Natural Compounds with BMI1 Promoter Inhibitory Activity from Mammea siamensis and Andrographis paniculata.

A new coumarin derivative (1) and 30 known compounds were isolated from Mammea siamensis and Andrographis paniculata, guided by B cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) promoter inhibitory activity. Among the isolated compounds, 15 compounds showed BMI1 promoter inhibitory activity, and five compounds were found to be cytotoxic. 14-Deoxy-11,12-dehydroandrographolide (18) was highly cytotoxic to DU145 cells with an IC50 value of 25.4 µM. Western blotting analysis of compound 18 in DU145 cells suggested that compound 18 suppresses BMI1 expression.

Isolation of Various Flavonoids with TRAIL Resistance-Overcoming Activity from Blumea lacera.

Eighteen compounds, including fourteen flavonoids (1-14), one steroid (15), two fatty acids (16,17), and one nitrogen-containing compound (18), were isolated from the methanol extract of the whole Blumea lacera plant collected in Thailand. Compounds 1-11 and 15-17 exhibited tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance-overcoming activity. Among them, bonanzin (2) and cirsilineol (7) had particularly strong TRAIL resistance-overcoming activity, where the IC50 values against the human gastric adenocarcinoma cell line AGS in the presence of TRAIL (100 ng/mL) were 10.7 µM and 5.9 µM, respectively.

Evaluation of Naturally Occurring HIF-1 Inhibitors for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a rare and severe progressive disorder characterized by high pulmonary artery pressure. Chronic hypoxia causes a metabolic disorder and the Warburg effect in pulmonary arterial smooth muscle cells (PASMCs). Pyruvate dehydrogenase kinase 1 (PDK1) is a key enzyme in Warburg effect increased by hypoxia-inducible factor (HIF-1). We constructed a cell-based luciferase assay system for HIF-1 inhibitors. Using this system, six HIF-1 inhibitors were identified. Among these inhibitors, the effect of tagitinin C (1) on PASMC was investigated. Tagitinin C (1) clearly decreased the amount of HIF-1ß and the HIF-1 target PDK1. This result indicates that HIF-1 inhibitors effectively decrease PDK1 activity, which is a cause of the metabolic disorder and Warburg effect observed in PASMCs. Identifying naturally occurring HIF-1 inhibitors could provide novel insights into the development of PAH medications.

Identification of BMI1 promoter inhibitors from Streptomyces sp. IFM-11958.

B-cell-specific Moloney murine leukemia virus region 1 (BMI1) is a central component of polycomb repressive complex 1 (PRC1), which maintains epigenetic repression of genes expression via chromatin condensation. BMI1 overexpression downregulates the expression of tumor suppressor genes, such as p16Ink4a and PTEN. BMI1 expression is upregulated in cancer stem cells (CSCs). Therefore, inhibitors of BMI1 expression have potential as therapeutic agents for cancer. This study aimed to identify BMI1 promoter inhibitors from actinomycetes. Using a recently constructed BMI1 promoter assay, we isolated three known compounds, elaiophylin (1), 2-methylelaiophylin (2), and nocardamin (3), from Streptomyces sp. IFM-11958 that inhibited BMI1 promoter activity with IC50 values of 30 nM, 447 nM, 22 µM, respectively. Elaiophylin (1) was the most potent. Western blot and PCR analyses revealed that elaiophylin (1) inhibited BMI1 expression at the mRNA level in human prostate cancer cells (DU145). Elaiophylin (1) also inhibited the sphere-forming activity of human hepatocellular carcinoma cells (Huh7), indicating that elaiophylin (1) suppresses the self-renewal capacity of CSCs. Elaiophylin (1) is the first BMI1 promoter inhibitor isolated from actinomycete metabolites.

Two new coumarins and a new xanthone from the leaves of Rhizophora mucronata.

