A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-induced paw edema in mice

Article abstract of DOI:10.1016/j.bcp.2013.06.021

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase is a key enzyme in the biosynthesis of 1-O-alkyl-2-acetyl-sn- glycero-3-phosphocholine (PAF) in inflammatory cells. Sub-stances which inhibit this enzyme are of therapeutic inte

Full text of DOI:10.1016/j.bcp.2013.06.021

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Contents lists available at SciVerse ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit
acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase
(
a key enzyme in platelet-activating factor biosynthesis) and
carrageenan-induced paw edema in mice
a
a
a
a
a
Yasuhiro Yamazaki , Kengo Yasuda , Tensei Matsuyama , Takuya Ishihara , Ryoko Higa ,
b
a
b
b
c
Taira Sawairi , Masahiko Yamaguchi , Masahiro Egi , Shuji Akai , Toshio Miyase ,
Akira Ikari , Masao Miwa , Junko Sugatani *
a
a
a,
a
Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
b
Department of Synthetic Organic Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
c
Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase is a key enzyme in the
biosynthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) in inflammatory cells. Sub-
stances which inhibit this enzyme are of therapeutic interest. In this study, we screened for new
inhibitors of lyso-PAF acetyltransferase with anti-inflammatory effects. In a metabolite from Penicillium
sp. F33, we isolated an acetyltransferase inhibitor identified as dihydrofumigatin (2-methoxy-1,3,4-
trihydroxy-5-methylbenzene) from high resolution mass spectrometer and NMR data. Dihydrofumi-
gatin had strong acetyltransferase inhibitory activity, but was not stable in aqueous solution. Thus, we
chemically synthesized its oxidized form fumigatin (3-hydroxy-2-methoxy-5-methyl-1,4-benzoqui-
none) and derivatives thereof, and evaluated their inhibitory effects. Strong inhibitory activity was
observed for saturated fatty acid esters of fumigatin; the order of inhibition was 3-decanoyloxy-2-
Received 23 March 2013
Accepted 20 June 2013
Available online xxx
Keywords:
Platelet activating factor
PAF biosynthesis
Lyso-PAF acetyltransferase
Fumigatin
Carrageenan-induced edema
methoxy-5-methyl-1,4-benzoquinone (termed FUD-7, IC50 = 3
canoyloxy-1,4-benzoquinone (termed FUD-8, IC50 = 20 M) > 3-hexanoyloxy-2-methoxy-5-methyl-
,4-benzoquinone (IC50 = 139 M). Interestingly, these compounds also significantly suppressed the
mM) > 2-methoxy-5-methyl-3-tetrade-
m
1
m
gene expression of lyso-PAF acetyltransferase/LPCAT2 in mouse bone marrow-derived macrophages
stimulated by lipopolysaccharide (LPS). We further evaluated the effect of these substances on anti-
inflammatory activity in vivo using the carrageenan-induced mouse paw edema test. FUD-7 and FUD-8
at 2.5 mg/kg showed significant, 47.9–51.7%, inhibition stronger than that of prednisolone at 10 mg/kg
(41.9%). These results suggest that FUD-7 and FUD-8 are potent inhibitors with anti-inflammatory
activity.
ß 2013 Elsevier Inc. All rights reserved.
chemical mediator in a diverse range of both normal and
pathophysiological responses including anaphylactic, inflamma-
tory and allergic responses [1–3] through its G protein-coupled
receptor (PAF receptor) [4,5]. Accordingly, many PAF antagonists
1
. Introduction
Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-
3
-phosphocholine) has been shown to function as a potent
Abbreviations: PAF, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; c-PAF, 1-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine;
lyso-PAF, 1-O-alkyl-sn-glycero-3-phosphocholine; LPCAT2, lysophosphatidylcholine acyltransferase 2; HPLC, high-performance liquid chromatography; HRMS, high
resolution mass spectrometer; DART, direct analysis in real time; TLC, thin layer chromatography; TCA, trichloroacetic acid; LPS, lipopolysaccharide; BMDMs, bone marrow-
derived macrophages; NDGA, nordihydroguaiaretic acid; FUD-1, 3,4-dihydroxy-1,2-dimethoxy-5-methylbenzene; FUD-2, 2-methoxy-5-methyl-1,3,4-triacetoxybenzene;
FUD-3, 1,4-diacetoxy-2-methoxy-5-methyl-3-tetradecanoyloxybenzene; FUD-4, 3-hydroxy-2-methoxy-5-methyl-1,4-benzoquinone (fumigatin); FUD-5, 3-acetoxy-2-
methoxy-5-methyl-1,4-benzoquinone; FUD-6, 3-hexanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone; FUD-7, 3-decanoyloxy-2-methoxy-5-methyl-1,4-benzoquinone;
FUD-8, 2-methoxy-5-methyl-3-tetradecanoyloxy-1,4-benzoquinone; FUD-9, 2-methoxy-5-methyl-3-octadecanoyloxy-1,4-benzoquinone.
*
Corresponding author. Tel.: +81 54 264 5779; fax: +81 54 264 5779/+81 54 264 5773.
E-mail address: sugatani@u-shizuoka-ken.ac.jp (J. Sugatani).
0006-2952/$ – see front matter ß 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bcp.2013.06.021
Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

G Model
BCP-11676; No. of Pages 13
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Y. Yamazaki et al. / Biochemical Pharmacology xxx (2013) xxx–xxx
have been developed, and several have been reported to inhibit
vascular disease [6], asthma [7], renal ischemia [8], pancreatic
disease [9], and the systemic inflammatory response syndrome
(Uppsala, Sweden), pyridine and 2,6-lutidine from Kishida
Chemical (Osaka, Japan), decanoyl chloride, tetradecanoyl chlo-
ride, and octadecanoic anhydride from TCI (Tokyo, Japan), acetic
[
10]. However, inhibitors of PAF synthesis as anti-allergic and/or
anhydride from Kanto Chemical (Tokyo, Japan), l-carrageenan
anti-inflammatory drugs have until now not been commercially
available.
from Nacalai Tesque (Kyoto, Japan), bacto malt extract, bacto
yeast extract, bacto peptone from BD (Sparks, MD) and
lipopolysaccharide (LPS) Escherichia coli 0111:B4 from Difco
Laboratories (Detroit, MI).
PAF activates numerous types of cells that may be involved in
inflammatory and allergic processes, such as neutrophils, baso-
phils, eosinophils, monocytes/macrophages, mast cells and endo-
thelial cells. These cells also synthesize PAF in response to
extracellular stimuli via a remodeling pathway. In this pathway,
2.2. Animals
1
-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF), the precursor
of PAF, is synthesized from 1-O-alkyl-2-arachidonoyl-sn-glycero-
-phosphocholine (1-alkyl phosphatidylcholine; PC) by the actions
of phospholipase A . Lyso-PAF is subsequently converted to PAF by
All the experiments followed protocols approved by the
Institutional Animal Care and Use Committee, University of
Shizuoka. Male Wistar rats, ddY mice and C57BL/6 mice were
obtained from Nihon SLC (Hamamatsu, Japan) and acclimatized for
1 week before the experiments. The animals were housed in
stainless-steel hanging cages with free access to food and water,
and were maintained on a 12 h light/dark cycle.
3
2
acetyl-CoA:lyso-PAF acetyltransferase, a key enzyme in PAF
biosynthesis. Recently, two endogenous lyso-PAF acetyltrans-
ferases were identified: the constitutively expressed enzyme
LPCAT1 (lysophosphatidylcholine acyltransferase 1) [11,12], and
the inducible enzyme, LPCAT2 [13,14]. In these studies, the
endogenous lyso-PAF acetyltransferase in inflammatory cells was
activated by prophlogistic stimuli [15], and the LPCAT2 in mouse
peritoneal macrophages and RAW264.7 cells was indeed activated
by lipopolysaccharide (LPS) through toll-like receptor 4 [13,14].
