Published papers

In the present study, we developed a high-throughput screening system for small molecule-inhibitors of the basic helix-loop-helix (bHLH) transcriptional repressor factor Hes1. Successful dimerization of Hes1 immobilized on a microplate and fluorophore (Cy3)-labelled Hes1 was confirmed. Using this system, several natural products were identified as the first Hes1 dimer inhibitors. Of these, two compounds which were isolated from myxomycetes (true slime molds) inhibited Hes1 from N box-dependent suppression of the gene expression in C3H10T1/2 cells. (C) 2009 Published by Elsevier Ltd.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling pathway induces apoptosis in cancer cells but not in normal cells. Therefore, this pathway has attracted attention regarding possible clinical treatment of cancer. However, many cancer cells demonstrate TRAIL resistance. To overcome this problem, small molecules that sensitize cancer cells to TRAIL are desired. Heterocyclic derivatives of the natural product, fuligocandin B (2), with activity for overcoming TRAIL resistance were synthesized, and their activity was evaluated. Of the synthetic molecules, the quinoline derivative (10g) showed potent activity against TRAIL-resistant gastric adenocarcinoma cells. After a docking study of the target protein valosin-containing protein, 7'-amino fuligocandin B (10m) was designed and synthesized. Compound 10m also showed good activity for overcoming TRAIL resistance. 10m produced a 49.7% difference in viability with TRAIL at 30 µM compared to without TRAIL. This activity was better than that of fuligocandin B (2).

Wnt signaling plays important roles in proliferation, differentiation, development of cells, and various diseases. Activity-guided fractionation of the MeOH extract of the Ricinus communis stem led to the isolation of four compounds (1-4). The TCF/beta-catenin transcription activities of 1 and 3 were 2.2 and 2.5 fold higher at 20 and 30 mu M, respectively. Cells treated with ricinine (1) had higher beta-catenin and lower of p-beta-catenin (ser 33, 37, 45, Thr 41) protein levels, whereas glycogen synthase kinase 3 beta (GSK3 beta) and casein kinase 1 alpha (CK1 alpha) protein levels remained unchanged. Cells treated with pyrvinium, an activator of CK1 alpha, had lower beta-catenin levels. However, the combined treatment of pyrvinium and 1 led to higher beta-catenin levels than those in cells treated with pyrvinium alone, which suggested that 1 inhibited CK1 alpha activity. Furthermore, 1 increased beta-catenin protein levels in zebrafish embryos. These results indicated that 1 activated the Wnt signaling pathway by inhibiting CK1 alpha. (C) 2014 Elsevier Ltd. All rights reserved.

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased beta-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of beta-catenin induced by 1. The 1-induced degradation of beta-catenin was also abrogated in the presence of pifithrin-alpha, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of beta-catenin, which resulted in the inhibition of TCF/beta-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

During our studies related to the search for bioactive natural products, we recently examined extracts of various natural resources including unexplored myxomycetes, marine organisms, as well as several medicinal plants collected in the north-eastern part of Thailand and Bangladesh. Here we describe our recent results from our screening programs targeting signaling molecules in cancer-related biological pathways such as TRAIL, Writ, and Hedgehog signalings.

The ability of TRAIL to selectively induce apoptosis in cancer cells while sparing normal cells makes it an attractive target for the development of new cancer therapy. In search of bioactive natural products for overcoming TRAIL-resistance from natural resources, we previously reported a number of active compounds. In our screening program on natural resources targeting overcoming TRAIL-resistance, activity-guided fractionations of the extract of Xanthium strumarium led to the isolation of five sesquiterpene compounds (1-5). 11 alpha, 13-dihydroxanthinin (2) and 11 alpha, 13-dihydroxanthuminol (3) were first isolated from natural resources and xanthinosin (1), desacetylxanthanol (4), and lasidiol p-methoxybenzoate (5) were known compounds. All compounds (1-5) showed potent TRAIL-resistance overcoming activity at 8, 20, 20, 16, and 16 mu M, respectively, in TRAIL-resistant AGS cells. Compounds 1 and 5 enhanced the levels of apoptosis inducing proteins DR4, DR5, p53, CHOP, Bax, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 and also decreased the levels of cell survival protein Bcl-2 in TRAIL-resistant AGS cells in a dose-dependent manner. Compound 1 also enhanced the levels of DR4 and DR5 proteins in a time-dependent manner. Thus, compounds 1 and 5 were found to induce both extrinsic and intrinsic apoptotic cell death. Compound 1 also exhibit TRAIL-resistance overcoming activity in DLD1, DU145, HeLa, and MCF7 cells but did not decrease viability in non-cancer HEK293 cells up to 8 mu M. (C) 2015 Elsevier Ltd. All rights reserved.

