Department of Natural Products Chemistry

in Japanese

Graduate School of Pharmaceutical Sciences and
Plant Molecular Science Center
Chiba University
1-8-1 Inohana, Chuo-ku, Chiba 260-8675, JAPAN

Prof. Masami ISHIBASHI, Ph.D.
　　TEL/FAX: +81-43-226-2923 　　 e-mail: mish＠chiba-u.jp “＠” should be replaced by “@”

Assoc. Prof. Akiko TAKAYA, Ph.D.
　　TEL: +81-43-226-2924 　　 e-mail: akiko＠faculty.chiba-u.jp “＠” should be replaced by “@”

Assist. Prof. Yasumasa Hara, Ph.D.
　　TEL: +81-43-226-2925 　　 e-mail: yhara＠chiba-u.jp “＠” should be replaced by “@”

Comprehensive Studies on Natural Products Chemistry: Chemical Biology based on Natural Resources

Natural products continue to play an important role in the discovery of low-molecular weight lead compounds for new-drug developments. Research interests of our laboratory are all concerned with chemistry of natural products, mainly based on the search for new naturally occurring molecules from a variety of terrestrial resources. Although extensive studies have been made on isolation and identification of bioactive substances from plants and microorganisms for more than a century, natural products chemistry is still of great importance as a basic science since natural products have made significant contributions to development of new drugs as well as progress of basic studies of life sciences.

Research projects of our group are aiming at (i) discovering bioactive small molecules useful for development of new drugs and (ii) providing new molecular tools applicable to basic biological sciences. These research projects have to be carried out on the basis of modern organic chemistry, and advanced skills are essentially required in isolation and structure elucidation of natural organic compounds using current chromatographic techniques, spectroscopic analyses, and chemical syntheses of key molecules.

Our current research interests:

1) Basic studies on development of unexplored organisms such as myxomycetes which are expected to be useful as new natural-products resources.

2) Search for new molecules with significant biological functions and unprecedented chemical structures from a variety of natural resources.

3) Synthesis of bioactive natural products and their modified or fragmentary compounds for the purpose of rigid determination of fine stereochemical structures and application to the analysis of the mechanism of actions.

4) Search for bioactive small molecules from natural products through several screening systems targeting mainly cancer-related signaling molecules using cell-based luciferase or fluorescent assays.

5) Construction of small molecule library based on natural products framework by using diversity-oriented synthesis and solid phase synthesis.

6) Development of high-throughput analyzing system of protein-protein interaction with small molecule based on target protein immobilizing microplate.

Publications: Original Articles (1998-)

A summary for recent studies in our group:

During our studies on search for bioactive natural products from unexplored natural resources targeting singaling molecules, here we describe two subjects: 1) Isolation of natural products from myxomycetes; and 2) Search for bioactive natural products targeting signaling molecules in cancer-related biological pathways.

1. Natural products from myxomycetes:

The Myxomycetes (true slime molds) are an unusual group of primitive organisms that may be assigned to one of the lowest classes of eukaryotes. Spore germination experiments were studied of hundreds of field-collected myxomycetes collected in Japan and succeeded in laboratory culture of plasmodia of several myxomycetes in a practical scale for natural products chemistry studies.　Pyrroloiminoquinones, polyene yellow pigments, and a peptide lactone were isolated from cultured plasmodia of myxomycetes, while new antimicrobial naphthoquinone pigments, tyrosine-kinase inhibitory bisindole alkaloids, a cytotoxic triterpenoid aldehyde lactone with a reversal effect of drug resistance, a cycloanthranilylproline with sensitizing effect of TRAIL-induced apoptosis through activation of COX2, a dibenzofuran glycoside, and sterols with a 2,6-dioxabicyclo[2.2.2]octan-3-one ring system were also isolated from field-collected fruit bodies of myxomycetes (Figure 1).^1-3)

Figure 1 New natural products isolated from myxomycetes in our group

2. Search for bioactive natural products targeting signaling molecules in cancer-related biological pathways:

During our studies on search for bioactive natural products, we recently examined extracts of various natural resources including unexplored myxomycetes,^1-3) marine organisms,⁴⁾ as well as several medicinal plants collected in north-east part (Khon Kaen area) of Thailands.⁵⁾ Here we describe our recent results on our screening programs targeting signaling molecules in cancer-related biological pathways such as TRAIL, Wnt, and Hedghog signaling pathways.

