Intriguing Natural Products from Marine Sources
Daisuke Uemura
Faculty of Science, Kanagawa University, Japan
uemurad@kanagawa-u.ac.jp
Many compounds with extraordinary chemical structures and brilliant bioactivities have been
identified from marine organisms. In this presentation, I will describe the fascinating natural
products I have been investigating to explore novel frontiers in both chemistry and bioscience.
My presentation covers three main topics.
1.
Halichondrin and metagenome-assisted production.
2.
Luminaolide, a metamorphosis enhancer in coral larvae
3.
Lyngbyacyclamides A and B, structure and total synthesis
1. Halichondrin and metagenome-assisted production
Halichondrin B was isolated from the black sponge Halichondria okadai, in 1986.
Interestingly, this polyether macrolide exhibited antitumor activity both in vitro and in vivo.
The mechanism of action of halichondrin B was shown to be a novel one that disrupts the
polymerization dynamics of tubulin, which makes this natural product an interesting
candidate for the treatment of cancer. However, the difficulty of collecting sufficient material
(only 12.5mg from 600kg of sponge) impaired studies for its development. The complete
synthesis of halichondrin B in 1992 represented a breakthrough. The total synthesis also
revealed that the activity resides in the macrocyclic lactone C1-C38 moiety. Ultimately, the
moiety derivative was approved for the treatment of breast cancer in several countries and is
now available on the market as the drug Halaven.
Although useful natural products such as halichondrins and okadaic acid have been isolated
from extracts of Halichondria okadai, it is not clear whether the black sponge itself
synthesizes these compounds. Recent studies have suggested that some as-yet-uncultivable
symbiotic microorganisms are true sources of these compounds. Therefore, we isolated the
genes of symbiotic bacteria in this black sponge and established a 160,000 fosmid library. I
will discuss our marine meta-genome project in detail.
2. Luminaolide, a metamorphosis enhancer in coral larvae
The settlement and metamorphosis of larvae of many marine invertebrates are known to be
influenced by crustose coralline algae (CCA). Some of these algae inhibit, while others
induce, settlement and/or metamorphosis. In our search for bioactive substances in CCA, we
found that fragments of coral rubble with the CCA Hydrolithon reinboldii induced larval
metamorphosis in the scleractinian coral Leptastrea purpurea. Based on these observations,
we isolated a novel macrodiolide, luminaolide, from H. reinboldii as a natural enhancer of
larval metamorphosis by a simple bioassay with larvae of L. purpurea. This is the first
example of a macrolide that enhances the metamorphosis of scleractinian coral larvae, and
could help to protect coral reefs.
3.
Lyngbyacyclamides A and B, structures and total synthesis
Cyanobacteria are photosynthetic prokaryotes and that are widely distributed throughout
marine and terrestrial environments. Members of the marine cyanobacteria genus Lyngbya are
known to produce structurally interesting and biologically active secondary metabolites. We
have purified new compounds lyngbyacyclamides A and B. The biological activities of these
compounds are quite unique, since they show toxicity against B16 melanoma cells, but not
brine shrimp. Efforts to synthesize these molecules are currently underway so that we can
elucidate the mechanism of action of Lyngbyacyclamides.
References
1.
Exploratory research on bioactive natural products with a focus on biological phenomena.,
D. Uemura, Proc. Jpn. Acad., Ser. B, 86, 190-201 (2010)
2.
Construction of a metagenomic library for the marine sponge, Halichondria okadai., T.
Abe, F.P. Sahin, K. Akiyama, T. Naito, M. Kisigami, K. Miyamoto, Y. Sakakibara, and D.
Uemura, Biosci. Biotechnol. Biochem., 76, 1233-1315 (2012)
3.
Halichondrins-antitumor polyether macrolides from a marine sponge., Y. Hirata and D.
Uemura, Pure Appl. Chem., 58, 701, (1986)
4.
Luminaolide, a novel metamorphosis-enhancing macrodiolide for scleractinian coral
larvae from crustose coralline algae., M. Kitamura, P. Schupp, Y. Nakano, D. Uemura,
Tetrahedron Lett., 50, 6606-6609 (2009)
5.
Lyngbyacyclamides A and B, novel cytotoxic peptides from marine cyanobacteria
Lyngbya sp., Tetrahedron Lett., 51, 6384-6387 (2010)
The Achievements of Professor Youji Sakagami, a Pioneer of Plant
and Microbial Peptide Hormone Researches
Makoto Ojika
Graduate School of Bioagricultural Sciences, Nagoya University, Japan
ojika@agr.nagoya-u.ac.jp
Dr. Youji Sakagami, professor of Nagoya University and Yongqian chair professor of
Zhejiang University, passed away on April 9, 2012. This deeply disappointed me, as I had
been a member of his laboratory for 6 and a half year. Here, I wish to introduce his
extraordinary achievements with my deepest condolences.
He had tried to solve many issues on endogenous bioactive substances such as hormones and
pheromones. When he started his academic research, he was surprised at the fact that little
endogenous peptides had been discovered from plants and microorganisms and made this
puzzle his life work. He finally clarified that such endogenous peptides actually existed as
uniquely modified forms, contributing to the recent development in the field of the natural
peptide research. One of his biggest achievements is the discovery of the plant peptide
hormone phytosulfokine (PSK),1 the first example of sulfated peptides, laying the foundations
for the general idea of "plant peptide hormones". Furthermore, the biosynthesis and mode of
action of PSK have been
revealed (Fig. 1).2 He then
identified a morphogenesis
inducer of shoot apices3
and
a
stomatal
differentiation
enhancer.4
He
discovered
also
microorganism
peptides,
proliferation
differentiation
somatic embryogenesis
PSK gene
prePSK
sulfation
signal
transduction
processing
SO3H SO3H
Tyr-Ile-Tyr-Thr-Gln
PSK
PSK receptor
PSK
Fig. 1 Biosynthesis and cellular signaling of the plant peptide
hormone phytosulfokine (PSK)
e.g., a basidiomycete sex
pheromone, tremelogen A-10, as a peptide with a terminal S-farnesylcysteine,5 and one of the
Bacillus subtilis quorum sensing pheromones ComX as the first peptide with a unusually
modified tryptophan.6 Though non-peptides, he identified sexual hormones of the crop
pathogen Phytophthora in collaborations with professor Jianhua Qi and me.7,8 These
achievements were published in a number of excellent journals such as Science (6 papers) and
Nature Chemical Biology (3 papers), and appears to be created from his outstanding research
stance: to focus on the issues that had not been reported in reviews and books yet and to
purify genuine functional molecules by using originally developed bioassays (but not to
predict from genomic data bases).
References
1. Matsubayashi, Y. et al. PNAS, 93, 7623 (1996).
2. Matsubayashi, Y. et al. Science, 296, 1470 (2002).
3. Kondo, T. et al. Science, 313, 845 (2006).
4. Kondo, T. et al. Plant Cell Physiol. 51, 1 (2010).
5. Sakagami, Y. et al. Science, 212, 1525 (1981).
6. Okada, M. et al. Nat. Chem. Biol. 1, 23 (2005).
7. Qi, J. et al. Science, 309, 1828 (2005).
8. Ojika, M. et al. Nat. Chem. Biol. 7, 591 (2011).
Ojika’s representative publications
1. Ojika, M.; Molli, S. D.; Kanazawa, H.; Yajima, A.; Toda, K.; Nukada, T.; Mao, H.; Murata,
R.; Asano, T.; Qi, J.; Sakagami, Y. The second Phytophthora mating hormone defines
interspecies biosynthetic crosstalk, Nat. Chem. Biol. 7, 591-593 (2011).
2. Ojika, M.; Inukai, Y.; Kito, Y.; Hirata, M.; Iizuka, T.; Fudou, R. Miuraenamides:
antimicrobial cyclic depsipeptides isolated from a rare and slightly halophilic
myxobacterium, Chem. Asian J. 3, 126-133 (2008).
3. Ojika, M.; Kigoshi, H.; Yoshida, Y.; Ishigaki, T.; Nisiwaki, M.; Tsukada, I.; Arakawa, M.;
Ekimoto, H.; Yamada, K. Aplyronine A, a potent antitumor macrolide of marine origin,
and the congeners aplyronines B and C: isolation, structures, and bioactivities,
Tetrahedron 63, 3138-3167 (2007).
4. Qi, J.; Asano, T.; Jinno, M.; Matsui, K.; Atsumi, K.; Sakagami, Y.; Ojika, M.
Characterization of a phytophthora mating hormone, Science, 309, 1828 (2005).
5. Onodera, K.; Nakamura, H.; Oba, Y.; Ohizumi, Y.; Ojika, M., Zooxanthellamide Cs:
vasoconstrictive polyhydroxylated macrolides with the largest lactone ring size from a
marine dinoflagellate of Symbiodinium sp. J. Am. Chem. Soc. 127, 10406-10411 (2005).
Sexual Reproduction Inducers of Phytophthora
Jianhua Qi
College of Pharmaceutical Sciences, Zhejiang University, China
qijianhua@zju.edu.cn
(Dedicated to the Late Professor Youji Sakagami.)
Members of the genus Phytophthora cause devastating plant diseases that threaten crops
worldwide. The ability of these species to reproduce sexually might account for their genetic
diversity and aggressive pathogenicity. Therefore, characterization of the endogenous factors
(-hormones) that stimulate sexual reproduction of the Phytophthora should be essential to
control the pathogen.
Since their existence was first proposed by Ashby in 1929, the mating hormones that induced
sexual reproduction in Phytophthora have received great attention.1) However several decades
pasted, numerous attempts have been made to isolate and determine the structure of these
hormones, their details are still unknown.2-4) We spent 11 years on this project. In 2005, 1.2
mg of pure hormone 1 was obtained from supernatant of culture broths (approximately 1,830
L) of the A1 mating type. 5) The full planar structure of hormone 1 was determined by the
combination analysis of MS, one- and two-dimensional NMR spectra.5) The structure was
then confirmed by chemical derivation methods.
To support future synthesis of
1, we determined the absolute configuration of the two
terminal stereogenic centers, C-3 and C-15, by NMR analysis of the Mosher’s esters of1
and the synthetic model compound.6) We then synthesized four optically pure diastereomers
with the fixed 3R and 15R configurations.7) Very small differences among synthesized
samples were observed by 13C NMR. Thus, it was difficult to distinguish the natural hormone
1 from the synthetic samples. Consequently, determination of the absolute configurations of
the natural hormone 1 by NMR analyses alone could be impossible for this linear diterpene.
Therefore, the oospore-inducing activities of the synthesized isomers were tested in
comparison with that of the natural hormone α1. The isomer 3R,7R,11R,15R induced
significant oospore formation on the A2 mating type of P. nicotianae at a dose of 30 ng,
which was similar to that of natural hormone 1.7) On the other hand, no remarkable oospore
formation was induced by other three isomers at the tested doses. These results indicated that
the natural hormone 1 possesses the 3R,7R,11R,15R absolute configurations.7) Recently, the
absolute stereostructure of the second mating hormone 2 was defined by spectroscopic
analysis and total synthesis8). We have uncovered not only the interspecies universality of 
hormones but also the pathway by which 2 is biosynthesized from phytol by A2 strains and
metabolized to 1 by A1 strains as shown in the figure below.
References
1. Ashby, S.F.: Trans. Br. Mycol. Soc., 14, 18-38 (1929).
2. Ko, W.H.: J. Gen. Microbiol., 107, 15-18 (1978).
3. Chern, L.L., Tang, C.S., Ko, W.H.: Bot. Bull. Acad. Sin., 40, 79-85 (1999).
4. Jee, H.J., Tang, C.S., Ko, W.H.: Bot. Bull. Acad. Sin., 43, 203-210 (2002).
5. Qi, J., Asano, T., Jinno, M., Matsui, K., Atsumi, K., Sakagami, Y., Ojika, M.: Science, 309,
1828-1828 (2005).
