Yushi Futamura

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

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.

Morphobase, an Encyclopedic Cell Morphology Database, and Its Use for Drug Target Identification

Visual observation is a powerful approach for screening bioactive compounds that can facilitate the discovery of attractive druggable targets following their chemicobiological validation. So far, many high-content approaches, using sophisticated imaging technology and bioinformatics, have been developed. In our study, we aimed to develop a simpler method that focuses on intact cell images because we found that dynamic changes in morphology are informative, often reflecting the mechanism of action of a drug. Here, we constructed a chemical-genetic phenotype profiling system, based on the high-content cell morphology database Morphobase. This database compiles the phenotypes of cancer cell lines that are induced by hundreds of reference compounds, wherein those of well-characterized anticancer drugs are classified by mode of action. Furthermore, we demonstrate the applicability of this system in identifying NPD6689, NPD8617, and NPD8969 as tubulin inhibitors.

Vipirinin, a Coumarin-based HIV-1 Vpr Inhibitor, Interacts with a Hydrophobic Region of VPR

The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) is an accessory protein that has been shown to have multiple roles in HIV-1 pathogenesis. By screening chemical libraries in the RIKEN Natural Products Depository, we identified a 3-phenyl coumarin-based compound that inhibited the cell cycle arrest activity of Vpr in yeast and Vpr-dependent viral infection of human macrophages. We determined its minimal pharmacophore through a structure-activity relationship study and produced more potent derivatives. We detected direct binding, and by assaying a panel of Vpr mutants, we found the hydrophobic region about residues Glu-25 and Gln-65 to be potentially involved in the binding of the inhibitor. Our findings exposed a targeting site on Vpr and delineated a convenient approach to explore other targeting sites on the protein using small molecule inhibitors as bioprobes.

Trierixin, a Novel Inhibitor of ER Stress-induced XBP1 Activation from Streptomyces sp. : I. Taxonomy, Fermentation, Isolation, and Biological Activities

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, trierixin, from a culture broth of Streptomyces sp. AC 654. Trierixin was purified by column chromatography on silica gel and by HPLC. The molecular formula of trierixin is C37H52N2O8S. Trierixin inhibited thapsigargin-induced XBP1-luciferase activation in HeLa/XBP1-luc cells and endogenous XBP1 splicing in HeLa cells with an IC50 of 14 ng/ml and 19 ng/ml, respectively. Moreover, in the process of isolating trierixin, we isolated structurally related mycotrienin II and trienomycin A as inhibitors of ER stress-induced XBP1 activation from a culture broth of a trierixin-producing strain. This study provides the first observation that triene-ansamycins have a novel inhibitory effect against XBP1 activation.

Discovery of Incednine as a Potent Modulator of the Anti-apoptotic Function of Bcl-xL from Microbial Origin

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-overexpress...

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

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. (Cancer Sci 2009)

Trierixin, a Novel Inhibitor of ER Stress-induced XBP1 Activation from Streptomyces sp. : II. Structure Elucidation

Trierixin, a new member of the triene-ansamycin group, has been isolated from the fermentation broth of Streptomyces sp. AC654 as an inhibitor of ER stress-induced XBP1 activation. The structure of trierixin was determined on the basis of its spectroscopical and chemical properties. Trierixin possessed a 21-membered macrocyclic lactam, which contains a methylthio-benzenediol structure, and a cyclohexanecarbonylalanine moiety. Trierixin is thus elucidated as 21-thiomethylmycotrienin II.

