Isolation of the protein tyrosine phosphatase 1B inhibitory metabolite from the marine-derived fungus Cosmospora sp. SF-5060

Article abstract of DOI:10.1016/j.bmcl.2009.09.025

In the course of bioassay-guided study on the EtOAc extract of a culture broth of the marine-derived fungus Cosmospora sp. SF-5060, aquastatin A (1) was isolated as a protein tyrosine phosphatase 1B (PTP1B) inhibitory component produced by the fungus. The

Full text of DOI:10.1016/j.bmcl.2009.09.025

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6095–6097
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isolation of the protein tyrosine phosphatase 1B inhibitory metabolite
from the marine-derived fungus Cosmospora sp. SF-5060
b
b,
*
Changon Seo a, Jae Hak Sohn a, Hyuncheol Oh a, , Bo Yeon Kim , Jong Seog Ahn
*
a College of Medical and Life Sciences, Silla University, San 1-1 Gwaebeop-dong, Sasang-gu, Busan 617-736, Republic of Korea
b Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Eoun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2009
Revised 3 September 2009
Accepted 8 September 2009
Available online 12 September 2009
In the course of bioassay-guided study on the EtOAc extract of a culture broth of the marine-derived fun-
gus Cosmospora sp. SF-5060, aquastatin A (1) was isolated as a protein tyrosine phosphatase 1B (PTP1B)
inhibitory component produced by the fungus. The compound was isolated by various chromatographic
methods, and the structure was determined mainly by analysis of NMR spectroscopic data. Compound 1
exhibited potent inhibitory activity against PTP1B with IC50 value of 0.19 lM, and the kinetic analyses of
PTP1B inhibition by compound 1 suggested that the compound is inhibiting PTP1B activity in a compet-
itive manner. Aquastatin A (1) also showed modest but selective inhibitory activity toward PTP1B over
other protein tyrosine phosphatases, such as TCPTP, SHP-2, LAR, and CD45. In addition, the result of
hydrolyzing aquastatin A (1) suggested that the dihydroxypentadecyl benzoic acid moiety in the mole-
cule is responsible for the inhibitory activity.
Keywords:
Marine-derived fungus
Cosmospora sp.
Protein tyrosine phosphatase 1B (PTP1B)
Fungal metabolite
Ó 2009 Elsevier Ltd. All rights reserved.
Competitive inhibitor
Marine microorganisms have been recognized as a rich source of
structurally novel and pharmacologically active secondary metabo-
lites.1,2 Particularly, fungi from marine environment have shown to
produce diverse secondary metabolites which are more or less sim-
ilar to those produced by terrestrial fungi.3,4 As a part of our ongoing
studies on PTP1B inhibitors from marine microorganisms collected
from Korea, a fungal isolate Cosmospora sp. SF-5060 was selected
for further investigation on the basis of its potent inhibitory effect
against PTP1B.
Protein tyrosine phosphatases (PTPs) constitute a large family
of enzymes, crucial modulators of tyrosine phosphorylation-
dependent cellular events such as growth, proliferation and differ-
entiation, metabolism, immune response, cell–cell adhesion, and
cell-matrix contacts.5–7 Among them, protein tyrosine phosphatase
1B (PTP1B), an intracellular non-receptor type PTP, is considered as
a well-validated target for drug development. The negative regula-
tion of insulin- and leptin-receptor mediated signaling pathways
by PTP1B has been demonstrated by a number of biochemical
and genetic studies.8–10 Several independent studies have shown
that PTP1B-knockout mice display increased insulin sensitivity,
improved glycemic control, and resistant to diet-induced obes-
ity.10,11 Accordingly, inhibition of PTP1B is predicted to be an excel-
lent, novel therapy to target type 2 diabetes and obesity. Given the
compelling biochemical and genetic evidences linking PTP1B to
several human diseases, a number of efforts have been conducted
to develop PTP1B inhibitors.8,12 However, the development of
small-molecule PTP1B-based drug candidates possessing target
selectivity and bioavailability remains an important challenge.13
The marine-derived fungus Cosmospora sp. SF-5060 was iso-
lated from an inter-tidal sediment collected at Gejae Island (April,
2007), and characterized based on analysis of the ribosomal RNA
(rRNA) sequences. Cosmospora sp. SF-5060 was cultivated in liquid
media at 25 °C for 14 days. The filtered culture broth was extracted
with EtOAc, and the resulting organic extract was subjected to C18
functionalized silica gel flash column chromatography, eluting
with a stepwise gradient consisting of MeOH in H2O (20% to
100% MeOH with 20% increment for each step; 400 mL each). The
fraction eluted at 100% MeOH was then subjected to semi-prepara-
tive reversed-phase HPLC to yield 1.
The structure of 1 was inferred to be a family of the glycosylated
depside base on analysis of the 1H and 13C NMR data. In the NMR
spectra of 1, the signals corresponding to two sets of meta-coupled
aromatic protons, twelve aromatic carbons, and two ester carbonyl
carbons suggested the presence of depside-type moiety in the mol-
ecule. In addition, the presence of sugar moiety in the molecule was
evident by the observation of the signals corresponding to a number
of oxymethines, an oxymethylene, and an anomeric methine in the
NMR spectra. Eventually, the planar structure of 1 was determined
to be aquastatin A by analysis of the 1D and 2D NMR and MS data,
along with in comparison with literature values.14 However, the
absolute configuration of the b-galactopyranoside aquastatin A
was not previously assigned. Therefore, compound 1 was subjected
* Corresponding authors. Tel.: +82 51 999 5026; fax: +82 51 999 5458 (H.O.); tel.:
+82 42 860 4312; fax: +82 42 860 4595 (J.S.A.).
E-mail addresses: hoh@silla.ac.kr (H. Oh), jsahn@kribb.re.kr (J.S. Ahn).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.025