6. Ojika, M., Qi, J., Kito, Y., Sakagami, Y.: Tetrahedron Asymmetry, 18, 1763–1765 (2007).
7. Yajima, A., et al.: Nature Chem. Biol., 4, 235-237 (2008).
8. M. Ojika, et al.: Nature Chem. Biol., 7, 591-593 (2011).
Representative Publications
1) Cerebroside-A provides potent neuroprotection after cerebral ischemia through reducing
glutamate release and Ca2+ influx of NMDA receptors. L. Li, Y. Bai, R. Yang, Z. Zhang, B.
Xu,
Z.
Qi,
J.
Qi*,
and
L.
Chen*,
The
International
Journal
of
Neuropsychopharmacology, 15, 497-507 (2012).
2) Structure-activity relationships of neuritogenic gentiside derivatives. Y. Luo, K. Sun, L. Li,
L. Gao, G. Wang, Y. Qu, L. Xiang, L. Chen, Y. Hu, and J. Qi,* ChemMedChem, 6,
1986-1989 (2011).
3) The second Phytophthora mating hormone defines interspecies biosynthetic crosstalk. M.
Ojika, S. Molli, H. Kanazawa, A. Yajima, K.Toda T. Nukada, H. Mao, R. Murata, T.
Asano, J. Qi, and Y. Sakagami, Nature Chemical Biology, 7, 591-593 (2011).
4) Synthesis and absolute configuration of hormone 1. A. Yajima,* Y. Qin,* X. Zhou, N.
Kawanishi, X. Xiao, J. Wang, D. Zhang, Y. Wu, T. Nukada, G. Yabuta, J. Qi,* T. Asano,
and Y. Sakagami, Nature Chemical Biology, 4, 235-237 (2008).
5) Granulatoside A, a starfish steroid glycoside, enhances PC12 Cell neuritogenesis induced
by nerve growth factor through an activation of MAP Kinase. J. Qi, C. Han, Y. Sasayama,
H. Nakahara, T. Shibata, K. Uchida, and M. Ojika, ChemMedChem, 1, 1351-1354 (2006).
6) Characterization of a Phytophthora mating hormone. J. Qi, T. Asano, M. Jinno, K. Matsui,
K. Atsumi, Y. Sakagami, and M. Ojika, Science, 309, 1828 (2005).
Peptide Isoprenylation
Masahiro Okada
College of Bioscience and Biotechnology, Chubu University, Japan
okada@isc.chubu.ac.jp
Bacillus subtilis and related bacilli produce a posttranslationally modified oligopeptide,
ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing.
Our studies revealed that the ComXRO-E-2 pheromone from B. subtilis strain RO-E-2 had a
unique modified tryptophan residue with a geranyl group, forming a tricyclic structure (Figure
1).1) Also, the ComXRO-C-2 pheromone from B. mojavensis strain RO-C-2 was confirmed to
have a farnesyl-modified tryptophan residue similar to that of the ComX RO-E-2 pheromone.2)
Together with its phylogenetic resemblance to ComX, these findings suggested that
posttranslational isoprenoidal modifications of ComX pheromones were classified into two
types: geranylation and farnesylation, the ComX pheromones are formed by geranylation or
farnesylation on a tryptophan residue at the 3 position of its indole ring. This results in the
formation of a tricyclic structure including, a newly formed five-membered ring, similar to
proline.
Figure 1. Chemical structure of ComXRO-E-2 pheromone.
Sakagami et al. first reported that the peptide pheromones, tremerogen a-13 and tremerogen
A-10, of basidiomycetous yeasts were modified with a farnesyl group on cysteine (Figure 2).3)
Posttranslational farnesylation or geranylgeranylation at C-terminal cysteine residue via a
thioether linkage is widely observed in eukaryotes and plays a critical role in protein function.
Figure 2. Chemical structure of tremerogen a-13.
Isoprenylation of ComX to form ComX pheromones is also essential for pheromonal activity.
However, only the modified tryptophan residue plays a determinative role in the activity of
the ComXRO-E-2 pheromone.4) The activity spectrum of the ComXRO-E-2 pheromone strongly
contrasts with that of tremerogen A-10, in which removal of the N-terminal amino acid
residue induced a loss of biological activity.
The ComX pheromone is the first example of isoprenoidal modifiations of tryptophan
residues in living organisms and posttranslational isoprenylation of any amino acid in
prokaryotes. Especially, because the presence of geranylated compounds is unusual in primary
and secondary metabolites outside the plant kingdom, posttranslational geranylation in bacilli
is unprecedented in nature.
References
1) M. Okada, et al., Nat. Chem. Biol., 2005, 1, 23.
2) M. Okada, et al., Biosci. Biotechnol. Biochem., 2008, 72, 914.
3) Y. Sakagami, et al., Science, 1981, 212, 1525.
4) M. Okada, et al., Bioorg. Med. Chem. Lett., 2007, 17, 1705.
Mining Functional Organic Compounds from Nature
Renxiang Tan
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, China
rxtan@nju.edu.cn
All plants and microorganisms are able to produce an array of functional natural products
essential for their survival in nature. A collection of evidences has demonstrated that active
biomolecules play multiple physiological and ecological roles in the process of microbe-host
and symbiont-host-environment interactions. Furthermore, some symbionts have been
disclosed as to be capable to improve or initiate the host growth through improving the
tolerance of the host to environmental stresses such as drought, salinity, heavy metals as well
as attacks of or consumptions by microbial pathogens, nematodes, insects and mammal
herbivores. Biochemically, a growing pile of data has demonstrated that the ‘host-helping’
effects of symbionts are ascribable to the production of bioactive compounds. As re-affordable
bio-resource, symbionts could be accepted as a promising reservoir of ‘special
microorganisms’ that could produce chemically inspiring and pharmacologically important
natural products.
This talk mainly deals with the new findings about the topic, particularly the full
characterization of the drug-like natural products with unique architectures. The significance of
the functional phytochemicals and symbiont metabolites will be discussed in brief.
Representative Publications
1.
W. Fang, S. Ji, N. Jiang, W. Wang, G. Y. Zhao, S. Zhang, H. M. Ge,
Q.
Xu,
A.
H.
Zhang, Y. L. Zhang, Y. C. Song, J. Zhang, R. X. Tan. Nat. Commun. 2012, 3, 1039
doi:10.1038/ncomms2031.
2. H. M. Ge, H. Sun, N. Jiang, Y. H. Qin, H. Dou, T. Yan, Y. Y. Hou, C. Griesinger, R. X. Tan.
Chem. Eur. J., 2012, 18, 5213.
3. L. Lin and R. X. Tan. Chem. Rev., 2011, 111, 2734.
4. Y. L. Zhang, H. M. Ge, W. Zhao, H. Dong, Q. Xu, S. H. Li, J. Li, J. Zhang, Y. C. Song and
R. X. Tan. Angew. Chem. Int. Ed., 2008, 47, 5823.
5. Y. L. Zhang, J. Zhang, N. Jiang, Y. H. Lu, L. Wang, S. H. Xu, W. Wang, G. F. Zhang, Q.
Xu, H. M. Ge, J. Ma, Y. C. Song and R. X. Tan. J. Am. Chem. Soc., 2011, 133, 5931.
6. A. H. Zhang, N. Jiang, W. Gu, J. Ma, Y. R. Wang, Y. C. Song and R. X. Tan. Chem. Eur. J.,
2010, 16, 14479.
7. H. M. Ge, W. H. Yang, Y. Shen, N. Jiang, Z. K. Guo, Q. Luo, Q. Xu, J. Ma and R. X. Tan.
Chem. Eur. J., 2010, 16, 6338.
8. H. M. Ge, W. Yan, Z. K. Guo, Q. Luo, R. Feng, L. Y. Zang, Y. Shen, R. H. Jiao, Q. Xu and
R. X. Tan. Chem. Commun., 2011, 47, 2321.
9. H. M. Ge, W. Y. Zhang, G. Ding, P. Saparpakorn, Y. C. Song, S. Hannongbua and R. X.
Tan. Chem. Commun. 2008, 5978.
10. H. M. Ge, C. H. Zhu, D. H. Shi, J. Yang, S. W. Ng, R. X. Tan. Chem. Eur. J., 2008, 14,
376.
Integrative Approach for Target Identification of Bioactive
Compounds
Hiroyuki Osada
Chemical Biology Core Facility, RIKEN ASI, Japan
hisyo@riken.jp
We have been exploring novel bioactive compounds from natural products and chemically
synthesized chemical libraries for many years. The screening of bioactive compounds based
on the cytotoxicity is still useful; however, its further target identification remains as an
obstacle. To accelerate the prediction of mechanism of action of the bioactive compounds
compound, we have constructed the target identification systems based on specific changes in
cell morphology and in cell proteome induced by an exposure.
The “Morphobase” compiles the phenotypes of cancer cell lines induced by hundreds of
reference compounds, wherein those of well-characterized antitumor compounds are
classified by the mode of action.
The other database, “Proteobase”, contains proteomic images of the changes in protein
expression after treatment with test compounds. The compounds targeting the same protein
form
at
least
similar
proteomic signatures on a
2D-DIGE
(a
kind
2-dimensional
of
gel
electrophoresis), which are
matched with the patterns
of reference compounds
characterized
recognized
by
targets
well
and
mechanisms of action. The
results derived from the
“Morphobase” were consistent with the “Proteobase”; depending on the case both techniques
can be used separately or supplementary, if necessary.
If the target molecule cannot be predicted by these methods, we use the affinity beads bearing
the aimed compounds. In this presentation, I will talk on the target identification systems for
newly discovered bioactive compounds.
References
1.
Futamura Y, Kawatani M, Kazami S, Tanaka K, Muroi M, Shimizu T, Tomita K,
Watanabe N, and Osada H: "Morphobase, an encyclopedic cell morphology database, and
its use for drug target identification." Chem Biol in press.
2.
Muroi M, Kazami S, Noda K, Kondo H, Takayama H, Kawatani M, Usui T, and Osada H:
"Application of proteomic profiling based on 2D-DIGE for classification of compounds
according to the mechanism of action." Chem Biol 17, 460-470 (2010).
3.
Kawatani M, Takayama H, Muroi M, Kimura S, Maekawa T, and Osada H:
"Identification of a small-molecule inhibitor of DNA topoisomerase II by proteomic
profiling." Chem Biol 18, 743-751 (2011).
4.
Kawatani M, Okumura H, Honda K, Kanoh N, Muroi M, Dohmae N, Takami M,
Kitagawa M, Futamura Y, Imoto M, and Osada H: "The identification of an
osteoclastogenesis inhibitor through the inhibition of glyoxalase I." Proc Natl Acad Sci,
USA 105, 11691-11696 (2008).
5.
Sasazawa Y, Kanagaki S, Tashiro E, Nogawa T, Muroi M, Kondoh Y, Osada H, and Imoto
M: "Xanthohumol impairs autophagosome maturation through direct inhibition of
valosin-containing protein." ACS Chem Biol 7, 892-900 (2012).
Selected publications
1. Osada H, Nogawa T: "Systematic isolation of microbial metabolites for natural products
depository (NPDepo) (Review article)." Pure Appl Chem, 84, 1407-1420 (2012)
2. Kato N, Takahashi S, Nogawa T, Saito T, and Osada H: "Construction of a microbial
natural product library for chemical biology studies (Review article)." Curr Opn Chem
Biol, 16, 101-108 (2012).
