Mycophenolic acid as a latent agonist of PPARγ

Article abstract of DOI:10.1016/j.bmcl.2007.06.059

Mycophenolic acid (MPA), known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), was found to inhibit the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes. Although the effect of MPA was attributed to inhibition of IMPDH, we u

Full text of DOI:10.1016/j.bmcl.2007.06.059

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4767–4770
Mycophenolic acid as a latent agonist of PPARc
Makoto Ubukata,a,* Hitomi Takamori,a Misaki Ohashi,a Shinya Mitsuhashi,a
Kaoru Yamashita,b Tomohisa Asada,b Noriyuki Nakajima,b Nobuyasu Matsuura,c
Mie Tsuruga,d Keiko Takid and Junji Magaed
aDivision of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9,
Nishi-9, Kita-ku, Sapporo 060-8589, Japan
bBiotechnology Research Center, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan
cDepartment of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
dDepartment of Biotechnology, Institute of Research and Innovation, 1201 Takada, Kashiwa 277-0861, Japan
Received 22 March 2007; revised 18 June 2007; accepted 20 June 2007
Available online 26 June 2007
Abstract—Mycophenolic acid (MPA), known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), was found to
inhibit the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes. Although the effect of MPA was attributed to inhibition
of IMPDH, we uncovered a hidden biological property of MPA as an agonist of peroxisome proliferator activated receptor c
(PPARc).
ꢀ 2007 Elsevier Ltd. All rights reserved.
Obesity, an excessive accumulation of adipose tissue, is a
common disorder,1 and white adipose tissue (WAT)
mass is a reflection of the number of adipocytes and
their volume.2 Adipocyte differentiation from pre-adipo-
cytes is therefore one of the major causes of the develop-
ment of obesity. The model cell line, 3T3-L1 cells, can be
induced to differentiate into mature adipocytes in cell
culture.3 Lovastatin,4 ritonavir, nelfinavir,5 6-ethoxyzo-
lamide,6 and prostaglandin F2a, fluprostenol,7 wortman-
nin8 have been reported as inhibitors of adipocyte
differentiation. Recently we reported several germacr-
anolides from Calea urticifolia as inhibitors of 3T3-L1
pre-adipocyte differentiation.9 In this study, we report
new biological properties of mycophenolic acid (MPA)
(1) as a latent agonist of peroxisome proliferator acti-
vated receptor c (PPARc) and a direct evidence of
molecular interaction between 1 and PPARc.
broth of an unidentified fungus BAUA 4031 (donated
by Kaken Pharmaceutical Co., Ltd), block insulin-in-
duced differentiation of 3T3-L1 pre-adipocytes into ma-
ture adipocytes (Fig. 1).
Terrein (2) showed weak inhibitory activity with mini-
mal inhibitory concentration at 81 lM, which was ob-
served by diminishing oil-red O stained cells under a
microscope. However, 1 showed potent inhibitory activ-
ity. The minimal effective concentration of a highly puri-
fied sample was 0.2 lM.
To clarify structure–activity relationship among the
derivatives of 1, we prepared 17 derivatives of 1. Scheme
1 shows the synthesis of some important derivatives, of
which C-7-OH, C-14 carboxylic acid, and C-5-OMe
were modified to several other functional groups.
During the course of our screening program for the
modulator of 3T3-L1 cell differentiation, we found that
two natural compounds, MPA (1) isolated from a broth
of Penicillium sp., BAUA 2775, and terrein (2)10 from a
OH
CH3
O
HO
CH3
COOH
HO
O
OCH3
O
CH3
Mycophenolic acid (1)
Keywords: Mycophenolic acid; IMPDH; PPARc; 3T3-L1; Inhibitor;
SPR.
Terrein (2)
*
Figure 1. Structures of inhibitors of 3T3-L1 pre-adipocyte cell
differentiation, mycophenolic acid (MPA) (1) and terrein (2).
