Fusarisetin A, an acinar morphogenesis inhibitor from a soil fungus, Fusarium sp. FN080326

Article abstract of DOI:10.1021/ja1110688

An acinar morphogenesis inhibitor named fusarisetin A (1) that possesses both an unprecedented carbon skeleton and a new pentacyclic ring system has been identified from an in-house fractionated fungal library using a three-dimensional matrigel-induced acinarmorphogenesis assay system. The structure of 1 was determined in detail by NMR and circular dichroism spectroscopy, X-ray analysis, and chemical reaction experiments.

Full text of DOI:10.1021/ja1110688

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Fusarisetin A, an Acinar Morphogenesis Inhibitor from a Soil Fungus,
Fusarium sp. FN080326
Jae-Hyuk Jang,†,r Yukihiro Asami,†,‡,r Jun-Pil Jang,†,r Sun-Ok Kim,† Dong Oh Moon,† Kee-Sun Shin,||
Daisuke Hashizume,^ Makoto Muroi,‡ Tamio Saito,‡ Hyuncheol Oh,# Bo Yeon Kim,*,§ Hiroyuki Osada,*,‡
and Jong Seog Ahn*,†
†Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro,
Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea
‡Chemical Biology Department, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
§World Class Institute, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea
Biological Resources Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea
^Cooperative Support Team, Advanced Technology Support Division, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi,
Saitama 351-0198, Japan
#College of Medical and Life Sciences, Silla University, 100 Silladaehak-gil, Sasang-gu, Busan 617-736, Republic of Korea
S Supporting Information
b
purified using flash reversed-phase (ODS) column chromatogra-
ABSTRACT: An acinar morphogenesis inhibitor named
fusarisetin A (1) that possesses both an unprecedented
carbon skeleton and a new pentacyclic ring system has been
identified from an in-house fractionated fungal library using
a three-dimensional matrigel-induced acinar morphogenesis
assay system. The structure of 1 was determined in detail by
NMR and circular dichroism spectroscopy, X-ray analysis,
and chemical reaction experiments.
phy followed by reversed-phase HPLC to afford 15 mg of a novel
bioactive fungal metabolite named fusarisetin A (1; Chart 1).
Compound 1 was obtained as a white powder. The molecular
formula C22H31NO5 was determined by analysis of high-resolu-
tion electrospray ionization mass spectrometry and NMR data.
The IR spectrum displayed absorptions due to hydroxyl groups
at 3325 cmꢀ1 and carbonyl groups at 1734 and 1666 cmꢀ1
.
Analysis of 13C, DEPT, and HMQC NMR spectra of 1 confirmed
the presence of 22 carbons, including two carbonyl carbons
(δC 170.9 and 212.9), three quaternary carbons (δC 108.5, 75.3,
and 55.2), three methyl carbons (δC 21.7, 16.6, and 13.2), one
N-methyl carbon (δC 28.8), four methylene carbons (δC 60.6,
42.1, 35.3, and 25.4), and nine methine carbons (Table S1 in
econdary metabolites produced by microorganisms are a
1
S
valuable repository of natural bioactive compounds, many
of which have been identified as useful research reagents and
potential drug candidates. It is often time-consuming and
expensive to identify new bioactive molecules using conventional
screening strategies. As an alternative approach, we fractionated
and screened various fungal extracts using a three-dimensional
(3D) epithelial culture system, which allows for the visualization
of unique phenotypes such as outgrowth potential, motility,
differentiation ability, and morphology formation.1ꢀ3 This
screening system uses a gel of reconstituted basement membrane
proteins containing growth factors.4ꢀ10 Since the tissue structure
and function of epithelial tumors depend on coordinated cues
from the extracellular matrix (ECM), neighboring cells, and
growth factors11,12 and because the acquisition of malignancy
is generally accompanied by tumor-cell motility, such as acinar
morphogenesis, migration, and invasion, this screening approach
should aid in the discovery of antitumor reagents related to
tumor-cell motility.
the Supporting Information). The H NMR spectrum of 1 in
CD3OD indicated the presence of two doublet methyl protons
(δH 1.47 and 0.94), one singlet methyl proton (δH 0.98), one
N-methyl proton (δH 2.97), two mutually coupled olefinic pro-
tons (δH 5.83 and 5.58, J = 10.0 Hz), two oxymethylene protons
(δH 3.89 and 3.84), and one oxymethine proton (δH 4.37).
