論文 - 井本　正哉

Isolation and structure elucidation of a novel androgen antagonist, arabilin, produced by Streptomyces sp. MK756-CF1

Kawamura T., Fujimaki T., Hamanaka N., Torii K., Kobayashi H., Takahashi Y., Igarashi M., Kinoshita N., Nishimura Y., Tashiro E., Imoto M.

Journal of Antibiotics （Journal of Antibiotics）  63 （ 10 ） 601 - 605 2010年10月

研究論文（学術雑誌）, 査読有り,  ISSN  00218820

　概要を見る

In the course of screening for a new type of androgen receptor (AR) antagonist, we isolated a novel compound, arabilin, with two structural isomers, spectinabilin and SNF4435C, produced by Streptomyces sp. MK756-CF1. Structure elucidation on the basis of the spectroscopic properties showed that arabilin is a novel polypropionate-derived metabolite with a p-nitrophenyl group and a substituted γ-pyrone ring. Arabilin competitively blocked the binding of androgen to the ligand-binding domain of AR in vitro. In addition, arabilin inhibited androgen-induced prostate-specific antigen mRNA expression in prostate cancer LNCaP cells. © 2010 Japan Antibiotics Research Association All rights reserved.

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Metabolomic identification of the target of the filopodia protrusion inhibitor glucopiericidin A

Kitagawa M., Ikeda S., Tashiro E., Soga T., Imoto M.

Chemistry and Biology （Chemistry and Biology）  17 （ 9 ） 989 - 998 2010年09月

研究論文（学術雑誌）, 査読有り,  ISSN  10745521

　概要を見る

Identifying the targets of bioactive compounds is a major challenge in chemical biological research. Here, we identified the functional target of the natural bioactive compound glucopiericidin A (GPA) through metabolomic analysis. We isolated GPA while screening microbial samples for a filopodia protrusion inhibitor. Interestingly, GPA alone did not inhibit filopodia protrusion, but synergistically inhibit protrusion with the mitochondrial respiration inhibitor, piericidin A (PA). These results suggested that GPA might inhibit glycolysis. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) provided strong evidence that GPA suppresses glycolysis by functionally targeting the glucose transporter. GPA may therefore serve as a glucose transporter chemical probe. Simultaneous inhibition of both glycolysis and mitochondrial respiration dramatically decreased intracellular ATP levels, indicating that GPA inhibits ATP-dependent filopodia protrusion with PA. Our results represent a challenge of molecular target identification using metabolomic analysis. © 2010 Elsevier Ltd. All rights reserved.

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Glyoxalase-I is a novel target against Bcr-Abl leukemic cells acquiring stem-like characteristics in a hypoxic environment

Takeuchi M., Kimura S., Kuroda J., Ashihara E., Kawatani M., Osada H., Umezawa K., Yasui E., Imoto M., Tsuruo T., Yokota A., Tanaka R., Nagao R., Nakahata T., Fujiyama Y., Maekawa T.

Cell Death and Differentiation （Cell Death and Differentiation）  17 （ 7 ） 1211 - 1220 2010年07月

研究論文（学術雑誌）, 査読有り,  ISSN  13509047

　概要を見る

Abl tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib are ineffective against Bcr-Abl leukemic stem cells. Thus, the identification of novel agents that are effective in eradicating quiescent Bcr-Abl stem cells is needed to cure leukemias caused by Bcr-Abl cells. Human Bcr-Abl cells engrafted in the bone marrow of immunodeficient mice survive under severe hypoxia. We generated two hypoxia-adapted (HA)-Bcr-Abl sublines by selection in long-term hypoxic cultures (1.0% O 2). Interestingly, HA-Bcr-Abl cells exhibited stem cell-like characteristics, including more cells in a dormant, increase of side population fraction, higher Β-catenin expression, resistance to Abl TKIs, and a higher transplantation efficiency. Compared with the respective parental cells, HA-Bcr-Abl cells had higher levels of protein and higher enzyme activity of glyoxalase-I (Glo-I), an enzyme that detoxifies methylglyoxal, a cytotoxic by-product of glycolysis. In contrast to Abl TKIs, Glo-I inhibitors were much more effective in killing HA-Bcr-Abl cells both in vitro and in vivo. These findings indicate that Glo-I is a novel molecular target for treatment of Bcr-Abl leukemias, and, in particular, Abl TKI-resistant quiescent Bcr-Abl leukemic cells that have acquired stem-like characteristics in the process of adapting to a hypoxic environment. © 2010 Macmillan Publishers Limited All rights reserved.

