Induction of Fungal Secondary Metabolites by Co-Culture with Actinomycete Producing HDAC Inhibitor Trichostatins.

A recently bioinformatic analysis of genomic sequences of fungi indicated that fungi are able to produce more secondary metabolites than expected. Despite their potency, many biosynthetic pathways are silent in the absence of specific culture conditions or chemical cues. To access cryptic metabolism, 108 fungal strains isolated from various sites were cultured with or without Streptomyces sp. 13F051 which mainly produces trichostatin analogues, followed by comparison of metabolic profiles using LC-MS. Among the 108 fungal strains, 14 produced secondary metabolites that were not recognized or were scarcely produced in mono-cultivation. Of these two fungal strains, Myrmecridium schulzeri 15F098 and Scleroconidioma sphagnicola 15S058 produced four new compounds (1-4) along with a known compound (5), demonstrating that all four compounds were produced by physical interaction with Streptomyces sp. 13F051. Bioactivity evaluation indicated that compounds 3-5 impede migration of MDA-MB-231 breast cancer cells.

Ulleungdolin, a Polyketide-Peptide Hybrid Bearing a 2,4-Di-O-methyl-ß-d-antiarose from Streptomyces sp. 13F051 Co-cultured with Leohumicola minima 15S071.

A new secondary metabolite, ulleungdolin (1), was isolated from the co-culture of an actinomycete, Streptomyces sp. 13F051, and a fungus, Leohumicola minima 15S071. Based on the NMR, UV, and MS data, it was deduced that the planar structure of 1 comprised an isoindolinone (IsoID) with an octanoic acid, a tripeptide, and a sugar. The tripeptide has the unprecedented amino acids norcoronamic acid, 3-hydroxy-glutamine, and 4-hydroxy-phenylglycine and is linked by a C-N bond with IsoID. The absolute configurations were determined by chemical derivatization, extensive spectroscopic methods, and electronic circular dichroism calculations and supported by bioinformatic analyses. Bioactivity evaluation studies indicated that 1 had an antimigration effect on MDA-MB-231 breast cancer cells.

Jejuketomycins A and B, polyketide glycosides with cancer cell migration inhibitory activity from Streptomyces sp. KCB15JA151.

Two new polyketide glycosides jejuketomycins A (1) and B (2), were isolated from a culture of Streptomyces sp. KCB15JA151. Their chemical structures including the absolute configurations were determined by detailed analyses of the NMR and HRMS data and ECD calculations and spectral data. Compounds 1 and 2 possess an unusual 6/6/8 tricyclic ring system. Biological evaluation with the wound healing assay and time-lapse cell tracking analysis revealed that compounds 1 and 2 have significant inhibitory activities against cancer cell migration with low cytotoxicity.

Near-Infrared Fluorescence Probe for Specific Detection of Acetylcholinesterase and Imaging in Live Cells and Zebrafish.

Acetylcholinesterase (AChE) is a pivotal enzyme that is closely related with multiple neurological diseases, such as brain disorders or alterations in the neurotransmission and cancer. The development of convenient methods for imaging AChE activity in biological samples is very important to understand its mechanisms and functions in a living system. Herein, a fluorescent probe exhibiting emission in the near-infrared (NIR) region is developed to detect AChE and visualize biological AChE activities. This probe exhibits a quick response time, reasonable detection limit, and a large Stokes shift accompanied by the NIR emission. The probe has much better reactivity toward AChE than butyrylcholinesterase, which is one of the significant interfering substances. The outstanding specificity of the probe is proved by cellular imaging AChE activity and successful mapping in different regions of zebrafish. Such an effective probe can greatly contribute to ongoing efforts to design emission probes that have distinct properties to assay AChE in biological systems.

Kurarinone induced p53-independent G0/G1 cell cycle arrest by degradation of K-RAS via WDR76 in human colorectal cancer cells.

Kurarinone (KR), a naturally occurring flavonoid in Sophora flavescens Aiton and a traditional herbal medicine, reportedly has anti-cancer activity against various cancer types both in vitro and in vivo. However, the cellular mechanism of KR remains unknown. Therefore, we aimed to elucidate the mechanism of cell cycle arrest induced by KR in human colorectal cancer cells. KR not only reduced cell proliferation but also induced G0/G1 arrest of colorectal cancer cell lines. The results of western blotting analysis showed that KR reduced the protein levels of cyclin D1/D3 and CDK4/6 by downregulating signaling proteins such as K-RAS, c-MYC, and p-extracellular signal-regulated kinase. Additionally, KR arrested the cell cycle in the G0/G1 phase in a p53-independent manner, and decreased the protein level of K-RAS by proteasomal degradation dependent on WDR76, an E3 ubiquitin ligase. From these results, we propose that KR could be a potent anti-cancer agent, acting through the degradation of K-RAS dependent on WDR76, regardless of the p53 status.