Corresponding author. Tel.: +81 11 706 3638; fax: +81 11 706
3843; e-mail: m-ub@for.agr.hokudai.ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.059

4768
M. Ubukata et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4767–4770
OR1
CH3
CH3
OAc
CH3
OH
CH3
O
O
O
a
b
COOH
COOCH3
c
COOCH3
d
1
O
O
O
OR2
OCH3
OCH3
CH3
3
CH3
4
5: R1 = Ts, R2 = CH3
6: R1 = Ts, R2 = H
OCH3
CH3
OCH3
CH3
O
O
f
COOH
COOCH3
COOH
e
g
O
1
O
OCH3
OCH3
CH3
8
CH3
7
OMEM
CH3
OH
CH3
OH
CH3
O
O
O
h
i
COOCH3
COOCH3
CH3
O
O
O
OH
OH
OH
CH3
11
CH3
10
CH3
9
OR1
CH3
CH3
OR
O
O
n
COOCH3
COOCH3
11
O
O
OR2
O
O
CH3
j
O
12: R1 = MEM, R2 = nicotinoyl
13: R1 = H, R2 = nicotinoyl
k
16: R = MEM
17: R = H
o
CH3
l
14: R1 = MEM, R2 = isonicotinoyl
15: R2 = H, R2 = isonicotinoyl
m
Scheme 1. Reagents and conditions: (a) Ac2O, DMAP, Py, rt, 62%; (b) TsOH, MeOH, rt, 9 h, 78%; (c) TsCl, Et3N, CH2Cl2, rt, 4 h, 96%; (d) Lil, Py/
collidine (1:5), 150 ꢁC, 15 h, 23%; (e) TMSCHN2, MeOH, rt, 12 h, 90%; (f) LiOH, H2O/acetone (1:4), 2 h, 33%; (g) Lil, Py/collidine (1:5), 120 ꢁC,
18 h, 74%; (h) TsOH, MeOH, rt, 9 h, 78%; (i) MEMCl, NaH, THF, 0 ꢁC–rt, 9 h, 44%; (j) nicotinoyl chloride hydrochloride, py, rt, 4 h, 56%; (k) TFA,
CH2Cl2, rt, 12 h, 83%; (l) isonicotinoyl chloride hydrochloride, DMAP, Py, rt, 4 h, 67%; (m) TFA, CH2Cl2, rt, 12 h, 81%; (n) ethylbromoacetate,
NaH, THF, 0 ꢁC–rt, 95%; (o) TFA, CH2Cl2, rt, 12 h, 61%.
Among the derivatives, only two compounds, 7-O-acetyl
MPA (3) and methyl ester derivative (4), showed the
inhibitory activity of 3T3-L1 pre-adipocyte cell differen-
tiation at 2 lM, while 1 showed the same activity at
0.2 lM. Compounds 5–17 did not inhibit the adipocyte
differentiation of 3T3-L1 cells at 2 lM. 7-O-Acetyl MPA
(3) was prepared from 1 with Ac2O in the presence of
pyridine and DMAP, and methyl ester derivative of
MPA (4) was prepared from 1 with p-toluenesulfonic
acid in methanol. 7-O-Methyl MPA (8) was prepared
via trimethyl derivative (7), which was derived from 1
by using TMSCHN2. Replacements of 5-OMe func-
tional group to 5-O-nicotinoyl (13), 5-O-isonicotinoyl
(15) functional moieties were achieved by demethylation
of 1 using modified protocol of a previously reported
method,11 followed by conversion of carboxylic acid
into methyl ester, regioselective protection of C-7-OH
with MEMCl, esterification with nicotinoyl chloride,
or isonicotinoyl chloride and deprotection of MEM
group. 5-O-Ethoxycarbonylmethyl derivative (17) was
synthesized using similar protocol (Scheme 1).
derivative (15) and 5-O-ethoxycarbonylmethyl deriva-
tive (17) did not inhibit at the same concentration, which
could be used for further biological investigation as
inactive derivatives. Conversion of the phenolic hydroxy
group at C-7 position into 7-O ether functional group
resulted in a decrease in the inhibitory activity, thus 7-
OMe MPA (8) and methyl ester derivative of 7-O-
MEM MPA (11) showed weak inhibitory activity (14%
and 10%, respectively) of the [1-14C]sodium acetate up-
take at 2 lM (Table 1).