3. Wierzba K, Muroi M, and Osada H: "Proteomics accelerating the identification of the
target molecule of bioactive small molecules (Review article)." Curr Opn Chem Biol, 15,
57-65 (2011).
4. Takahashi S, Toyoda A, Sekiyama Y, Takagi H, Nogawa T, Uramoto M, Suzuki R,
Koshino H, Kumano T, Panthee S, Dairi T, Ishikawa J, Ikeda H, Sakaki Y, and Osada H:
"Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal
formation." Nature Chem Biol, 7, 461-468 (2011).
5. Jang JH, Asami Y, Jang JP, Kim SO, Moon DO, Shin KS, Hashizume D, Muroi M, Saito
T, Oh H, Kim BY, Osada H, and Ahn JS: "Fusarisetin a, an acinar morphogenesis
inhibitor from a soil fungus, fusarium sp. Fn080326." J Am Chem Soc, 133, 6865-6867
(2011).
6. Khan MM, Simizu S, Lai NS, Kawatani M, Shimizu T, and Osada H: "Discovery of a
small molecule pdi inhibitor that inhibits reduction of hiv-1 envelope glycoprotein
gp120." ACS Chem Biol, 6, 245-251 (2011).
7.
Ong E.B.B, Watanabe N, Saito A, Futamura Y, Abd El Galil K.H, Koito A, Najimudin N,
and Osada H: "Vipirinin, a coumarin-based HIV-1 Vpr Inhibitor, interacts with a
hydrophobic region of Vpr." J Biol Chem, 286, 14049-14056 (2011).
8. Miyazaki I, Simizu S, Okumura H, Takagi S, and Osada H: "A small-molecule inhibitor
shows that pirin regulates migration of melanoma cells." Nature Chem Biol, 6, 667-673
(2010).
9. Sun Y, Hahn F, Demydchuk Y, Chettle J, Tosin M, Osada H, and Leadlay PF: "In vitro
reconstruction of tetronate rk-682 biosynthesis." Nature Chem Biol, 6, 99-101 (2010).
10. Yano A, Tsutsumi S, Soga S, Lee MJ, Trepel J, Osada H, and Neckers L: "Inhibition of
hsp90 activates osteoclast c-src signaling and promotes growth of prostate carcinoma cells
in bone." Proc Natl Acad Sci, USA, 105, 15541-15546 (2008).
Biosynthesis-Based Natural Product Discovery
Wen Liu
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China
wliu@mail.sioc.ac.cn
Natural products (NPs) are small molecules of incredible structural diversity that have long
been appreciated for their critical role in drug discovery and development. Production of NPs
from biosynthetic gene clusters depends on the coordinated transcription of relevant genes to
messenger RNAs (mRNAs), the translation of these mRNAs to polypeptide chains, and the
correct folding of these polypeptides to create functional biosynthetic machineries that
cooperate to catalyze the formation of complex structures from simple precursor molecules.
Sequencing of numerous actinomycete genomes during the past decade has revealed a
stunning number of NP biosynthetic gene clusters, only about 10% of which have been linked
to characterized NPs. While bioinformatics-guided efforts have been successful in correlating
some of these NP gene clusters to new metabolites, which include those produced by
polyketide synthases, nonribosomal peptide synthetases, and terpene synthases, it is clear that
our ability to mine these new gene clusters to uncover the chemical potential hidden within
them has not kept pace with DNA sequencing technology. We herein choose a few examples
from our current research regarding the biosynthesis of pharmaceutical NPs, to show our
efforts in methodology development by incorporating NP-forming chemistry into rational
mining from strains for which the genome sequence is known or even unknown..
References
1. Qu, X.; Pang, B.; Zhang, Z.; Chen, M.; Wu, Z.; Zhao, Q.; Zhang, Q.; Wang, Y.; Liu, Y.;
Liu, W., Caerulomycins and collismycins share a common paradigm for 2,2’-bipyridine
biosynthesis via an unusual hybrid polyketide-peptide assembly logic. J. Am. Chem. Soc.
2012, 134, 9038-9041.
2. Yan, Y.; Zhang, L.; Ito, T.; Qu, X.; Asakawa, Y.; Awakawa, T.; Abe, I.; Liu, W.,
Biosynthetic pathway for high structural diversity of a common dilactone core in
antimycin production. Org. Lett. 2012, 14, 4142-4145.
3. Li, J.; Qu, X.; He, X.; Duan, L.; Wu, G.; Bi, D.; Deng, Z.; Liu, W.; Ou, H. Y., ThioFinder:
a web-based tools for the identification of thiopeptide gene clusters in DNA sequence.
PloS One 2012, 7, e45878.
4. Qu, X. D.; Lei, C.; Liu, W., Transcriptome mining of active biosynthetic pathways and
their associated products in Streptomyces flaveolus. Angew. Chem. In. Ed. 2011, 50,
9651-9654.
5. Liao, R. J.; Duan, L.; Lei, C.; Pan, H. X.; Ding, Y.; Zhang, Q.; Chen, D. J.; Shen, B.; Yu,
Y.; Liu, W., Thiopeptide Biosynthesis Featuring Ribosomally Synthesized Precursor
Peptides and Conserved Posttranslational Modifications. Chem. & Biol. 2009, 16,
141-147.
Representative Publications
1. Wu, Q.; Wu, Z.; Qu, X.; Liu, W., Insights into pyrroindomycin biosynthesis reveal a
uniform paradigm for tetramate/tetronate formation. J. Am. Chem. Soc. 2012, DOI: org/
10.1021/ja304829g.
2. Qu, X.; Pang, B.; Zhang, Z.; Chen, M.; Wu, Z.; Zhao, Q.; Zhang, Q.; Wang, Y.; Liu, Y.;
Liu, W., Caerulomycins and collismycins share a common paradigm for 2,2’-bipyridine
biosynthesis via an unusual hybrid polyketide-peptide assembly logic. J. Am. Chem. Soc.
2012, 134, 9038-9041.
3. Duan, L.; Wang, S.; Liao, R.; Liu, W., Insights into quinaldic acid formation in
thiostrepton biosynthesis facilitating fluorinated thiopeptide generation. Chem. & Biol.
2012, 19, 443-448.
4. Zhang, Q,; van der Donk, W. A.; Liu, W., Radical-mediated enzymatic methylation: a tale
of two SAMS. Acc. Chem. Res. 2012, 45, 555-564.
5. Zhang, Q.; Li, Y.; Chen, D.; Yu, Y.; Duan, L.; Shen, B.; Liu, W., Radical-mediated
enzymatic carbon chain fragmentation- recombination. Nat. Chem. Biol. 2011, 7,
154-160.
6. Qu, X. D.; Lei, C.; Liu, W., Transcriptome mining of active biosynthetic pathways and
their associated products in Streptomyces flaveolus. Angew. Chem. In. Ed. 2011, 50,
9651-9654.
7. Liao, R.; Liu, W., Thiostrepton Maturation Involving a Deesterification-Amidation Way
To Process the C-Terminally Methylated Peptide Backbone. J. Am. Chem. Soc. 2011, 133,
2852-2855.
8. Zhang,
Q.;
Liu,
W.,
Complex
biotransformations
catalyzed
by
radical
S-adenosylmethionine enzymes. J. Biol. Chem. 2011, 286, 30245-30252.
9. Yu, Y.; Guo, H.; Zhang, Q.; Duan, L.; Ding, Y.; Liao, R.; Lei, C.; Shen, B.; Liu, W., NosA
Catalyzing Carboxyl-Terminal Amide Formation in Nosiheptide Maturation via an
Enamine Dealkylation on the Serine-Extended Precursor Peptide. J. Am. Chem. Soc.
2010, 132, 16324-16326.
10. Ding, W.; Lei, C.; He, Q. L.; Zhang, Q. L.; Bi, Y. R.; Liu, W., Insights into Bacterial
6-Methylsalicylic Acid Synthase and Its Engineering to Orsellinic Acid Synthase for
Spirotetronate Generation. Chem. & Biol. 2010, 17, 495-503.
Biology-oriented Study of Natural Products: Unnatural
Dual-functional abeo-Taxanoids
Zhu-Jun Yao,*1 Yu Zhao,2 Qinshi Zhao2
1
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical
Engineering, Nanjing University, Nanjing, Jiangsu 210093;
2
State key laboratory of phytochemistry and Plant Resources in West China, Kunming
Institute of Botany, Chinese Academy of Science, Kunming 650204, China.
yaoz@nju.edu.cn or yaoz@sioc.ac.cn
Paclitaxel (Figure 1, Taxol, 1a) and its semisynthetic analogue docetaxel (1b) are two
powerful anticancer drugs, both of which induce tubulin polymerization to microtubules and
stabilize microtubules, cause the arrest of cell cycle at G2/M phase, and result in cell
apoptosis. Besides 380 taxoids with normal skeleton, 139 taxoids with an 11(15→1)
abeo-taxane skeleton have been discovered primarily from Taxus Chinensis, T. yunnanensis
and T. wallichiana. However, few advances have been made in structural modification of the
later type of taxoids, as well as the corresponding biological studies. To discover new dual- or
multi-functional
anticancer
agents,
our
laboratory
recently
explored
a
drug-fragment-embedment modification of taxchinin A (Figure 1, 2), an abundant but
biologically inactive taxoids with the abeo-taxane skeleton from the leaves of T. chinensis,
and investigated the corresponding biological actions.
References
1.
P. B. Schiff, J. Fant, S. B. Horwitz, Nature 1979, 277, 665-667.
2.
(a) Y. Zhao, Q. S. Zhao, et al. Bioorg. Med. Chem. 2008, 16, 4860-4871; (b) Y. Zhao, Z.-J.
Yao, Q.-S. Zhao, et al. Tetrahedron Lett. 2011, 52, 139-142; (c) Y. Zhao, Z.-J. Yao, Q.-S.
Zhao, et al. unpublished work (2012).
Synthetic Studies on Natural Products by Means of RCM
Tohru Fukuyama
Graduate School of Pharmaceutical Sciences, Nagoya University, Japan
Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan
fukuyama@ps.nagoya-u.ac.jp
When the ring-closing metathesis (RCM) was first recognized as a powerful tool for
constructing a variety of carbocyclic as well as heterocyclic systems, I was not particularly
interested in incorporating the reaction into our projects simply because I am an old-fashioned
synthetic chemist who was rather reluctant in jumping on the bandwagon. I did not like to
prepare olefins from the corresponding carbonyl compounds for the sake of carrying out the
RCM reactions. Nor did I like to construct 5- or 6-membered rings using RCM because there
are a plenty of conventional ways to access these compounds. However, when we were trying
to come up with a practical, asymmetric synthesis of kainic acid (1), an irresistible temptation
to use RCM emerged as illustrated in the retrosynthetic analysis. In this case, the requisite
olefin for RCM (2) would become available as a result of the asymmetric aldol reaction of the
readily available crotonic acid derivative (3) This idea worked like a charm and a reasonably
efficient total synthesis of kainic acid could be achieved.1
Our kainic acid synthesis played an important role in removing our psychological block about
RCM reactions. We next applied the RCM reaction to the total synthesis of (+)-manzamine A2
which we do not have time to discuss at this symposium. The second topic I would like to
discuss is the total synthesis of (–)-isoschizogamine (4) in which the RCM reaction used
twice in this case played a very important role.3
Several projects using RCM are currently in progress in our labs and I have to confess that the
RCM reaction has dramatically changed a way how we construct organic molecules.
References
1. “Stereocontrolled Total Synthesis of (–)-Kainic Acid,” H. Sakaguchi, H. Tokuyama, and T.
Fukuyama, Org. Lett., 9, 1635 (2007).