Two new coumarins (1, 2) and a new xanthone (3), together with 14 known compounds-eight coumarins (4, 5, 9, 10, 12-15), three xanthones (11, 16, 17), a benzoic acid (6) and two flavonones (7, 8)-were isolated from the leaves of Rhizophora mucronata. The structures of the compounds were elucidated by spectroscopic (IR, MS, and NMR) analyses. The isolated compounds were tested for cytotoxicity against human cancer cell lines HL-60 and HeLa. Among these compounds, only compound 16 inhibited the growth of both HeLa (IC50 = 4.8 µM) and HL-60 (IC50 = 1.0 µM) cells. Compounds 4, 7, 10, and 12 exhibited moderate activity against HeLa cells (IC50 = 3.8-8.3 µM). Compounds 5, 9, 11, and 17 showed moderate activity against HL-60 cells (IC50 = 2.2-6.3 µM). Higher selectivity against HL-60 cell lines was observed for compounds 5, 9, 11, and 16 with SI values (NIH 3T3/HL-60) of 8.6, 19.2, 9.4, and 10.2, respectively.

The Notch Inhibitors Isolated from Nerium indicum.

Notch signaling plays a crucial role in differentiation and cell maintenance, but once aberrantly activated, it contributes to cancer progression. Notch inhibitors were isolated from plant extracts and tested using an originally constructed cell-based assay system. We isolated eight compounds from Nerium indicum that showed inhibition of the Notch signaling pathway. HES1 and HES5 are target genes of the Notch signaling pathway, and oleandrin (1) decreased the protein levels of HES1 and HES5 in assay cells. Oleandrin (1) showed potent cytotoxicity against HPB-ALL cells and decreased HES1 and the Notch intracellular domain in these cells. The main mechanism of action of 1 appears to be inhibition of Notch signaling by acceleration of Notch intracellular domain degradation.

Synthesis and Evaluation of Fuligocandin B Derivatives with Activity for Overcoming TRAIL Resistance.

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).

Isolation of Nabscessin C from Nocardia abscessus IFM 10029T and a Study on Biosynthetic Pathway for Nabscessins.

A new aminocyclitol derivative, designated nabscessin C (1), was isolated from Nocardia abscessus IFM 10029T. Nabcessin C is an isomer of nabscessins A (2) and B (3) with different positioning of the acyl group. Absolute configuration of nabscessin A was determined by conversion into the 2-deoxy-scyllo-inosamine pentaacetyl derivative (4) by hydrolysis and acetylation of 2. The biosynthetic pathway of nabscessins is proposed based on gene expression analysis.

Total synthesis of agalloside, isolated from Aquilaria agallocha, by the 5-O-glycosylation of flavan.

Agalloside (1) is a neural stem cell differentiation activator isolated from Aquilaria agallocha by our group using Hes1 immobilized beads. We conducted the first total synthesis of agalloside (1) via the 5-O-glycosylation of flavan 25 using glycosyl fluoride 20 in the presence of BF3·Et2O. Subsequent oxidation with DDQ to flavanone 2 and deprotection successively provided agalloside (1). This synthetic strategy holds promise for use in the synthesis of 5-O-glycosylated flavonoids. The synthesized agalloside (1) accelerated neural stem cell differentiation, which is a result comparable to that for the naturally occurring compound 1.

Nabscessins A and B, Aminocyclitol Derivatives from Nocardia abscessus IFM 10029T.

Two new aminocyclitol amide derivatives, nabscessins A (1) and B (2), were isolated from the culture broth of a pathogenic actinomycete species, Nocardia abscessus IFM 10029T. The structures of nabscessins A and B were elucidated by spectral studies, and the compounds showed antifungal activity against Cryptococcus neoformans, with IC50 values of 32 and 16 µg/mL, respectively.

Hes1-Binding Compounds Isolated by Target Protein Oriented Natural Products Isolation (TPO-NAPI).