New phenalenone derivatives from the Hawaiian volcanic soil-associated fungus Penicillium herquei FT729 and their inhibitory effects on indoleamine 2,3-dioxygenase 1 (IDO1).

Phenalenone derivatives sourced from fungi are polyketides that have attracted significant interest because of their diverse chemical structures and potential bioactivities. As part of our ongoing quest to discover novel natural products with biological properties from diverse natural resources, three unreported phenalenone derivatives (1-3), named ent-12-methoxyisoherqueinone (1), (-)-scleroamide (2), and (+)-scleroamide (3), together with four known phenalenone derivatives, ent-atrovenetinone (4), isoherqueinone (5), herqueinone (6), and ent-peniciherquinone (7) were isolated from the Hawaiian soil fungus Penicillium herquei FT729, collected on the Big Island, Hawaii. Compounds 2 and 3 were enantiomers, which were separated using a chiral-phase HPLC column, which provided optically pure compounds 2 and 3. The structures of the novel compounds were established by extensive spectroscopic analyses, including 1D and 2D NMR and high-resolution ESIMS. Their absolute configurations were determined using quantum chemical electronic circular dichroism (ECD) calculations. The inhibitory activity of the isolated compounds (1-7) against indoleamine 2,3-dioxygenase 1 (IDO1) was assessed. Compounds 1, 5-7 inhibited IDO1, with IC50 values of 32.59, 36.86, 19.05, and 24.18 µM, respectively. These findings demonstrated that the phenalenone derivatives 1 and 5-7, as IDO1 inhibitors, are promising anticancer immunotherapeutic agents.

RK-270D and E, Oxindole Derivatives from Streptomyces sp. with Anti-Angiogenic Activity.

A chemical investigation of a culture extract from Streptomyces sp. RK85-270 led to the isolation and characterization of two new oxindoles, RK-270D (1) and E (2). The structures of 1 and 2 were determined by analyzing spectroscopic and spectrometric data from 1D and 2D NMR and High-resolution electrospray ionization mass spectrometry (HRESIMS) experiments. Compound 1 exhibited anti-angiogenic activities against human umbilical vein endothelial cells (HUVECs) without cytotoxicity. Results of Western blot analysis revealed that 1 inhibits VEGF-induced angiogenesis in the HUVECs via VEGFR2/ p38 MAPK-mediated pathway.

Streptooctatins A and B, fusicoccane-type diterpenoids with autophagic activity from Streptomyces sp. KCB17JA11.

Two new fusicoccane-type diterpenoids, streptooctatins A (1) and B (2), together with a known compound cyclooctatin (3) were isolated from Streptomyces sp. KCB17JA11. The structures of 1 and 2 were determined by analyzing spectroscopic and spectrometric data from 1D and 2D NMR and HRESIMS experiments. Compounds 1 and 2 induced EGFP-LC3 puncta indicating autophagic activities against HeLa cells without cytotoxicity.

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling.

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-ß and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.

Thiolactomide: A New Homocysteine Thiolactone Derivative from Streptomyces sp. with Neuroprotective Activity.

A new homocysteine thiolactone derivative, thiolactomide (1), was isolated along with a known compound, N-acetyl homocysteine thiolactone (2), from a culture extract of soil-derived Streptomyces sp. RK88-1441. The structures of these compounds were elucidated by detailed NMR and MS spectroscopic analyses with literature study. In addition, biological evaluation studies revealed that compounds 1 and 2 both exert neuroprotective activity against 6-hydroxydopamine (6-OHDA)-mediated neurotoxicity by blocking the generation of hydrogen peroxide in neuroblastoma SH-SY5Y cells.

Ulleunganilines A-C, Trichostatin Analogues Bearing a Modified Side Chain from Streptomyces sp. 13F051.