Pyrrolizilactone, a new pyrrolizidinone metabolite produced by a fungus

In the course of our screening program to find structurally unique metabolites from microorganisms on the basis of spectral data collected through LC/MS analysis, a new pyrrolizidinone metabolite, pyrrolizilactone (1) (Figure 1), was discovered and isolated from an uncharacterized fungus. The structure of 1 was determined from spectroscopic results. Compound 1 showed moderate cytotoxic activity against HL-60 and HeLa cells. Microorganisms have a tremendous capacity for producing structurally diverse metabolites, which show various activities.1 They are important sources of pharmaceutical leads and therapeutic agents,2,3 and are also used as bioprobes in chemical biology for the exploration of biological functions.4,5 To search for and discover such structurally unique metabolites efficiently and rapidly, we have constructed a microbial metabolite fraction library with a spectral database on the basis of photodiode array detectorattached LC/MS analysis.6,7 Through our methodology for the construction of this fraction library, we discovered and identified a 16-membered macrolactam with an unusual b-keto-amide moiety, verticilactam,8 6,6-spiroacetal polyketide, spirotoamides, A and B,9 the new fraquinocins, I and J,10 and 6-dimethylallylindole-3carbaldehyde.11 These results demonstrate the advantage of the fraction library in isolating novel metabolites from natural sources. We report herein the isolation of a novel compound from a fungi fraction library. An 18-liter culture broth of an uncharacterized fungus was cultivated to obtain 16.1 g of an ethyl acetate-soluble extract. This was separated into eight fractions through a silica-gel column chromatography, with a stepwise gradient of CHCl3/MeOH. The second fraction eluted with CHCl3/MeOH (100:1) was further separated by chromatography using a Sephadex LH-20 column with CHCl3/MeOH (1:1) to afford three fractions. The second fraction, showing an unidentified peak in LC/MS analysis, was purified by C18HPLC to afford a colorless amorphous solid (1, 5.5 mg). Colorless amorphous; [a]589þ 5.91 (c 0.08, MeOH); UV (MeOH) lmax (log e) 209 (3.34), 236 (3.01) nm; IR (ATR) nmax (cm 1) 3410, 2920, 2875, 1790, 1715, 1685, 1575, 1450, 1375, 1335, 1280, 1160, 1110, 1020; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z: 416.2433 [MþH]þ (calcd for C24H34NO5: 416.2437). Compound 1 had the molecular formula C24H33NO5, as determined by HRESIMS. The IR spectrum implied the presence of hydroxyl (3410 cm 1) and carbonyl (1685, 1715 and 1790 cm 1) groups. The 1H NMR spectrum showed five methyl signals, which included a singlet, three doublets and a doublet of doublet (1.71 p.p.m., dd, J1⁄4 0.9, 0.9 Hz) branched at an sp2 carbon, and an olefin signal (5.12 p.p.m., br s), suggesting that 1 contained a double bond (Supplementary Figure S1). The 13C NMR spectrum showed 24 signals including five methyls, four methylenes, nine methines (including oxygenated and olefin carbons: 81.6 and 131.3 p.p.m.),

Identification of a molecular target of a novel fungal metabolite, pyrrolizilactone, by phenotypic profiling systems.

In the course of screening our microbial metabolite fraction library, we identified a novel pyrrolizidinone compound, pyrrolizilactone. In this study, we report the identification and characterization of a molecular target for pyrrolizilactone by using two phenotypic profiling systems. Cell morphology‐based profiling analysis using an imaging cytometer (MorphoBase) classified pyrrolizilactone as a proteasome inhibitor. Consistently, proteome‐based profiling analysis using 2D difference gel electrophoresis (DIGE; ChemProteoBase) also demonstrated that pyrrolizilactone is associated with proteasome inhibition. On the basis of these predictions, we determined that pyrrolizilactone is a novel type of proteasome inhibitor inhibiting the trypsin‐like activity of the proteasome.

High-throughput screening identifies artesunate as selective inhibitor of cancer stemness: Involvement of mitochondrial metabolism

Cancer stem cells (CSCs) have robust systems to maintain cancer stemness and drug resistance. Thus, targeting such robust systems instead of focusing on individual signaling pathways should be the approach allowing the identification of selective CSC inhibitors. Here, we used the alkaline phosphatase (ALP) assay to identify inhibitors for cancer stemness in induced cancer stem-like (iCSCL) cells. We screened several compounds from natural product chemical library and evaluated hit compounds for their efficacy on cancer stemness in iCSCL tumorspheres. We identified artesunate, an antimalarial drug, as a selective inhibitor of cancer stemness. Artesunate induced mitochondrial dysfunction that selectively inhibited cancer stemness of iCSCL cells, indicating an essential role of mitochondrial metabolism in cancer stemness.