Stylissamide X, a new proline-rich cyclic octapeptide as an inhibitor of cell migration, from an Indonesian marine sponge of Stylissa sp.

Article abstract of DOI:10.1016/j.bmcl.2011.10.023

A new proline-rich cyclic octapeptide named stylissamide X (1) was isolated from an Indonesian marine sponge of Stylissa sp. as an inhibitor of cell migration from the guidance of wound-healing assay. The chemical structure of stylissamide X (1) was determined on the basis of spectroscopic analysis, and stereostructure of the amino acids were deduced by Marfey's method. Compound 1 showed inhibitory activity against migration of HeLa cells in the ranges of 0.1-10 μM concentration through both wound-healing assay and chemotaxicell chamber assay, while the cell viability was maintained more than 75% up to 10 μM concentration of 1.

Full text of DOI:10.1016/j.bmcl.2011.10.023

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1818–1821
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Stylissamide X, a new proline-rich cyclic octapeptide as an inhibitor of cell
migration, from an Indonesian marine sponge of Stylissa sp.
Masayoshi Arai a, , Yoshi Yamano a, Mayumi Fujita a, Andi Setiawan b, Motomasa Kobayashi a,
⇑
⇑
a Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6, Suita, Osaka 565-0871, Japan
b Department of Chemistry, Faculty of Science, Lampung University, Jl. Prof. Dr. Sumantri Brodjonegoro No. 1, Bandar Lampung 35145, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history:
A new proline-rich cyclic octapeptide named stylissamide X (1) was isolated from an Indonesian marine
sponge of Stylissa sp. as an inhibitor of cell migration from the guidance of wound-healing assay. The
chemical structure of stylissamide X (1) was determined on the basis of spectroscopic analysis, and
stereostructure of the amino acids were deduced by Marfey’s method. Compound 1 showed inhibitory
Received 6 September 2011
Revised 5 October 2011
Accepted 7 October 2011
Available online 13 October 2011
activity against migration of HeLa cells in the ranges of 0.1–10
wound-healing assay and chemotaxicell chamber assay, while the cell viability was maintained more
than 75% up to 10 M concentration of 1.
lM concentration through both
Keywords:
l
Stylissamide X
Marine sponge
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
Migration
Metastasis
Cancer metastasis is responsible for over 90% of cancer deaths1
and consists of the multi-step process involving epithelial–
mesenchymal transition (EMT), degradation of matrix, cell migra-
tion, and cell adhesion.2 Then, cell migration is attracting much
attention as one of the alternative strategies for development of
anti-cancer chemotherapies. So far, several natural products such
as withaferin A,3 prodigiosin,4 and migrastatin5 have been re-
ported to inhibit cell migration.
In the course of our study of pharmaceutical valuable sub-
stances from marine organisms, we started a search for anti-cell
migration substances and isolated a new proline-rich cyclic octa-
peptide named stylissamide X6 (1) from an Indonesian marine
sponge of Stylissa sp. on the basis of bioassay-guided separation.
In this paper, the structure elucidation of stylissamide X (1) and
its anti-cell migration activity are presented.
The dried marine sponge of Stylissa sp. 05A05 (500 g), which
was collected in 2005 at Biak, Indonesia, was extracted with MeOH
to obtain a MeOH extract. The MeOH extract (125 g), which
showed anti-cell migration activity through wound-healing assay,7
was partitioned into a water–EtOAc mixture (1:1). The EtOAc sol-
uble portion was further partitioned with hexane–90% aq MeOH
mixture (1:1) to furnish a 90% MeOH extract (62 g) and hexane
extract (36 g). On the guidance of bioassay, the 90% MeOH extract
[62 g, minimum inhibitory concentration (MIC) of anti-cell
Figure 1. Chemical structure of stylissamide X (1).
migration activity = 10 lg/mL] was further fractionated by SiO2
gel column chromatography [CHCl3/MeOH/H2O = 65:3:1 (lower
phase)] to give 10 fractions (Fr. M1–Fr. M10). The active Fr. M5
⇑
Corresponding authors. Tel.: +81 66879 8215; fax: +81 66879 8219 (M.K.); tel./
fax: +81 66879 8217 (M.A.).