Hairy and enhancer of split 1 (Hes1) is a transcription factor that acts in neural stem cells to inhibit differentiation. We recently developed target protein oriented natural products isolation (TPO-NAPI) using Hes1-immobilized beads to identify activators of neural stem cells. Isomicromonolactam (1), staurosporin (2), and linarin (3) were isolated as Hes1-binding compounds using the TPO-NAPI method. Of these, compound 1 enhanced neural stem cell differentiation. Using truncated Hes1 proteins, the binding region of Hes1 for 1 was estimated to be in the C-terminal half that includes a TLE/Grg binding site. The differentiation-promoting activity of inohanamine (4) is also reported.

Identification of BMI1 Promoter Inhibitors from Beaumontia murtonii and Eugenia operculata.

B-Cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) is a core component of the polycomb repressive complex 1 (PRC1). Abnormal expression of BMI1 is associated with a number of human malignances and cancer stem cells (CSCs), which cause chemotherapy resistance. Therefore, small molecules that inhibit BMI1 expression are potential candidates for cancer therapy. In this study, a cell-based reporter gene assay was developed that allowed BMI1 promoter activity to be measured in 293T human embryonic kidney cells based on luciferase expression levels. Using this screening assay, the methanol-soluble extracts of Beaumontia murtonii and Eugenia operculata were selected as leads. Bioassay-guided fractionation of the extracts led to the isolation of three known cardenolides (1-3) and one new compound (4) from B. murtonii and two known triterpenoids (5 and 6) and one new compound (7) from E. operculata. These seven compounds inhibited BMI1 promoter activity (IC50 range 0.093-23.0 µM), and the most active compound, wallichoside (1), was further evaluated. Western blot analysis revealed that wallichoside (1) decreases BMI1 protein levels in HCT116 human colon carcinoma cells, and flow cytometry analysis showed that it significantly reduced levels of the CSC biomarker epithelial cell adhesion molecule. Wallichoside (1) also inhibited sphere formation of Huh7 human hepatocellular carcinoma cells, indicating that it diminished the self-renewal capability of CSCs.

Notch Inhibitors from Calotropis gigantea That Induce Neuronal Differentiation of Neural Stem Cells.

Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease occur due to loss of the structure and function of neurons. For the potential treatment of neurodegenerative diseases, accelerators of neuronal differentiation of neural stem cells (NSCs) have been focused on and a cell-based assay system for measuring Notch signaling pathway activity was constructed. Using this assay system, eight compounds isolated from Calotropis gigantea were identified as inhibitors of the Notch signaling pathway. Hes1 and Hes5 are target genes of the Notch signaling pathway, and compound 1, called uscharin, decreased the protein levels of Hes1 and Hes5 in assay cells and MEB5 cells (mouse NSCs). Furthermore, uscharin (1) enhanced the differentiation of MEB5 cells into neurons. The mechanism of uscharin (1) for the Notch signaling inhibitory activity would be acceleration of the degradation of the Notch intracellular domain (NICD) in the MEB5 cells.

Sucupiranins A-L, Furanocassane Diterpenoids from the Seeds of Bowdichia virgilioides.

Twelve new furanocassane diterpenoids, sucupiranins A-L (1-12), and three known compounds (13-15) were isolated from the seeds of Bowdichia virgilioides. The structures of the compounds were elucidated via 1H and 13C NMR analysis, including 2D NMR (1H-1H COSY, HSQC, HMBC, and NOESY); HRMS data; and X-ray crystallographic analysis. The absolute configurations were defined using their electronic circular dichroism (ECD) spectra by applying the exciton chirality method to the bis-p-bromobenzoate of compound 13. Sucupiranin J (10) inhibited lipopolysaccharide-induced nitric oxide production (IC50 30.6 µM), whereas sucupiranins J (10), K (11), and 13 exhibited weak antimalarial activity against Plasmodium falciparum K1 with IC50 values of 32.2, 23.5, and 22.9 µM and selectivity indices of 4.3, 1.9, and >12.0 (MRC-5/K1), respectively.

Efficient Synthesis of Heterocyclic Flavonoids with Hedgehog Signal Inhibitory Activity.