Three new trichostatin analogues, ulleunganilines A-C (1-3), and seven known trichostatins (4-10) were isolated from cultures of Streptomyces sp. 13F051. NMR, UV, and MS data indicated that the planar structures of 1-3 consisted of modified side chains in the trichostatic acid moiety. The absolute configuration of the 2,4-dimethyl-branched carbon chains in 1 and 2 was determined by the PGME method, while the amino acid group in 3 was identified by advanced Marfey's method. Based on the structure of the modified side chains, the origin of 1-3 is proposed. Further experiments indicated that 1 and 3 displayed moderate histone deacetylase inhibitory activity, suggesting that not only the hydroxamate group but also the N,N-dimethyl group were essential for the inhibitory activity.

Angucyclines containing ß-á´ -glucuronic acid from Streptomyces sp. KCB15JA151.

Two angucyclines, pseudonocardones D (1) and E (2), were isolated from Streptomyces sp. KCB15JA151. The planar structure was elucidated by comprehensive spectroscopic analysis. The absolute configuration of the sugar unit was determined based on the basis of coupling constants, ROESY, chemical derivatization and HPLC analysis. The biological activities of compounds 1 and 2 were examined by performing a computational target prediction, which led to tests of the antiestrogenic activity. The result suggested that compound 1 might be an ERα antagonist.

Dutomycin Induces Autophagy and Apoptosis by Targeting the Serine Protease Inhibitor SERPINB6.

Autophagy plays an important role in maintaining tumor cell progression and survival in response to metabolic stress. Thus, the regulation of autophagy can be used as a strategy for anticancer therapy. Here, we report dutomycin (DTM) as a novel autophagy enhancer that eventually induces apoptosis due to excessive autophagy. Also, human serine protease inhibitor B6 (SERPINB6) was identified as a target protein of DTM, and its novel function which is involved in autophagy was studied for the first time. We show that DTM directly binds SERPINB6 and then activates intracellular serine proteases, resulting in autophagy induction. Inhibitory effects of DTM on the function of SERPINB6 were confirmed through enzyme- and cell-based approaches, and SERPINB6 was validated as a target protein using siRNA-mediated knockdown and an overexpression test. In a zebrafish xenograft model, DTM showed a significant decrease in tumor area. Furthermore, the present findings will be expected to contribute to the expansion of novel basic knowledge about the correlation of cancer and autophagy by promoting active further research on SERPINB6, which was not previously considered the subject of cancer biology.

Jejucarbazoles A-C, carbazole glycosides with indoleamine 2,3-dioxygenase 1 inhibitory activity from Streptomyces sp. KCB15JA151.

A bioassay-guided investigation led to the isolation of three new carbazole glycosides, jejucarbazoles A-C (1-3), from Streptomyces sp. KCB15JA151. Their planar structures were elucidated by detailed NMR and MS spectroscopic analysis with a literature study. Their relative and absolute configurations were established by ROESY correlations, coupling constants, LC-MS analysis of thiocarbamoyl-thiazolidine carboxylate derivatives, and ECD calculation. Compounds 1-3 showed indoleamine 2,3-dioxygenase 1 (IDO1) inhibitory activity with IC50 values of 18.38, 9.17, and 8.81 µM. The molecular docking analysis suggested that all compounds act as heme-displacing inhibitors against IDO1 enzyme.

A pipecolic acid-rich branched cyclic depsipeptide ulleungamide C from a Streptomyces species induces G0/G1 cell cycle arrest in promyelocytic leukemia cells.

In this study, screening by LC-MS and cytotoxicity-guided isolation led to the identification of ulleungamide C (1), a previously unknown pipecolic acid-rich branched cyclic depsipeptide, from a soil actinobacterium Streptomyces sp. KCB13F003. The structure of 1 was determined by interpretation of spectroscopic and spectrometric data from 1D and 2D NMR and HRESIMS experiments. Antiproliferative assays using mammalian cancerous cells revealed that 1 inhibits the proliferation of HL-60 human promyelocytic leukemia cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with 1. Results of immunoblotting assays revealed that 1 reduced the expression levels of cyclin-dependent kinase 4 (CDK4), CDK6, retinoblastoma protein (Rb), and phosphorylated Rb, whereas it induced cyclin-dependent kinase inhibitor 1B (p27/Kip1) expression.

Ent-Peniciherqueinone Suppresses Acetaldehyde-Induced Cytotoxicity and Oxidative Stress by Inducing ALDH and Suppressing MAPK Signaling.