Heronamides are a class of potent antifungal metabolites produced by marine-derived actinomycetes. The number of hydroxy groups and the stereochemistry of the two hydroxylated methine carbons are important for the activity of heronamide C, whereas the effect of the hydrocarbon chains is not known. In this study, the stereochemistry and the biological activity of BE-14106, another member of the heronamide class of antibiotics, isolated from an actinomycete Actinoalloteichus cyanogriseus IFM 11549 was investigated. Spectroscopic analysis coupled with photo- and O-2-induced conversion revealed that BE-14106 and the heronamides had the same stereochemistry. BE-14106 showed potent growth inhibition against fission yeast cells with an MIC value of 0.50 mu M (0.21 mu g/mL), being 4 times less potent than heronamide C, which revealed the importance of the structure of the hydrocarbon tail for the activity.

Two derivatives of 14-epi-1 alpha,25-Dihydroxy-19-norvitamin D-3, 14-epi-2 alpha-(3-hydroxypropyl)-1 alpha,25-dihydroxy-19-norvitamin D-3 (14-epi-MART-10) and its 2-epimeric analog (14-epi-MART-11), were synthesized using Julia coupling reaction to connect between the C5 position (steroidal numbering) of an A-ring precursor ketone derived from (-)-quinic acid and the C6 position of a protected 14-epi-CD-ring benzothiazole sulfone. The coupling and deprotection reactions generated a mixture of 14-epi-MART-10 and 14-epi-MART-11 in a moderate yield. The C2-isomers were then separated as their pivalate forms. The C2-stereochemistry of 2 alpha- and 2 beta-isomers was determined by H-1 NMR studies including NOE experiments. The pivaloyl group was removed under basic conditions to obtain the target molecules of 14-epi-MART-10 and 14-epi-MART-11. The metabolism of these two new analogs was further studied in a reconstituted cell-free human CYP24A1 system to elucidate the potential mechanism of their super agonistic action on vitamin D receptor. Our results indicate that epimerization at C14 makes the analogs less susceptible to CYP24A1 degradation and therefore more bio-available, leading to enhanced biological activities.

The 14-epimer of MART-10, namely 14-epi-MART-10 (14-epi-2 alpha-(3-hydroxypropyl)-1 alpha,25-dihydroxy-19-norvitamin D(3)) and its 2-epimeric analog (14-epi-MART-11) were efficiently synthesized using the Julia coupling reaction to connect between the C5 and C6 positions (steroid numbering). An A-ring precursor was prepared from (-)-quinic acid as shown in the previous MART-10 synthesis. The novel 14-epi-CD-ring coupling partner with an elongated two carbon unit as a sulfone was synthesized from 14-epi-25-hydroxy Grundmann's ketone in good yield. The subsequent coupling reaction followed by a deprotection step afforded a mixture of 14-epi-MART-10 and 14-epi-MART-11 in 40% yield. To separate 14-epi-MART-10 and 14-epi-MART-11, each primary hydroxyl group was esterified with a pivaloyl group and the resulting pivalates 2 alpha and 2 beta were separated by high performance liquid chromatography. After the separation, the C2-stereochemistry of each (2 alpha or 2 beta) was determined by (1)H NMR (nuclear magnetic resonance) studies including NOE (nuclear Overhauser effect) experiments. The pivaloyl group was removed under basic conditions to obtain the target molecules of 14-epi-MART-10 and 14-epi-MART-11, respectively. The VDR (vitamin D receptor)-binding affinity, HL-60 (human promyelocytic leukemia) cell differentiation activity, antiproliferative activity in PZ-HPV-7 (immortalized normal prostate) cells and transactivation activity of the osteocalcin promoter in HOS (human osteoblast cell line) cells (serum-free conditions) were investigated. In addition, the effects on bone mineral density (BMD) and the blood and urine calcium concentrations of ovariectomized (OVX) rats were examined. 14-epi-MART-10 has much greater antiproliferative and cell differentiation activities compared to 1 alpha,25-dihydroxyvitamin D(3) (1 alpha,25(OH)(2)D(3)).

Two stereoisomers of melleumin B (1), seco acid methyl ester of melleumin A (2), which are novel peptide compounds isolated from the myxomycete Physarum melleum, were synthesized and evaluated for their inhibition of Wnt signal transcription. The 3R-epimer and (3R, 4S, 10R, 11R)-isomer of 1 showed moderate inhibition of the Wnt signal.