The hedgehog (Hh) signaling pathway performs important roles in embryonic development and cellular proliferation and differentiation. However, in many cancer cells Hh signaling is aberrantly activated, which has provided a strong impetus for the development of Hh pathway inhibitors. To address this, we synthesized a series of heterocyclic flavonoids and evaluated their Hh signaling inhibitory activity on cancer cell lines using our cell-based assay system. Of the synthetic flavonoids, compounds 4a and g showed good inhibitory activity (IC50 was 16.8 and 21.8 µM, respectively), and were cytotoxic toward human pancreatic (PANC1) and prostate (DU145) cancer cells in which Hh signaling was activated. Compounds 4a and g had moderate selectivity against PANC1 cells. Western blotting analyses revealed that PTCH and GLI1 expression was reduced after treatment with these compounds. Overall, these synthetic flavonoids represent promising new additions to our expanding panel of Hh pathway inhibitors, and with further development these molecules may ultimately be considered for clinical use.

Synthesis of rocaglamide derivatives and evaluation of their Wnt signal inhibitory activities.

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.

Stereochemical Assignment and Biological Evaluation of BE-14106 Unveils the Importance of One Acetate Unit for the Antifungal Activity of Polyene Macrolactams.

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 O2-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 µM (0.21 µg/mL), being 4 times less potent than heronamide C, which revealed the importance of the structure of the hydrocarbon tail for the activity.

Boesenberols, Pimarane Diterpenes with TRAIL-Resistance-Overcoming Activity from Boesenbergia pandurata.

TRAIL is a potent and selective inducer of apoptosis in most cancer cells while sparing normal cells, which makes it an attractive target for the development of new cancer therapies. In a screening program on natural resources with the ability to abrogate TRAIL resistance, the bioassay-guided fractionation of Boesenbergia pandurata rhizomes resulted in the isolation of 17 pimarane diterpenes and a monoterpene. Among these, compounds 1-8, named boesenberols A-H, are new pimarane diterpenes. All compounds exhibited TRAIL-resistance-overcoming activity in TRAIL-resistant AGS cells. Subtoxic doses of the major compound 9 sensitized AGS cells to TRAIL-induced apoptosis by up-regulating apoptosis-inducing proteins, such as DR4, DR5, p53, Fas, CHOP, Bak, and cleaved caspases-3, -8, and -9, and down-regulating the levels of cell survival proteins, such as Bcl-2, c-FLIP, and GSK-3ß, in TRAIL-resistant AGS cells. Furthermore, compound 9 did not decrease the viability of noncancerous (HEK293) cells at concentrations up to 30 µM.

Cerasoidine, a Bis-aporphine Alkaloid Isolated from Polyalthia cerasoides during Screening for Wnt Signal Inhibitors.

A new bis-aporphine alkaloid, cerasoidine (1), was isolated from the root extract of Polyalthia cerasoides together with the known bis-aporphine bidebiline E (2) during screening for compounds with Wnt signal inhibitory activities. The structure of cerasoidine (1) was established by X-ray analysis and shown by chiral HPLC analyses and electronic circular dichroism to be a 57:43 mixture of R(-)- and S(+)-atropisomers. Bidebiline E (2) exhibited inhibition of transcriptional activity of TCF/ß-catenin with an IC50 value of 20.2 µM and was also found to inhibit Wnt signaling by decreasing nuclear ß-catenin.

Bio-active Natural Products with TRAIL-Resistance Overcoming Activity.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent as it selectively kills cancer cells. However, TRAIL resistance limits its use as a therapeutic agent. An understanding the mechanisms responsible for TRAIL resistance and strategies to overcome it are important for its effective use as an anticancer agent. During our studies to screen natural products from medicinal plants, we identified a number of compounds with synergistic effects on TRAIL-induced apoptosis in tumor cells. This review describes our recent studies on the isolation of bioactive compounds with TRAIL-resistance overcoming activity.