2. “Total Synthesis of (+)-Manzamine A,” T. Toma, Y. Kita, and T. Fukuyama, J. Am.
Chem. Soc., 132, 10232-10235 (2010).
3. “Total Synthesis of (–)-Isoschizogamine,” Y. Miura, N. Hayashi, S. Yokoshima, and T.
Fukuyama,
J. Am. Chem. Soc., 134, 11995 (2012).
Representative Publications of Tohru Fukuyama
1.
“Practical Total Synthesis of (±)-Mitomycin C,” T. Fukuyama, and L.-H. Yang, J. Am.
Chem. Soc., 111, 8303 (1989).
2.
“Facile Reduction of Ethyl Thiol Esters to Aldehydes: Application to a Total Synthesis
of (+)-Neothramycin A Methyl Ether,” T. Fukuyama, S.-C. Lin, and L.-P. Li, J. Am.
Chem. Soc., 112, 7050 (1990).
3.
“Total Synthesis of (+)-Leinamycin,” Y. Kanda, and T. Fukuyama, J. Am. Chem. Soc.,
115, 8451 (1993).
4.
“2- and 4-Nitrobenzenesulfonamides: Exceptionally Versatile Means for Preparation of
Secondary Amines and Protection of Amines.,” T. Fukuyama, C.-K. Jow, and M.
Cheung, Tetrahedron Lett.,36, 6373 (1995).
5.
“Stereocontrolled Total Synthesis of (±)-Gelsemine,” T. Fukuyama and G. Liu, J. Am.
Chem. Soc., 118, 7426 (1996).
6.
“Stereocontrolled Total Synthesis of (+)-Vinblastine,” S. Yokoshima, T. Ueda, S.
Kobayashi, A. Sato, T. Kuboyama, H. Tokuyama, and T. Fukuyama, J. Am. Chem. Soc.,
124, 2137 (2002).
7.
“Total Synthesis of Ecteinascidin 743,” A. Endo, A. Yanagisawa, M. Abe, S. Tohma, T.
Kan, and T. Fukuyama, J. Am. Chem. Soc., 124, 6552 (2002).
8.
“A Practical Synthesis of (–)-Oseltamivir,” N. Satoh, T. Akiba, S. Yokoshima and T.
Fukuyama, Angew. Chem. Int. Ed., 46, 5734 (2007).
9.
“Concise Total Synthesis of (+)-Lyconadine A,” T. Nishimura, A. K. Unni, S.
Yokoshima, and T. Fukuyama, J. Am. Chem. Soc., 133, 418 (2011).
10.
“Total Synthesis of Gelsemoxonine,” J. Shimokawa, T. Harada, S. Yokoshima, and T.
Fukuyama
J. Am. Chem. Soc., 133, 17634 (2011).
Total Synthesis of 13-Oxyingenol and Its Natural Derivative
Hideo Kigoshi
Department of Chemistry, Graduate School of Pure and Applied Sciences, University of
Tsukuba, Japan
kigoshi@chem.tsukuba.ac.jp
13-Oxyingenol derivative 1 and ingenol are diterpenoids isolated from the plants of
Euphorbia sp. The main structural features of ingenols are a bicycle [4.4.1] undecane skeleton
with inside–outside, intrabridgehead stereochemistry and a high degree of oxygenation. They
and their analogs showed strong bioactivities such as protein kinase C activation and anti-HIV
activity. We have achieved the formal synthesis of ingenol (13-deoxy analog of 2) by using
ring-closing olefin metathesis efficiently.
Herein the first total synthesis of (–)-13-oxyingenol (2) and its natural derivative 1 is
described. The efficient functionalization of the A- and B-ring parts was established by using
C-2 and C-7 hydroxy groups as clues. This approach is highlighted by ring-closing olefin
metathesis for the construction of
an inside–outside framework, regio- and stereoselective dihydroxylation for the
functionalization of the A-ring, and [2,3]-sigmatropic rearrangement for the functionalization
of the B-ring.
References
1) H. Kigoshi, Y. Suzuki, K. Aoki, and D. Uemura, Synthetic Studies of Ingenol: Synthesis
of in,out-Tricyclo[7.4.1.01,5]tetradecan-14-one, Tetrahedron Lett., 41, 3927-3930 (2000).
2) K. Watanabe, Y. Suzuki, K. Aoki, A. Sakakura, K. Suenaga, and H. Kigoshi, Formal
Synthesis of Optically Active Ingenol via Ring-Closing Olefin Metathesis, J. Org. Chem.,
69, 7802-7808 (2004).
3) I. Hayakawa, Y. Asuma, T. Ohyoshi, K. Aoki, and H. Kigoshi, Synthetic Study on
13-Oxyingenol: Construction of the Full Carbon Framework, Tetrahedron Lett., 48,
6221-6224 (2007).
4) I. Hayakawa, Y. Miyazawa, T. Ohyoshi, Y. Asuma, K. Aoki, and H. Kigoshi, Synthetic
Studies toward Optically Active 13-Oxyingenol via Asymmetric Cyclopropanation,
Synthesis, 5, 769–777 (2011).
5) T. Ohyoshi, S. Funakubo, Y. Miyazawa, K. Niida, I. Hayakawa, and H. Kigoshi, Total
Synthesis of (–)-13-Oxyingenol and Its Natural Derivative, Angew. Chem. Int. Ed., 51,
4972–4975 (2012).
Synthesis of Guanidine-Containing Natural Products
Toshio Nishikawa
Graduate School of Bioagricultural Sciences, Nagoya University, Japan
nisikawa@agr.nagoya-u.ac.jp
Many guanidine-containing biologically active natural products have been isolated from
nature, in particular from marine sources. Among them, tetrodotoxin (TTX, 1) is one of the
most well-known as a toxic principle of puffer fish intoxication. This small natural product
exerts the potent toxicity through specific blockage of sodium ion influx through
voltage-gated sodium channels (VGSC) on neuro-cell membrane.
Due to the unique
biological property, tetrodotoxin has been employed as an important biochemical tool in
neurophysiological experiments.
In 1975, Mosher and his co-workers isolated a
tetrodotoxin-like compound from skin of a dart frog (Atelopus Chiriquiensis) living in Costa
Rica.1) However, the structures had not been elucidated until 1990, when Yotsu-Yamashita
and Yasumoto elucidated the structure as below by extensive analysis of NMR spectra.2)
The toxicity was reported to be the same as that of tetrodotoxin, however, the details of
inhibitory activity against VGSC and other ion channels have not been investigated yet,
because of the difficult availability from natural sources.
O
O
HO
HO
H
N
H2N
H
N
O
O
OH
H2N
H
HO
OH
O
OH
H
N
N O
OH
O
O
H
OH
tetrodotoxin (TTX, 1)
O
OH
NH3
chiriquitoxin (CHTX, 2)
Our research group has been interested in the synthesis of tetrodotoxin as well as biological
issues associated with this natural product.3)
In the course of our synthetic studies on
tetrodotoxin, we embarked on the synthesis of chiriquitoxin (CHTX) in order to confirm the
structure, and also to supply the molecule for biological studies.
In this symposium, the first
total synthesis of chiriquitoxin will be presented.
The synthesis began with the compound 4, prepared from the Diels-Alder reaction between an
isoprenol derivative and bromolevoglucosenone, a chiral dienophile prepared from a
carbohydrate.4)
This compound was recently designed as a common intermediate for
synthesis of a wide variety of TTX analogue in our laboratory, and the synthetic route in a 100
g scale was established.
The intermediate 4 was transformed to fully functionalized cyclohexane intermediate 5 in an
analogous manner as the previous synthesis of TTX in our laboratory.3) The vinyl group was
elaborated to -hydroxycarboxylic acid, which opened the epoxide to form lactone
intermediate 6. This compound was employed for the synthesis of TTX (1). On the other
hand, 6 was transformed to aldehyde 7 after several protective group transformations.
The
aldehyde 7 was coupled with a chiral glycine equivalent (the structure is not shown) followed
by guanidinylation to provide a fully protected compound of CHTX. Careful removal of all
the protective groups furnished CHTX (2). The NMR spectra were good agreement with the
literature data, confirming the structure of CHTX including configurations.
O
O
O
O
COCCl3
NH
PMBO
OTIPS
COCCl3
NH
HO
O
Cl3C
OH
OTBS
O
OH
TBSO
4
O
O
NO
H
TBS
5
O
O
OTBS
OH
6
OTIPS
O
O
RO
EtO
N
H O
TBS
O
O
COOH
deprotection
O
NH2
H
7
OP1
CHTX (2)
TTX (1)
a chiral
glycine unit
References
1)
Kim, Y. H.; Brown, G. B.; Mosher H. S.; Fuhrman, F. A. Science 1975, 189, 151-152.
2) Yotsu, M.; Yasumoto, T.; Kim, Y. H.; Naoki, H.; Kao, C. H. Tetrahedron Lett. 1990, 31,
3187-3190.
3) a) Nishikawa, T.; Urabe, U.; Yoshida, K.; Iwabuchi, T.; Asai, M.; Isobe, M. Pure & Appl.
Chem., 2003, 75, 247-253; b) Urabe, D.; Nishikawa, T.; Isobe, M. Chem. Asian J. 2006,
1, 125-135.
4)
Satake, Y.; Nishikawa, T.; Hiramatsu, T.; Araki, H.; Isobe, M. Synthesis 2010,
1992-1998.
Chemical Diversity and the Bioactive Compounds from Marine
Invertebrates Inhabited in South China Sea
Wenhan Lin
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China
whlin@bjmu.edu.cn
China holds a long coastline (approximately 18,000 km) and covers three different climatic
zones ranging from temperate to tropical regions. Its coastal waters are known to hold a vast
but hitherto largely unexplored and even undescribed biodiversity of marine macro- and
microorganisms that have hardly been studied so far in a systematic manner for bioactive
compounds in the field of anti-cancer agents.
Chinese marine resources still hold a plethora
of uninvestigated organisms with uninvestigated natural products that need to be tapped and
evaluated systematically for bioactive natural products. Only in recent years has China started
to become aware of its natural marine resources and is now launching major research
programs directed at the discovery of novel bioactive compounds from marine organisms. In
this presentation several examples of bioactive natural products recently discovered by our
group will be presented. These include the alkaloids derived from marine sponge and their
targeting on HIV-1 proteins and antivirus activity, various terpenoids obtained from marine
soft corals, the antitumor active diterpenoids from marine mangrove plant. The examples
chosen will highlight the structural diversity of compounds that are obtained from various
marine sources and that can be made available for detailed pharmacological studies.
References
1. Y. Li, S. Yu, D. Liu, P. Proksch, W. Lin* “inhibitory effects of polyphenols toward HCV
from the mangrove plant Excoecaria agallocha L.” Bioorg. Med. Chem. Lett. 2012, 22,
1099-1102.
2. J. Li, H. Zhu, J. Ren, Z. Deng, N. J. de Voogd, P. Proksch, W. Lin*, “Globostelletins J-S,
isomalabaricanes with unusual cyclopentane sidechains from the marine sponge
Rhabdastrella globostellata”, Tetrahedron, 2012, 68, 559-565.
3. D. Chen, S. Yu, L. Ofwegen, P. Proksch, W. Lin*, “Anthogorgienes A−O, New
Guaiazulene-Derived Terpenoids from a 2 Chinese Gorgonian Anthogorgia Species, and
Their Antifouling and Antibiotic Activities”, J. Agr. Food Chem. 2012, 60, 112-123.
4.
W. Jiang, D. Liu, Z. Deng, N. J. de Voogd, P. Proksch, W. Lin*, “Brominated
polyunsaturated lipids and their stereochemistry from the Chinese marine sponge
Xestospongia testudinaria” Tetrahedron 2011, 67, 58-68.