(1) TRAIL signaling

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) induces apoptosis in many transformed cells but not in normal cells and, hence, has been expected as a new anticancer strategy. We recently identified several natural products which exhibited activities related to TRAIL signaling (Figure 2).⁶⁾ A dimeric sesquiterpenoid, parviflorene F (1),⁷⁾ isolated from Zingiberaceous plant, Curcuma parviflora, showed enhancement activity of gene expression of TRAIL-receptor (TRAIL-R2) and TRAIL-R2 protein level (Figure 3). Apoptosis was induced by 1 as revealed by the distribution of DNA and Annexin V/PI staining using flow cytometry. In addition, 1-induced apoptosis was inhibited by human recombinant TRAIL-R2/Fc chimera protein, TRAIL-neutralizing fusion protein. We also found that 1 induced the activation of caspase-8, caspase-9, and caspase-3, indicating that the cytotoxic effect of 1 is correlated with apoptosis by a caspase-dependent mechanism through TRAIL-R2. In addition, 1 enhanced TRAIL-induced cell death against HeLa and TRAIL-resistant DLD1 cells. Thus, it was suggested that up-regulation of TRAIL-R2 by 1 may contribute to sensitization of TRAIL-induced cell death.⁸⁾

Several new isoflavone natural products, named brandisianins (e.g., brandisianin D (2)),⁹⁾ were isolated from Leguminosaeous plant, Millettia brandisiana, by our screening study targeting TRAIL receptor expression enhancement activity by a luciferase assay system using DLD-1/SacI cells. A dihydroflavonol (BB-1, 3)¹⁰⁾ that was extracted from Compositaeous plant, Blumea balsamifera, and fuligocandin B (4),¹¹⁾ a new anthranilylproline-indole alkaloid isolated from myxomycete were found to exhibit reversal effect of TRAIL resistance activity.

Figure 2. Natural products having effects on TRAIL signaling

(*New compounds isolated in our group)

Figure 3. Enhancement of TRAIL-R2 protein levels in DLD1/TRAIL-R cells treated with parviflorene F (1) at 4 and 8 μg/mL (n = 3).

(2) Wnt signaling

The Wnt/β-catenin signaling pathway plays key roles in cell morphology, motility, proliferation, and differentiation. When inappropriately activated, the pathway has been linked to colorectal cancer and melanoma. In these cells, the presence of Wnts or mutations in APC, Axin, etc. cause activation of Wnt signaling and result in the stablization of β-catenin. Then β-catenin enters into the nucleus and associates with transcriptional factors of TCF/LEF, then this complex binds to the TCF/LEF binding sites and leads to the overexpression of target genes and finally contributes to tumorgenesis. Therefore, compounds down-regulating Wnt/β-catenin signaling were expected in colon cancer therapy. To investigate small molecules down-regulating Wnt/β-catenin signaling, natural extracts and compounds were tested using stably transfected cells (STF cells), in which luciferase reporter plasmids with TCF/LEF binding sites were transfected. We screened extracts of plants collected from Thailand, and five of them were judged as active.¹²⁾ We also screened natural compounds from myxomycetes isolated or synthesized by our laboratory. Lycogarubin B (5), cis-dihydroarcyriarubin C (6, prepared by synthesis),^13,14) and 10-epi-melleumin B (7, prepared by synthesis)^15,16) were found to exhibit significant inhibition in TCF/β-catenin transcriptional activity (Figures 4 and 5).