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Terpenoids produced by actinomycetes: Isolation, structural elucidation and biosynthesis of new diterpenes, gifhornenolones A and B from Verrucosispora gifhornensis YM28-088

Shirai M., Okuda M., Motohashi K., Imoto M., Furihata K., Matsuo Y., Katsuta A., Shizuri Y., Seto H.

Journal of Antibiotics （Journal of Antibiotics）  63 （ 5 ） 245 - 250 2010年05月

研究論文（学術雑誌）, 査読有り,  ISSN  00218820

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New terpenoids named gifhornenolones A (1) and B (2) were isolated from the culture broth of Verrucosispora gifhornensis YM28-088, and their structures were established as hydroxylated isopimaradiene derivatives on the basis of extensive NMR and MS spectral analyses. In addition, a known sesquiterpene compound cyperusol C (3) was isolated. The absolute configuration of 1 was determined by nuclear Overhauser effect spectroscopy (NOESY) and CD spectra as 4R, 5S, 9R, 10S, 13R, and that of 2 was determined by NOESY experiments as 3R, 4R, 5R, 9R, 10S, 13R. Labeling experiments with 1-13 Cglucose and U-13 C6 glucose confirmed that the MEP (2-C-methyl-D-erythritol-4-phosphate) pathway was used for the biosynthesis of terpenoids in this organism. 1 showed potent inhibitory activity to the androgen receptor with an IC50 of 2.8 μg ml-1. © 2010 Japan Antibiotics Research Association All rights reserved.

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Distinct subcellular localization in the cytosol and apicoplast, unexpected dimerization and inhibition of Plasmodium falciparum glyoxalases

Urscher M., Przyborski J., Imoto M., Deponte M.

Molecular Microbiology （Molecular Microbiology）  76 （ 1 ） 92 - 103 2010年04月

研究論文（学術雑誌）, 査読有り,  ISSN  0950382X

　概要を見る

The ubiquitous glyoxalase system removes methylglyoxal as a harmful by-product of glycolysis. Because malaria parasites have drastically increased glycolytic fluxes, they could be highly susceptible to the inhibition of this detoxification pathway. Here we analysed the intracellular localization, oligomerization and inhibition of the glyoxalases from Plasmodium falciparum. Glyoxalase I (GloI) and one of the two glyoxalases II (cGloII) were located in the cytosol of the blood stages. The second glyoxalase II (tGloII) was detected in the apicoplast pointing to alternative metabolic pathways. Using a variety of methods, cGloII was found to exist in a monomer-dimer equilibrium that might have been overlooked for homologues from other organisms and that could be of physiological importance. The compounds methyl-gerfelin and curcumin, which were previously shown to inhibit mammalian GloI, also inhibited P. falciparum GloI. Inhibition patterns were predominantly competitive but were complicated because of the two different active sites of the enzyme. This effect was neglected in previous inhibition studies of monomeric glyoxalases I, with consequences for the interpretation of inhibition constants. In summary, the present work reveals novel general glyoxalase properties that future research can build on and provides a significant advance in characterizing the glyoxalase system from P. falciparum. © 2010 Blackwell Publishing Ltd.

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Mesenchyme cells can function to induce epithelial cell proliferation in starfish embryos

Hamanaka G., Matsumoto M., Imoto M., Kaneko H.