6096
C. Seo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6095–6097
to acid hydrolysis with 6 N HCl, and the liberated sugar was identi-
fied as -galactose by TLC analysis and comparison of its specific
rotation with those of authentic samples.
As shown in Table 1, compounds 1 strongly inhibited the hydro-
lysis of the p-nitrophenyl-phosphate (pNPP) catalyzed by PTP1B in a
dose-dependent manner with IC50 values of 0.19 lM. The character-
istics of the inhibition of PTP1B by compound 1 were then analyzed
in detail. PTP1B was incubated with increasing concentrations of
compound 1 and full velocity curves were determined (Fig. 1).
Non-linear regression analysis showed that the data best fit a com-
petitive model of inhibition, and re-plotting of the data as Linewe-
aver–Burk transformations confirmed this result, displaying the
characteristic intersecting line pattern for competitive inhibition
15
10
5
no compound
0.075 µM
0.12 µM
D
0.16 µM
0
0.00
0.25
0.50
pNPP (mmol-1
0.75
1.00
1.25
)
with a Ki value of 0.10 lM. Therefore, it was shown that aquastatin
900
A (1) binds to the active site within PTP1B. Next, compound 1 was
converted to the corresponding methyl ester (compound 2) to eval-
uate the significance of the carboxylic acid group for potency in
PTP1B inhibition. As shown in Table 1, the IC50 value of compound
2 was significantly lower than that of compound 1. This observation
was consistent with the previous results; that is the carboxylic acid
group plays a major role in the inhibition mechanism.15,16
no compound
0.075 µM
0.12 µM
800
700
600
500
400
300
200
100
0.16 µM
OH
OH
3'
3'
OH
O
OH
O
3
3
O
O
O
O
1' 7'
8'
OH
7
1' 7'
8'
7
O
HO
HO
OH
6''
1''
O
OR
1
5'
1
5'
5
OH
-1
0
1
2
3
4
5
5
22
8
8
22
HO
3''
1/[p
NPP], mM-1
1 R = H
3
Figure 1. Substrate titration studies using the small substrate pNPP revealed that
compound 1 is a classical competitive inhibitor that inhibits substrate binding
(constant Km), but not substrate catalysis (Vmax). (a) Velocity measurements
performed with PTP1B in the presence of increasing concentrations of compound
1. (b) The Lineweaver–Burk transformations of the data from (a). Concentrations
2 R = CH3
OH
3
OH
OH
7
O
HO
6''
1''
O
1
O
OH
5
22
8
HO
3''
(lM) of 1 are indicated in the figure. Data are expressed as mean initial velocity for
n = 3 replicates at each substrate concentration.
4
Aquastatin A (1) belongs to the orcinol p-depsides family, and
only a few of the orcinol p-depsides conjugated with sugars have
been found in nature.17 Chemically, they consist of 2,4-dihy-
droxy-6-alkylbenzoic acid joined by sugars and another 2,4-dihy-
droxy-6-alkylbenzoic acid through two potentially labile bonds
to hydrolysis (i.e., ester and glycoside bonds). Therefore, a couple
of hydrolyzing studies on compound 1 were conducted to check
if all these three structural units are required for the inhibitory
mechanism. When compound 1 was subjected to enzymatic
hydrolysis with b-galactosidase, the aglycone moiety of compound
1 was obtained, and the structure was identified by analysis of its
MS and 1H NMR data. The resulting hydrolysis product 3 was then
evaluated for its PTP1B inhibitory effect, and the observed IC50
showed only 45% inhibition of PTP1B activity at the 178 lM level.
Taken these observations together, it was suggested that 2,4-dihy-
droxy-6-pentadecylbenzoic acid is a critical pharmacophore of
aquastatin A (1) in the inhibition of PTP1B activity. 2,4-Dihy-
droxy-6-pentadecylbenzoic acid, named corticiolic acid, has been
reported as a constituent of several fungal species,18 but no biolog-
ical activity of the compound has been reported.
In the development of PTP1B inhibitors from natural products
or synthetic counter parts, selective inhibition of PTP1B over other
PTPs is one of the biggest issues. Therefore, the specific inhibitory
activities of compound 1 toward a small panel of PTPs including
cytosolic PTPs, T-cell protein tyrosine phosphatase (TCPTP) and
src homology phosphatase-2 (SHP-2), and receptor-like PTP, leuko-
cyte antigen-related phosphatase (LAR), and CD45 were evaluated.
As shown in Table 2, compound 1 displayed significant selectivity
for PTP1B over SHP-2, LAR, and CD45 tyrosine phosphatase (CD45),
but only ꢀ2.5-fold over TCPTP. It has been reported that PTP1B and
TCPTP share a high degree of homology within the catalytic active
site cleft, imposing a major problem in finding selective inhibitors
for PTP1B.
value (0.22 lM) suggested that the galactose moiety in compound
1 does not play any role in the inhibition mechanism. Another
hydrolysis product 4 was obtained from the basic hydrolysis con-
dition (1 N NaOH), and the structure was identified by analysis of
its MS and 1H NMR data. Compound 4 also strongly inhibited the
hydrolysis of the pNPP catalyzed by PTP1B in a dose-dependent
manner with IC50 values of 0.59 lM. On the other hand, the other
basic hydrolysis product, 2,4-dihydroxy-6-methylbenzoic acid,
As noted previously, most of reported PTP1B inhibitors suffer
from drawbacks, such as lack of selectivity and bioavailability.
Table 1
PTP1B inhibitory activity of compounds 1–4
Table 2
Compound
PTP1B inhibitory activity
(IC50, lM)
Selectivity of compound 1 against a panel of PTPs
PTPs
IC50 (lM)
1
2
3
4
0.19
17
0.22
0.59
2.5
PTP1B
TCPTP
SHP-2
LAR
0.19
0.51
>44
>44
>44
Ursolic acida
CD45
a
Positive control.

