Discovery of incednine as a potent modulator of the anti-apoptotic function of Bcl-xL from microbial origin

Article abstract of DOI:10.1021/ja710124p

Anti-apoptotic oncoproteins Bcl-2 and Bcl-xL are overexpressed in many cancers and play a crucial role in cancer initiation, progression, and resistance to chemotherapy. Therefore, the discovery of a functional inhibitor for these proteins and improved understanding of the molecular mechanisms of these proteins will be an aid to novel anti-tumor therapies. Here, using chemical-genetic cell-based screening, we have discovered a chemically and biologically unique substance, incednine, as a novel functional modulator of Bcl-2/Bcl-xL from the fermentation broth of Streptomyces sp. ML693-90F3. This compound was isolated as a HCl salt by solvent extraction and using centrifugal liquid-liquid partition chromatography. Its structure was elucidated by spectroscopic analysis, X-ray crystallographic analysis, and computational studies. Incednine has a molecular formula, C₄₂H₆₃N₃O₈, and consists of a novel skeletal structure, enol-ether amide in a 24-membered macrolactam core, with two aminosugars. Bcl-xL-overexpressing Ms-1 cells displayed resistance to several anti-tumor agents; however, anti-tumor agent-induced cell death was observed only when cells were treated with incednine. Overexpression of Bcl-xL inhibited cell death in Bax-overexpressing HEK293T cells by forming a complex with Bax, whereas Bcl-xL failed to inhibit cell death in the presence of incednine without affecting the heterodimerization of Bcl-xL and Bax. These findings suggest that incednine serves as a potent modulator of the anti-apoptotic Bcl-2/Bcl-xL distinct from the other known Bcl-2 inhibitors and could provide a chemical probe to study the underlying mechanisms of Bcl-2/Bcl-xL. Copyright

Full text of DOI:10.1021/ja710124p

Published on Web 01/19/2008
Discovery of Incednine as a Potent Modulator of the Anti-apoptotic Function
of Bcl-xL from Microbial Origin
Yushi Futamura,† Ryuichi Sawa,‡ Yoji Umezawa,‡ Masayuki Igarashi,‡ Hikaru Nakamura,‡
Kimiko Hasegawa,§ Mikio Yamasaki,§ Etsu Tashiro,† Yoshikazu Takahashi,‡ Yuzuru Akamatsu,‡ and
Masaya Imoto*,†
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio UniVersity, 3-14-1 Hiyoshi,
Kohoku-ku, Yokohama 223-8522, Japan, Microbial Chemistry Research Center, 3-14-23 Kamiosaki, Shinagawa-ku,
Tokyo 141-0021, Japan, and Rigaku Corporation, 3-9-12 Matsubara-cho, Akishima-shi, Tokyo 196-8666, Japan
Received November 7, 2007; E-mail: imoto@bio.keio.ac.jp
Anti-apoptotic oncoproteins Bcl-2 and Bcl-xL are overexpressed
in many cancers,1 resulting in the expansion of a transformed
population and the advancement of the multidrug-resistant stage.
Consequently, Bcl-2/Bcl-xL have stood out among molecular targets
in oncology, and the functional blockade of these proteins will be
an aid to novel anti-tumor therapies. Since these proteins are known
to show an anti-apoptotic effect partly through forming a het-
erodimer with pro-apoptotic Bcl-2 members, such as Bax and Bak,2
several researchers have rationally designed and synthesized
compounds that target their binding pocket and have reported
several compounds such as HA14-1 and ABT-737 as Bcl-2/Bcl-
xL inhibitors.3 Currently, several lines of evidence indicate that
Bcl-2/Bcl-xL clearly have other functions related to their abilities
to interact physically with many other proteins; however, the
underlying mechanisms for the regulation of apoptosis by Bcl-2/
Bcl-xL through interacting with such proteins still remain unclear.4
For further understanding of the regulation of apoptosis by Bcl-2/
Bcl-xL, the development of a new class of chemical tools is
required. Cell-based chemical-genetic screens have been used to
help discover small, cell-permeable bioactive molecules that induce
phenotypic changes, and through subsequent identification of their
target proteins, they can contribute to reveal the molecular basis
of biological processes. Therefore, we constructed a cell-based
chemical-genetic screening system to discover small molecules
that induce apoptosis in Bcl-xL-overexpressing human small cell
lung carcinoma Ms-1 cells when combined with anti-tumor drugs.
In the course of our screening, we isolated a structurally and
functionally unique compound, named incednine (1), from the
culture broth of Streptomyces sp. ML694-90F3. Here, we describe
the isolation, structure elucidation, and biological activities of 1.
(λmax ) 294.5, 309.5, 322.5, 356.0 nm) was indicative a polyene
moiety. The positive color reaction to GL reagent, negative reaction
to ninhydrin, and the typical IR absorption at 1650, 1510 cm-1
suggested the presence of an amido group.