Thus, in addition to the development of specific PAF antagonists,
substances which are capable of inhibiting these enzymes
selectively would be useful for inhibiting the pathological
functions of PAF under inflammatory conditions.
2.3. Cultivation of the strain of Penicillium sp. F33
The ITS-5.8S rDNA sequence of a fungus in which lyso-PAF
acetyltransferase inhibitory activity had been detected in the
culture supernatant was identical to that of Penicillium sp. F33
(GenGank accession No. JF439500) identified by TechnoSuruga
Laboratory Co.,Ltd. The culture medium used had the following
composition: Bacto malt extract, 20 g; Bacto yeast extract, 1 g;
Bacto peptone, 1 g; glucose, 20 g in 1000 ml of distilled water, and
the pH was adjusted to 6.0 with NaOH. This medium was
distributed in 400 ml portions, sterilized, inoculated with spores
and cultivated at 30 8C.
We previously developed a simple and reproducible method
(the TCA precipitation assay) for screening substances which
inhibit acetyltransferase activity, and showed that major compo-
nents from the cortex of Magnoliae (magnolol and honokiol), and
the polyphenols from green tea and black tea (catechins and
theaflavins) were potent inhibitors of PAF biosynthesis [16,17]. By
using this method, we newly found strong inhibitory activity for an
acetyltransferase in the culture broth of a fungus from among 8000
species of actinomyces and fungi. In this study, we isolated an
inhibitor from Penicillium sp. F33, and identified it as dihydrofu-
migatin (2-methoxy-1,3,4-trihydroxy-5-methylbenzene). The
substance was unstable in aqueous solution, and easily inter-
converted to its oxidized and more stable form fumigatin (3-
hydroxy-2-methoxy-5-methyl-1,4-benzoquinone). Therefore, we
chemically synthesized fumigatin and its derivatives, and exam-
ined their effect on the activity of lyso-PAF acetyltransferase in rat
spleen microsomes and the enzyme induction by LPS in mouse
bone marrow-derived macrophages. Further, to evaluate the
efficacy of these compounds in vivo, we performed the carrageen-
an-induced mouse paw edema test.
2.4. Isolation of PAF synthesis inhibitors
The culture broth (about 48 l in total) was separated by
filtration from the mycelium with four layers of gauze. The culture
filtrate was extracted with ethyl acetate by shaking at least five
times. The ethyl acetate solution was evaporated and the reddish
brown residue (20.4 g) was dissolved with chloroform. The
chloroform solution was applied to a column packed with 230 g
of PSQ100B silica gel (Fuji silysia, Japan), then eluted with
chloroform–methanol (98.5:1.5). A total of 49 fractions (200 ml
each) were collected. Similar fractions were combined after TLC
analysis to yield seven fractions (A1: 0.1 g, A2: 0.4 g, A3: 1.3 g, A4:
5.3 g, A5: 2.6 g, A6: 3.3 g, A7: 0.6 g) (Fig. 2). The subfraction A5
(0.1 g), which had the highest specific activity for inhibition of PAF
synthesis, was redissolved in 50% acetonitrile in water and
injected onto the COSMOSIL 5PE-MS HPLC column (20 ꢀ 250 mm,
Nacalai tesque, Japan). The HPLC column was eluted with 4%
acetonitrile in water containing 0.05% trifluoroacetic acid at a flow
rate of 6.5 ml/min. The UV detector was set at 250 nm. Fractions
were collected as indicated in Fig. 2C, dried in vacuo, and then
subfractions B1 (2.5 mg), B2 (59.7 mg), and B3 (30.5 mg) were
obtained.
2
. Materials and methods
2.1. Chemicals
1
-Hexadecyl-sn-glycero-3-phosphocholine (lyso-PAF) was
obtained from Cayman Chemical (Ann Arbor, MI), 1-hexadecyl-
-N-methylcarbamyl-sn-glycero-3-phosphocholine (c-PAF) from
2
2.5. Stability of fumigatin derivatives
Merck Millipore (Billerica, MA), WEB2086 from Boehringer
3
Ingelheim (Ingelheim am Rhein, FRG), [ H]Acetyl-CoA
(
The stability of fumigatin and dihydrofumigatin was analyzed
by HPLC separation. The sample solution dissolved in 50%
acetonitrile in water (1.25 mg/ml) was stored in sealed glass
containers at room temperature for 0, 24 and 72 h. The stored
92.5 GBq/mmol) from Perkin Elmer (Boston, MA), acetyl-CoA,
sodium fluoride (NaF), trichloroacetic acid (TCA), trifluoroacetic
acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic
acid, stearic acid, hexanoic anhydride, zinc, and sodium acetate
from Wako Chemicals (Tokyo, Japan), 4-dimethylaminopyridine,
nordihydroguaiaretic acid, 6-p-toluidino-2-naphthalene sulfonic
acid, prednisolone and BSA (fractionV, essentially fatty acid free)
from Sigma (St Louis, MO), ficoll–paque from GE Healthcare
samples (10
ml) were injected onto the Mightysil RP-18GP column
(4.6 ꢀ 250 mm, Kanto reagent, Japan). The HPLC column was
eluted with 20% acetonitrile in water containing 0.05% trifluor-
oacetic acid at a flow rate of 1.0 ml/min. The UV detector was set at
250 nm.
Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

G Model
BCP-11676; No. of Pages 13
Y. Yamazaki et al. / Biochemical Pharmacology xxx (2013) xxx–xxx
3
Scheme 1.
2.6. NMR
2.9. Synthesis of fumigatin and its derivatives
1
13
H and C NMR spectra were recorded on a JEOL JNM-
a
-400
3,4-Dihydroxy-1,2-dimethoxy-5-methylbenzene (FUD-1), 3-hy-
droxy-2-methoxy-5-methyl-1,4-benzoquinone (fumigatin, FUD-4),
and 3-acetoxy-2-methoxy-5-methyl-1,4-benzoquinone (FUD-5)
were synthesized according to procedures (Scheme 1) described
previously, and the spectroscopic data were in good agreement with
those in the literature, except for the C NMR data for 3-acetoxy-2-
methoxy-5-methyl-1,4-benzoquinone (FUD-5). There seems to be a
typographical error in the reported data [18,19]. FUD-2, 3, and 6–9
were prepared as shownin Scheme 1, whose details are given below.
FT-NMR or JEOL JNM-ECA 500 spectrometer and chemical shifts
were given as values with TMS as the internal standard in
acetone-d or CDCl
d
6
3
.
1
3
2
.7. Preparation of rat spleen microsomal fractions
Spleens were obtained from male Wistar rats. The tissue was
minced in 9 volumes of a 0.25 M sucrose solution, 1 mM
ethylenediamine tetraacetic acid (EDTA) and 25 mM NaF, and
homogenized with four strokes of a glass–Teflon homogenizer. The
homogenization and subsequent fractionation procedures were
carried out at 4 8C. Nuclei and cell debris were removed by
centrifugation at 900g for 10 min. Microsomes were pelleted from
the 9000g (10 min) supernatant by centrifugation at 105,000g for
0 min. The pellet was suspended in homogenization medium
containing no EDTA. The microsomal preparations were stored at
80 8C.
