seem to
be useful synthetic compounds to evaluate the molecular mechan-
ism of conformational changes of ATX/lysoPLD leading to
inhibition and activation of the enzymatic activity. Consistent
with this present result, ATX is not stereoselective in its recogni-
tion of glycerol-based phospholipids because none of the enan-
tiomeric pairs of alkylglycerophosphonocholines (C16:0 and
C18:0), analogues of natural alkyl-LPC, showed statistically
different inhibitions of lysoPLD activity of ATX (45). Consider-
ing these findings together, the lysoPLD inhibitors found in this
study were classified into three groups: inhibitors that exert their
effect mainly in a competitive manner, a noncompetitive manner,
or both (mixed-type inhibition).
LysoPLD/ATX is known to be up-regulated in different types
of cancers and is considered to be an endogenous promoter of
tumor metastasis. Thus, it is an attractive target for a therapy
suppressing tumor aggressiveness. In the present study, we
systematically investigated several groups of compounds as
potential lysoPLD inhibitors and found that flavonols are the
best candidates among those tested in this study. Unfortunately,
however, the effective concentrations shown in this study are
not easily reached under normal physiological conditions because
the in vivo plasma concentration of total metabolites of flavo-
noids was estimated to vary from 0.1 to 10 μM even after the
consumption of a flavonoid-rich meal (46, 47). However, our
results on the structure-activity relationships of flavonols
and related substances will be useful for exploring more effec-
tive food components or developing compounds with higher

6062 J. Agric. Food Chem., Vol. 58, No. 10, 2010
Ueda et al.
therapeutic potential. Further work is needed to extend the
present results.
(8) Tokumura, A.; Miyake, M.; Nishioka, Y.; Yamano, S.; Aono, T.;
Fukuzawa, K. Production of lysophosphatidic acids by lysopho-
spholipase D in human follicular fluids of in vitro fertilization
patients. Biol. Reprod. 1999, 61, 195–199.
The lysoPLD activity of plasma ATX is constitutively active,
but may be negatively regulated to slowly generate LPA in vivo,
although the in vivo plasma level of LPC, the predominant
physiological substrate for lysoPLD in human beings, is as high
as its Km value for highly purified lysoPLD/ATX from human
plasma in vitro (2). In this study, we obtained useful information
about the conformational structures of ATX/lysoPLD that are
involved in the physiological regulation of the lysoPLD activity of
ATX by using the inhibitory compounds as a useful tool for
investigating physiological and pathophysiological significances
of LPA production by ATX/lysoPLD in humans. Furthermore,
our present findings will be useful for future identification of
natural regulatory mechanisms and structural factors of ATX
involved in lysoPLD activity in human biological fluids.
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ABBREVIATIONS USED
ATX, autotaxin; BHA, 3-tert-butyl-4-hydroxyanisole; BHT,
2,6-di-tert-butyl-p-cresol; BSA, bovine serum albumin; DMSO,
dimethyl sulfoxide; FA, fatty acid; FBS, fetal bovine serum;
LC-MS/MS, liquid chromatography-tandem mass spectrome-
try; LPA, lysophosphatidic acid; LPN, lysophosphatidyl-p-nitro-
phenol; lysoPLD, lysophospholipase D; NAT, N-acyltyrosine;
NPP, ecto-nucleotide pyrophosphatase/phosphodiesterase; PA,
phosphatidic acid; TLC, thin-layer chromatography. The fatty
acyl moieties of lysophospholipids, free fatty acids, and N-acyl
tyrosines are designated in terms of the numbers of carbon atoms
and double bonds.
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ACKNOWLEDGMENT
(16) Kishi, Y.; Okudaira, S.; Tanaka, M.; Hama, K.; Shida, D.; Kitayama,
J.; Yamori, T.; Aoki, J.; Fujimaki, T.; Arai, H. Autotaxin is over-
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We thank Dr. T. Kishimoto, Alfresa Pharma, Osaka, Japan,
and Dr. J. Aoki, Tohoku University, Sendai, Japan, for the
generous gifts of LPN and human recombinant ATX, respectively.
(17) Baumforth, K. R.; Flavell, J. R.; Reynolds, G. M.; Davies, G.; Pettit,
T. R.; Wei, W.; Morgan, S.; Stankovic, T.; Kishi, Y.; Arai, H.;
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