Additionally, two exchangeable proton signals were observed in
DMSO-d6 at δ 6.69 (s) and 4.57 (s) and assigned as hydroxyl
protons (Figure S10 in the Supporting Information). Interpreta-
tion of the 2D NMR data, including COSY, HMQC, and HMBC
spectra, led to the construction of the planar structure of 1.
Compound 1 possesses an unprecedented carbon skeleton
with a pentacyclic ring system comprising a decalin moiety (6/6)
and a tricyclic moiety (5/5/5). The decalin moiety was deter-
1
1
mined by Hꢀ H COSY and HMBC correlations to be con-
nected from C-7 to C-16, with methyl groups (H3-21 and H3-22)
attached at C-12 and C-16, respectively.
The COSY cross-peaks (H3-20/H-5/H-6 and H-3/H-18) and
HMBC correlations (H3-20/C-5 and C-6; H-5/C-20; H-6/C-1,
C-3, C-5, and C-7; H3-22/C-15, C-16, and C-17; NꢀCH3/C-2
In the course of screening our fractionated library, we discov-
ered a fraction derived from the fungus Fusarium sp. FN080326
that exhibited inhibitory activity on 3D matrigel-induced acinar
morphogenesis of MDA-MB-231 cells. The fungus Fusarium sp.
FN080326 was isolated from a soil sample collected in Daejeon,
Korea. The EtOAc-soluble portion of the fungal extract was
Received: December 9, 2010
Published: April 18, 2011
r
2011 American Chemical Society
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Chart 1. Structures of Compounds 1ꢀ6
Figure 2. X-ray crystal structure of fusarisetin A (1).
Figure 1. 2D NMR correlations of fusarisetin A (1).
and C-3; H-3/C-2 and C-4; H-18/C-3 and C4) were established
as a tricyclic moiety, 3a-hydroxy-3-(hydroxymethyl)-2,5-dimeth-
ylhexahydro-1H-cyclopenta[3,4]furo[2,3-c]pyrrole-1,8(2H)-dione.
Furthermore, the HMBC spectrum in DMSO-d6 confirmed the
location of two hydroxyl groups. C-4 was assigned as a tertiary
alcohol on the basis of its carbon chemical shift and an HMBC
coupling to an exchangeable proton (δ 6.69 s; 4-OH), which was
further coupled to C-3 and C-1. The other hydroxyl group
(δ 4.57) was assigned as attached to C-18. Two subunits were
connected on the basis of the following 2D NMR correlations:
COSY (H-6/H-7/H-8) and HMBC (H3-22/C-15, C-16 and
C-17). Accordingly, the planar structure of 1 was elucidated as
shown in Figure 1. The relative stereochemistry of 1 was deduced
Figure 3. Fusarisetin A (1) inhibits 3D matrigel culture-induced colony
formation in MDA-MB-231 cells: (A) 3D matrigel control culture; (B)
LY294002 (30 μM); (C, D) MDA-MB-231 cells treated with (C) 10 and
(D) 30 μg/mL concentrations of 1 for 48 h. Circled areas in the
photographs of the control and experimental groups indicate acinar
morphogenesis.
1
on the basis of 1Hꢀ H coupling constants and NOESY spectro-
scopic data (Figure 1). The NOESY correlations of H-10 with
H-12 and H3-22 as well as the correlations of H-7 with H3-20 and
H3-22 indicated that these groups are positioned on the same
face, while the correlations of H-15 with H-6 and H-5 with H-6
and H-3 revealed that they are on the opposite face of the
molecule. These NOESY data and the relevant coupling con-
stants confirmed the trans junction of the decalin ring system and
the cis junction between the tricyclic and decalin ring systems.
The configurations of 1 were thereby established except for the
configuration at C-1.
Fortunately, 1 was crystallized by slow evaporation from a
solution in a 1:1 EtOAc/n-hexane (1:1) mixture over 2 weeks.