Studies on ethanol-induced stress and acetaldehyde toxicity are actively being conducted, owing to an increase in alcohol consumption in modern society. In this study, ent-peniciherqueinone (EPQ) isolated from a Hawaiian volcanic soil-associated fungus Penicillium herquei FT729 was found to reduce the acetaldehyde-induced cytotoxicity and oxidative stress in PC12 cells. EPQ increased cell viability in the presence of acetaldehyde-induced cytotoxicity in PC12 cells. In addition, EPQ reduced cellular reactive oxygen species (ROS) levels and restored acetaldehyde-mediated disruption of mitochondrial membrane potential. Western blot analyses revealed that EPQ treatment increased protein levels of ROS-scavenging heme oxygenase-1 and superoxide dismutase, as well as the levels of aldehyde dehydrogenase (ALDH) 1, ALDH2, and ALDH3, under acetaldehyde-induced cellular stress. Finally, EPQ reduced acetaldehyde-induced phosphorylation of p38 and c-Jun N-terminal kinase, which are associated with ROS-induced oxidative stress. Therefore, our results demonstrated that EPQ prevents cellular oxidative stress caused by acetaldehyde and functions as a potent agent to suppress hangover symptoms and alcohol-related stress.

Herqueilenone A, a unique rearranged benzoquinone-chromanone from the Hawaiian volcanic soil-associated fungal strain Penicillium herquei FT729.

The study of a Hawaiian volcanic soil-associated fungal strain Penicillium herquei FT729 led to the isolation of one unprecedented benzoquinone-chromanone, herqueilenone A (1) and two phenalenone derivatives (2 and 3). Their structures were determined through extensive analysis of NMR spectroscopic data and gauge-including atomic orbital (GIAO) NMR chemical shifts and ECD calculations. Herqueilenone A (1) contains a chroman-4-one core flanked by a tetrahydrofuran and a benzoquinone with an acetophenone moiety. Plausible pathways for the biosynthesis of 1-3 are proposed. Compounds 2 and 3 inhibited IDO1 activity with IC50 values of 14.38 and 13.69 µM, respectively. Compounds 2 and 3 also demonstrated a protective effect against acetaldehyde-induced damage in PC-12 cells.

Highly oxygenated angucycline from Streptomyces sp. KCB15JA014.

LC/MS-based chemical screening of culture extract led to a new highly oxygenated angucycline derivative, grecocycline D (1), from Streptomyces sp. KCB15JA014, isolated from a soil sample of Oedolgae in Jeju Island, Korea. The planar structure was determined on the basis of spectroscopic analysis, including 1D and 2D NMR techniques as well as HRESIMS and comparison with data from the literature. A relative and absolute configuration of 1 was assigned by ROESY experiment and electronic circular dichroism calculation. Compound 1 showed weak inhibitory activity against indoleamine 2,3-dioxygenase.

Cytotoxic Activities and Molecular Mechanisms of the Beauvericin and Beauvericin G1 Microbial Products against Melanoma Cells.

Melanoma is the most serious type of skin cancer and remains highly drug-resistant. Therefore, the discovery of novel effective agents against melanoma is in high demand. Herein, we investigated the cytotoxic activities in melanoma cells and underlying molecular mechanisms of beauvericin (BEA) and its analogue beauvericin G1 (BEA G1), which are cyclohexadepsipeptides isolated from fungi. BEA and BEA G1 significantly suppressed the growth, clonogenicity, migration, and invasion of A375SM human melanoma cells and promoted caspase-dependent apoptosis through upregulation of death receptors, as well as modulation of pro- and anti-apoptotic Bcl-2 family members. Furthermore, the effects of BEA and BEA G1 were associated with the suppression of multiple molecular targets that play crucial roles in melanoma oncogenesis, including ERK, JNK, p38, NF-κB, STAT3, and MITF. Notably, the cytotoxic efficacy of BEA G1 against A375SM cells was stronger than that of BEA. These findings suggest that BEA and BEA G1 can be further investigated as potent cytotoxic natural compounds for the suppression of melanoma progression.

Xyloneside A: A New Glycosylated Incisterol Derivative from Xylaria sp. FB.

Xylaria species are prolific natural product producers. Here, we report the characterization of a new glycosylated incisterol derivative, called xyloneside A (1) and two known lignans (2 and 3) from the ascomycetous Xylaria sp. FB. The structure of xyloneside A (1) was determined by 1D and 2D NMR spectroscopy, high-resolution electrospray ionization mass spectrometry and electronic circular dichroism measurements. Xyloneside A is composed of a 1,2,3,4,5,10,19-heptanorergosterane skeleton and a ß-D-mannopyranose moiety. This is the first report of an incisterol derivative from an Ascomycete. The biological effects of the isolated metabolites on cytotoxicity, autophagy, cell-migration, and angiogenesis were evaluated.