Hirocidins, Cytotoxic Metabolites from Streptomyces hiroshimensis, Induce Mitochondrion-Mediated Apoptosis.

Recent advances in whole genome sequencing have revealed an immense microbial potential for the production of therapeutic small molecules, even from well-known producers. To access this potential, we subjected prominent antimicrobial producers to alternative antiproliferative assays using persistent cancer cell lines. Described herein is our discovery of hirocidins, novel secondary metabolites from Streptomyces hiroshimensis with antiproliferative activities against colon and persistent breast cancer cells. Hirocidin A is an unusual nine-membered carbocyclic maleimide and hirocidins B and C are relatives with an unprecedented, bridged azamacrocyclic backbone. Mode of action studies show that hirocidins trigger mitochondrion-dependent apoptosis by inducing expression of the key apoptotic effector caspase-9. The discovery of new cytotoxins contributes to scaffold diversification in anticancer drug discovery and the reported modes of action and concise total synthetic route for variant A set the stage for unraveling specific targets and biochemical interactions of the hirocidins.

Non-peptide secondary metabolites from poisonous mushrooms: overview of chemistry, bioactivity, and biosynthesis.

Covering: up to June 2021A wide variety of mushrooms have traditionally been recognized as edible fungi with high nutritional value and low calories, and abundantly produce structurally diverse and bioactive secondary metabolites. However, accidental ingestion of poisonous mushrooms can result in serious illnesses and even death. Chemically, mushroom poisoning is associated with secondary metabolites produced in poisonous mushrooms, causing specific toxicity. However, many poisonous mushrooms have not been fully investigated for their secondary metabolites, and the secondary metabolites of poisonous mushrooms have not been systematically summarized for details such as chemical composition and biosynthetic mechanisms. The isolation and identification of secondary metabolites from poisonous mushrooms have great research value since these compounds could be lethal toxins that contribute to the toxicity of mushrooms or could provide lead compounds with remarkable biological activities that can promote advances in other related disciplines, such as biochemistry and pharmacology. In this review, we summarize the structures and biological activities of secondary metabolites identified from poisonous mushrooms and provide an overview of the current information on these metabolites, focusing on their chemistry, bioactivity, and biosynthesis.

GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga3+ -complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99.

Induction of Diverse Cryptic Fungal Metabolites by Steroids and Channel Blockers.

Fungi offer a deep source of natural products but remain underutilized. Most biosynthetic gene clusters (BGCs) that can be detected are silent or "cryptic" in standard lab cultures and their products are thus not interrogated in routine screens. As genetic alterations are difficult and some strains can only be grown on agar, we have herein applied an agar-based high-throughput chemical genetic screen to identify inducers of fungal BGCs. Using R. solani and S. sclerotiorum as test cases, we report 13 cryptic metabolites in four compound groups, including sclerocyclane, a natural product with a novel scaffold. Steroids were the best elicitors and follow-up studies showed that plant-steroids trigger sclerocyclane synthesis, which shows antibiotic activity against B. plantarii, an ecological competitor of S. sclerotiorum. Our results open new paths to exploring the chemical ecology of fungal-plant interactions and provide a genetics-free approach for uncovering cryptic fungal metabolites.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus.

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from "drained" strains.

Structural Elucidation of Cryptic Algaecides in Marine Algal-Bacterial Symbioses by NMR Spectroscopy and MicroED.

Microbial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, biosynthetic precursors are likely provided by symbionts or hosts. In the current work, we report eight novel and architecturally unusual secondary metabolites synthesized by the bacterial symbiont Phaeobacter inhibens from precursors that, in a native context, would be provided by their algal hosts. Three of these were produced at low titres and their structures were determined de novo using the emerging microcrystal electron diffraction method. Some of the new metabolites exhibited potent algaecidal activity suggesting that the bacterial symbiont can convert algal precursors, tryptophan and sinapic acid, into complex cytotoxins. Our results have important implications for the parasitic phase of algal-bacterial symbiotic interactions.

Bioactive Phytochemicals from Mulberry: Potential Anti-Inflammatory Effects in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