(3.6 g, MIC = 1.0
matography (MeOH–H2O) to obtain five fractions (Fr. M51–Fr.
M55). The active Fr. M53 (180 mg, MIC = 0.1 g/mL) was further
lg/mL) was then separated by ODS column chro-
E-mail addresses: araim@phs.osaka-u.ac.jp (M. Arai), kobayasi@phs.osaka-u.ac.jp
l
(M. Kobayashi).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.023

M. Arai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1818–1821
1819
purified by ODS HPLC (Cosmosil MS-II, MeOH–H2O = 8:2) to afford
stylissamide X (1) (16 mg, 0.013% yield from the MeOH extract)
(Fig. 1).
Stylissamide X (1) was obtained as a colorless solid. The ESI-TOF
MS of 1 showed a pseudomolecular ion peak at m/z 974 [M+Na]+.
The molecular formula was determined as C51H69N9O9 by high-res-
olution (HR-) ESI-TOF MS. The IR absorptions at 3490, 3320, and
1670 cmꢀ1 suggested the presence of hydroxyl group, amino group,
and amide carbonyl group, respectively. The observation of the sig-
nals at 168–170 ppm in the 13C NMR spectrum and the downfield
signals (7.5–10.8 ppm) of the 1H NMR spectrum indicated peptide
structure of 1. To confirm the amino acid composition of 1, com-
pound 1 was treated by dabsyl chloride following to acid hydroly-
sis.8 HPLC analysis of the dabsylated amino acids revealed that
compound 1 contained three mol of Pro, one mol each of Leu, Ile,
Thr, Trp, and Phe in the structure. The existing of eight amino acids
was also confirmed by NMR analysis including TOCSY, HMQC, and
HMBC experiments (Fig. 2). The connectivity of these amino acids
was figured out by HMBC and ROESY analysis. As shown in Figure 3,
the ROESY correlations between Trp1-NH (dH 8.24) and Pro8-H
3.90); Phe2-H (dH 5.00) and Pro3-Hd1 (dH 3.90), Pro3-Hd2 (dH 3.60);
Pro3-H (dH 4.00) and Leu4-NH (dH 8.70); Thr5-H
(dH 4.90) and
Pro6-Hd1 (dH 3.70); Thr5-Hb (dH 4.10) and Pro6-Hd2 (dH 3.57);
a (dH
a
a
a
Figure 2. COSY and HMBC correlations for stylissamide X (1).
Pro6-H
a a (dH 4.20) and
(dH 4.40) and Ile7-NH (dH 8.30); Ile7-H
Pro8-Hd1 (dH 3.96), Pro8-Hd2 (dH 3.53) revealed the presence of the
sequences of Phe2-Pro3-Leu4 and Thr5-Pro6-Ile7-Pro8-Trp1. In addi-
tion, the HMBC correlations (Fig. 2) between Phe2-NH (dH 8.10)
and Trp1-CO (dC 170.1); Trp1-H
a
(dH 3.68) and Trp1-CO; Thr5-NH
(dH 3.56) and Leu4-CO
(dH 7.50) and Leu4-CO (dC 170.1); Leu4-H
a
proved that stylissamide X (1) has cyclo-(Trp-Phe-Pro-Leu-Thr-
Pro-Ile-Pro) structure. Stereostructure of amino acids was con-
firmed by HPLC analysis of the derivatized amino acids by 5-flu-
oro-2,4-dinitrophenyl-
result of HPLC analysis indicated that all the constituting amino
acids in 1 were found to be -amino acids. In addition, geometry
L
-alanine-amide (Marfey’s reagent).9 The
L
of the peptidic linkages at the proline residues was figured out on
the basis of the 13C chemical shift difference between the proline
b and
Pro6
peptide bonds were trans geometry.10,11,12c The ROESY correlations
between Phe2-H and Pro3-Hd1, Pro3-Hd2; Ile7-H and Pro8-Hd1,
Pro8-Hd2; Thr5-H and Pro6-Hd1, Pro6-Hd2 also supported the trans
c
D
carbons (
D Ddbꢀ = 5.0,
dbꢀ ). The small differences (Pro3
c
c
dbꢀ = 4.4, Pro8
Ddbꢀ = 3.6) indicated that all the proline
c
c
a
a
a
geometry of these proline peptidic linkages. Taken together, the
gross structure of stylissamide X (1) was elucidated as shown in
Figure 1. All the proton- and carbon-signals were assigned as shown
Figure 3. ROESY correlations for stylissamide X (1).