Vitamin D-3 analogues have been developed for a mutant vitamin D receptor (VDR), Arg274Leu. The mutant VDR has a Mutation at Arg274, which forms an important hydrogen bond with 1 alpha-OH of 1 alpha,25-dihydroxyvitamin D3 to anchor the ligand tightly in the VDR ligand binding pocket. Stereoselective synthesis of the A-ring part of the novel vitamin D analogue, 2 alpha-(3-hydroxypropyl)-1 alpha-methyl-25-hydroxyvitamin D3 (4), from D-galactose was accomplished with the key steps of the introduction of the methyl and allyl groups to the chiral building blocks. The new analogue 4 is ca. 7.3-fold more active than the natural hormone 1 alpha,25-dihydroxyvitamin D-3 (1). (C) 2009 Elsevier Ltd. All rights reserved,

Rocaglamides are bioactive natural compounds which have a cyclopenta[b]benzofuran core structure. The total synthesis of a reported natural product, 3'-hydroxymethylrocaglate (5), was achieved using [3 + 2] cycloaddition between 3-hydroxyflavone and methyl cinnamate. We also describe the synthesis of rocaglamide heterocycle derivatives and evaluate their Wnt signal inhibitory activities. Compounds 4, 5, 22a, 22b, 22c and 23c showed potent Wnt signal inhibitory activity.

To determine the stereochemistry of dihydroarcyriarubin C (1), new bisindole alkaloid isolated from the myxomycete Arcyriajerruginea, cis- (2) and trans-dihydroarcyriarubin C (3) were synthesized. Comparison of their NMR characteristics allowed the trans stereochemistry of the natural product to be confirmed. Moreover, the Writ signal inhibitory activities of 2 and 3 were compared with that of arcyriaflavin C (4), which is a natural product containing a bond between C-2 and C-T. The cis-dihydroar-cyriarubin C (2) showed moderate inhibition of Wnt signal transcription, which suggests that bisindole frameworks might be useful as small-molecule Writ signal inhibitors. (c) 2007 Elsevier Ltd. All rights reserved.

In the courses of our search for bioactive natural products on TRAIL resistance-overcoming activity, the active EtOAc extract from the bark of Celtis tetrandra was chemically investigated. The fractionation of the extract resulted in the isolation of flavanols; (+)-afzelechin (1), (-)-epiafzelechin (2), (-)-catechin (3), and two new flavanol dimers; (-)-epiafzelechin-(4α8)-(-)-catechin (4) and (-)-epiafzelechin-(4α 8)-(-)-epicatechin (5). All isolated compounds 1-5 were tested for their TRAIL resistance-overcoming activity against AGS cells at the concentrations from 5-20 µM. It is likely that the flavanol dimers 4 and 5 exhibited weak activity in the range of the concentration of 10-20 µM as compared with the standard.

© The Author(s) 2019 Upon screening for compounds with B cell-specific Moloney murine leukemia virus region 1 (BMI1) promoter inhibitory activity, 3 nonactic acid derivatives (1-3) were isolated from Streptomyces werraensis IFM12104. Although spectral data of compound 1 were identical with those reported in literature for 4α-(3, 5-dihydroxy-heptyl)-3α-methyl-2-oxetanone (4), compound 1 was revealed to be homononactic acid by X-ray analysis. Compounds 2 and 3 were identified as bishomononactic acid and nonactic acid using spectral data. The 3 compounds 1 to 3 inhibited BMI1 promoter activities with IC50 values of 240, 160, and 180 μM, respectively.

A stereoselective synthetic route to chiral C3-spirooxindole- and C2-spiropseudoindoxyl-pyrrolidines was accomplished by an enantioselective organocatalyzed Pictet-Spengler reaction of tryptamines and isotryptamines followed by a diastereoselective oxidative rearrangement using eco-friendly oxidants (i. e., NaOCl ⋅ 5H2O and Oxone®). This sequential reaction enables rapid access to chiral C3-spirooxindole- and C2-spiropseudoindoxyl-pyrrolidines in a one-pot process. A Wnt signaling inhibitory assay of the prepared enantioenriched spiro compounds demonstrated that they exhibited moderate activities. (Figure presented.).

The thermotolerant strain Streptomyces sp. HR41 was found to produce compound 1 only in a 45 °C culture, and not at the standard temperature. We previously designated this type of compound as a “heat shock metabolite” (HSM). NMR and MS analytical techniques were used to determine that the chemical structure of 1 comprised a methylated-oxazole ring and a linear chain moiety modified with a terminal amide group. Thus, 1 was shown to be a new curromycin analog, which we have designated noaoxazole (1). Compound 1 weakly activated Notch signal reporter activity without exhibiting cytotoxicity against assay cells at the same concentration.

The Wnt signalling pathway is essential in many biological processes. The Wnt signal is associated with several diseases, particularly cancer and neurodegenerative diseases. Recently, high-throughput screening systems have been developed to rapidly identify compounds, including natural compounds, that target the Wnt signal. Some studies on natural modulators of the Wnt signal have also suggested their possible target. This review highlights some important natural compounds reported to regulate Wnt activity and describes their possible mechanism of action.