Although the resulting crystals were not suitable in size and
quality for single-crystal X-ray analysis using a laboratory source,
data collections performed using a synchrotron source (SPring-8)
furnished single-crystal X-ray data that established the relative
3D structure of 1 (Figure 2). The absolute configuration of the
chiral centers in 1 was determined by the exciton chirality method13
using the reduced di-p-bromobenzoate derivative of 1. After 1
was acetylated, the diacetyl derivative 3 was subjected to OsO4
oxidation to afford the 8,9-diol derivative (4), which was then
treated with p-bromobenzoyl chloride in pyridine to furnish the
8-mono-p-bromobenzoate derivative (5) and the 8,9-di-p-bro-
mobenzoate derivative (6). The magnitudes of the coupling
constants of H-8 (δ 5.85, t, J = 3.2 Hz) and H-9 (δ 5.29, dd,
J = 11.2, 3.2 Hz) of 6 confirmed that the p-bromobenzoyl groups
at C-8 and C-9 are equatorial and axial, respectively. The circular
dichroism (CD) spectrum of 6 exhibited negative first and
positive second Cotton effects [Δε ꢀ5.88 (253 nm), þ1.86
(238 nm)] (Figure S33). The observed negative exciton chirality
indicated the counterclockwise screw sense between the two long
axes of the benzoate chromophores, leading to the assignment of
the 8S and 9R configurations in 6. Therefore, the absolute
stereochemistries of all the stereogenic centers in 1 were deter-
mined as 1S, 3R, 4S, 5R, 6R, 7R, 11R, 12S, 15S, and 16R.
After the structure of fusarisetin A (1) was established, the
effect of 1 on the cell-based bioassay system was investigated. 1
suppressed the 3D matrigel-induced acinar morphogenesis at a
concentration of 30 μg/mL (Figure 3 and Figure S34). In
addition, 1 reduced tube formation at the same concentration
(Figure S34B,C). To further elucidate the biological effects of 1,
we performed cell migration, cell-growth inhibition, and cell
invasion assays using MDA-MB-231 cells. The cell migration was
suppressed by 1 at a concentration of 3 μg/mL without any
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phomopsichalasin15 and chaetochalasin A.16 Our results show
that 1 inhibits a key molecule involved in the development of
acinar morphogenesis, cell migration, and invasion in MDA-MB-
231 cells. Further studies aimed at the identification of this
molecule will provide key insights into the development of
antitumor drugs.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details, spectral
b
data for compounds, and biological data. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
bykim@kribb.re.kr; osadahiro@riken.jp; jsahn@kribb.re.kr
Figure 4. Fusarisetin A (1) inhibits cell migration and invasion without
cytotoxicity to MDA-MB-231 cells. (A) 1 inhibited cell migration
without cytotoxicity at a concentration of 3 μg/mL. Bold bars and open
circles show the numbers of migrated cells and extent of cell-growth
inhibition, respectively. (B) 1 inhibited serum-induced cell invasion at a
concentration of 10 μg/mL. Bold bars show the numbers of invaded
cells. LY294002 (30 μM) was used as a positive control.
Author Contributions
rThese authors contributed equally.
’ ACKNOWLEDGMENT
This research supported by a grant from the Global R&D
Center (GRDC) and World Class Institute (WCI) Program
through the National Research Foundation of Korea (NRF) and
the 21st Century Frontier for Microbial Genomic Application
Center funded by the Ministry of Education, Science, and
Technology of Korea and by a grant from the KRIBB Research
Initiative Program. We thank Mr. Michael Molstad for proof-
reading the manuscript.