Table 1
1H and 13C NMR data for stylissamide X (1)
a
b
a
b
Residue
dc
dH
Residue
dc
dH
Trp1
a
Leu4
a
b
c
d1 CH3
d2 CH3
CO
57.0
23.4
3.68 (m)
3.64 (m)
3.16 (dd, 12.5, 3.0)
6.96 (s)
52.9
36.1
24.7
23.3
18.5
170.1
3.56 (m)
1.67 (m), 2.15 (m)
1.47 (m)
0.88 (d, 7.0)
0.85 (d, 6.5)
b
2
3
4
5
6
7
8
9
123.5
111.2
136.0
111.3
120.8
118.1
117.9
127.0
170.1
7.30 (d, 8.0)
7.00 (t, 7.5)
6.92 (dd, 8.0, 7.0)
7.40 (d, 7.5)
NH
8.70 (d, 6.0)
Thr5
a
56.3
67.6
4.90 (dd, 9.0, 3.0)
4.10 (m)
5.50 (d, 12.5)
0.94 (d, 6.0)
b
CO
b OH
NH (indole)
NH
10.8 (d, 1.5)
8.24 (d, 6.5)
c
CH3
21.0
168.9
CO
NH
7.50 (d, 9.5)
(continued on next page)

1820
M. Arai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1818–1821
Table 1 (continued)
a
b
a
b
Residue
dc
dH
Residue
dc
dH
Phe2
a
51.0
36.4
5.00 (m)
2.96 (dd, 14.5, 4.0)
2.86 (dd, 14.5, 9.0)
Pro6
a
b
59.2
29.1
24.7
47.0
4.40 (dd, 8.5, 4.0)
1.95 (m), 2.06 (m)
1.74 (m)c, 1.84 (m)c
3.70 (m)
b
1
138.1
129.2
127.8
125.9
169.8
c
2/6
3/5
4
CO
NH
7.25 (m)
7.21 (m)
7.10 (t, 7.5)
d1
d2
CO
3.57 (m)
170.6
8.10 (d, 9.5)
Ile7
a
53.9
35.0
24.6
14.4
9.7
4.20 (t, 9.5)
1.98 (m)
0.86 (d, 7.0)
1.03 (m), 1.30 (m)c
0.65 (t, 7.5)
Pro3
a
b
c
d1
d2
CO
b
60.6
29.7
24.7
47.8
4.00 (t, 7.5)
b CH3
c
d CH3
CO
NH
1.74 (m)c, 2.13 (m)
1.84 (m)c, 2.05 (m)
3.90 (m)c
170.4
3.60 (m)
8.30 (d, 8.0)
170.7
Pro8
a
b
c
d1
d2
CO
60.9
28.4
24.8
47.5
3.90 (m)c
1.94 (m), 2.00 (m)
1.30 (m)c, 1.77 (m)
3.96 (m)
3.53 (m)
170.9
a
13C NMR: dC (ppm), (150 MHz, DMSO-d6).
1H NMR: dH (ppm, J in Hz), (600 MHz, DMSO-d6).
Overlapping resonances.
b
c
Figure 5. Effect of stylissamide X (1) on the migration of HeLa cells induced by EGF.
HeLa cells (3 ꢁ 105 cells) were treated with the indicated concentrations of 1 and
stimulated with EGF (50 ng/mL). After 24 h, the migrated cells to the reverse side of
the membrane filter were counted in the six different microscopic fields. Repre-
sentative figure of the migrated HeLa cells are shown (A). Data were shown by
percentage based on the migrated cell number of the EGF-stimulated HeLa cells (B).
Figure 4. Anti-migration activity and anti-proliferative activity of stylissamide X
(1). (A) Anti-migration activity of 1 by wound-healing assay, (B) anti-proliferative
activity of 1 by MTT method.