The fruits of the mulberry tree (Morus alba L.), known as white mulberry, have been consumed in various forms, including tea, beverages, and desserts, worldwide. As part of an ongoing study to discover bioactive compounds from M. alba fruits, the anti-inflammatory effect of compounds from M. alba were evaluated in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages. Phytochemical analysis of the ethanol extract of the M. alba fruits led to the isolation of 22 compounds. Among the isolated compounds, to the best of our knowledge, compounds 1, 3, 5, 7, 11, 12, and 14-22 were identified from M. alba fruits for the first time in this study. Inhibitory effects of 22 compounds on the production of the nitric oxide (NO) known as a proinflammatory mediator in LPS-stimulated RAW 264.7 macrophages were evaluated using NO assays. Western blot analysis was performed to evaluate the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5). We evaluated whether the anti-inflammatory effects of cyclo(L-Pro-L-Val) (5) following LPS stimulation in RAW 264.7 macrophages occurred because of phosphorylation of IκB kinase alpha (IKKα), IκB kinase beta (IKKß), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB) and activations of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Cyclo(L-Pro-L-Val) (5) significantly suppressed phosphorylations of IKKα, IKKß, IκBα, and NF-κB and activations of iNOS and COX-2 in a concentration-dependent manner. Taken together, these results indicate that cyclo(L-Pro-L-Val) (5) can be considered a potential therapeutic agent for the treatment of inflammation-associated disorders.

Antioxidant and Anti-Inflammatory Effects of White Mulberry (Morus alba L.) Fruits on Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

In this study, the protective effects of white mulberry (Morus alba) fruits on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were investigated. The ethanol (EtOH) extract of white mulberry fruits and its derived fractions contained adequate total phenolic and flavonoid contents, with good in vitro antioxidant radical scavenging activity. The extract and fractions also markedly inhibited ROS generation and antioxidant activity. After treatment with the EtOH extract and its fractions, LPS stimulation-induced elevated nitric oxide (NO) production was restored, which was primarily mediated by downregulation of inducible NO synthase expression. A total of 20 chemical constituents including flavonoids, steroids, and phenolics were identified in the fractions using ultra-high-performance liquid chromatography (UHPLC)-quadrupole time-of-flight (QTOF) high-resolution mass spectrometry (HRMS). These findings provide experimental evidence of the protective effects of white mulberry fruit extract against oxidative stress and inflammatory responses, suggesting their nutraceutical and pharmaceutical potential as natural antioxidant and anti-inflammatory agents.

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.

Absolute Configuration and Corrected NMR Assignment of 17-Hydroxycyclooctatin, a Fused 5-8-5 Tricyclic Diterpene.

The absolute configuration and corrected NMR assignment of 17-hydroxycyclooctatin isolated from Streptomyces sp. M56 recovered from a nest of South African Macrotermes natalensis termites are reported. 17-Hydroxycyclooctatin is a unique tricyclic diterpene (C20) consisting of a fused 5-8-5 ring system, and in this study, its structure was unambiguously determined by a combination of HR-ESIMS and 1D and 2D NMR spectroscopic experiments to produce corrected NMR assignments. The absolute configuration of 17-hydroxycyclooctatin is reported for the first time in the current study using chemical reactions and quantum chemical ECD calculations. The corrected NMR assignments were verified using a gauge-including atomic orbital NMR chemical shifts calculation, followed by DP4 probability. To understand the pharmacological properties of 17-hydroxycyclooctatin, a network pharmacological approach and molecular docking analyses were used, which also predicted its effects on human breast cancer cell lines. Cytotoxicity and antiestrogenic activity of 17-hydroxycyclooctatin were determined, and it was found this compound may be an ERα antagonist.

(±)-Kituramides A and B, pairs of enantiomeric dopamine dimers from the two-spotted cricket Gryllus bimaculatus.

Two-spotted cricket Gryllus bimaculatus is one of many cricket species, and it is widely used as a food source for insectivorous animals. Moreover, this species is one of the edible insects approved by the Korea Food and Drug Administration (KFDA). (±)-Kituramides A (1) and B (2), which are pairs of novel enantiomeric dopamine dimers possessing a formamide group, were isolated from the two-spotted cricket, together with four other known biosynthetically related compounds (3-6). The chemical structures of 1 and 2 were elucidated using a combination of 1D and 2D NMR spectroscopic experiments and HR-ESIMS data. Compounds 1 and 2 were identified as racemic mixtures; the enantiomers (+)-1/2 and (-)-1/2 were successfully separated by utilizing a chiral HPLC column. The absolute configurations of (±)-1 and (±)-2 were unambiguously delineated by the application of quantum chemical ECD calculations. Further, these insect-derived substances were evaluated to understand their effects on cytokine expression in adipocytes. Treatment with (-)-1, (+)-2, and (-)-2 during adipocyte differentiation significantly promoted the expression of Leptin and IL-6, which resembles the actions of dopamine.

Macrocyclic Trichothecene Mycotoxins from a Deadly Poisonous Mushroom, Podostroma cornu-damae.