2.9.1. 3-Acetoxy-2-methoxy-5-methyl-1,4-benzoquinone (FUD-5)
A brown solid, mp 89–91 8C (lit. mp 92–94 8C) [19]. IR (KBr)
n
ꢁ
1 1
2926, 1778, 1663, 1618 cm ; H NMR (500 MHz, CDCl
3
)
d
2.05 (d,
13
3H, J = 1.5 Hz), 2.34 (s, 3H), 4.08 (s, 3H), 6.50 (d, 1H, J = 1.5 Hz);
NMR (125 MHz, CDCl 15.5, 20.4, 60.4, 131.3, 134.9, 144.7, 147.6,
168.1, 181.2, 182.8; high resolution mass spectrometer (HRMS)
C
3
) d
6
(direct analysis in real time. DART) m/z calculated for C10
[M + H] : 211.0601, found: 211.0588.
H
11
O
5
+
ꢁ
2
.8. Assay for acetyl-CoA: Lyso-PAF acetyltransferase activity (TCA
2.9.2. Preparation of 3-hexanoyloxy-2-methoxy-5-methyl-1,4-
benzoquinone (FUD-6) using fumigatin and hexanoic anhydride
precipitation assay)
Hexanoic anhydride (0.14 ml, 0.60 mmol) and pyridine (0.10 ml,
1.2 mmol) were added to a mixture of fumigatin (50.0 mg,
0.30 mmol) and 4-dimethylaminopyridine (1.8 mg, 0.015 mmol)
The activity of the acetyltransferase in rat spleen microsomes
was measured as follows. Rat spleen microsomes (2.5
m
g protein)
M lyso-PAF
l of 0.1 M Tris–HCl buffer (pH 6.9) containing 25 mM NaF
3
were incubated with 300
in 100
m
M [ H]acetyl-CoA and 40
m
in CH
temperature, the reaction mixture was quenched with saturated aq.
NaHCO and extracted with CH Cl . The combined organic layer was
washed with brine, dried over Na SO , and evaporated in vacuo. The
residue was purified by column chromatography (hexane/ethyl
acetate = 5:1) to give FUD-6 (51.0 mg, 64%). A brown oil. IR (KBr)
2 2
Cl (1 ml) at room temperature. After stirring for 1.5 h at room
m
and 0.1% BSA in the presence of various concentrations of
inhibitors dissolved in dimethyl sulfoxide (final concentration
3
2
2
2
4
5
%) or dimethyl sulfoxide alone as a control at 37 8C for 15 min as
described before [16]. The reaction was stopped by the addition of
l of a 5.2% BSA saline solution. After 75 l of a 30%
n
ꢁ1 1
2
5
m
m
2958, 2932, 1769, 1663, 1618 cm ; H NMR (500 MHz, CDCl ) d
3
trichloroacetic acid (TCA) solution was added, the reaction
mixture was centrifuged at 750g for 2 min to obtain [ H]acetyl-
0.92 (t, 3H, J = 7.0 Hz), 1.32–1.44 (m, 4H), 1.72–1.80 (m, 2H), 2.06 (s,
3H), 2.60 (t, 2H, J = 7.5 Hz), 4.07 (s, 3H), 6.50 (s, 1H); C NMR
3
13
PAF bound to the denatured BSA. The resulting pellet was
3
(125 MHz, CDCl ) d 13.8, 15.5, 22.2, 24.3, 31.0, 33.7, 60.3, 131.2,
dissolved in 200
% SDS (pH 8.0), and mixed with 4 ml of scintillation cocktail
clearsol I, Nacalai Tesque, Japan). Radioactivity was measured in
ml of 0.1 M sodium phosphate buffer containing
135.0,144.6, 147.6,171.0, 181.3,182.8;HRMS(DART)m/zcalculated
+
1
(
19 5
for C14H O [M + H] : 267.1227; found: 267.1242.
an Alloka LSC-3100 liquid scintillation counter. The IC50 value was
determined from a regression analysis of the concentration-PAF
production curve. Protein content was determined using a
bicinchoninic acid protein assay kit (Pierce, Rockford, IL) with
BSA as a standard.
2.9.3. Preparation of 3-decanoyloxy-2-methoxy-5-methyl-1,4-
benzoquinone (FUD-7) using fumigatin and decanoyl chloride
A mixture of fumigatin (52.0 mg, 0.31 mmol), decanoyl chloride
(0.090 ml, 0.45 mmol), and 2,6-lutidine (0.080 ml, 0.69 mmol) in
CH
O, the reaction mixture was extracted with CH
layer was washed with brine, dried over Na SO , and evaporated in
2
Cl
2
(6 ml) was stirred for 45 min at 0 8C. After quenching with
H
2
2
2
Cl . The organic
2
4
Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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Y. Yamazaki et al. / Biochemical Pharmacology xxx (2013) xxx–xxx
vacuo. The residue was purified by column chromatography
2.9.8. 1,4-Diacetoxy-2-methoxy-5-methyl-3-
tetradecanoyloxybenzene (FUD-3)
(
hexane/ethyl acetate = 5:1) to give FUD-7 (61.0 mg, 61%). A yellow
ꢁ1
ꢁ1
1
solid, mp 53–54 8C. IR (KBr)
n
d
2928, 2857, 1769, 1663, 1618 cm
0.88 (t, 3H, J = 7.0 Hz), 1.22–1.45 (m,
2H), 1.71–1.78 (m, 2H), 2.06 (d, 3H, J = 1.5 Hz), 2.60 (t, 2H,
;
Yield: 83%, a brown oil. IR (KBr)
n
2927, 2855, 1775 cm
; H
1
H NMR (500 MHz, CDCl
3
)
NMR (500 MHz, CDCl 0.88 (t, 3H, J = 7.0 Hz), 1.22–1.44 (m,
3
) d
1
20H), 1.71–1.78 (m, 2H), 2.12 (s, 3H), 2.29 (s, 3H), 2.31 (s, 3H), 2.55
(t, 2H, J = 7.5 Hz), 3.79 (s, 3H), 6.85 (s, 1H); C NMR (125 MHz,
CDCl ) d 14.1, 15.9, 20.3, 20.7, 22.7, 25.1, 29.1, 29.2, 29.3, 29.4,
3
29.56, 29.60, 29.63, 31.9, 33.8, 61.1, 121.6, 126.5, 136.8, 139.7,
13
13
J = 7.5 Hz), 4.07 (s, 3H), 6.50 (d, 1H, J = 1.5 Hz); C NMR (125 MHz,
CDCl 14.1, 15.5, 22.7, 24.7, 29.0, 29.19, 29.22, 29.4, 31.8, 33.8,
0.4, 131.2, 135.0, 144.7, 147.6, 171.1, 181.3, 182.9; HRMS (DART)
3
)
d
6
+
m/z calculated for C18
H
27
O
5
[M + H] : 323.1853; found: 323.1851.
141.2, 142.9, 167.6, 168.7, 170.6; HRMS (DART) m/z calculated for
+
C
26
H
41
O
7
[M + H] : 465.2847; found: 465.2836.
2.9.4. Preparation of 2-methoxy-5-methyl-3-tetradecanoyloxy-1,4-
benzoquinone (FUD-8)
2.10. Assay for cyclooxygenase-1, and -2, and lipoxygenase activity
Similarly to the preparation of FUD-7, FUD-8 (93.0 mg, 62%)
was prepared from fumigatin (66.6 mg, 0.40 mmol), tetradecanoyl
chloride (0.12 ml, 1.0 mmol), and 2,6-lutidine (0.12 ml,
The effect of FUD-7 and NDGA on the activity of the
cyclooxygenase-1 and -2, and 15-lipoxygenase were measured
enzymatically with commercial kits from Cayman Chemical (Ann
Arbor, MI).