The assignable NMR spectra were obtained when the sample
was dissolved in CD3OH/H2O (3:1) and measured at -5 °C. The
13C NMR and DEPT spectra revealed that 1 contained 42 carbons,
including one carbonyl, four quaternary sp2, one quaternary sp3,
fourteen sp2 methines, nine sp3 methines, four methylenes, and nine
methyl carbons. One carbonyl signal and eighteen sp2 carbons
require the presence of three rings from the unsaturation numbers
of the formula of 1.
Partial structures of 1 were determined by 1H-1H COSY,
TOCSY, and HMBC (Figure 1). A detailed analysis of the COSY
spectrum of 1 revealed five partial proton-proton connectivities:
(a) H-5 to H-9 and methyl group H-24, (b) from H-11 to H-13, (c)
from H-21 to NH and methyl group H-27 and H-28 and (A) from
H-1′ to H-5′, and (B) H-1′′ to H-6′′. The linkage of the partial
structures between (a) and (b) was established by HMBC correla-
tions from H-25 to C-9, C-10, and C-11, which were bridged by
the quaternary oxygenated carbon of C-10. The overlapping olefin
region (δ 6.1-6.3) was assigned by the following experiments. The
connectivities from H-13 to H-15, H-17 to H-19 in TOCSY spectra
and those from H-26 to C-15, C-16, and C-17 and from H-27 to
C-19, C-20, and C-21 in HMBC clarified the linkage of the partial
structures (b) and (c) to a pentaene structure. The correlations from
the NH proton in the partial structure (c) to C-1, from H-3 to C-1
and C-2, from H-24 to C-3, C-4, and C-5, and from H-29 to C-2
constructed a 24-membered macrocyclic lactam ring. The remaining
partial structures (A) and (B) were deduced from HMBC experi-
ments. The correlations from H-5′ to C-1′, from H-5′′ to C-1′′, and
from the N-methyl group of H-6′ to C-2′ and H-7′′ to C-4′′
established the presence of two types of aminosugars: a pentopy-
ranose (A) and a hexopyranose (B), respectively. Furthermore, the
HMBC correlations from H-1′ to C-11 and from H-1′′ to C-4′
revealed the planar structure of 1: the linkage of aminosugar (A)
and the macrocyclic lactam ring at C-11 and the glycoside linkage
of C-1′′ of aminosugar (B) and C-4′ of aminosugar (A).
Incednine (1) was obtained (18.9 mg/L) as a pale-yellow powder
from the cultured broth of the producing strain by centrifugal
liquid-liquid partition chromatography for two reasons: (1) this
compound decomposes easily under acidic conditions or when
exposed to light, and (2) the isolation using a solid carrier such as
silica gel was inefficient. The molecular formula of 1 was found
to be C42H63N3O8 by HRESIMS. The characteristic UV absorption
The stereochemistry of incednine aglycon was elucidated as
follows. The geometry of the double bonds was determined by the
proton coupling constants and NOEs (Figure S8). The coupling
constants of J6,7 ) 14.4 Hz, J8,9 ) 15.8 Hz, J12,13 ) 9.0 Hz revealed
6E, 8E, and 12Z, respectively. NOEs observed between H-24 and
H-29, H-6 and H-24, H-18 and H-26, H-18 and H-27, and H-19
and H-21 showed 2Z, 4E, 16E, 18E, and 20E, respectively. The
† Keio University.
1
geometry of H-14 was inferred from H-1H J-resolved spectros-
‡ Microbial Chemistry Research Center.
§ Rigaku Corporation.
copy to be 14E (J14,15 ) 14.0 Hz, Figure S9). The relative
9
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J. AM. CHEM. SOC. 2008, 130, 1822-1823
10.1021/ja710124p CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Incednine overcame the anti-apoptotic function of Bcl-xL against
adriamycin-induced apoptosis. Cells were treated with drugs as indicated
(ADM, adriamycin; 0.3 µg/mL, ICN, incedenine; 100 nM). After 48 h, cells
were observed under a phase-contrast microscope.
Figure 1. Gross structure of 1 determined by 2D-NMR spectra (bold lines,
COSY; arrows, selected HMBC correlations; dashed squares, TOCSY
connectivities; single squares, partial structures labeled as indicated).
apoptosis in HEK293T cells, and it was inhibited by coexpression
of Bcl-xL (Figure S17). Upon treatment with 60 nM of incednine,
HEK293T cells overexpressing both Bax/Bak and Bcl-xL underwent
apoptosis, indicating that incednine restored the pro-apoptotic ability
of Bax or Bak that was inhibited by Bcl-xL. However, incednine
did not inhibit the binding capacity of Bcl-xL to Bax (Figure S18).