M. Arai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1818–1821
1821
Parafilm. The samples were then heated at 70 °C for 10 min. After dabsylation,
the samples were analyzed by reversed-phase HPLC under the following
condition; Cosmosil 5C18 AR, (4.6 mm i.d. ꢁ 250 mm), a 50 min linear gradient
from acetonitrile–25 mM sodium acetate buffer (pH 6.5) = 3:7 to 1:1, 30 °C,
1 mL/min, and detection at 436 nm. Authentic dabsylated Thr, Pro, Ile, Leu, Trp,
and Phe were eluted with retention times of 24.8, 30.8, 37.2, 38.4, 40.4, and
42.4 min, respectively.
in Table 1. Stylissamide X (1) is structurally classified to a group of
the proline-rich cyclic peptides, which are characterized to consist
of seven or eight amino acids containing two or three proline resi-
dues and have been isolated from various genera of marine sponges
such as Axinella,12 Stylotella,13 Phakellia,14 Hymeniacidon,15 and Sty-
lissa.16 So far, these cyclic peptides have been found to exhibit mod-
erate cytotoxic activity and weak antimicrobial activity.
9. The crude amino acids from stylissamide X (1) and
acids were dissolved in 100 L of 0.5 M sodium bicarbonate in 3-mL screw-cap
glass tubes, respectively. Then, 100 L of 5-fluoro-2,4-dinitrophenyl- -alanine
amide ( -FDAA, Marfey’s reagent, 10 mg/mL in acetone) was added to each
sample. The samples were sealed with caps and incubated at 70 °C for 60 min.
After addition of 20 L of 1 N HCl aq, the reaction mixture was diluted with
methanol to suitable volumes (10–15 fold dilution). An aliquot of the -FDAA
derivatives was analyzed by HPLC under the following condition; Cosmosil
NAP, (4.6 mm i.d. ꢁ 250 mm), a 80 min linear gradient from acetonitrile–H2O
containing 0.1% TFA = 1:9 to 1:1, 1.5 mL/min, and detection at 340 nm.
Authentic derivatized -Thr, -Thr, -Pro, -Pro, -Ile, -Ile, -Leu, -Leu, -Phe,
-Phe, -Trp, and -Trp were eluted with retention times of 39.6, 43.2, 47.6,
L- and D-authentic amino
l
l
L
Stylissamide X (1) inhibited migration of HeLa cells in the con-
L
centration ranging from 0.1
assay, whereas cell viability of HeLa cells were maintained more
than 75% up to 10 M of 1 as shown in Figure 4. In addition, stylis-
samide X (1) also inhibited EGF-induced migration of HeLa cells17
from 0.1 M to 10 M concentration of 1 through chemotaxicell
lM to 10 lM through wound-healing
l
L
l
p
l
l
L
D
L
D
L
D
L
D
L
chamber assay (Fig. 5). Further detailed evaluation of the anti-
migration activity of tumor cells is under way.
D
L
D
51.2, 62.8, 68.8, 63.6, 68.8, 66.4, 71.6, 64.8, and 68.4 min, respectively.
10. (a) Dorman, D. E.; Bovey, F. A. J. Org. Chem. 1973, 38, 1719; (b) Dorman, D. E.;
Bovey, F. A. J. Org. Chem. 1973, 38, 2379.
11. McDonald, L. A.; Foster, M. P.; Phillips, D. R.; Ireland, C. M.; Lee, A. Y.; Clardy, J. J.
Org. Chem. 1992, 57, 4616.
Acknowledgements
This study was financially supported by the Hoansha Founda-
tion, the Uehara Memorial Foundation and Grant-in-Aid for scien-
tific research from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan.
12. (a) Pettit, G. R.; Herald, C. L.; Boyd, M. R.; Leet, J. E.; Dufresne, C.; Doubek, D. L.;
Schmidt, J. M.; Cerny, R. L.; Hooper, J. N. A.; Rutzler, K. C. J. Med. Chem. 1991, 34,
3339; (b) Pettit, G. R.; Gao, F.; Cerny, R. L.; Doubek, D. L.; Tackett, L. P.; Schmidt,
J. M.; Chapuis, J. C. J. Med. Chem. 1994, 37, 1165; (c) Randazzo, A.; Piaz, F. D.;
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Schmidt, J. M.; Tackett, L. P.; Bakus, G. J. J. Org. Chem. 1992, 57, 7217; (b)
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van den Bosch, J. J.; Jaspars, M. Tetrahedron 2002, 58, 7863.