0
.87 mmol). A yellow solid, mp 63–65 8C. IR (KBr)
n
2926, 2855,
ꢁ
1 1
1
769, 1663, 1618 cm
; H NMR (500 MHz, CDCl ) d 0.88 (t, 3H,
3
J = 7.0 Hz), 1.22–1.45 (m, 20H), 1.71–1.79 (m, 2H), 2.06 (d, 3H,
J = 1.5 Hz), 2.60 (t, 2H, J = 7.5 Hz), 4.08 (s, 3H), 6.50 (d, 1H,
2.11. Preparation of bone marrow-derived macrophages
13
J = 1.5 Hz); C NMR (125 MHz, CDCl
3
)
d
14.1, 15.5, 22.7, 24.7, 29.0,
9.2, 29.36, 29.42, 29.59, 29.63, 29.67, 31.9, 33.8, 60.4, 131.3, 135.1,
44.7, 147.6, 171.1, 181.3, 182.9; HRMS (DART) m/z calculated for
2
1
C
Primary bone marrow-derived macrophages (BMDMs) were
harvested from the femurs (8 to 9-week old mice) using standard
procedures [20]. These cells were cultured with RPMI/10%FCS/
penicillin and streptomycin. Thirty percent cultured supernatant
from a mouse fibroblast cell line (L929, RIKEN BRC, Ibaraki, Japan)
was added to the medium as a source of M-CSF. Seven-day-
cultured BMDMs were used for functional assays.
+
22
H
35
O
5
[M + H] : 379.2479; found: 379.2458.
2.9.5. Preparation of 2-methoxy-5-methyl-3-octadecanoyloxy-1,4-
benzoquinone (FUD-9)
Similarly to the preparation of FUD-6, FUD-9 (67.5 mg, 52%)
was prepared from fumigatin (48.6 mg, 0.29 mmol), octadecanoic
anhydride (246 mg, 0.45 mmol), pyridine (0.070 ml, 0.87 mmol),
and 4-dimethylaminopyridine (6.1 mg, 0.050 mmol). A yellow
2.12. Cell culture
ꢁ
1
5
solid, mp 74–75 8C. IR (KBr)
n
2926, 2855, 1769, 1663, 1618 cm
0.88 (t, 3H, J = 7.0 Hz), 1.22–1.45 (m,
8H), 1.71–1.79 (m, 2H), 2.06 (d, 3H, J = 1.0 Hz), 2.60 (t, 2H,
;
BMDMs (6 ꢀ 10 cells) seeded onto six-well plates and cultured
1
H NMR (500 MHz, CDCl
3
)
d
for 24 h were pretreated with compounds or vehicle for 30 min and
stimulated with 50 ng/ml of LPS. At 24 h after the stimulation, total
RNA was extracted.
2
13
J = 7.5 Hz), 4.07 (s, 3H), 6.50 (d, 1H, J = 1.0 Hz); C NMR (125 MHz,
CDCl 14.1, 15.5, 22.7, 24.7, 29.0, 29.2, 29.37, 29.42, 29.58, 29.63,
9.65, 29.69, 31.9, 33.8, 60.4, 131.3, 135.0, 144.7, 147.6, 171.1,
3
) d
2
1
4
2.13. Quantitative Real-Time PCR
+
43 5
81.3, 182.9; HRMS (DART) m/z calculated for C26H O [M + H] :
35.3105; found: 435.3091.
Total RNA was extracted using TRIzol reagent from Invitrogen
(
Carlsbad, CA), and cDNAs were synthesized with a PrimeScript RT
reagent kit (Takara Bio, Ohtsu, Japan) according to the manu-
facturer’s instructions. cDNA synthesized from 0.5 g of total RNA
2.9.6. Typical procedure for synthesis of triacyl compounds
Zinc (20 equiv) and sodium acetate (3 equiv) were added to a
m
solution of 3-acetoxy- or 3-tetradecanoyloxy-2-methoxy-5-meth-
yl-1,4-benzoquinone (1 equiv) in acetic anhydride (0.1 M) at room
temperature. After stirring for 1 h at room temperature, the
was subjected to quantitative real-time PCR with an Mx300P
Sequence Detector (Stratagene, La Jolla, CA) using SYBR Premix Ex
Taq reagent (Takara Bio, Ohtsu, Japan) for LPCAT2 (GenBank
accession number NM173014) and 18S rRNA (NR003278). The
reaction mixture was quenched with H
CH Cl . The organic layer was washed with saturated aq. NaHCO
and brine, dried over Na SO , and evaporated in vacuo. The residue
2
O and extracted with
0
2
2
3
primers for mouse LPCAT2 were 5 -GTCCAGCAGACTACGAT-
0
0
0
2
4
CAGTG-3 (forward) and 5 -CTTATTGGATGGGTCAGCTTTTC-3
0
was diluted with pyridine (100 equiv) and acetic anhydride (250
equiv), and the reaction mixture was stirred at room temperature
(reverse). Those for 18S rRNA were 5 -TTCTGGCCAACGGTCTAGA-
0
0
0
CAAC-3 (forward) and 5 -CCAGTGGTCTTGGTGTGCTGA-3 (re-
verse). The thermal profile was as follows: hold for 10 s at 95 8C,
then two-step PCR for 40 cycles of 95 8C for 5 s followed by 60 8C for
30 s. 18S rRNA was used to normalize gene expression in all
samples. Fold induction values were calculated by subtracting the
mean difference of LPCAT2 and 18S rRNA cycle threshold Ct
numbers for each treatment group from the mean difference of
LPCAT2 and 18S rRNA Ct numbers for the vehicle group and raising
overnight. After quenching with saturated aq. NaHCO
reaction mixture was extracted with diethyl ether. The organic
layer was washed with saturated aq. NaHCO and brine, dried over
Na SO , and evaporated in vacuo. The residue was purified by
3
, the
3
2
4
column chromatography (hexane/ethyl acetate) to give the desired
products.
ꢁ
xxCt
2
.9.7. 2-Methoxy-5-methyl-1,3,4-triacetoxybenzene (FUD-2)
the difference to the power of 2 (2
).
ꢁ1 1
Yield: 81%, a brown oil. IR (KBr)
n
2931, 1775 cm
; H NMR
(
3
6
500 MHz, CDCl 2.13 (s, 3H), 2.31 (s, 6H), 2.32 (s, 3H), 3.79 (s,
3
)
d
2.14. Biosynthesis and quantitation of PAF in BMDMs
1
3
H), 6.85 (s, 1H); C NMR (125 MHz, CDCl
3
) d 15.8, 20.2, 20.3, 20.7,
6
1.1, 121.6, 126.5, 136.8, 139.6, 141.3, 142.9, 167.6, 167.8, 168.7;
BMDMs (6 ꢀ 10 cells) were pretreated with compounds or
+
HRMS (DART) m/z calculated for C14
found: 297.0974.
H
17
O
7
[M + H] : 297.0969;
vehicle for 30 min and stimulated with 50 ng/ml of LPS. At 15 min
after the stimulation, the reaction was stopped by the addition of
methanol and chloroform. After centrifugation for 5 min at 1000g,
total lipids were extracted from the samples by the method of Bligh
and Dyer and separated on a TLC plate (TLC plates silica gel 60,
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alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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Merck, Whitehouse Station, NJ) using
a
solvent system of
1.2
A
2
cloroform:methanol:H O (65:35:6, by volume). The plate was
exposed to 6-p-toluidino-2-naphthalene sulfonic acid, and PAF,
located on the TLC plate between the areas corresponding to
sphingomyelin and lysophosphatidylcholine, was scraped off and
extracted by the method of Bligh and Dyer. The sample obtained
after TLC was used to determine the PAF concentration. PAF was
quantified by bioassay using rabbit platelet aggregation [16,17],
i.e., by comparison through platelet suspension stimulated by
experimental samples with that through a suspension stimulated
by a known quantity of 1-hexadecyl-2-N-methylcarbamyl-sn-
glycero-3-phosphocholine (c-PAF). Platelet aggregation activity
was measured as the change in light transmission using an
aggregometer (NBS Hematracer 601, Nikko Bioscience, Tokyo,
Japan).