Developmental Dynamics （Developmental Dynamics）  239 （ 3 ） 818 - 827 2010年03月

研究論文（学術雑誌）, 査読有り,  ISSN  10588388

　概要を見る

Here, we show that mesenchyme cells have a novel morphogenetic function in epithelial cell proliferation in starfish embryos. Blastula embryos were injected with pure populations of mesenchyme cells and the total cell numbers in the treated embryos were subsequently determined at different developmental stages. When a total of 40-50 mesenchyme cells was injected, total cells numbers in mid-gastrula embryos and 3-day-old bipinnaria larvae increased significantly (by 1.3-fold) compared with controls, with no indication of any mitotic activity in the injected mesenchyme cells. However, injection of more than 150 mesenchyme cells failed to induce proliferation of the epithelial cells and, moreover, interfered with normal morphogenesis. These developmental abnormalities occurred concomitantly with a severe condensation of the fibrous component of the extracellular matrix. Our data suggest that epithelial cell proliferation is induced by an appropriate number of mesenchyme cells in concert with the fibrous component of the extracellular matrix. © 2010 Wiley-Liss, Inc.

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Chemical Biology of Cell Motility Inhibitors

Kawamura T., Kitagawa M., Imoto M.

Protein Targeting with Small Molecules: Chemical Biology Techniques and Applications （Protein Targeting with Small Molecules: Chemical Biology Techniques and Applications）     169 - 187 2009年08月

研究論文（学術雑誌）, 査読有り,  ISSN  9780470120538

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Oligomycin induced the proteasomal degradation of cyclin D1 protein

Kanai M., Iba S., Okada R., Tashiro E., Imoto M.

Journal of Antibiotics （Journal of Antibiotics）  62 （ 8 ） 425 - 429 2009年08月

研究論文（学術雑誌）, 査読有り,  ISSN  00218820

　概要を見る

We searched for compounds that affect the cyclin D1/retinoblastoma protein pathway from the in-house natural product library using a recombinant adenovirus with the Cre/loxP-regulated cyclin D1 overexpression system, and we found that oligomycin inhibited cell growth more effectively in cyclin D1-overexpressing SW480 cells than in control SW480 cells. We also found that oligomycin reduced the expression levels of cyclin D1 protein and that this reduction is, at least in part, mediated by Thr-286 phosphorylation-dependent proteasomal degradation. © 2009 Japan Antibiotics Research Association.

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Vacuolar H⁺-ATPase inhibitors overcome Bcl-xL-mediated chemoresistance through restoration of a caspase-independent apoptotic pathway

Sasazawa Y., Futamura Y., Tashiro E., Imoto M.

Cancer Science （Cancer Science）  100 （ 8 ） 1460 - 1467 2009年07月

研究論文（学術雑誌）, 査読有り,  ISSN  13479032

　概要を見る

The anti-apoptotic oncoproteins Bcl-2 and Bcl-xL play crucial roles in tumorigenesis and chemoresistance, and are thus therapeutic cancer targets. We searched for small molecules that disturbed the anti-apoptotic function of Bcl-2 or Bcl-xL, and found vacuolar H+-ATPase (V-ATPase) inhibitors, such as bafilomycin A1 (BMA), that showed such activity. Bcl-xL-overexpressing Ms-1 cells displayed resistance to anticancer drugs, but underwent apoptosis following treatment with a combination of V-ATPase inhibitors at doses similar to those that caused inhibitory activities of V-ATPase. We investigated the apoptosis mechanism induced by cotreatment of Bcl-xL-overexpressing Ms-1 cells with BMA as a V-ATPase inhibitor and taxol (TXL) as an anticancer drug. With BMA, TXL triggered mitochondrial membrane potential loss and cytochrome c release, whereas downstream caspase activation was not observed. In contrast, pronounced nuclear translocation of mitochondrial apoptosis-inducing factor and endonuclease G, known as effectors of caspase-independent apoptosis, was observed with BMA and TXL cotreatment. Moreover, depletion of apoptosis-inducing factor and endonuclease G using each siRNA significantly rescued cells from BMA- and TXL-induced apoptosis. Hence, the apoptosis-inducing factor- and endonuclease G-dependent pathway was critical for apoptosis induction by BMA and TXL cotreatment. Our data suggest that V-ATPase inhibitors could not only suppress anti-apoptotic Bcl-2 nor Bcl-xL but could also facilitate the caspase-independent apoptotic pathway. V-ATPase inhibition will be a promising therapeutic approach for Bcl-2- or Bcl-xL-overexpressing malignancies. © 2009 Japanese Cancer Association.