C. Seo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6095–6097
6097
One reason for this is that most of these compounds were developed
Supplementary data
to target the positively charged active site of PTP1B, thus leading to
low cell permeability.13 Second, most PTPs are known to share a
highly conserved catalytic domain that can be broadly inhibited
by nonspecific inhibitors.8 Therefore, the development of novel
PTP1B inhibitors with improved target-specificity and bioavailabil-
ity is still necessary. In this study, aquastatin A (1) was identified as
competitive inhibitor of PTP1B, and the hydrolyzing studies on the
compound led to the identification of dihydroxypentadecyl benzoic
acid as a key pharmacophore. Aquastatin A (1) showed modest but
selective inhibitory activity for PTP1B over other PTPs such as
TCPTP, SHP-2, LAR and CD45. In addition, the pentadecyl moiety
in the molecule could aid in its ability to diffuse into target cells
to inhibit intracellular PTPs. Therefore, aquastatin A (1) and/or the
essential structural feature identified in this study could be viewed
as potential lead compounds for the treatment of diabetes and obes-
ity. Further optimization of the structures with aiming their selec-
tive potency and efficacy in vivo would be beneficial.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.09.025.
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This research was supported in part by the grants from Global
Partnership Program (No. M60602000001-06E0200-00100) of Kor-
ea Foundation for International Cooperation of Science & Technol-
ogy (KICOS) through the grant provided by the Korean Ministry of
Education, Science & Technology (MEST), and KRIBB Research Initi-
ation Program. We gratefully acknowledge S.-Y. Kim for her techni-
cal support of this project.