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
5
10
15
20
25
30
Cultivation period (days)
2.15. Carrageenan-induced mouse foot edema
B
0.4
The anti-inflammatory activity of acetyltransferase inhibitors
was determined using a carrageenan-induced paw edema test.
Seven-week-old male ddY mice were divided into groups (n = 6–9
each group) and anti-inflammatory reagents dissolved in saline
containing 10% ethanol were administered intravenously for three
prednisolone) or one (other agents) hour before the subcutaneous
injection of carrageenan. Each group of animals were anesthetized
with diethyl ether before, receiving a subplantar administration of
0
0
0
.3
.2
.1
0
(
15 ml of saline to the left paw or 15 ml of carrageenan 2% (w/v) in
saline to the right paw. The paw thickness was measured with a
micrometer caliper immediately before the subplantar injection
and at 1, 2, 3, 4 h after the injection. The increase in paw volume
was calculated as the difference between the right paw thickness
0
5
10
15
20
25
30
(
carrageenan) and the left paw thickness (saline) measured at each
point. The assessment of paw thickness was performed always in a
triple blind manner by the same operator.
Cultivation period (days)
Fig. 1. Relationship between production of PAF acetyltransferase inhibitors and
changes in color of culture broth. The fungus was cultivated on a malt extract
medium at 30 8C for 30 days. The culture broth (10 ml) was removed at intervals of
2.16. Statistical analysis
5
days. (A) Time course of changes in the inhibitory activity toward PAF
The data were analyzed by means of Student’s t test for
acetyltransferase in culture broth. The culture broth was extracted with ethyl
acetate to obtain the PAF acetyltransferase inhibitor. Acetyltransferase activity was
measured by the TCA precipitation method as described in Section 2. Relative
inhibitory activities are expressed taking the control value obtained for the culture
broth of 20 days’ cultivation as 1. (B) Changes in absorbance of the culture broth at
unpaired samples with p < 0.05 considered significant.
. Results
.1. Isolation and structural determination of a acetyl-CoA:lyso-PAF
3
5
50 nm. The results are expressed as the mean ꢂ S.E. for three independent
3
experiments.
acetyltransferase inhibitor from Penicillium sp. F33 metabolites
To discover new inhibitors for lyso-PAF acetyltransferase, we
isolated and identified target substances from metabolites of
Penicillium sp. F33. This microorganism was cultivated on a malt
extract medium at 30 8C. The incubation period was determined by
examining the culture supernatant for a change in color and
inhibitory activity for PAF synthesis with a microsomal fraction
from rat spleen. As shown in Fig. 1, the inhibitory activity declined
at 25 d or later, at which a large change in the absorbance of the
supernatant at 550 nm occurred. In consideration of these results,
we cultivated this fungus until the absorbance at 550 nm exceeded
system of 4% acetonitrile in water containing 0.05% trifluoroacetic
acid with a flow rate of 6.5 ml/min, yielding subfraction B2
(Fig. 2C).
On an octadecyl silica HPLC column (Mightysil RP-18GP
column, Kanto reagents) using a solvent system of 20% acetonitrile
in water containing 0.05% trifluoroacetic acid with a flow rate of
1.0 ml/min, the fraction B2 was eluted as a single peak at 5.1 min
(Fig. 2D). The component of the fraction was identified as
dihydrofumigatin
(2-methoxy-1,3,4-trihydroxy-5-methylben-
zene), which had inhibitory effects on the acetyltransferase
activity in rat spleen microsomes (Table 1). Its structure was
0
.2.
Fig. 2 shows the method, used to separate the acetyltransferase
8 11 4
elucidated by HRMS analysis (m/z calculated for C H O [M + H]+:
1
13
inhibitor. The culture filtrate (48 l) was extracted with ethyl
acetate and the solvent was evaporated. The ethyl acetate extract
171.0657; found:171.0681) as wellas Hand CNMR spectroscopic
assignments using heteronuclear multiple bond correlation and
heteronuclear multiple quantum correlation experiments (Table 2).
(20.4 g) was chromatographed on a silica gel column. Based on TLC
1
analysis (Fig. 2B), the eluate was separated into seven fractions, A1
to A7. The inhibitory effect on PAF synthesis by the fractions was
determined using the TCA precipitation method described in the
Section 2. Fraction A5 (100 mg), in which the strongest activity was
detected, was further chromatographed using a preparative HPLC
system equipped with the 5PE-MS HPLC column using a solvent
More solid evidence was obtained by the direct comparison of its H
NMR chemical shifts with those of the compound synthesized by
reduction of fumigatin (3-hydroxy-2-methoxy-5-methyl-1,4-ben-
zoquinone) with sodium thiosulfate (Table 2).
Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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Y. Yamazaki et al. / Biochemical Pharmacology xxx (2013) xxx–xxx
Fig. 2. Isolation of the acetyl-CoA:lyso-PAF acetyltransferase inhibitors from the metabolites of Penicillium sp. F33. (A) Schematic diagram of the isolation of inhibitors. One
unit is defined as the amount of sample that inhibited 50% of PAF synthesis in the TCA precipitation assay described in Section 2. The specific activity is expressed as the
mean ꢂ S.E. for three independent experiments. (B) The eluate from the silica gel column chromatography was monitored by TLC, and separated into A1–A7 fractions. (C) HPLC
chromatogram of subfraction A5. The subfraction was applied to a COSMOSIL 5PE-MS HPLC column (20 ꢀ 250 mm). The column was eluted with 4% acetonitrile in water containing
0.05% trifluoroacetic acid at a flow rate of 6.5 ml/min. The eluate was separated into B1–B3 fractions. (D) HPLC chromatogram of subfraction B2. The subfraction was applied to a
Mightysil RP-18GP column (4.6 ꢀ 250 mm). The column was eluted with 20% acetonitrile in water containing 0.05% trifluoroacetic acid at a flow rate of 1.0 ml/min. The UV detector
was set at 250 nm.
Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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7
Table 1
Effect of inhibitors on acetyl-CoA: lyso-PAF acetyltransferase activity in rat spleen microsomes.
Compound
Inhibition of PAF synthesis IC50 (mM)
1
2
3
Dihydrofumigatin
FUD-1
R =R =R =H
101 ꢂ 7
166 ꢂ 8
>500
1
2
3
3
R =CH , R =R =H
1
2
3
FUD-2
3
R =R =R =CH CO
1
3
2
FUD-3
R =R =CH
3
CO, R =n-C13
H
27CO
>500
2
FUD-4 (fumigatin)
FUD-4 + decanoic acid
FUD-5
R =H
109 ꢂ 11
108 ꢂ 17
121 ꢂ 5
139 ꢂ 0
IC50
2
3
R =CH CO
2
FUD-6
R =n-C
5
H
H
11CO
19CO
2
FUD-7
R =n-C
9
3
ꢂ 0
2
FUD-8
R =n-C13
H
27CO
35CO
20 ꢂ 0
2
FUD-9
R =n-C17
H
391 ꢂ 53
The acethyltransferase activity was measured by the TCA precipitation method as described in Section 2. Rat spleen microsomes (2.5
mg protein) were incubated with various
3
concentrations of inhibitors dissolved in dimethyl sulfoxide (final concentration, 5%) in 100 l of 0.1 M Tris buffer (pH 6.9) containing 300 mM [ H]acetyl-CoA, 40 mM lyso-
m
PAF, 25 mM NaF and 0.1% bovine serum albumin at 37 8C for 15 min. FUD-4 + decanoic acid means equimolar mixture of these compounds. The results are expressed as the
mean ꢂ S.E. for three separate experiments.