cytotoxicity, and 1 inhibited serum-induced cell invasion at a
concentration of 10 μg/mL (Figure 4 and Figure S35). Addi-
tionally, we tested the broad toxic activity of 1 on the same cell
line using a cell-growth inhibition assay (data not shown) and
flow cytometry analysis (Figure S36). The results showed that 1
did not exhibit any significantinhibitionof cellgrowth orinduction
of cell death at a concentration of 30 μg/mL. Next, proteomic
profiling analysis using the 2D fluorescence difference gel electro-
phoresis (2D-DIGE) drug targets prediction system was con-
ducted to analyze the key molecule involved in the inhibition of
acinar morphogenesis, cell migration, and cell invasion.14 Inter-
estingly, no similar proteomic profiles were identified when the
profile of 1 was compared with those of 19 reference compounds
(Figure S37). In addition, no similar patterns of protein variation
were observed in comparison with 31 other compounds in our
database (data not shown). 1 did not show any inhibitory effect on
the phosphorylationof ERK1/2, AKT, p38, orc-Jun in response to
epidermal growth factor (EGF) in MDA-MB-231 cells (Figure
S38). These results suggested that the molecular target of 1 might
be different from those of the reference compounds and is not
significantly related to well-known signal pathways for bioactiv-
ities. Our findings suggest that 1 inhibits a key process involved in
the induction of acinar morphogenesis, cell migration, and inva-
sion in MDA-MB-231 cells.
’ REFERENCES
(1) Li, J. W.; Vederas, J. C. Science 2009, 325, 161–165.
(2) Bugni, T. S.; Richards, B.; Bhoite, L.; Cimbora, D.; Harper, M. K.;
Ireland, C. M. J. Nat. Prod. 2008, 71, 1095–1098.
(3) Bugni, T. S.; Harper, M. K.; McCulloch, M. W.; Reppart, J.;
Ireland, C. M. Molecules 2008, 13, 1372–1383.
(4) Krause, S.; Maffini, M. V.; Soto, A. M.; Sonnenschein, C. BMC
Cancer 2010, 10, 263.
(5) Dhimolea, E.; Maffini, M. V.; Soto, A. M.; Sonnenschein, C.
Biomaterials 2010, 31, 3622–3630.
(6) Coppock, H. A.; Gilham, D. E.; Howell, A.; Clarke, R. B. Cell
Proliferation 2007, 40, 721–740.
(7) Shaw, K. R.; Wrobel, C. N.; Brugge, J. S. J. Mammary Gland Biol.
Neoplasia 2004, 9, 297–310.
(8) Debnath, J.; Walker, S. J.; Brugge, J. S. J. Cell Biol. 2003, 163,
315–326.
(9) Debnath, J.; Muthuswamy, S. K.; Brugge, J. S. Methods 2003,
30, 256–268.
(10) Muthuswamy, S. K.; Li, D.; Lelievre, S.; Bissell, M. J.; Brugge,
In conclusion, an acinar morphogenesis inhibitor named
fusarisetin A (1) that possesses both an unprecedented carbon
skeleton and new ring system has been identified from an in-
house fractionated fungal library. A literature search identified
two previously reported cytochalasin-type fungal metabolites,
phomopsichalasin15 and chaetochalasin A,16 that might have
biosynthetic pathways similar to that of 1 on the basis of their
similar pentacyclic ring systems. However, 1 possesses different
and unique structural characteristics, such as an N-methyl group,
a conjugated hexahydrofuro[2,3-c]pyrrole unit, a primary alcohol
group, and an oxygen-containing isoindol tricyclic moiety (5/5/5)
instead of the isoindole tricyclic unit (5/6/5) found in both
J. S. Nat. Cell Biol. 2001, 3, 785–792.
(11) Wang, F.; Hansen, R. K.; Radisky, D.; Yoneda, T.; Barcellos-
Hoff, M. H.; Petersen, O. W.; Turley, E. A.; Bissell, M. J. J. Natl. Cancer
Inst. 2002, 94, 1494–1503.
(12) Debnath, J.; Brugge, J. S. Nat. Rev. Cancer 2005, 5, 675–688.
(13) Harada, N.; Nakanishi, K. Acc. Chem. Res. 1972, 5, 257–263.
(14) Muroi, M.; Kazami, S.; Noda, K.; Kondo, H.; Takayama, H.;
Kawatani, M.; Usui, T.; Osada, H. Chem Biol. 2010, 17, 460–470.
(15) Horn, W. S.; Simmonds, M. S. J.; Schwartz, R. E.; Blaney, W. M.
Tetrahedron 1995, 51, 3969–3978.
(16) Oh, H.; Swenson, D. C.; Gloer, J. B. Tetrahedron Lett. 1998,
39, 7633–7636.
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