Three new macrocyclic trichothecenes (1-3) and five known related compounds (4-8) were isolated from the MeOH extract of a plate culture of the fungus Podostroma cornu-damae, a deadly poisonous mushroom. Miophytocen D (1) is a rearranged macrocyclic type D trichothecene, featuring a bicyclo-[6.5]dodecahydrocyclopenta[ b]chromene scaffold, and the structures of new compounds (1-3) were delineated by the combination of 1D and 2D NMR spectroscopic experiments and HRESIMS, modified Mosher's esterification, and quantum chemical ECD calculations. The isolated compounds (1-8) were evaluated for cytotoxicity against four human breast cancer cell lines (Bt549, HCC70, MDA-MB-231, and MDA-MB-468). Compounds 4, 6, and 8 exhibited significant cytotoxic effects against the breast cancer cell lines, with IC50 values in the range of 0.02-80 nM, which is stronger than doxorubicin, the positive control, and a structure-activity relationship was suggested.

Pantheric Acids A-C from a Poisonous Mushroom, Amanita pantherina, Promote Lipid Accumulation in Adipocytes.

Amanita pantherina is a poisonous mushroom that causes muscle cramps, insanity, and audiovisual disorders. As part of our systematic study on Korean mushrooms, a chemical investigation of A. pantherina fruiting bodies resulted in the isolation and structural identification of three new fatty acid derivatives, pantheric acids A-C (1-3), and a known compound, 1,10-dimethyl ester-2-decenedioic acid (4). Although 1,10-dimethyl ester-2-decenedioic acid (4) was previously reported as a synthetic product, it was structurally identified from a natural source for the first time. The structures of the new compounds were established by detailed analysis of 1D and 2D (1H-1H COSY, HSQC, and HMBC) NMR, HRMS, and LC/MS/MS data. The absolute configurations of compounds 1 and 2 were unambiguously determined by a recently developed method using competing enantioselective acylation coupled with LC/MS analysis. The isolated compounds (1-4) were evaluated for their effects on lipid accumulation during adipocyte maturation. Pantheric acids A-C (1-3) promoted the enlargement of lipid droplets in 3T3-L1 adipocytes and altered lipid metabolism by inducing lipogenesis and inhibiting lipolysis. Our findings provide experimental evidence suggesting the potential adverse effects of pantheric acids A-C from a poisonous mushroom on lipid metabolism.

Sesquiterpenes from Curcuma zedoaria rhizomes and their cytotoxicity against human gastric cancer AGS cells.

Curcuma zedoaria rhizome (Zingiberaceae) is a well-known traditional medicinal plant used in Ayurvedic and traditional Chinese medicine to treat various cancers. This study aimed to identify the cytotoxic components from C. zedoaria rhizomes that act against gastric cancer, which is the third leading cause of death from cancer worldwide because the MeOH extract of C. zedoaria rhizome was found to show a cytotoxic effect against gastric cancer AGS cells. Repeated column chromatography and semi-preparative HPLC purification were used to separate the components from the C. zedoaria MeOH extract. Two new sesquiterpenes, curcumenol-9,10-epoxide (1) and curcuzedoalide B (2), and 12 known related sesquiterpenes (3-14) were isolated from the C. zedoaria MeOH extract. The structures of new compounds were determined by 1D and 2D NMR spectroscopic experiments and HR-ESIMS, and quantum chemical ECD calculations. The cytotoxic effects of the isolated compounds were measured in human gastric cancer AGS cells using an MTT cell viability assay. Compounds 9, 10, and 12 exhibited cytotoxic effects against gastric cancer AGS cells, with IC50 values in the range of 212-392 µM. These findings provide further experimental scientific evidence to support the traditional use of C. zedoaria rhizomes for the treatment of cancer. Curcumenol (9), 4,8-dioxo-6ß-methoxy-7α,11-epoxycarabrane (10), and zedoarofuran (12) were identified as the main cytotoxic components in C. zedoaria rhizomes.

Bioactivity-based analysis and chemical characterization of anti-inflammatory compounds from Curcuma zedoaria rhizomes using LPS-stimulated RAW264.7 cells.

Inflammation is not only a self-defense response of the innate immune system, but also the pathogenesis mechanism of multiple diseases such as arthritis, neurodegeneration, and cancer. Curcuma zedoaria Roscoe (Zingiberaceae), an indigenous plant of India, has been used traditionally in Ayurveda and folk medicine. As part of our ongoing efforts to screen traditional medicinal plants exhibiting pharmacological potential and to characterize the compounds involved, we examined the anti-inflammatory effects of the MeOH extract of C. zedoaria rhizomes using lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage cells and found that MeOH extract inhibited the synthesis of nitric oxide (NO) in a dose-dependent manner (IC50: 23.44 ±â€¯0.77 µg/mL). In our efforts to characterize the compounds responsible for these anti-inflammatory effects, bioactivity-guided fractionation of the MeOH extract and chemical investigation of its active hexane-soluble fraction led to the successful isolation of five sesquiterpenes (1-5), the structures of which were elucidated by NMR spectroscopic analysis and LC/MS analysis. Among them, curcuzedoalide (5) exhibited potent inhibitory effects on NO synthesis (IC50: 12.21 ±â€¯1.67 µM) and also suppressed pre-inflammatory protein expression of iNOS and COX-2. Curcuzedoalide (5) was thus determined to be a contributor to the anti-inflammatory effect of C. zedoaria rhizomes and could be a potential candidate for therapeutic applications.