5. D. Lai, Y. Li, M. Xu, Z. Deng, L. Ofwegen, P. Qian, P. Proksch, W. Lin* “Sinulariols
AeS, 19-oxygenated cembranoids from the Chinese soft coral Sinularia rigida”
Tetrahedron 2011, 67, 6018-6029.
6.
D. Liu, J. Xu, W. Jiang, Z. Deng, N. J. de Voogd, P. Proksch, W. Lin* “Xestospongienols
A–L, brominated acetylenic acids from the Chinese marine sponge Xestospongia
testudinaria” Helv. Chim. Acta 2011, 94, 1600-1607.
7.
P. Yan, Z. Deng, L. Ofwegen, P. Proksch, W. Lin* “Lobophytones U–Z1,
biscembranoids from the Chinese soft coral Lobophytum pauciflorum”, Chem. Biodiver.
2011, 8, 1724-1734.
8．D. Lai, S. Yu, L. Ofwegen, F. Totzke, P. Proksch, W. Lin* “9,10-Secosteroids, protein
kinase inhibitors from the Chinese gorgonian Astrogorgia sp.” Bioorg. Med. Chem. 2011,
19, 6873-6880.
9.
X. Feng, W. Zhao, S. Ban, C. Zhao, Q. Li, W. Lin* “Structure–Activity Relationship of
Halophenols as a New Class of Protein Tyrosine Kinase Inhibitors” Int. J. Mol. Sci. 2011,
12, 6104-6115.
10.
H. Liu, R. Edrada-Ebel, R. Ebel, Y. Wang, B. Schulz, S. Draeger, W. Muller, V. Wray, W.
Lin*, P. Proksch* “Ophiobolin sesterterpenoids and pyrrolidine alkaloids from the
sponge-derived fungus Aspergillus ustus.”. Helv. Chim. Acta 2011, 94, 623-631.
Anti-parasitic Agents from Marine Organisms
Yoichi Nakao
School of Advanced Science and Engineering, Waseda University, Japan
ayocha@waseda.jp
Leishmaniasis is caused by the intracellular protozoa belonging to the genus Leishmania and
threats to 350 million people, with 1.5–2 million new cases annually. Pentavalent antimony
compounds have been used for the first line drugs, while amphotericin B and other antifungal
agents for the second line.
However, toxicity and high cost for the development of
antileishmanial drugs pose serious problem for controlling leishmaniasis. Thus, new drugs
should be urgently developed. More than 90 marine natural products have been reported so far,
but none of them has reached clinical trials.1
In the course of our efforts to discover potential drug leads from marine invertebrates, we
have screened extracts of marine organisms against the recombinant L. amazonensis doped
with green fluorescence protein (La/egfp).2
From the extracts showing promising activities
in this screening, we have isolated several anti-leishmanial compounds.3-6
In this presentation, isolation, structure elucidation, and biological activities of these
compounds will be introduced.
References
1.
Tempone, A. G.; de Oliveira, C. M.; Berlinck, R. G. S. Planta Med. 77, 572-585, (2011).
2.
Okuno, T.; Goto, Y.; Matsumoto, Y.; Otsuka, H.; Matsumoto, Y. Exp. Anim. 52, 109-118,
(2003).
3.
Ishigami, S.-T.; Goto, Y.; Inoue, N.; Kawazu, S.-I.; Matsumoto, Y.; Imahara, Y.; Tarumi,
M.; Nakai, H.; Fusetani, N.; Nakao, Y. Cristaxenicin A, an antiprotozoal xenicane
diterpenoid from the deep sea gorgonian Acanthoprimnoa cristata, J. Org. Chem. in
press.
4.
Ueoka, R.; Nakao, Y.; Kawatsu, S.; Yaegashi, J.; Matsumoto, Y.; Matsunaga, S.; Furihata,
K.; van Soest, R. W. M.; Fusetani, N. Gracilioethers A-C, Anti-malarial Metabolites from
the Marine Sponge Agelas gracilis, J. Org. Chem. 74, 4204-4207, (2009).
5.
Nakao, Y.; Kawatsu, S.; Okamoto, C.; Okamoto, M.; Matsumoto, Y.; Matsunaga, S.; van
Soest, R. W. M.; Fusetani, N. Ciliatamides A-C, Bioactive Lipopeptides from the
Deep-sea Sponge Aaptos ciliata, J. Nat. Prod. 71, 469-472, (2008).
6.
Nakao, Y.; Shiroiwa, T.; Murayama, S.; Matsunaga, S.; Goto, Y.; Matsumoto, Y.; Fusetani,
N. Identification of Renieramycin A as an Antileishmanial Substance in a Marine Sponge
Neopetrosia sp., Marine Drugs. 2, 55-62, (2004).
Representative Publications
1.
Yamashita, T.; Nakao, Y.; Matsunaga, S.; Oikawa, T.; Imahara, Y.; Fusetani, N. A New
Antiangiogenic C24 Oxylipin from the Soft Coral Sinularia numerosa, Bioorg. Med.
Chem. 17, 2181-2184, (2009).
2.
Nakao, Y.; Narazaki, G.; Hoshino, T.; Maeda,S.; Yoshida, M.; Maejima, H.; Yamashita, J.
K. Evaluation of Antiangiogenic Activity of Azumamides by the in vitro Vascular
Organization Model Using Mouse Induced Pluripotent Stem (iPS) Cells, Bioorg. Med.
Chem. Lett. 18, 2982-2984, (2008).
3.
Nakao, Y.; Yoshida, S.; Matsunaga, S.; Shindoh, N.; Terada, Y.; Nagai, K.; Yamashita, J.
K.; Ganesan, A.; van Soest, R. W. M.; Fusetani N. Azumamides A-E, New HDAC
Inhibitory Cyclic Tetrapeptides from the Marine Sponge Mycale izuensis, Angew. Chem.
Int. Ed. 45, 7553-7557, (2006).
4.
Takada, K.; Uehara, T.; Nakao, Y.; Matsunaga, S.; van Soest, R. W. M.; Fusetani, N.
Schulzeines A-
-Glucosidase Inhibitors from the Marine Sponge Penares
schulzei, J. Am. Chem. Soc. 126, 187-193, (2004).
5.
Fujita, M.; Nakao, Y.; Matsunaga, S.; Seiki, M.; Itoh, Y.; Yamashita, J. van Soest, R. W.
M.; Fusetani, N. Ageladine A : an anti-angiogenic matrixmetalloproteinase inhibitor from
the marine sponge Agelas nakamurai, J. Am. Chem. Soc. 125, 15700-15701, (2003).
6.
Nakao, Y.; Fujita, M.; Warabi, K.; Matsunaga, S.; Fusetani, N. Miraziridine A, a novel
cysteine protease inhibitor from the marine sponge Theonella aff. mirabilis, J. Am. Chem.
Soc. 122, 10462-10463, (2000).
7.
Nakao, Y.; Masuda, A.; Matsunaga, S.; Fusetani, N., Pseudotheonamides, serine protease
inhibitors from the marine sponge Theonella swinhoei, J. Am. Chem. Soc. 121,
2425-2431, (1999).
8.
Reese, M. T.; Gulavita, N. K.; Nakao, Y.; Hamann, M. T.; Yoshida, W. Y.; Coval, S. J.;
Scheuer, P. J. Kulolide, a cytotoxic depsipeptide from a cephalaspidean mollusk,
Philinopsis speciosa, J. Am. Chem. Soc. 118, 11081-11084, (1996).
9.
Yeung, B. K. S.; Nakao, Y.; Kinnel, R. B.; Carney, J. R.; Yoshida, W. Y.; Scheuer, P. J.
The kapakahines, cyclic peptides from the marine sponge Cribrochalina olemda, J. Org.
Chem. 61, 7168-7173 (1996).
10. Nakao, Y.; Yeung, B. K. S.; Yoshida, W. Y.; Scheuer, P. J.; Kelly-Borges, M. Kapakahine
-carboline ring system from the marine sponge
Cribrochalina olemda, J. Am. Chem. Soc. 117, 8271-8272, (1995).
The Angel’s Wing Mystery
Attempt to Disclose the Molecular Mechanism of Acute
Encephalopathy Caused by Eating Angel’s Wing Oyster
Mushroom (Sugihiratake)
Hirokazu Kawagishi
Graduate School of Science and Technology, Shizuoka University, Japan
achkawa@ipc.shizuoka.ac.jp
The mushroom Pleurocybella porrigens (Angel’s wings in English; Sugihiratake in Japanese)
is widespread and common throughout temperate regions of the world. It had been eaten for a
long time all over the world. However, in autumn 2004 in Japan, fifty-five people got
poisoned by eating this mushroom, and seventeen people among them died of acute
encephalopathy. There had been no report regarding toxicity of the fruiting bodies until the
incident. Under these circumstances, we tried to isolate the principle(s) of the disease.
Purification of a glycoprotein showing lethal activity against mouse
The mushroom was extracted with water and boiling water. After repeated chromatography of
the water-soluble fractions, a glycoprotein was purified. The substance showed lethal toxicity
toward mice.
Purification, characterization, and cDNA cloning of a lectin (PPL)
PPL was purified from this mushroom. The results of SDS-PAGE, gel filtration and
MALDI-TOF-mass of PPL indicated that its molecular mass was 56 kDa, and it was
composed of four 14 kDa subunits with no disulfide bonds. The complete amino acid
sequence was determined by amino acid sequencing. The cDNA of PPL was cloned from
RNA extracted from the mushroom. The open reading frame of the cDNA of the protein
consisted of 411 bp encoding 137 amino acids. Intravenous (i.v.) (50 mg/kg) or intraperitoneal
(i.p.) (150 mg/kg) administration of PPL to mice did not show any toxicity. However, i.v. (9
mg/kg) administration of the protein to rats exhibited lethal toxicity.1)
Unusual amino acids showing cytotoxicity
Six amino acid derivatives including three novel ones were isolated from the mushroom.
These compounds were cytotoxic to mouse glial cells.2) The structural novelty and analogy of
the amino acids is such that each acid has the β-hydroxyvaline unit adducted to endogenous
molecules, which inspired us to conclude the occurrence of an aziridine-amino acid as the
common precursor of the six compounds. We synthesized this compound and proved its
occurrence in this mushroom. The aziridine (we named it pleucybellaziridine) showed
specific toxicity against rat CG4-16 oligodendrocyte cells.3)
Mechanism of the acute encephalopathy
We found that a mixture of the lethal glycoprotein and PPL showed protease activity and
disrupted the blood-brain barrier (BBB) in mice. We speculated that pleucybellaziridine or its
derivatives caused demyelinating symptom after disruption of BBB. Verification of the
hypothesis is now on progress.
References
1. Suzuki, T., Amano, Y., Fujita, M., Kobayashi, Y., Dohra, H., Hirai, H., Murata, T., Usui, T.,
and Kawagishi, H. Purification, characterization and cDNA cloning of a lectin from the
mushroom Pleurocybella porrigens, Biosci. Biotechnol. Biochem., 73, 702-709 (2009).
2. Kawaguchi, T., Suzuki, T., Kobayashi, Y., Kodani, S., Hirai, H., Nagai, K., and Kawagishi,
H., Unusual amino acid derivatives from the mushroom Pleurocybella porrigens,
Tetrahedron, 66, 504–507 (2010).
3. Wakimoto, T., Asakawa, T., Akahoshi, S., Suzuki, T., Nagai, K., Kawagishi, H., and Kan, T.:
Proof of the existence of an unstable amino acid, pleurocybellaziridine, in Pleurocybella
porrigens (angel’s wing mushroom), Angew. Chem., Int. Ed. Engl., 50, 1168-1170 (2011).