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Transcriptional regulation of human fibroblast growth factor receptor 1 by E2F-1

Kanai M., Tashiro E., Maruki H., Minato Y., Imoto M.

Gene （Gene）  438 （ 1-2 ） 49 - 56 2009年06月

研究論文（学術雑誌）, 査読有り,  ISSN  03781119

　概要を見る

Overexpression of fibroblast growth factor receptors (FGFRs) has been observed in many types of human tumors; however, the regulatory mechanism of human FGFR expression is still largely unknown. In the present study, we first identified the transcriptional initiation site in the human FGFR 1 gene by 5′-RACE. Furthermore, we show that the expression of human FGFR 1 is regulated by E2F-1. Characterization of the human FGFR 1 promoter demonstrated that two non-consensus E2F binding sequences at positions + 4 to + 22 and + 25 to + 43 relative to our identified transcriptional initiation site in the human FGFR 1 gene were critical for E2F-1-mediated transactivation of human FGFR 1 promoter. Mutations of these sites completely abolished the response of human FGFR 1 promoter to E2F-1 as well as E2F-1 binding in electrophoretic mobility-shift assays. Furthermore, chromatin immunoprecipitation assay showed that E2F-1 was able to bind in vivo to the human FGFR 1 promoter. Moreover, human FGFR 1 protein expression was up-regulated by the overexpression of E2F-1, but down-regulated by the overexpression of pRB in situ, suggesting that the expression of human FGFR 1 is regulated by the pRB/E2F pathway. Because disruption of the pRB/E2F pathway is frequently observed in tumor cells, our findings provide valuable information for studying the role of FGFR 1 in tumor progression. © 2009 Elsevier B.V. All rights reserved.

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Integrating statistical predictions and experimental verifications for enhancing protein-chemical interaction predictions in virtual screening

Nagamine N., Shirakawa T., Minato Y., Torii K., Kobayashi H., Imoto M., Sakakibara Y.

PLoS Computational Biology （PLoS Computational Biology）  5 （ 6 ） e1000397 2009年06月

研究論文（学術雑誌）, 査読有り,  ISSN  1553734X

　概要を見る

Predictions of interactions between target proteins and potential leads are of great benefit in the drug discovery process. We present a comprehensively applicable statistical prediction method for interactions between any proteins and chemical compounds, which requires only protein sequence data and chemical structure data and utilizes the statistical learning method of support vector machines. In order to realize reasonable comprehensive predictions which can involve many false positives, we propose two approaches for reduction of false positives: (i) efficient use of multiple statistical prediction models in the framework of two-layer SVM and (ii) reasonable design of the negative data to construct statistical prediction models. In two-layer SVM, outputs produced by the first-layer SVM models, which are constructed with different negative samples and reflect different aspects of classifications, are utilized as inputs to the second-layer SVM. In order to design negative data which produce fewer false positive predictions, we iteratively construct SVM models or classification boundaries from positive and tentative negative samples and select additional negative sample candidates according to pre-determined rules. Moreover, in order to fully utilize the advantages of statistical learning methods, we propose a strategy to effectively feedback experimental results to computational predictions with consideration of biological effects of interest. We show the usefulness of our approach in predicting potential ligands binding to human androgen receptors from more than 19 million chemical compounds and verifying these predictions by in vitro binding. Moreover, we utilize this experimental validation as feedback to enhance subsequent computational predictions, and experimentally validate these predictions again. This efficient procedure of the iteration of the in silico prediction and in vitro or in vivo experimental verifications with the sufficient feedback enabled us to identify novel ligand candidates which were distant from known ligands in the chemical space. © 2009 Nagamine et al.

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Bioprobe generation from microbial origin for chemical biology study

Imoto M.