Table 2
dihydrofumigatin was identified as fumigatin. Actually, the
stability test under the conditions used for dihydrofumigatin
NMR chemical shifts of fraction B2 and reduced fumigatin.
Fraction B2
Reduced fumigatin
showed that the peak corresponding to fumigatin remained
unchanged and other peaks could not be detected after 72 h
storage (Fig. 3C and 3D). As shown in Fig. 4 and Table 1, the
inhibitory activity of fumigatin toward the acetyltransferase was
equivalent to that of dihydrofumigatin. These results indicate that
fumigatin has a very stable conformation, and a similar inhibitory
effect on lyso-PAF acetyltransferase as dihydrofumigatin.
1
13
1
H (ppm)
C (ppm)
H (ppm)
1
2
3
4
5
6
7
8
–
143.6
135.0
137.3
139.0
120.1
108.4
15.7
–
–
–
–
–
–
–
–
–
6.18
2.10
3.77
6.15
2.08
3.75
3.3. Inhibition of acetyl-CoA:lyso-PAF acetyltransferase by synthetic
60.7
fumigatin derivatives
1
13
6
H and C NMR measurements were performed in acetone-d at 35 8C.
Using fumigatin as a starting material, we further synthesized
derivatives chemically, and evaluated them. Modifying the
hydroxyl moieties of dihydrofumigatin resulted in three com-
pounds, FUD-1, FUD-2, and FUD-3 (Table 1). As shown in Fig. 4 and
Table 1, FUD-1 had a slightly weaker inhibitory effect on acetyl-
CoA:lysoPAF acetyltransferase than dihydrofumigatin, while FUD-
2
and FUD-3 did not have a significant effect. These results indicate
that the C-1 and/or C-4 hydroxy groups are essential to inhibit the
acetyltransferase activity.
We found that several commercially available saturated fatty
acids (Table 3) exhibited inhibitory activity toward the acetyl-
transferase. This prompted us to examine whether fatty acid
esters of fumigatin, termed FUD-5, FUD-6, FUD-7, FUD-8, and
FUD-9 (Table 1), inhibit the acetyltransferase activity. As shown in
Fig. 4 and Table 1, esterification of the C-3 hydroxyl group of
fumigatin with decanoic acid and tetradecanoic acid remarkably
enhanced the inhibitory effect. FUD-5, FUD-6, and equimolar
mixture of fumigatin and decanoic acid showed similar levels of
activity to fumigatin, whereas FUD-9 exhibited weak activity.
Nordihydroguaiaretic acid (NDGA) has been reported to be the first
potentinhibitorforacetyl-CoA:lyso-PAFacetyltransferases[22], and
was used as a standard substance in our previous reports [16,17]. In
our assay, the IC50 value of NDGA against the acetyltransferase
activity in rat spleen microsomes was 123 ꢂ 6 mM, quite a bit larger
than that of FUD-7 or FUD-8 (Fig. 4 and Table 1).
3
.2. Fumigatin, an oxidized and highly stable form of
dihydrofumigatin, has a similar level of lyso-PAF acetyltransferase
inhibitory activity as dihydrofumigatin
The stability test of purified dihydrofumigatin was carried out
as described in Section 2. The chromatographs show that the peak
at 5.1 min corresponding to dihydrofumigatin decreased dramati-
cally (18.8% and 17.4% after 24 h and 72 h storage, respectively).
Alternatively, a main additional peak at 8.8 min, a moderate peak
at 2.9 min, and several minor other peaks were detected (Fig. 3A
and D). The decrease in the inhibitory activity of the stored sample
was small (Fig. 3B). These observations suggest the altered
substances to be potent inhibitors.
Since dihydrofumigatin is reported to readily interconvert to its
oxidized form fumigatin [21], we chemically synthesized fumiga-
tin (termed FUD-4, Table 1) and evaluated its inhibitory activity
and stability. The peak at 8.8 min in the chromatograph of
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alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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2
Fig. 3. Stability of fumigatin derivatives. Fumigatin derivatives (1.25 mg/ml) were dissolved in acetonitrile-H O (50:50, v/v). After 0, 24 and 72 h storage at room temperature,
each sample was analyzed as described in Section 2. (A) HPLC chromatogram of purified dihydrofumigatin. Arrows (5.1 min) and arrowheads (8.8 min) indicate the peak of
dihydrofumigatin and fumigatin, respectively. Right upper images are enlargements of the peak of dihydrofumigatin (5.5 min). (B) Acetyltransferase activity of each sample
was measured by the TCA precipitation method. Relative inhibitory activity is expressed taking the control value obtained for the sample at 0 h as 1. Data are shown as the
mean ꢂ S.E. for three independent experiments. *p < 0.05. (C) HPLC chromatogram of synthesized fumigatin. Arrowheads (8.8 min) indicate the peak of fumigatin. (D) Surviving
samples were calculated from the area under the curve (AUC) of the corresponding peaks. Relative AUCs are expressed taking the control value obtained for the sample at 0 h as 1.
Data are shown as the mean ꢂS.E. for three independent experiments.
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alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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Table 3
Effect of saturated fatty acids on acetyl-CoA: lyso-PAF acetyltransferase activity in
rat spleen microsomes.
Compound
Inhibition of PAF
synthesis IC50 (mM)
n-C
n-C
7
H
15COOH
19COOH
>500
9
H
>500
n-C11
n-C13
n-C15
H
23COOH
27COOH
31COOH
419 ꢂ 13
183 ꢂ 5
>500
H
H
The acethyltransferase activity was measured by the TCA precipitation method as
described in Section 2. Rat spleen microsomes (2.5 g protein) were incubated with
various concentrations of fatty acids dissolved in dimethyl sulfoxide (final
concentration, 5%) in 100 l of 0.1 M Tris buffer (pH 6.9) containing 300
M lyso-PAF, 25 mM NaF and 0.1% bovine serum albumin at
7 8C for 15 min. The inhibitory activity of palmitic acid was measured dispersed in
m
m
mM
3
[
H]acetyl-CoA, 40 m
3
solution because of poor solubility. The results are expressed as the mean ꢂ S.E. for
three separate experiments.
Fig. 4. Effects of PAF acetyltransferase inhibitors on acetyltransferase activity in rat
spleen microsomes. Acetyltransferase activity was measured by the TCA
precipitation method as described in Section 2. Rat spleen microsomes (2.5
mg
protein) were incubated with various concentration of inhibitors;
nordihydroguaiaretic acid (NDGA) (A, B, &), dihydrofumigatin (DF) (A, &), FUD-
1
(A, *), FUD-2 (A, *), FUD-3 (A, ~), fumigatin (FUD-4) (A, ~), FUD-5 (B, &), FUD-6
B, *), FUD-7 (B, *), FUD-8 (B, ~), FUD-9 (B, ~) dissolved in dimethyl sulfoxide
final concentration, 5%) in 100 l of 0.1 M Tris buffer (pH 6.9) containing 300
M lyso-PAF, 25 mM NaF and 0.1% BSA at 37 8C for 15 min.
(
(
[
m
mM
3
H]acetyl-CoA, 40
m
Values are expressed as the mean ꢂ S.E. for one of three experiments, in triplicate,
that gave similar results.