Representative Publications
1. Kawagishi, H. et al., Hericenones C, D and E, stimulators of nerve growth factor
(NGF)-synthesis, from the mushroom Hericium erinaceum. Tetrahedron Lett., 32,
4561-4564 (1991).
2. Kawagishi, H. et al., Erinacines A, B and C, strong stimulators of nerve growth factor
(NGF)-synthesis, from the mycelia of Hericium erinaceum. Tetrahedron Lett., 35,
1569-1572 (1994).
3. Kawagishi, H. et al., Erinacines E, F, and G, stimulators of nerve growth factor
(NGF)-synthesis, from the mycelia of Hericium erinaceum. Tetrahedron Lett., 37,
7399-7402 (1996).
4. Sano, Y. et al., Ustalic acid as a toxin and related compounds from the mushroom
Tricholoma ustale. Chem. Commun., (13), 1384 -1385 (2002).
5. Kobayashi, Y. et al., Purification, characterization and sugar-binding specificity of an
N-glycolylneuraminic acid-specific lectin from the mushroom Chlorophyllum molybdites. J.
Biol. Chem., 279, 53048-53055 (2004).
6. Choi, J-H. et al., Disclosure of the “fairy” of fairy-ring forming fungus Lepista sordida,
ChemBioChem, 11, 1373-1377 (2010)
7. Choi, J-H. et al., Plant-growth regulator, imidazole-4-carboxamide produced by fairy-ring
forming fungus Lepista sordida. J. Agric. Food Chem., 58, 9956-9959 (2010).
8. Wakimoto, T. et al., Proof of the existence of an unstable amino acid, pleurocybellaziridine,
in Pleurocybella porrigens (angel’s wing mushroom), Angew. Chem., Int. Ed. Engl., 50,
1168-1170 (2011).
9. Kobayashi1, Y. et al., A novel core fucose-specific lectin from the mushroom Pholiota
squarrosa, J. Biol. Chem., 287, 33973-33978 (2012).
10. Wu, J. et al., Strophasterols A to D with an unprecedented steroid skeleton: from the
mushroom Stropharia rugosoannulata, Angew. Chem., Int. Ed. Engl., 51, 10820 -10822
(2012).
Ajmaline Biosynthesis:
from Alkaloid Structure to Enzyme Structure
Joachim Stöckigt
College of Pharmaceutical Sciences, Zhejiang University, China
joesto2000@yahoo.com
One of the most impressive groups of plant natural products are alkaloids, because of their
divers and complex carbon skeletons and their various therapeutic applications for the
treatment of human diseases.
Enormous efforts have been made over many decades to unravel their multi-step pathways at
the enzyme level and to obtain deep knowledge on their biosynthetic networks.
Alkaloid biosynthetic pathways have been elucidated in detail in plants of Papaver, Taxus and
Rauvolfia concerning the enzymatic formation of isoquinoline alkaloids (morphine), diterpene
alkaloids (taxol) and monoterpenoid
indole alkaloids (ajmaline) (see Figure).
Enzymatic biosynthesis of monoterpenoid indole alkaloids in cell suspension cultures of the Indian and Chinese
medicinal plant Rauvolfia. Single steps were elucidated by isolation, characterization and partial sequencing of
individual enzymes, cloning and expression of corresponding cDNAs by “reverse genetics” followed by
crystallization and 3D X-ray analysis of the major enzymes. (STR1, strictosidine synthase; SG, strictosidine
glucosidase; PNAE, polyneuridine aldehyde esterase; VS, vinorine synthase; VH ,vinorine hydroxylase; CPR,
cytochrome P 450 reductase; and RG , raucaffricine glucosidase). Figure taken from Xia et al., ACS Chem. Biol.,
in press.
The biosynthesis of ajmaline, with a chemical structure which harbours six rings and nine
chiral carbons, is catalyzed by enzymes belonging to various families, such as synthases,
glucosidases, esterases, reductases, oxidases or transferases as illustrated in the Figure.
Only during recent years (since 2004) a much thorough understanding of the most important
enzymes involved in the Rauvolfia biosynthetic network has been gained through structural
biology techniques leading to successful crystallization and 3D X-ray analysis. This
knowledge has been also successfully applied for the generation of rational designed enzyme
mutants useful for development of novel alkaloid libraries.
The lecture will focus on a few examples of these enzymes, including strictosidine synthase,
catalyzing the Pictet-Spengler reaction between tryptamine and secologanin as the key
reaction for the biosynthesis of about 2000 monoterpenoid indole alkaloids.
References
1. Ma, X., Koepke, J., Panjikar, S., Fritzsch, G., Stöckigt, J.: Crystal structure of vinorine
synthase, the first representative of the BAHD superfamily. J. Biol. Chem. 2005, 280,
13576-13583.
2. Ma, X., Panjikar, S., Koepke, J., Loris, E., Stöckigt, J.: The structure of Rauvolfia
serpentina strictosidine synthase is a novel six-bladed beta-propeller fold in plant proteins.
Plant Cell 2006, 18, 907-920.
3. Barleben, L., Panjikar, S., Ruppert, M., Koepke, J., Stöckigt, J.: Molecular Architecture of
Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole
Alkaloid Family.
Plant Cell 2007, 19, 2886-2897.
4. Maresh, J. J., Giddings, L.-A., Friedrich, A., Loris, E. A., Panjikar, S., Trout, B. L.,
Stöckigt, J., Peters, B., O'Connor, S. E.: Strictosidine Synthase: Mechanism of a
Pictet-Spengler Catalyzing Enzyme. J. Am. Chem. Soc. 2008, 130, 710-723.
5. Yang L. Q., Hill, M., Wang, M., Panjikar, S., Stöckigt, J.: Structural basis and enzymatic
mechanism of the biosynthesis of C9- from C10- Monoterpenoid indole alkaloids.
Angew.
Chem. Intern. Ed. 2009, 48, 5211-5213.
6. Stöckigt, J., Antonchick, A. P., Wu F., Waldmann H.: The Pictet-Spengler Reaction in
Nature and in Organic Chemistry.
Angew. Chem. Intern. Ed. 2011, 123, 8538-8564.
7. Xia, L., Ruppert M., Wang, M., Panjikar, S., Lin, H., Rajendran, C.,Barleben L., Stöckigt,
J.: Structures of Alkaloid Biosynthetic Glucosidases Decode Substrate Specificity.
ACS
Chem. Biol. 2012 , 7, 226-234.
8. Wu, F., Zhu, H., Sun, C., Rajendran, C., Wang, M., Ren, X., Cherkasov, A., Zou,H. ,
Stoeckigt, J.: Scaffold Tailoring by a newly Detected Pictet-Spenglerase
Activity of
Strictosidine
the
Synthase : From the Common Tryptoline
Skeleton
to
Piperazino-indole Framework. J. Amer. Chem. Soc. 2012, 134, 1498 – 1500.
Rare
Small Molecules That Block Fat Synthesis
Motonari Uesugi
Institutes for Integrated Cell-Material Sciences (WPI-iCeMS) and for Chemical Research
(ICR), Kyoto University, Japan
uesugi@scl.kyoto-u.ac.jp
In human history, bioactive small molecules have had three primary uses: as medicines,
agrochemicals, and biological tools. Among them, the focus in our laboratory has been the
discovery and use of biological tools. Our laboratory has been discovering and designing
small organic molecules with unique activities to understand and control human cells.
This presentation provides an overview of the recent results regarding one of these molecules
we call “fatostatin.” Fatostatin was originally discovered from our in-house chemical library
as a molecule that inhibits the insulin-induced adipogenesis of mouse 3T3-L1 cells and
represses the serum-independent growth of DU145 human prostate cancer cells. Cell
biological and molecular biological analyses indicated that this synthetic diarylthiazole
derivative inhibits the activation process of the sterol regulatory element binding proteins
(SREBPs), a master transcription factor for lipid biosynthesis in cells. Chemical biological
studies allowed us to identify its direct molecular
target: SREBP-cleavage activating protein (SCAP), an
escort protein for SREBP. Fatostatin is a unique,
simple small molecule that shutdowns lipogenesis in
Fatostatin
cells by blocking the activation of SREBP, a master
switch of lipogenesis.
The structure of fatostatin provides a model that may help direct the design of small molecule
tools to investigate metabolic diseases, including fatty liver disease. In fact, fatostatin
downregulated the expression of lipogenic enzymes and blocked increases in body weight,
blood glucose, and hepatic fat accumulation in obese ob/ob mice, even under uncontrolled
food intake. However, fatostatin showed only moderate potency in mice, and its utility was
limited by low aqueous solubility.
Our laboratory synthesized a number of fatostatin derivatives and compared their potency and
physicochemical properties, with the goal of identifying an analog with improved
characteristics for use in in vivo evaluation in a variety of disease models. Our
structure-activity relationship studies led to the identification of FGH10019 as the most potent
molecule among the analogs tested. FGH10019
has higher aqueous solubility and membrane
permeability,
and may serve as a tool for in vivo studies.
Other efforts regarding fatostatin, including
FGH10019
those for discovering endogenous natural ligands that control SREBP, may be discussed in the
presentation.
References
1. Minami, I., Yamada, K., Otsuji, T.G., Yamamoto, T., Shen, Y., Otsuka, S., Kadota, S.,
Morone, N., Barve, M., Asai, Y., Tenkova-Heuser, T., Heuser, J. E., Uesugi, M.,* Aiba, K.,*
Nakatsuji, N. A small molecule that promotes cardiac differentiation of human pluripotent
stem cells under defined, cytokine- and xeno-free conditions. Cell Reports, in press (2012).
2. Kamisuki, S., Shirakawa T., Kugimiya, A., Abu-Elheiga, L., Choo, H.-Y., Yamada, K.,
Shimogawa, H., Wakil, S. J., Uesugi, M. Synthesis and evaluation of diarylthiazole
derivatives that inhibit activation of sterol regulatory element-binding proteins. J. Med.
Chem. 54, 4923-4927 (2011).
3. Kawazoe, Y., Shimogawa, H., Sato, A., Uesugi, M. A mitochondrial surface-specific
fluorescent probe activated by bioconversion. Angew. Chem. Int. Ed. 50, 5478-5481
(2011).
4. Sumiya, E., Shimogawa, H., Sasaki, H., Tsutsumi, M., Yoshita, K., Ojika, M., Suenaga, K.,
Uesugi, M. Cell-morphology profiling of a natural product library identifies
bisebromoamide and miuraenamide A as actin-filament stabilizers. ACS Chem. Biol. 6,
425-431 (2011).
5. Sato, S. Murata, A., Orihara, T., Shirakawa, T., Suenaga, K., Kigoshi, H., Uesugi, M.
Marine natural product aurilide activates the OPA1-mediated apoptosis by binding to
prohibitin. Chem. Biol. 18, 131-139 (2011).
6. Kamisuki, S., Mao, Q., Abu-Eliheiga, L., Gu, Z., Kugimiya, A., Kwon, Y., Shinohara, T.,
Kawazoe, Y., Sato, S. Asakura, K., Choo, H., Sakai, J., Wakil, SJ., Uesugi, M. A small
molecule that blocks fat synthesis by inhibiting the activation of SREBP. Chem. Biol. 16,
882-892 (2009).
7. Yamazoe, S., Shimogawa, H., Sato, S., Esko, J. D., Uesugi, M. A dumbbell-shaped small
molecule that promotes cell adhesion and growth. Chem. Biol. 16, 773-782 (2009).