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J. N. A.; Bakus, G. J. J. Nat. Prod. 1993, 56, 260; (b) Pettit, G. R.; Tan, R.; Williams,
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References and notes
1. Germanov, E.; Berman, J. N.; Guernsey, D. L. Int. J. Mol. Med. 2006, 18, 1025.
2. Gavert, N.; Ben-Ze’ev, A. Trends Mol. Med. 2008, 14, 199.
3. Lee, J.; Hahm, E. R.; Singh, S. V. Carcinogenesis 2010, 31, 1991.
4. Zhang, J.; Shen, Y.; Liu, J.; Wei, D. Biochem. Pharmacol. 2005, 69, 407.
5. Nakae, K.; Yoshimoto, Y.; Sawa, T.; Homma, Y.; Hamada, M.; Takeuchi, T.;
Imoto, M. J. Antibiot. 2000, 53, 1130.
6. Stylissamide X (1): Colorless solid. ½a D20
ꢂ
ꢀ77 (c = 0.2, CHCl3). IR
m
max (KBr) cmꢀ1
e): 209 (7700). ESI-MS: m/z 974
:
3490, 3320, 1670. UV kmax (MeOH) nm (
[M+Na]+. High resolution ESI-MS: Calcd for C51H69N9O9Na: m/z 974.5116.
Found 974.5107. 1H NMR (600 MHz, DMSO-d6, dH), 13C NMR (150 MHz, DMSO-
d6, dC) spectra: as shown in Table 1.
7. (a) Liang, C. C.; Park, A. Y.; Guan, J. L. Nat. Protoc. 2007, 2, 329; (b) Wound-
healing assay was performed according to the method described by Liang, et al.
[Ref. 7a]. Briefly, the suspension of HeLa cells in DMEM supplemented with 10%
FBS were plated into each well of 96-well plates (1 ꢁ 105 cells/well/100
lL),
and were incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C to
allow the forming of confluent monolayer. After 24 h, the monolayer was
scratched with a pipette tip, washed with culture medium to remove floating
cells, and photographed. Then, the cells were treated with testing samples for
24 h. The cells were photographed again to evaluate whether the cells treated
with testing samples were able to migrate from the cutting edge of wound or
17. A polycarbonate filter of the inner chamber (Chemotaxicell chamber, 8
lm)
was soaked in fibronectin solution (1.3 g/mL) for 1 h at 37 °C and dried in
l
vacuo. Then, HeLa cells (3.0 ꢁ 105 cells) were suspended in FBS free-DMEM
containing the indicated concentration of 1 and seeded in the inner chamber.
The inner chamber was put into the outer chamber (24-well plate), which was
filled with FBS free-DMEM containing Epidermal Growth Factor (EGF) (50 ng/
mL). After 24 h incubation at 37 °C, the non-migrated cells on the upper surface
of the filter were removed by wiping with cotton swabs, and the filter was
fixed with 70% EtOH and stained with Giemsa. The cells, which migrated
through the filter to the reverse side, were counted manually at six different
areas under a microscope. Data were shown by percentage based on the
migrated cell number of EGF-stimulated HeLa cells.
not. The anti-migration activity of testing samples was determined as
minimum concentration of testing samples to inhibit the migration of cells
completely.
a
8. Stylissamide X (1) (0.5 mg) were treated with 2 mL of 1.2 N HCl aq and heated
at 110 °C for 12 h. The reaction mixture was dried under reduced pressure to
obtain crude amino acids. The crude amino acids from stylissamide X (1) and
authentic amino acids were dissolved in 40 lL of 50 mM sodium bicarbonate
(pH 8.1) in glass tubes respectively. Then, 80 lL of freshly prepared 4-
dimethylaminoazobenzene-40-sulfonyl chloride (DABS-Cl) solution (4 mM in
acetonitrile) was added to each sample. The samples were sealed with caps and