Next we focused our attention on the molecular target
of 1. Differentiation of HL60 cells by 1 is associated with
substantial decreases in both the guanylate and adenyl-
ate pools and appeared to be dependent on the state of
depletion of intracellular GTP.12 Simultaneous addition
of guanosine or guanine to 1-treated cells restored the
GTP pool and prevented differentiation from occurring.
Table 1. Inhibitory activities of [1-14C]sodium acetate uptake into
3T3-L1 cells by MPA (1) and its analogs
Compound
Inhibitory activity
(%) at 2 lM
To determine quantitative inhibitory activities of 1, 3, 4,
8, 11, 15, and 17, we measured the inhibitory activity
against [1-14C]sodium acetate uptake into 3T3-L1 cells,
which is one of the markers of lipid biosynthesis.3
Although, the inhibitory activities on the sodium acetate
uptake are not wholly parallel to the inhibitory activities
of 3T3-L1 cell differentiation, 1 and its analogs 3 and 4
clearly inhibited at 2 lM, whereas 5-O-isonicotinoyl
1
3
74
86
67
14
10
3
4
8
11
15
17
0

M. Ubukata et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4767–4770
4769
7
6
5
4
3
2
1
0
Recently we found that 4-O-carboxymethyl-2-O-methy-
lascochlorin and 4-O-isonicotinoyl-2-O-methylascochlo-
rin are potent agonists of PPARc.16 From the
standpoint of structural similarities between 1 and asco-
chlorin, we next estimated the effect on the peroxisome
proliferator activated receptor c (PPARc), which was
determined as a critical nuclear receptor on adipocyte
differentiation. Transcriptional activity of PPARc stim-
ulated with pioglitazone was inhibited by 1, 3, or 4 even
at the dose of 1 lM (Fig. 2). These observations suggest
that 1 is a ligand of PPARc.
1 μM Pioglytazone
To obtain direct evidence that 1 is a ligand of PPARc,
characterization of the affinity and rate of the molecular
interaction between 1 and PPARc were performed by
surface plasmon resonance (SPR) method.17 Molecular
interaction between 1 and PPARc was recorded on a
Biacore X (Biacore, Sweden). A recombinant human
PPARc GST fusion protein was immobilized on a Sen-
sor Chip CM5 with amine coupling using PBS buffer
(pH 4.0). Excess reactive groups on the surface were
deactivated with 1 M ethanolamine hydrochloride. The
amount of immobilized PPARc GST fusion protein
was adjusted to 13,000RU. The same equivalent molar
of GST protein (300RU) was captured on a Sensor Chip
CM5 as a reference using a similar amine coupling meth-
od. A recombinant human PPARa GST fusion was
immobilized by similar manner. The amount of immobi-
lized protein was 15,600RU. The same equivalent of
GST protein (390RU) was immobilized as a reference.
We determined the analytical conditions such as concen-
tration of DMSO, selection of buffer, and sample prep-
arations using ciglitazone (KD = 1.91 · 10ꢀ4 M, PBS-N
buffer), which is known as an agonist of PPARc. Ana-
lyte (1) dissolved in 5% DMSO in PBS-N buffer
(67 mM Na2HPO4, 12.5 mM KH2PO4, 70 mM NaCl)
was loaded on the Sensor Chip. The kinetics of the ana-
lyte–ligand interaction was determined by a nonlinear
fitting method using BIAevaluation software (Biacore,
Sweden). Using an indirect method for kinetic analysis,
the dissociation constant (KD) between 1 and PPARc
was determined to be 1.10 · 10ꢀ4 M (Rmax = 17.7,
v2 = 2.04 · 10ꢀ4) as shown in Figure 3, whereas the
Compound 1 (μM)
Compound 3 (μM)
Compound 4 (μM)
1
10
1
10
1
10
Figure 2. Partial antagonistic activities of MPA (1) and its derivatives (3
and 4) against pioglitazone (Pio) on PPARc activation. U2OS cells were
transfected with expression plasmids for nuclear receptor, together with
the corresponding reporter plasmids and pCMV-b-galactosidase as
internal control. Values are means of triplicate cultures.