Figure 4. Natural products having inhibitory effects on TCF/β-catenin transcriptional activity

(**Stereoisomers of new compounds isolated in our group and prepared by synthesis)

Figure 5. Inhibition of TCF/β-catenin transcriptional activity of lycogarubin B (5)

(3) Hedghog signaling

The hedgehog (Hh)/GLI signaling pathway has been implicated not only in a variety of developmental processes in wide range of organisms, but also in the formation and development of different tumors including skin, brain, prostate, upper gastrointestinal tract, pancreas and lung. Targeting Hh/GLI signaling has been expected as an effective cancer therapeutic strategy. To find specific inhibitors of Hh/GLI signaling pathway from natural resources, a cell-based screening assay system targeting transcriptional activator GLI1, which constitutes the final step in the Hh signaling pathway, was constructed. A pGL4-Luc reporter vector inserted with 12´GLI binding sites was stably transfected into HaCaT cell line expressing GLI1 under tetracycline repressor control. By using this assay system, we identified six active compounds; staurosporinone (8), 6-hydroxystaurosporinone (9),¹⁷⁾ arcyriaflavin C (10), 5,6-dihydroxyarcyriaflavin A (11),¹⁷⁾ zerumbone (12), and zerumbone epoxide (13). Their IC₅₀ values of GLI1 transcriptional inhibitory activity were 1.8, 3.6, 11.3, 6.9, 3.0 and 55 μM, respectively. Next, from the screening study of our natural plants extracts library, the extract of Physalis minima was found to be active. Repeated chromatography separations of the MeOH extracts of P. minima gave two active compounds, physalin F (14) and physalin B (15) with the IC₅₀ values of 0.66 and 0.62 μM, respectively. These compounds also inhibited GLI2-mediated transactivation. These inhibitors are the first natural products shown to be selective inhibition of GLI-mediated transcription. Western blotting analysis further revealed that 8, 12, 14, and 15 decreased the expression of GLI1 and PTCH proteins in HaCaT cells. It was also revealed for the first time that these selective GLI-mediated transactivation inhibitors directly reduced the level of the anti-apoptosis Bcl2 protein. Finally, physalins F and B were found to be cytotoxic against PANC1 pancreatic cancer cells (IC₅₀ values, 2.7 and 5.3 mM, respectively), which significantly express Hh/GLI components. These results strongly suggested that the cytotoxicity of these compounds against PANC1 cells may be correlated with their inhibition of GLI-mediated transcription (Figures 6 and 7).^18,19)

Figure 6. Natural products having inhibitory effects on GLI transcriptional activity

(*New compounds isolated in our group)

Figure 7. Decrease in protein levels of PTCH, a Hh/GLI signaling component, in PACN1 cells treated with physarin F (14)

References and Notes

1) Ishibashi, M. Yakugakuzasshi 2007, 127, 1369-1381.

2) Ishibashi, M. Medicinal Chemistry 2005, 1, 575-590

3) Ishibashi, M. Studies in Natural Products Chemistry, Atta-ur-Rahman, Ed.; Elsevier Science; Amsterdam, 2003, 29, 223-262.

4) Ishibashi, M.; Yamaguchi, Y.; Hirano, Y. J. Biomaterials from Aquatic and Terrestrial Organisms, M. Fingerman and R. Nagabhushanam, Eds.; Science Publishers, Inc.; Enfield, 2006, pp. 513-535.

5) Ishibashi, M.; Toume, K.; Yamaguchi, Y.; Ohtsuki, T. Recent Research Developments in Phytochemistry; Research Signpost; Trivandrum, India, 2004, 8, 139-156.

6) Ishibashi, M.; Ohtsuki, T. Med. Res. Rev. 2008, 28, 688-714

7) Toume, K.; Takahashi, M.; Koyano, T.; Kowithayakorn, T.; Yamaguchi, K.; Hayashi, M.; Komiyama, K.; Ishibashi, M. Tetrahedron 2004, 60, 10817-10824.

8) Ohtsuki, T.; Tamaki, M.; Toume, K.; Ishibashi, M. Bioorg. Med. Chem. 2008, 16, 1756-1763.