We further evaluated the effect of FUD-7 on the inhibitory
activity toward 15-lipoxygenase and cyclooxygenases. As shown in
Table 4, FUD-7 did not have significant effects.
3
.4. Inhibition of lipopolysaccharide (LPS)-induced PAF production
and gene expression of lysophosphatidylcholine acyltransferase
LPCAT2)/lyso-PAF acetyltransferase by synthetic fumigatin
derivatives
(
Bone marrow-derived macrophages (BMDMs) were pretreated
with the inhibitors or vehicle for 30 min and stimulated by 50 ng/
ml of LPS. The expression of LPCAT2 mRNA was then measured
Fig. 5. Effects of the fatty acid esters of fumigatin on LPS-stimulated transcription of
2
4 h later. As shown in Fig. 5A, FUD-7 significantly inhibited the
expression at 3 M. The inhibitory activity of FUD-7 was stronger
than that of NDGA at 25 M or WEB2086 at 5 M. Furthermore,
lyso-PAF acetyltransferase/LPCAT2 mRNA in bone marrow-derived macrophages
(
(
BMDMs).
m
A, B) LPCAT2 mRNA levels were measured by quantitative RT-PCR. The y-axis
m
m
shows relative levels expressed by taking the control vehicle only values obtained
from non-stimulated BMDMs as 1. Data are shown as the mean ꢂ S.E. for three to
four independent experiments. *p < 0.05 and **p < 0.01 compared with LPS-
stimulated control.
we evaluated the effect of other fumigatin derivatives. Fig. 5B
shows the inhibitory effect of FUD-6, FUD-7, and FUD-8 on LPCAT2
mRNA expression, indicating that it correlated with the lyso-PAF
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alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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Table 4
3
.5. Inhibition of carrageenan-induced mouse paw edema by fatty
Effect of FUD-7 and nordihydroguaiaretic acid (NDGA) on lipoxygenase and
cyclooxygenases.
acid esters of fumigatin
Inhibition of enzymes IC50
FUD-7
(mM)
To examine the anti-inflammatory effect of dihydrofumigatin
and the synthetic fumigatin derivatives in vivo, the carrageenan-
induced paw edema test was performed (Fig. 7). Prednisolone has
been generally employed for carrageenan-induced paw edema test
as a standard substance. To confirm this assay work properly and to
compare the anti-edematous effect of fumigatin derivatives with
those of other anti-inflammatory reagents, we used prednisolone
as a positive control. Prednisolone at 10 mg/kg significantly
reduced paw thickness compared to the control vehicle only.
Mice were administered various doses of dihydrofumigatin or the
fumigatin derivatives (1–10 mg/kg) intravenously 1 h before the
carrageenan administration. Dihydrofumigatin at 10 mg/kg sig-
nificantly decreased paw thickness, while at 5 mg/kg it had a
significant effect from at 3 h compared to the control. The anti-
edematous effect of the synthetic fatty acid esters of fumigatin was
examined at a dose of 2.5 or 1 mg/kg because of poor solubility.
FUD-7 and FUD-8 significantly (p < 0.05) suppressed paw edema
at 2.5 mg/kg, and tended to do so at 1 mg/kg, whereas FUD-6 at
both concentrations and co-administration of fumigatin and
decanoic acid at equimolar amount of 2.5 mg/kg as FUD-7 had
no effect. The maximum inhibition of edema by FUD-7 and FUD-8
at 2.5 mg/kg was 51.7% and 47.9%, respectively, which was greater
than that of prednisolone at 10 mg/kg (41.9%). The efficacy of
fumigatin derivatives was dose-dependent and consistent with the
NDGA
1
5-Lipoxygenase
>100
>100
>100
8.8 ꢂ 0.3
44.5 ꢂ 4.2
9.1 ꢂ 0.7
Cyclooxygenase-1
Cyclooxygenase-2
The enzyme activities were measured with commercially available kits as described
in Section 2. The results are expressed as the mean ꢂ S.E. for three separate
experiments.
acetyltransferase inhibitory activity in rat spleen microsomes.
LPCAT1 mRNA was not significantly induced by LPS in BMDMs,
indicating that the effect of FUD-7 on the gene expression of
LPCAT1 was not significant (data not shown).
The production of PAF 15 min after the LPS-stimulation was
quantified by bioassay using rabbit platelet aggregation. As shown
in Fig. 6B, the sample extracted from LPS-stimulated BMDMs
caused the shape transforming response of rabbit platelet similar
to that of c-PAF at 2.5 ꢀ 10ꢁ M, and pre-treatment of a PAF
antagonist WEB2086 suppressed this effect. Pre-incubation of
11
BMDMs with FUD-7 at concentration of 5
mM for 30 min resulted
in a significant inhibition of PAF biosynthesis at the level of non-
stimulated BMDMs (Fig. 6C and D).
6
Fig. 6. Effect of FUD-7 on PAF biosynthesis in mice bone marrow-derived macrophages (BMDMs) stimulated with LPS. BMDMs (6 ꢀ 10 cells) seeded onto dishes and cultured
for 24 h were pretreated with FUD-7 at 5
mM (C) or vehicle (B) for 30 min and stimulated with 50 ng/ml of LPS. At 15 min after the stimulation, the amount of PAF produced
was determined by bioassay using rabbit platelet aggregation as described in Section 2. PAF biosynthesis in non-stimulated BMDMs (D) was also examined. Platelets were
preincubated with vehicle (0.1% BSA/saline) or WEB2086 for 1 min and then exposed to a known quantity of c-PAF (A) or tenth part of the samples (B–D). a = vehicle (0.1% BSA
in saline); b = WEB2086; c = c-PAF or the samples.
Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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Y. Yamazaki et al. / Biochemical Pharmacology xxx (2013) xxx–xxx
11
A
B
C
D
E
F
Fig. 7. Effect of fumigatin derivatives on carrageenan-induced paw edema. Mice were pretreated with prednisolone ((A) dissolved in saline containing 10% ethanol, i.p., n = 9),
dihydrofumigatin ((B) dissolved in saline, i.v., n = 6), fatty acid esters of fumigatin ((C) FUD-7, (D) co-administration of fumigatin and decanoic acid, E:FUD-6, F:FUD-8
dissolved in saline containing 10% ethanol, i.v., n = 6), or vehicle (saline containing 10% ethanol or saline, i.p. or i.v., n = 6–7) 3 h (prednisolone) or 1 h (other substances) before
the local injection of carrageenan (300
mg/paw) at the indicated dose. Edema was expressed as increases in paw thickness (mm) 0 to 4 h following the carrageenan injection.
Data are expressed as the mean ꢂ S.E. *,** indicate p < 0.05 or p < 0.01 vs. vehicle, respectively.
Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

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BCP-11676; No. of Pages 13
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2
Y. Yamazaki et al. / Biochemical Pharmacology xxx (2013) xxx–xxx
results of the TCA precipitation assay. These results indicated that
fumigatin derivatives have a strong anti-edematous effect on
carrageenan-induced paw edema which correlated with the
inhibitory activity for lyso-PAF acetyltransferase in rat spleen
microsomes.
was stronger than that of these compounds. Although it still
remains to be clarified whether or not the inhibition of PAF
synthesis by fumigatin derivatives is due to direct effects on the
acetyltransferase enzyme, the inhibitory effect was not on the
oxidation–reduction reaction because the inhibitory activity of
fumigatin was similar to that of its reduction product dihydro-
fumigatin. In relation to the mode of action of fumigatin
derivatives, the quinone or dihydroquinone structure appeared
necessary for the inhibition of PAF synthesis because diester
derivative of FUD-8, i.e., FUD-3, abolished the inhibitory effect
(Fig. 4 and Table 1).