8. Jung, D., Shimogawa, H., Kwon, Y., Mao, Q., Sato, S., Kamisuki, S., Kigoshi, H., Uesugi,
M. Wrenchnolol derivative optimized for gene activation in cells. J. Am. Chem. Soc. 131,
4774-4782 (2009).
9. Sato, S., Kwon, Y., Kamisuki, S., Srivastava, N., Mao, Q., Kawazoe, Y., Uesugi, M.
Polyproline-rod approach to isolating protein targets of bioactive small molecules: isolation
of a new target of indomethacin. J. Am. Chem. Soc. 129, 873-880 (2007).
10. Kwon,Y., Arndt, H., Mao, Q., Choi, Y., Kawazoe, Y., Dervan, P. B., Uesugi, M. Small
molecule transcription factor mimic. J. Am. Chem. Soc. 126, 15940-15941 (2004).
Analogues of Cyclic ADP-ribose and Their Functions to Regulate
Calcium Signal Pathway
Liangren Zhang
School of Pharmaceutical Sciences, Peking University, China
liangren@bjmu.edu.cn
Cyclic ADP-ribose (cADPR, 1), a metabolite of NAD+ discovered by Lee in 1987, is a
signaling molecule to regulate calcium mobilization via ryanodine receptors (RyR) from
intracellular stores in a wide variety of biological systems. Due to the important biological
activities of cADPR, much effort has focused on the syntheses of structural derivatives to
elucidate the structure-activity relationship and to supply tools to investigate cellular Ca2+
signaling. A variety of cADPR analogues were synthesized based on the modification of
ribose, nucleo-base and pyrophosphate (2). The pharmacological activities of these analogues
were analyzed in intact and permeabilized human Jurkat T-lymphocytes. The results indicated
that the analogues permeated the plasma membrane and most of them were calcium signaling
agonists. They released Ca2+ from an intracellular cADPR-sensitive Ca2+ store, and
subsequently initiated Ca2+ release-activated Ca2+ entry. A novel fluorescent caged cADPR
analogue, coumarin caged isopropylidene protected cIDPRE (Co-i-cIDPRE, 3) was also
investigated, and found that it is a potent and controllable cell permeant cADPR analogue.
Moreover, we demonstrated that uncaging of Co-i-cIDPRE activates RyRs for Ca2+
mobilization and triggers Ca2+ influx via TRPM2.
X
OH
HO
O
O
O
O
OH
P
OH
OH
1 (cADPR)
Y
O
O
OH
OH
OH
R = H, Cl, Br, CF3, N3, NH2
X = O, NH
Y = O, S, Se
2
N
N
O P OR1
O
P
O
O
N
N
N
R
N
O P OH
N
O
N
N
O
N
N
N
O P OH
O
O
NH
O
O
O
O
P
O
O
OR2
O
O
1
2
R , R = H, H2C
R1 = R2
3 (Co-i-cIDPRE)
This work was supported by the National Natural Science Foundation of China.
O
OAc
Representative Publications
1.
N. Qi, K. Jung, M. Wang, et al. A novel membrane-permeant cADPR antagonist
modified in the pyrophosphate bridge. Chem Comm, 2011, 47, 9462-9464.
2.
Y. Ma, L. Qu, Z. Liu, et al. Synthesis of Salinosporamide A and Its Analogs as 20S
Proteasome Inhibitors and SAR Summarization.
Curr Top Med Chem, 2011, 11,
2906-2922.
3.
Z. Chen, A. K. Y. Kwong, Z. Yang, et al. Studies on the synthesis of nicotinamide
nucleoside and nucleotide analogues and their inhibitions towards CD38 NADase.
Heterocycles, 2011, 83, 2837-2850.
4.
Z. Wang, S. Zhang, H. Jin, et al. Angiotensin-I-converting enzyme inhibitory peptides:
Chemical feature based pharmacophore generation. Eur J Med Chem, 2011, 46,
3428-3433.
5.
Y. Zhou, K. Y. Ting, C. M. C. Lam, et al. Design, synthesis and biological evaluation of
novel non-covalent inhibitors of human CD38 NADase. ChemMedChem, 2012, 7,
223-228.
6.
T. Zuo, D. Liu, W. Lv, et al. Small-Molecule Inhibition of Human Immunodeficiency
Virus Type 1 Replication by Targeting of the Interaction between Vif and ElonginC. J
Viol, 2012, 8, 5497-5507.
7.
Z. Zhao, S. Gao, J. Wang, et al. Self-assembly nanomicelles based on cationic
mPEG-PLA-b-Polyarginine(R15) triblock copolymer for siRNA delivery. Biomaterials,
2012, 33, 6793-6807.
8.
P. Yu, Z. Zhang, B. Hao, et al. A Novel Fluorescent Cell Membrane Permeable Caged
cyclic ADP-Ribose Analogue. J Biol Chem, 2012, 287, 24774-24783.
Food Signals and Circadian Rhythm
Zhengwei Fu
College of Biological and Environmental Engineering, Zhejiang University of Technology,
China
azwfu2003@yahoo.com.cn
Circadian clocks are autonomous time-keeping mechanisms that allow living organisms to
predict and adapt to environmental time cues. In mammals, studies involving the circadian
response to external time cues indicate that the peripheral clocks are dominated mainly by
food cues. However, it is still largely unknown about the mechanism and physiological
function of peripheral clock’s response to food cues.
In the present study, we first investigated the resetting of peripheral clocks in the pineal
gland, liver, heart, and kidney of rats induced by the change of feeding schedule with or
without a change in the LD cycle [1-4]. Our findings indicate distinct mechanisms
underlying the peripheral clocks, for the observations of the tissue-specific resetting of
peripheral clocks and the different resetting modes of clock genes by feeding and lighting.
Daytime restricted feeding (RF) for 7 days had only weak effect on the circadian pattern of
clock gene expression in the pineal gland, whereas the same change of feeding schedule
could completely reset the liver clock within 3 days and largely shift the phases of clock
genes in the heart and kidney in 7 days. In contrast, the cooperative stimuli of light and food
cues can markedly facilitate the adjustment of peripheral clocks to a new environmental
condition by resetting the liver clock in 2 days and the other three peripheral clocks in 5-7
days. Thus, coupling the LD cycle and feeding schedule will promote the circadian resetting
of peripheral clocks in rats.
Secondly, we examined the molecular responses of clock genes to different feeding stimuli in
the feeding sensitive liver clock [5, 6]. A 30-min feeding stimulus is sufficient to
significantly induce the expression of Per2 and Dec1 within 1 h and alter the transcript levels
and circadian phases of other selected clock genes (Bmal1, Cry1, Per1, Per3, Dec2, and
Rev-erb) in the liver clock at longer time intervals. Moreover, among the examined clock
genes, Per2 was most sensitive to food cues which could be significantly induced by a
minimal amount of food. In contrast to the other clock genes, 12-h phase shift of Per2
induced by the feeding reversal could be rapidly and consistently accomplished, regardless
of the shift of the light/dark (LD) cycle. Thus, the feeding-induced resetting of the circadian
clock in the liver is associated with the acute induction of Per2 and Dec1 transcription,
which may serve as the main and secondary input regulators that initiate this feeding-induced
circadian resetting.
Thirdly, we tested the roles of daily three meals on the circadian system and physiological
function of rats [7]. We developed a model of daily three meals mimicing the feeding habit
of human, whereby animals were divided into three groups (three meals, TM; no first meal,
NF; no last meal, NL) all fed with the same amount of food every day. Rats in the NF group
displayed significantly decreased levels of plasma triglyceride (TG), total cholesterol (TC),
high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C),
and glucose in the activity phase, accompanied by delayed circadian phases of hepatic
peripheral clock and downstream metabolic genes. Rats in the NL group showed lower
concentration of plasma TC, HDL-C, and glucose in the rest phase, plus reduced adipose
tissue accumulation and body weight gain. An attenuated rhythm in the food-entraining
pathway, including down-regulated expression of the clock genes Per2, Bmal1, and
Rev-erb, was observed, which may further contribute to the delayed and decreased
expression of FAS in lipogenesis in this group. Thus, the daily first meal determines the
circadian phasing of peripheral clocks, such as in the liver, whereas the daily last meal
tightly couples to lipid metabolism and adipose tissue accumulation, which suggests
differential physiological function of the respective meal timings.
Finally, to study whether and how the function of circadian clock is impaired under the
diabetic condition, we examined not only the expression of circadian genes in the heart and
pineal gland but also the behavioral rhythm of type 2 diabetic and control rats in both the
nighttime restricted feeding (NRF) and daytime restricted feeding (DRF) conditions [8]. In
the NRF condition, the circadian expression of clock genes in the heart and pineal gland was
conserved in the diabetic rats, being similar to that in the control rats. DRF shifted the
circadian phases of peripheral clock genes more efficiently in the diabetic rats than those in
the control rats. Moreover, the activity rhythm of rats in the diabetic group was completely
shifted from the dark phase to the light phase after 5 days of DRF treatment, whereas the
activity rhythm of rats in the control group was still under the control of the suprachiasmatic
nucleus (SCN) after the same DRF treatment. Furthermore, the serum glucose rhythm of
type 2 diabetic rats was also shifted and controlled by the external feeding schedule, ignoring
the SCN rhythm. Therefore, DRF shows stronger effect on the re-entrainment of circadian
rhythm in the type 2 diabetic rats, suggesting that the circadian system in diabetes is unstable
and more easily shifted by feeding stimuli.
References
1. Wu T, Jin YX, Kato H and Fu ZW*. Light and food signals cooperate to entrain rat pineal
clock. Journal of Neuroscience Research, 2008, 86: 3246-255.
2. Wu T, Jin YX, Ni YH, Zhang DP, Kato H and Fu ZW*. Effects of light cues on
re–entrainment of the food–dominated peripheral clocks in mammals. Gene, 2008, 419:
27-34.
3. Wu T, Ni YH, Dong Y, Xu JF, Song XH, Kato H and Fu ZW*. Regulation of circadian
gene expression in the kidney by light and food cues in rats. American Journal of
Physiology-regulatory Integrative and Comparative Physiology, 2010, 298: R635-641.
4. Wu T, Dong Y, Yang ZQ, Kato H, Ni YH and Fu ZW*. Differential resetting process of
circadian system in rat pineal gland after the reversal of light/dark cycle via a 24-h light
or dark period transition. Chronobiology International, 2009, 26: 793-807.
5. Wu T, Ni YH, Kato H and Fu ZW*. Feeding-induced rapid resetting of the hepatic
circadian clock is associated with acute induction of Per2 and Dec1 transcription in rats.
Chronobiology International, 2010, 27: 1–18.
6. Wu T, Fu O, Yao L, Sun L, Zhuge F, Fu ZW*. Differential responses of peripheral
circadian clocks to a short-term feeding stimulus. Molecular Biology Reports, 2012, 39:
9783-9789.
7. Wu T, Sun L, ZhuGe F, Guo X, Zhao Z, Tang R, Chen Q, Chen L, Kato H and Fu ZW*.
Differential roles of breakfast and supper in rats of a daily three-meal schedule upon
circadian regulation and physiology. Chronobiology International, 2011, 28: 890-903.
8. Wu T, ZhuGe F, Sun L, Ni Y, Fu O, Gao G, Chen J, Kato H and Fu ZW*. Enhanced effect
of daytime restricted feeding on the circadian rhythm of streptozotocin-induced type 2
diabetic rats. American Journal of Physiology – Endocrinology and Metabolism, 2012,
302: E1027-1035.