Although the effect of 1 on 3T3-L1 cells is inhibitive and
not inductive, it was considered that inhibition of purine
and pyrimidine nucleotide biosynthesis is also important
in this case. To confirm this idea, we achieved similar
experimental tests for 1-treated 3T3-L1 cells. First of
all, we improved the culture conditions to obtain stable
results concerning the differentiation of 3T3-L1 cells in
our laboratory in Sapporo. Second, we actualized quan-
tification of differentiation of 3T3-L1 cells based on
quantitative determinations of triglyceride and DNA.13
Under these cultural conditions, it was finally suggested
that the depletion of guanine ribonucleotide is the oblig-
atory step toward the inhibition of differentiation in 1-
treated 3T3-L1 cells, and hidden biological properties
of 1 toward 3T3-L1 were uncovered as described later.
Ribavirin14 and oxanosine, the putative precursor of
oxanosine-50-phosphate,15 which are known as inhibi-
tors of IMPDH, did not show the same effects as 1 in
our experiments. Thus 1 could conceivably be a unique
molecule among the class of IMPDH inhibitors.
30
25
20
15
10
5
6.25 μM
12.5 μM
25 μM
50 μM
100 μM
200 μM
400 μM
0
-5
0
50
100
150
200
-50
Time (s)
Figure 3. Sensorgram showing the interaction of 1 with PPARc.

4770
M. Ubukata et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4767–4770
4
3
2
1
0
supported by the Grant-in-Aid for Scientific Research
on Priority Area ‘Creation of Biologically Functional
Molecules’ from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
References and notes
1. Ogden, C. L.; Carroll, M. D.; Curtin, L. R.; McDowell,
M. A.; Tabak, C. J.; Flegal, K. M. JAMA 2006, 295, 1549.
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Biol. 2000, 20, 1008.
3. Mackall, J. C.; Student, A. K.; Polakis, S. E.; Lane, M. D.
J. Biol. Chem. 1976, 251, 6462.
1
(μM)
0.01
0.1
1
0.01
0.1
1
Reporter Gene
None
PPARγ
PPARα
4. Nishio, E.; Tomiyama, K.; Nakata, H.; Watanabe, Y. Eur.
J. Pharmacol. 1996, 301, 203.
5. Zhang, B.; MacNaul, K.; Szalkowski, D.; Li, Z.; Berger,
J.; Moller, D. E. J. Clin. Endocrinol. Metab. 1999, 84,
4274.
Figure 4. Agonistic activities for PPARc and PPARa of MPA (1).
U2OS cells were transfected with expression plasmids for nuclear
receptors (PPARc or PPARa), together with the corresponding
reporter plasmids and pCMV-b-galactosidase as internal control.
6. Takahata, T.; Kumano, T.; Ookawa, K.; Hayakari, M.;
Kakizaki, I.; Tsuchida, S. Biochem. Pharmacol. 2004, 67,
1667.
7. Miller, C. W.; Casimir, D. A.; Ntambi, J. M. Endocrinol-
ogy 1996, 137, 5641.
8. Uehara, T.; Tokumitsu, Y.; Nomura, Y. Biochem. Bio-
phys. Res. Commun. 1995, 210, 574.
9. Matsuura, N.; Yamada, M.; Suzuki, H.; Hasegawa, N.;
Kurosaka, C.; Ubukata, M.; Tanaka, T.; Iinuma, M.
Biosci. Biotechnol. Biochem. 2005, 69, 2470.
