Misc

During our studies on a search for natural products from myxomycetes, we have isolated novel compounds, fuligocandin A and B containing cycloanthranilylproline skeleton, from the extract of fruit bodies of Fuligo candida. We studied the total synthesis of fuligocandin A, B and its derivatives. Fuligocandin B consists of three residues, anthranilic acid, L-proline, and indole-3-carboxyaldehyde. Total synthesis of fuligocandin A, B and their enantiomers and fuligocandin B derivatives were achieved.TRAIL-resistance overcoming activities of fuligocandin A, B and their enantiomers and fuligocandin B derivatives AGS cells were evaluated. Fuligocandin B and its derivatives showed effects on the TRAIL-resistance AGS cells.

An asymmetric Heck reaction-carbanion capture process was realized for the first time, making possible the catalytic asymmetric synthesis of various functionalized bicyclo[3.3.0]octane derivatives 6 in up to 94% ee. Sodium bromide had interesting effects on this asymmetric Heck reaction-carbanion capture process, and these effects were useful for improving the enantiomeric excess. Furthermore, the catalytic asymmetric synthesis of (-)-Delta(9(12))-capnellene (7) was achieved for the first time, using 6b as a key intermediate and a radical cyclization as a key step.

Wnt signaling pathway has a key roles in various cellular process such as cell proliferation and tissue maintenance. Aberrant activation of Wnt signal can initiate the development of several diseases including cancer, Alzheimer's disease, and diabetes. Using cell-based luciferase screening assay system for a Wnt signaling pathway (TCF/β-catenin transcription), activity-guided exploration of our natural resources extracts library were conducted. Detailed investigation on mode of action of isolated compounds were also carried out.Six cardenolides (1-6) were isolated from the methanol extract of Calotropis gigantea (Asclepiadaceae) exudate, and compounds 1, 3, 5, and 6 strongly inhibited TCF/β-catenin transcription activity (IC500.7-3.8 nM). Compounds 1, and 3-6showed potent cytotoxicity (IC50 1.8-7.0 nM) against Wnt dependent colon cancer cell (SW480, DLD1, HCT116), without cytotoxicity against RKO (Wnt independent colon cancer) cells. Calotropin (1) inhibited Wnt signaling by reducing both nuclear and cytosolic β-catenin levels in SW480 cells. Further investigation using inhibitors revealed that 1 potentiate the degradation of β-catenin by increasing the phosphorylation of β-catenin at Ser45 mediated by casein kinase 1α (CK1α). Moreover, 1 significantly enhanced CK1α protein and mRNA levels.Three limonoides (7-9) were isolated from methanol extract of Xylocarpus granatum (Meliaceae) leaves, including two new limonoids, named as xylogranin A (7) and B (8). Compounds 7 and 8 inhibited TCF/β-catenin transcription activity. Treatment of SW480 cells with 8 led to a significant decrease of β-catenin in nuclei but not in the cytosol, and c-myc and PPARδ, target proteins of Wnt signaling pathway. These results indicated that a decrease of β-catenin in nuclei by 8contributed the Wnt inhibition of 8.

Highly efficient screening method of naturally occurring products that bind to a specific target protein was demonstrated by using hVDR-magnetic beads. The native ligand 1α,25(OH)_2VD_3 was selectively bound by hVDR magnetic beads when present in a mixture of natural compounds. Furthermore, this method was proven to be applicable to the identification of natural products, which interact with a specific protein immoblized on the beads, from an extract of a natural resource. Two new natural compounds were isolated by this method. This approach will be helpful for the discovery of novel, neturally occurring products that bind to specific target proteins. A high-throughput screening system for small molecule-inhibitors of the basic helix-loop-helix (bHLH) transcriptional repressor factor Hes1 was construcrted. Successful demerization of Hes1 immobilized on a microplate and fluorophore (Cy3)-labeled 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.

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 the 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. A suitable protocol for the screening was then established. The screening of our natural products library with the above constructed system identified six active compounds; staurosporinone (1), 6-hydroxystaurosporinone (2), arcyriaflavin C (3), 5,6-dihydroxyarcyriaflavin A (4), zerumbone (5), and zerumbone epoxide (6). Their IC_<50> values of GLI1 transcriptional inhibitory activity were 1.8, 3.6, 11.3, 6.9, 3.0 and 55μM, respectively. The next screening of our natural plants extracts library gave 15 hit plants. Repeated chromatography separations of the MeOH extracts of Physalis minima and Zizyphus cambodiana gave active compounds. P minima gave two compounds; physalin F (10) and physalin B (11), and Z. combodiana a compound; colubrinic acid (13). Their IC_<50> values were 0.66, 0.62 and 29μM, respectively. It was reported that glil, ptchl, bcl2 are the Hh/GLI-mediated transcriptional targets. The influences of these inhibitors (1, 5, 10 and 11) on these protein expressions were investigated with western blotting analysis. These inhibitors decreased the expression of GLI1 and PTCH1, which are related Hh signaling pathway. Moreover, BCL2, which is related with apoptosis, was also decreased.

Natural products have their important meanings in biological systems. Isolation of new natural products and synthesis of small molecules based on their scaffolds would be of chemical relevance to living cells and organs. In this study, we focused on the Wnt signaling, which pathways show aberrant activation in many cancer cells. We synthesized natural products melleumin A (1) and B (2), and trans-dihydroarcyriarubin C, which were isolated by us from myxomycetes (true slime molds), and also synthesized their isomers. Melleumin A (1) and B (2) are novel peptide compounds isolated from the myxomycete Physarum melleum. Their syntheses have been achieved by coupling procedures of their components, L-threonine, glycine, and an unusual amino acid, a tyrosine-attached acetic acid. We found cis-dihydroarcyriarubin C (12), and isomers of melleumin B are moderate inhibitors of Wnt signal transcription. We also developed a high-throughput system for analyzing of inhibition of making a complex between TCF and β-catenin by using microplates. The regulation of transcription by TCF/β-catenin complex is a final step of the Wnt signal pathway. In this assay, cis-dihydroarcyriarubin C (12) showed inhibition, which indicates that a cell-based Wnt signal inhibition would be caused by disruption of TCF/β-catenin complex. We also found several natural products from our natural products library that show a cell-based Wnt signal inhibition and a protein-based TCF/β-catenin complex inhibition.