Folia Pharmacologica Japonica （Folia Pharmacologica Japonica）  132 （ 1 ） 26 - 30 2008年09月

研究論文（学術雑誌）, 査読有り,  ISSN  00155691

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The identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase I

Kawatani M., Okumura H., Honda K., Kanoh N., Muroi M., Dohmae N., Takami M., Kitagawa M., Futamura Y., Imoto M., Osada H.

Proceedings of the National Academy of Sciences of the United States of America （Proceedings of the National Academy of Sciences of the United States of America）  105 （ 33 ） 11691 - 11696 2008年08月

研究論文（学術雑誌）, 査読有り,  ISSN  00278424

　概要を見る

Osteoclasts, bone-resorptive multinucleated cells derived from hematopoietic stem cells, are associated with many bone-related diseases, such as osteoporosis. Osteoclast-targeting small-molecule inhibitors are valuable tools for studying osteoclast biology and for developing antiresorptive agents. Here, we have discovered that methyl-gerfelin (M-GFN), the methyl ester of the natural product gerfelin, suppresses osteoclastogenesis. By using M-GFN-immobilized beads, glyoxalase I (GLO1) was identified as an M-GFN-binding protein. GLO1 knockdown and treatment with an established GLO1 inhibitor in osteoclast progenitor cells interfered with osteoclast generation, suggesting that GLO1 activity is required for osteoclastogenesis. In cells, GLO1 plays a critical role in the detoxification of 2-oxoaldehydes, such as methylglyoxal. M-GFN inhibited the enzymatic activity of GLO1 in vitro and in situ. Furthermore, the cocrystal structure of the GLO1/M-GFN complex revealed the binding mode of M-GFN at the active site of GLO1. These results suggest that M-GFN targets GLO1, resulting in the inhibition of osteoclastogenesis. © 2008 by The National Academy of Sciences of the USA.

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Hot springs and cool natural products

Kwon H., Lee C., Osada H., Yoshida M., Imoto M.

Nature Chemical Biology （Nature Chemical Biology）  4 （ 8 ） 444 - 446 2008年08月

研究論文（学術雑誌）, 査読有り,  ISSN  15524450

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SAR study of a novel triene-ansamycin group compound, quinotrierixin, and related compounds, as inhibitors of ER stress-induced XBP1 activation I. Taxonomy, fermentation, isolation, biological activities and SAR study

Kawamura T., Tashiro E., Yamamoto K., Shindo K., Imoto M.

Journal of Antibiotics （Journal of Antibiotics）  61 （ 5 ） 303 - 311 2008年05月

研究論文（学術雑誌）, 査読有り,  ISSN  00218820

　概要を見る

In the course of screening for an inhibitor of ER stress-induced XBP1 activation, we isolated a new member of the triene-ansamycin group compound, quinotrierixin, from a culture broth of Streptomyces sp. PAE37. Quinotrierixin inhibited thapsigargin-induced XBP1 activation in HeLa cells with an IC 50 of 0.067 μM. We found that other triene-ansamycin group compounds such as demethyltrienomycin A and mycotrienin I also inhibited ER stress-induced XBP1 activation. Moreover, we performed SAR study of twelve triene-ansamycin group compounds. The study showed that OH group at C-13 was crucial, and CH3 group at C-14 would be important for the XBP1 inhibitory activity. © Japan Antibiotics Research Association.

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SAR study of a novel triene-ansamycin group compound, quinotrierixin, and related compounds, as inhibitors of ER stress-induced XBP1 activation.

Kawamura T, Tashiro E, Shindo K, Imoto M

The Journal of antibiotics 61 （ 5 ） 312 - 7 2008年05月

研究論文（学術雑誌）, 査読有り,  ISSN  0021-8820

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SAR study of a novel triene-ansamycin group compound, quinotrierixin, and related compounds, as inhibitors of ER stress-induced XBP1 activation.