10. Cole, R. J.; Cox, R. H. Handbook of Toxic Fungal
Metabolites; Academic Press, 1981, pp 866–869 myco-
phenolic acid; pp 769–772 terrein.
11. Jones, D. F.; Mills, S. D. J. Med. Chem. 1971, 14, 305.
12. Sokoloski, J. A.; Blair, O. C.; Sartorelli, A. C. Cancer Res.
1986, 46, 2314.
13. Precise assay conditions will be reported elsewhere.
Briefly, 3T3-L1 cells (purchased from Health Science
Research Resources Bank (Osaka, Japan)) were cultured
in the culture plate doubly coated with poly-L-lysine and
collagen, and measurement of triglyceride and genomic
DNA in the cell lysate was conducted using a triglyceride
E-test kit (Wako) and a PicoGreen dsDNA quantitation
Reagent and Kits (Molecular Probes), respectively.
14. Leyssen, P.; Balzarini, J.; De Clercq, E.; Neyts, J. J. Virol.
2005, 79, 1943.
dissociation constant between one of thiazolidinedione,
troglitazone,
and
PPARc
was
6.15 · 10ꢀ6 M
(Rmax = 12.7, v2 = 0.00893). The dissociation constant
between 1 and PPARa could not be deduced, because
of the adsorption of 1 to the reference cell. However,
it was determined to be 5.08 · 10ꢀ4 M (Rmax = 25.2,
v2 = 0.131) using PBS-P buffer (PBS-N + 0.005% Tween
20). The dissociation constant of troglitazone and
PPARa was 5.22 · 10ꢀ5 M (Rmax = 42.2, v2 = 0.192)
using PBS-N buffer.
The interaction between 1 and PPARc was comparable
to the dissociation constants of some of thiazolidinedi-
one analogs.18 These results showed the direct evidence
of the molecular interaction between 1 and PPARc.
To determine if 1 acts on PPARc as an agonist in vivo,
we conducted a reporter assay of 1 without pioglitazone.
MPA (1) activated the transcriptional activity of PPARc
at the concentrations of 0.01–1 lM as shown in Figure
4. Moreover, 1 stimulated adipocyte differentiation of
3T3-L1 cells at the concentration of 2–4 lM in the pres-
ence of 50 lM of guanosine, which diminishes IMPDH
activity of 1 (data not shown).
15. Uehara, Y.; Hasegawa, M.; Hori, M.; Umezawa, H.
Cancer Res. 1985, 45, 5230.
In conclusion, we succeeded in uncovering a molecular
target of 1 except IMPDH. These findings will provide
a new insight into the drug discovery utilizing 1.
16. Togashi, M.; Ozawa, S.; Abe, S.; Nishimura, T.; Tsuruga,
M.; Ando, K.; Tamura, G.; Kuwahara, S.; Ubukata, M.;
Magae, J. J. Med. Chem. 2003, 46, 4113.
17. Kretschmann, E.; Raether, H. Naturforcshung Teil A 1968,
23, 2135.
18. Interactions of PPARc with other thiazolidinediones are
Acknowledgments
as
follows.
Rosiglitazone:
KD = 1.42 · 10ꢀ3 M,
Rmax = 467, v2 = 9.24 in PBS-N buffer; ciglitazone:
KD = 8.62 · 10ꢀ4 M, Rmax = 1.18 · 103 for nonspecific
interaction,
The authors express their gratitude to S. Kato, N. H.
Heintz, and C. L. Wu for providing plasmid and a cell
line, and S. Kanazawa for providing fungi, and K.
Umezawa for providing oxanosine, and S. Maeda and
C. Furukawa for technical assistances. This work was
v2 = 0.392
in
PBS-N
buffer
and
KD = 3.51 · 10ꢀ3 M, Rmax = 47.1, v2 = 0.877 in PBS-N
buffer; pioglitazone with PPARc: KD = 1.53 · 10ꢀ5 M,
Rmax = 6.41, v2 = 0.392 in PBS-P buffer.