FUD-7 significantly inhibited not only the activity of acetyl-
transferase in rat spleen microsomes but also the PAF production
in LPS-stimulated bone marrow-derived macrophages (BMDMs)
(Fig. 6). In addition to the efficacy to inhibit the function of lyso-
PAF acetyltransferase/LPCAT2, FUD-6, FUD-7, and FUD-8 also
inhibited the expression of LPCAT2 mRNA (Fig. 5). Together with
the data that a PAF antagonist WEB2086 weakly inhibited, this may
be a secondary effect by attenuating PAF-induced PAF synthesis by
blocking the initial PAF synthesis rather than a direct effect by
inhibiting the signals which induce the LPCAT2 expression. It was
reported that PAF itself stimulated the lyso-PAF acetyltransferase
and PAF biosynthesis in rat and human neutrophils [35,36]. In
macrophages, inflammatory cytokines are produced in response to
PAF, and known to stimulate PAF synthesis [37]. Thus, the gene
expression of LPCAT2 may be involved in such a positive feedback
mechanism.
4
. Discussion
In inflammatory and allergic processes, PAF is biosynthesized in
specific enzymatic reactions, which involve acetyl-CoA:lyso-PAF
acetyltransferases (remodeling pathway). Inhibitors of these
acetyltransferases are expected to be of therapeutic interest. Here,
we screened for such inhibitors with anti-inflammatory effects.
There are several reports of substances which inhibit PAF
synthesis in intact cells such as human neutrophils, rat peritoneal
macrophages and human endothelial cells. Calmodulin antagonists
(trifluoperazine and N-6-aminohexyl-5-chloro-1-naphthalene sul-
fonamide) [23], antiflammins and luffariellolide which interfere
with the activation of phospholipase A2 [24,25], nitroprusside and
3
-morpholinosydnonimine which stimulate the production of
cGMP [26], and a protein kinase C inhibitor ( -sphingosine) [27]
D
have been shown to inhibit PAF biosynthesis. However, these
substances did not directly inhibit acetyltransferase activity. In
addition, the quinoline-based compounds PF-5901 and Wy-50295
[
28], ketotifen [29,30], the cyclooxygenase and 5-lipoxygenase
inhibitor nordihydroguaiaretic acid (NDGA) [22], magnolol,
honokiol [16], and galloyl compounds [17] are also reported to
be potent acetyltransferase inhibitors. In this study, we found
fumigatin and its derivatives to be novel inhibitors. The mould
Carrageenan-induced paw edema is a suitable animal model for
evaluating anti-edematous effects. Since the involvement of PAF
with the formation of paw edema was reported previously [38,39],
we employed this model in the present study. The edema that
develops following the injection of carrageenan serves as an index
of acute inflammatory changes, and can be determined from
differences in paw thickness measured immediately after the
injection and then every hour. The edema induced by carrageenan
is believed to be biphasic: the first phase (mouse: 0–4 h) involves
the release of serotonin and histamine and the second phase
(mouse: over 24 h) is mediated by prostaglandins, cyclooxygenase
products. Continuity between the two phases is provided by kinins
[40–42]. PAF is thought to be involved in the first phase, because
PAF production transiently increased 1 h after carrageenan
injections in rats and PAF antagonists inhibited the edema more
effectively in the first phase than the second phase [38,39]. Thus,
we examined the effect of the fumigatin derivatives on the first
phase (1–4 h) of carrageenan-induced paw edema in mice. All the
fumigatin derivatives used in this study except for FUD-6 showed a
significant reduction of paw edema at 1 h or more after the
carrageenan injection, and the efficacy was dose-dependent. The
order in terms of inhibition of carrageenan-induced edema was
FUD-7 > FUD-8 > FUD-6, which was similar that for the inhibition
of PAF synthesis in rat spleen microsomes (Figs. 4 and 7, and
Table 1). These results suggest that fumigatin derivatives produce
an anti-edematous effect during the first phase of carrageenan-
induced paw edema through a reduction of PAF synthesis. To date,
several PAF receptor antagonists have been reported to inhibit
carrageenan-induced paw edema: for example, kadsurenone
(50 mg/kg i.p.) and L-652731 (10 mg/kg p.o.) inhibited the first
phase of the edema by 27% and 38%, respectively [39], and
administration of WEB2086 (4–16 mg/kg, i.p.) and WEB2170 (2–
8 mg/kg, i.p.) achieved 34–50% and 30–60% inhibition at 4 h after
the injection, respectively [38]. The efficacy of FUD-7 and FUD-8,
whose administration (2.5 mg/kg i.v.) resulted in 47.9–51.7%
inhibition in the first phase (Fig. 7), was stronger than that of
PAF antagonists, although the difference in the route of adminis-
tration should be considered. Esterification of fumigatin with fatty
acids might be contributed to this strong effect by improving
metabolic product fumigatin, C
8 8 4
H O , and its reductive form
dihydrofumigatin, C , were reported to be isolated from
8 10 4
H O
cultures of Aspergillus fumigatus [21,31]. Whereas it is suggested
that these two substances act on the oxidation-reduction system in
the vital processes of this mould, their physiological function
against mammals remains to be clarified. Thus, this is the first
report on the physiologic activity of fumigatin and its derivatives.
Fumigatin was more stable, and its inhibitory activity toward
lyso-PAF acetyltransferase was similar to that of dihydrofumigatin
(
Fig. 3 and Table 1). In addition, some saturated fatty acids also had
inhibitory activity (Table 3). These observations prompted us to
produce fatty acid esters of fumigatin chemically as pro-drugs.
Carboxylic acid conjugates of drugs such as indomethacin farnesil,
flurbiprofen axetil, and docosahexaenoic acid ester of paclitaxel
have been shown to improve the therapeutic potential of drugs by
increasing absorption and half-lives, improving drug delivery, and
reducing side effects [32–34]. Since it has been proposed that these
compounds are cleaved by a carboxyesterase in vivo and converted
to an active form, it was expected that fatty acid esters of fumigatin
may be converted to two active compounds at inflamed sites and
exert additive effects. Among the fatty acid esters of fumigatin,
FUD-7 and FUD-8, the decanoic and tetradecanoic acid esters of
fumigatin, respectively, had strong inhibitory activity in rat spleen
microsomes (Fig. 4 and Table 1). Interestingly, the inhibitory
activities for the acetyltransferase and for the carrageenan-
induced paw edema were far stronger than the additive effect of
fumigatin and corresponding fatty acid (Fig. 7 and Table 1), and the
order of inhibitory activity of fatty acid esters did not correlate
with that of the fatty acids themselves. These results imply that the
enhancement of inhibitory activity does not result in the
hydrolysis of the compounds to fumigatin and fatty acids by a
carboxylesterase. The IC50 value of FUD-7, which had the strongest
activity in this study, was 40 times lower than that of NDGA. Since
IC50 values of honokiol, magnolol, theaflavins, and dimeric flavan-
3
-ols with a galloyl group at C-3 under our experimental
conditions have been reported to be 2–10 times lower than that
of NDGA [16,17], the inhibitory effect on PAF biosynthesis of FUD-7
Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021

G Model
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Y. Yamazaki et al. / Biochemical Pharmacology xxx (2013) xxx–xxx
13
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Please cite this article in press as: Yamazaki Y, et al. A Penicillium sp. F33 metabolite and its synthetic derivatives inhibit acetyl-CoA:1-O-
alkyl-sn-glycero-3-phosphocholine acetyltransferase (a key enzyme in platelet-activating factor biosynthesis) and carrageenan-
induced paw edema in mice. Biochem Pharmacol (2013), http://dx.doi.org/10.1016/j.bcp.2013.06.021