Kawamura T, Tashiro E, Yamamoto K, Shindo K, Imoto M

The Journal of antibiotics 61 （ 5 ） 303 - 11 2008年05月

研究論文（学術雑誌）, 査読有り,  ISSN  0021-8820

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SAR study of a novel triene-ansamycin group compound, quinotrierixin, and related compounds, as inhibitors of ER stress-induced XBP1 activation II. Structure elucidation

Kawamura T., Tashiro E., Shindo K., Imoto M.

Journal of Antibiotics （Journal of Antibiotics）  61 （ 5 ） 312 - 317 2008年05月

研究論文（学術雑誌）, 査読有り,  ISSN  00218820

　概要を見る

Four novel triene-ansamycin group compounds, quinotrierixin, demethyltrienomycin A, demethyltrienomycin B and demethyltrienomycinol, were isolated from the culture broth of Streptomyces sp. PAE37 as inhibitors of ER stress-induced XBP1 activation. The structures of quinotrierixin, demethyltrienomycin A, demethyltrienomycin B and demethyltrienomycinol were determined on the basis of their spectroscopical and chemical properties. All of four possessed 21-membered macrocyclic lactams including triene moieties. © Japan Antibiotics Research Association.

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Identification and characterization of mouse type II platelet-derived growth factor receptor α transcript

Minato Y., Nihei Y., Kodama Y., Tashiro E., Kanai M., Imoto M.

Bioscience, Biotechnology and Biochemistry （Bioscience, Biotechnology and Biochemistry）  72 （ 3 ） 759 - 766 2008年04月

研究論文（学術雑誌）, 査読有り,  ISSN  09168451

　概要を見る

Recently, we identified a novel human platelet-derived growth factor (PDGF) receptor α mRNA (type II) that differs from the known human PDGF receptor α mRNA (type I) only in its 5′-untranslated region and is regulated by E2F-1. In this study, we determined the nucleotide sequence of the mouse type II PDGF receptor α mRNA and found that the expression of this transcript was regulated by E2F-1 through an E2F-1-responsive sequence located +711/+718 downstream of the transcription start site of the type I transcript. Furthermore, we observed embryo-predominant expression of the type II transcript.

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

Futamura Y., Sawa R., Umezawa Y., Igarashi M., Nakamura H., Hasegawa K., Yamasaki M., Tashiro E., Takahashi Y., Akamatsu Y., Imoto M.

Journal of the American Chemical Society （Journal of the American Chemical Society）  130 （ 6 ） 1822 - 1823 2008年02月

研究論文（学術雑誌）, 査読有り,  ISSN  00027863

　概要を見る

Anti-apoptotic oncoproteins Bcl-2 and Bcl-xL are overexpressed in many cancers and play a crucial role in cancer initiation, progression, and resistance to chemotherapy. Therefore, the discovery of a functional inhibitor for these proteins and improved understanding of the molecular mechanisms of these proteins will be an aid to novel anti-tumor therapies. Here, using chemical-genetic cell-based screening, we have discovered a chemically and biologically unique substance, incednine, as a novel functional modulator of Bcl-2/Bcl-xL from the fermentation broth of Streptomyces sp. ML693-90F3. This compound was isolated as a HCl salt by solvent extraction and using centrifugal liquid-liquid partition chromatography. Its structure was elucidated by spectroscopic analysis, X-ray crystallographic analysis, and computational studies. Incednine has a molecular formula, C42H63N3O8, and consists of a novel skeletal structure, enol-ether amide in a 24-membered macrolactam core, with two aminosugars. Bcl-xL-overexpressing Ms-1 cells displayed resistance to several anti-tumor agents; however, anti-tumor agent-induced cell death was observed only when cells were treated with incednine. Overexpression of Bcl-xL inhibited cell death in Bax-overexpressing HEK293T cells by forming a complex with Bax, whereas Bcl-xL failed to inhibit cell death in the presence of incednine without affecting the heterodimerization of Bcl-xL and Bax. These findings suggest that incednine serves as a potent modulator of the anti-apoptotic Bcl-2/Bcl-xL distinct from the other known Bcl-2 inhibitors and could provide a chemical probe to study the underlying mechanisms of Bcl-2/Bcl-xL. Copyright © 2008 American Chemical Society.

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