9) Kikuchi, H.; Ohtsuki, T.; Koyano, T.; Kowithayakorn, T.; Sakai, T.; Ishibashi, M. J. Nat. Prod. 2007, 70, 1910-1914.

10) (a) Osaki, N.; Koyano, T.; Kowithayakorn, T.; Hayashi, M.; Komiyama, K.; Ishibashi, M. J. Nat. Prod. 2005, 68, 447-449. (b) Hasegawa, H.; Yamada, Y.; Komiyama, K.; Hayashi, M.; Ishibashi, M.; Yoshida, T.; Sakai, T.; Koyano, T.; Kam, T.-S.; Murata K.; Sugahara, K.; Tsuruda, K.; Akamatsu, N.; Tsukasaki, K.; Masuda, M.; Takasu, N.; Kamihira S. Blood 2006, 107, 679-688.

11) (a) Nakatani, S.; Yamamoto, Y.; Hayashi, M.; Komiyama, K.; Ishibashi, M. Chem. Pharm. Bull. 2004, 52, 368-370. (b) Hasegawa, H.; Yamada, Y.; Komiyama, K.; Hayashi, M.; Ishibashi, M.; Sunazuka, T.; Izuhara, T.; Sugahara, K.; Tsuruda, K.; Masuda, M.; Takasu, N.; Tsukasaki, K.; Tomonaga, M.; Kamihira, S. Blood 2007, 110, 1664-1674

12) Li, X.; Ohtsuki, T.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. Chem. Asian J. 2009, 4, 540-547

13) Kaniwa, K.; Arai, M. A.; Li, X.; Ishibashi, M. Bioorg. Med. Chem. Lett. 2007, 17, 4254-4257.

14) Nakatani, S.; Naoe, A.; Yamamoto, Y.; Yamauchi, T.; Yamaguchi, N.; Ishibashi, M. Bioorg. Med. Chem. Lett. 2003, 13, 2879-2881.

15) Hanazawa, S.; Arai, M. A.; Li, X.; Ishibashi, M. Bioorg. Med. Chem. Lett. 2008, 18, 95-98.

16) Nakatani, S.; Kamata, K.; Sato, M.; Onuki, H.; Hirota, H.; Matsumoto, J.; Ishibashi, M. Tetrahedron Lett. 2005, 46, 267-271.

17) Hosoya, T.; Yamamoto, Y.; Uehara, Y.; Hayashi, M.; Komiyama, K.; Ishibashi, M. Bioorg. Med. Chem. Lett. 2005, 15, 2776-2780.

18) (a) Hosoya, T.; Arai, M. A.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. ChemBioChem 2008, 9, 1082-1092. (b) Arai, M. A.; Tateno, C.; Hosoya, T.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. Bioorg. Med. Chem. 2008, 16, 9420-9424

19) Acknowledgment: Myxomycetes are collected by Yukinori Yamamoto (Ohtsu-ko, Kochi), and plant materials are provided through a collaboration project with Dr. Takashi Koyano (Temko Corporation) and Professor Thaworn Kowithayakorn (Khon Kaen Univeristy, Thailand) or a collaboration project with Professor S. K. Sadhu (Khulna University, Bangladesh). We thank Dr. Bingliang Fang (The University of Texas, MD, Anderson Cancer Center) for TRAIL-resistant DLD1 cells, Prof. T. Sakai (Kyoto Prefectural University of Medicine) for DLD-1/SacI cells, Prof. J. Nathans (John Hopkins Medical School) for the STF cells, Prof. F. Aberger (University of Salzburg) for tetracycline-regulated HaCaT cells, and Prof. R. Toftgård (Karolinska Institute) for the 12GLI-RE-TKO luciferase plasmid. We also thank Dr. Masaaki Sato for valuable discussions in the beginning of these studies. This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, from the Futaba Electronics Memorial Foundation and the Japan Science and Technology Agency.

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