Small Molecules Targeting Mitochondrial UQCRB
Ho Jeong KWON
Chemical genomics NRL, Department of Biotechnology, Yonsei University, Korea
kwonhj@yonsei.ac.kr
Natural products have served as drugs or templates for drugs as well as have contributed to a
better understanding of the targets and pathways involved in human disease processes.1,2 On
the line of these merits of natural products, my lab has done a large scale of phenotypic screen
of microbial or plant metabolites to identify novel small molecules that could perturb the
angiogenic responses of endothelial cells (ECs) to pro-angiogenic stimuli, such as tube
formation and invasive activity.3 As the result, a number of distinct small molecules in respect
with structure and activity have identified. Among these, terpestacin was identified from the
metabolites of the fungus Embellisia chlamydospora as a small molecule with a unique
bicyclo-sesterterpene structure capable of inhibiting the angiogenic response at concentrations
below the toxic threshold.4 Terpestacin effectively inhibited tube formation and EC invasion
induced by VEGF, bFGF, and hypoxia in vitro and angiogenesis within the embryonic chick
chorioallantoic membrane in vivo. To explore the molecular mechanisms underlying
angiogenesis inhibition by terpestacin, its cellular binding protein was identified through
phage display biopanning, an affinity-based target protein selection method with human
cDNA libraries expressed on the surface of bacteriophages using immobilized small
molecules as ligands. The terpestacin binding proteins were identified from T7
phage-displayed human cDNA libraries using biotinylated terpestacin derivatives as affinity
ligands. Ubiquinol-cytochrome c reductase binding protein (UQCRB), a 13.4-kDa subunit of
Complex III in the mitochondrial respiratory chain, was identified as a specific binding
protein of terpestacin.5 Furthermore, the molecular interaction between terpestacin and
UQCRB was validated through a variety of experiments, including biophysical, cell
biological, and genome-wide transcriptional profiling of cells treated with the small molecule
or subjected to genetic knockdown. Interestingly, this interaction dissipated the mitochondrial
membrane potential without disrupting mitochondrial respiration and Complex III functional
structure, implying that terpestacin regulates mitochondrial Complex III function without
affecting mitochondrial energy metabolism. Although several small molecules that regulate
the mitochondrial respiratory chain have been discovered, terpestacin is the first small
molecule targeting UQCRB, suggesting that this unique small molecule could be a useful tool
to explore the role of UQCRB in angiogenesis. Indeed, terpestacin suppressed mitochondrial
ROS generation and HIF-1 stabilization in tumor cells under hypoxic conditions.5 Notably,
the following studies showed that terpestacin inhibits protein synthesis and stability of
HIF-1 through suppression of Complex III-derived ROS generation during hypoxia.
Terpestacin inhibited hypoxia-induced tumor angiogenesis via inhibition of HIF-1-mediated
VEGF expression in a murine breast carcinoma xenograft model. Accordingly, it is proposed
that UQCRB may play an important role in modulating ROS- and HIF-mediated angiogenesis
during hypoxia. Regulation of UQCRB expression demonstrates that it plays a crucial role in
the oxygen sensing mechanism that regulates hypoxia responses.5 Overexpression of UQCRB
induced mitochondrial ROS generation in cells and increased HIF-1 and VEGF protein
levels, whereas its suppression using RNA interference inhibited hypoxia-induced tumor
angiogenesis via inhibition of HIF-1-mediated VEGF expression. Therefore, these data
clearly demonstrate that UQCRB is a critical mediator of hypoxia-induced tumor
angiogenesis via mitochondrial ROS-mediated signaling.
Intriguingly, unlike the Qo site inhibitors myxothiazol and stigmatellin that prevent ROS
generation by blocking electron entry into Complex III and thereby blocking mitochondrial
respiration and ATP generation, terpestacin suppressed hypoxia-induced ROS generation
without inhibiting mitochondrial respiration.5 Therefore, by targeting UQCRB and inducing a
conformational change in Complex III, terpestacin may accelerate the forward electron
transfer to cytochrome b, which shortens the lifetime of SQ at the Qo site to attenuate
hypoxia-induced ROS production without acting as a respiratory poison. In addition, the
ability of terpestacin, or its pharmacophore-based derivatives, to suppress tumor angiogenesis
in vivo without apparent systemic toxicity underscores its potential utility as a new anti-cancer
agent targeting UQCRB. Indeed, from a target-based screen with structural information on the
binding mode of terpestacin and UQCRB, a novel synthetic small molecule targeting UQCRB
(HDNT) was identified and exhibited potent anti-angiogenic activity without cytotoxicity by
modulating the oxygen-sensing function of UQCRB.6 Accordingly, HDNT can serve as a new
synthetic small molecule probe to explore the role of UQCRB in angiogenesis as well as a
potential lead compound for medical applications. Other possible translations of this
information on UQCRB may expand its applications into a pro-angiogenic factor using a cell
permeable form of the protein or conditional UQCRB gene expression.
Collectively, new biologically active small molecules (such as terpestacin) are powerful tools
to explore biology (such as angiogenesis) as well as to develop other small molecules
(HDNT) in a positive feedback manner based on information and experience.
References
1. D. J. Newman, G. M. Cragg and K. M. Snader, Nat. Prod. Rep., 2000, 17, 215−234.
2. I. Paterson and E. A. Anderson, Science., 2005, 21, 451-453.
3. Y. S. Cho and H. J. Kwon, Bioorg. Med. Chem., 2012, 20, 1922-1928.
4. H. J. Jung, H. B. Lee, C. J. Kim, J. R. Rho, J. Shin and H. J. Kwon, J. Antibiot., 2003, 56,
492–496.
5. H. J. Jung, J. S. Shim, J. Lee, Y. M. Song, K. C. Park, S. H. Choi, N. D. Kim, J. H. Yoon, P.
T. Mungai, P. T. Schumacker and H. J. Kwon, J. Biol. Chem., 2010, 285, 11584-11595.
6. H. J. Jung, K. H. Kim, N. D. Kim, G. Han and H. J. Kwon, Bioorg. Med. Chem. Lett., 2011,
21, 1052-1056.
Representative Publications
1. Kwon, HJ,* Owa, T., Hassig, C. A., Shimada, J., and Schreiber, S. L. (1998) Proc. Natl.
Acad. Sci., U.S.A., 95: 3356-3361. “highlighted issue”
2. Kim MS, Kwon HJ, and Kim KW et. al. (2001) Nature Medcine, 7: 437-443.
3. Shim JS, Kim JH, Cho HY, Yum YN, Kim SH, Park HJ, Shim BS, Choi SH, and Kwon HJ*
(2003) Chemistry & Biology, 10: 695-704. “cover issue”
4. Shim JS, Kim DH, and Kwon HJ*. (2004) Oncogene, 23: 1704-1711.
5. Shim JS, Lee J, Park HJ, Park SJ, and Kwon HJ*. (2004) Chemistry & Biology,
11:1455-1463.
6. Kwon HJ*. (2006) Curr. Drug Targets, 7:397-405.
7. Kwon HJ*, Lee CH, Osada H, Yoshida M, and Imoto M. (2008) Nat Chem Biol. 4:
444-446.
8. Kim JH, Kim JH, Oh M, Yu YS, Kim KW, and Kwon HJ.* (2009) Mol Pharm. 6:
513-519. “Most Accessed Paper in 2009”
9. Jung HJ, Shim JS, Park JC, Ha HJ, Kim JH, Kim JG, Kim ND, Yoon JH, and Kwon HJ.*
(2009) Proteomics Clin. Appl. 3: 423-432. “cover issue”
10. Jung HJ, Shim JS, Lee J, Song YM, Park KC, Choi SH, Kim ND, Yoon JH, Mungai PT,
Schumacker PT, and Kwon HJ* (2010) J Biol Chem. 285: 11584-11595. Highlighted by
“Faculty of 1000”
S-3, a Spiraea Diterpenoid Derivative with Potent Anti-tumor
Activity
Xiaojiang Hao1), Lin Li2), Quan Chen3)
1) The State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming, China
2) State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
200031, China
3) The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of
Zoology, Chinese Academy of Sciences, Beijing, China
haoxj@mail.kib.ac.cn
Spiraea japonica L. (Rosaceae) complex consisting of seven varieties are widespread in
Yunnan Province, China. The young leaves, fruits and roots of some of these plants have been
used as diuretic, detoxicant and analgesic agents and for the treatment of inflammation, cough,
headache and toothache in traditional Chinese medicine (TCM). Our group systematically
studied on the all seven varieties of the complex S. japonica, regarding new natural products,
chemical properties of diterpenoid alkaloids and diterpenoids, chemotaxonomy, biosynthesis
of Spiraea alkaloids, and bioactivities of the components of Spiraea japonica complex[1].
S-3 is a derivative of spiramilactone, a diterpenoid isolated from Spiraea japonica complex.
Cooperating with Chen Quan’s group of Institute of Zoology, Chinese Academy of Sciences,
found S-3 can induce apoptosis in bax/bak double knockout murine embryonic fibroblasts.
Further study indicated S-3 significantly increased expression of Bim, which migrated to
mitochondria, altering the conformation of resident Bcl-2 to induce cytochrome C release and
caspase activation. Thus, S-3 induces a structural and functional conversion of Bcl-2 through
Bim to permeabilize the mitochondrial outer membrane thereby inducing apoptosis
independent of Bax and Bak [2].
On the other hand, cooperating with Li Lin’s group of Shanghai Institutes of Biological
Science, Chinese Academy of Sciences, we found that S-3 inhibits Wnt3a or LiCl-stimulated
Top-flash reporter activity in HEK293T cells and growth of colon cancer cells, SW480 and
Caco-2. Treatment of SW480 cells with S-3 led to decreases in the mRNA and/or protein
expression of Wnt target genes Axin2, Cyclin D1 and Survivin, as well as decreases in the
protein levels of Cdc25c and Cdc2. S-3 did not affect the cytosol-nuclear distribution and
protein level of soluble β-catenin, but decreased β-catenin/TCF4 association and the
recruitment of β-catenin to the Axin2 promoter [3]. S-3 showed good inhibition activities
against several colon cancer cell lines such as SW480, Caco-2, HT29 and HCT116, and also
showed good inhibition against multidrug resistant tumor cells MCF-7/ADR and KB/VCR.
Collectively these studies demonstrate that S-3 may be a potential compound for treating
colorectal cancer. The result of structure-activity study showed that -unsaturated ketone
group is an essential group for anti-tumor activity, equally, lack of lactone ring, activity of
derivative will be disappeared.
To investigate structure-activity relationship of anti-tumor, more than 60 derivatives of S-3
have been synthesized by our group. The results of the Wnt inhibitions and cytotoxicities
indicated that the effects of the intramolecular hydrogen bond, the lactone ring, as well as
“Michael acceptor” moiety of S-3 derivatives.
References
[1]. Xiaojiang Hao*, Yuemao Shen, Ling Li, Hongping He, The Chemistry and Biochemistry
of Spiraea japonica Complex, Current Medicinal Chemistry, 10 (2003), 2253-2263.
[2]. Lixia Zhao, Feng He, Haiyang Liu, Yushan Zhu, Weili Tian, Ping Gao, Hongping He,
Wen Yue, Xiaobo Lei, Biyun Ni, Xiaohui Wang, Haijing Jing , Xiaojiang Hao*, Jialing
Lin*, Quan Chen*, Natural diterpenoid compound elevates expression of bim protein,
which interacts with antiapoptotic protein bcl-2, converting it to proapoptotic bax-like
molecule, Journal of Biological Chemistry, 287 (2012), 1054-1065.
[3]. Wei Wang, Haiyang Liu, Sheng Wang, Xiaojiang Hao*, Lin Li*, A diterepenoid
derivative 15-oxospiramilactone inhibits wnt/-catenin signaling and colon cancer cell
tumorigenesis, Cell Research, 21 (2011), 730-740.