In addition, incednine did not decrease the expression levels of Bcl-
xL (data not shown). These results clearly suggest that incednine
can inactivate the anti-apoptotic function of Bcl-2/Bcl-xL by a
distinct mode of action from other inhibitors that recognize the
surface pocket of Bcl-2/Bcl-xL.
In conclusion, we isolated a structurally unique compound,
incednine, as a modulator of Bcl-xL function through a cell-based
chemical-genetic screening from microbial origin. Because this
compound inhibits the anti-apoptotic function of Bcl-2/Bcl-xL
without affecting its binding to pro-apoptotic Bcl-2 family proteins,
it may target other proteins associated with the Bcl-2/Bcl-xL-
regulated apoptotic pathway. Therefore, incednine may be a useful
tool for further study of Bcl-xL function, and the identification of
its target protein could provide a new insight into the anti-apoptotic
mechanism of Bcl-2 family proteins.
stereochemistry of three chiral centers was established by NOE
experiments and computations. The geometry of double bonds in
aglycon and NOEs observed surrounding 4, 16, and 20-methyl
groups allowed four types of the ring conformers. The coupling
constant of J11,12 ) 9.0 Hz and NOEs observed between H-8 and
H-25, H-25 and H-11, and H-11 and H-14 indicated the relative
stereochemistry of C-10 and C-11 to be R* and S*, respectively.
The remaining asymmetric center, C-23, was elucidated by NOEs
observed surrounding the C-23 position to be S*, which was
supported by Discovery III programs (Figure S10).
The absolute configuration of the C-11 position of the aglycon
(2)5 was determined by the application of a modified Mosher’s
method.6 Both the (()-MTPA esters (3, 4) of 2 were prepared and
subjected to 1H NMR analysis. The ∆δ values of the protons
allowed the absolute configuration at C-11 to be S; thus, the
stereochemistry of the three asymmetric centers of the aglycon was
10R, 11S, and 23S (Figure S12).
The stereochemistry for two aminosugars was deduced as
follows. Large coupling constants of J1′,2′ax ) 7.0 Hz, J2′,3′ ) 7.0
Hz, J3′,4′ ) 8.0 Hz, J4′,5′ax ) 8.0 Hz, J1′′,2′′ax ) 9.1 Hz, and J4′′,5′′
)
Acknowledgment. We thank Dr. H. Osada and Dr. S. Simizu
(RIKEN) for kindly providing us with human bcl-2, bcl-xL, and
bax constructs. We also thank Dr. Y. Tsujimoto (Osaka University,
Japan) for the generous gift of human bak plasmid.
9.2 Hz indicated that the connected carbon, nitrogen, and oxygen
atoms were all equatorial. Moreover, the absolute stereochemistry
of H-1′ was elucidated to be S by NOEs observed between H-1′
and H-11, H-1′ and H-25, and H-6′ and H-25 (Figure S13). Thus,
the aminosugar (A) was 2-deoxy-2-methylamino-â-D-xylopyranose.7
To determine the only remaining absolute stereochemistry, the 4-N-
4-bromobenzoyl derivative of the aminosugar (B) was prepared.
Methanolysis of the 4′′-N-4-bromobenzoyl derivative of 18 gave
the corresponding R and â (5) methyl glycoside. Compound 5 was
recrystallized with hexane/acetone to give a colorless platelet.9 X-ray
crystallography for 5 elucidated the aminosugar (B) as 2,3,4,6-
tetradeoxy-4-methylamino-â-D-erythro-hexopyranose (N-monodem-
ethyl-D-forosamine, Figure S14).10 Thus, incednine (1) consists of
a novel skeletal structure, enol-ether amide in the 24-membered
macrolactam core, with two aminosugars.
Incednine was tested for its suppressive activity against the anti-
apoptotic function of Bcl-2/Bcl-xL. Bcl-xL-overexpressing Ms-1
cells displayed resistance to various types of anti-tumor agents, such
as adriamycin (Figure S15). However, this resistance was overcome
by the sequential combination of anti-tumor agents and 100 nM
incednine, whereas incednine alone did not induce apoptosis in these
cells (Figure 2, S16). The same results were obtained when Bcl-
2-overexpressing cells were used (data not shown). These findings
suggest that incednine significantly sensitizes Bcl-2/Bcl-xL-over-
expressing cells to chemotherapeutic treatment most likely through
dysfunction of Bcl-2/Bcl-xL.
Supporting Information Available: Experimental procedures,
NMR spectra data, crystallographic information, and results of Bcl-xL
inhibitory activities for 1. This material is available free of charge via
the Internet at http://pubs.acs.org.
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(8)°, V ) 782.31 (2) Å3, Z ) 2, T ) 296 K, R1 ) 0.0362, GOF ) 1.205.
See details in Supporting Information.
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Bak.11 Indeed, overexpression of either Bax or Bak induced
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