Hybrid Polyketides from a Hydractinia-Associated Cladosporium sphaerospermum SW67 and Their Putative Biosynthetic Origin.

Five hybrid polyketides (1a, 1b, and 2-4) containing tetramic acid core including a new hybrid polyketide, cladosin L (1), were isolated from the marine fungus Cladosporium sphaerospermum SW67, which was isolated from the marine hydroid polyp of Hydractinia echinata. The hybrid polyketides were isolated as a pair of interconverting geometric isomers. The structure of 1 was determined based on 1D and 2D NMR spectroscopic and HR-ESIMS analyses. Its absolute configuration was established by quantum chemical electronic circular dichroism (ECD) calculations and modified Mosher's method. Tetramic acid-containing compounds are reported to be derived from a hybrid PKS-NRPS, which was also proved by analyzing our 13C-labeling data. We investigated whether compounds 1-4 could prevent cell damage induced by cisplatin, a platinum-based anticancer drug, in LLC-PK1 cells. Co-treatment with 2 and 3 ameliorated the damage of LLC-PK1 cells induced by 25 µM of cisplatin. In particular, the effect of compound 2 at 100 µM (cell viability, 90.68 ± 0.81%) was similar to the recovered cell viability of 88.23 ± 0.25% with 500 µM N-acetylcysteine (NAC), a positive control.

Suppression of 6-Hydroxydopamine-Induced Oxidative Stress by Hyperoside Via Activation of Nrf2/HO-1 Signaling in Dopaminergic Neurons.

In our ongoing research to discover natural products with neuroprotective effects, hyperoside (quercetin 3-O-galactoside) was isolated from Acer tegmentosum, which has been used in Korean traditional medicine to treat liver-related disorders. Here, we demonstrated that hyperoside protects cultured dopaminergic neurons from death via reactive oxygen species (ROS)-dependent mechanisms, although other relevant mechanisms of hyperoside activity remain largely uncharacterized. For the first time, we investigated the neuroprotective effects of hyperoside on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in neurons, and the possible underlying mechanisms. Hyperoside significantly ameliorated the loss of neuronal cell viability, lactate dehydrogenase release, excessive ROS accumulation and mitochondrial membrane potential dysfunction associated with 6-OHDA-induced neurotoxicity. Furthermore, hyperoside treatment activated the nuclear erythroid 2-related factor 2 (Nrf2), an upstream molecule of heme oxygenase-1 (HO-1). Hyperoside also induced the expression of HO-1, an antioxidant response gene. Remarkably, we found that the neuroprotective effects of hyperoside were weakened by an Nrf2 small interfering RNA, which blocked the ability of hyperoside to inhibit neuronal death, indicating the vital role of HO-1. Overall, we show that hyperoside, via the induction of Nrf2-dependent HO-1 activation, suppresses neuronal death caused by 6-OHDA-induced oxidative stress. Moreover, Nrf2-dependent HO-1 signaling activation represents a potential preventive and therapeutic target in Parkinson's disease management.

Highly Sensitive, Simple, and Cost- and Time-Effective Method to Determine the Absolute Configuration of a Secondary Alcohol Using Competing Enantioselective Acylation Coupled with LC/MS.

The absolute-configuration determination of natural products and synthetic compounds with stereogenic centers is very important because stereoisomers dramatically and differentially affect many crucial properties, such as physical behaviors and biological functions. Despite several established methods for determining the absolute configuration, significant unmet needs for new methods still exist owing to the specific limitations of established methodologies. Here, we present a simple, optimized, new chemical-derivative method that utilizes competing enantioselective acylation followed by LC/MS analysis, and we demonstrate its successful application in determining the absolute configuration of a secondary alcohol in natural products with multiple reactive functional groups. This new development relies on the enantiomeric pair of homobenzotetramisole (HBTM) catalysts exhibiting adequate kinetic resolution for acylation of the secondary alcohol, and then the fast reaction was quantitatively confirmed via LC/MS as the characterization technique for the enantioselective transformations. Our new approach was successfully applied to determine the absolute configuration of one secondary alcohol in compound 1, which has other hydroxyl groups to be reacted. The identified stereocenter of 1 was verified by previously established methods including quantum chemical electronic-circular-dichroism (ECD) calculations, computational NMR-chemical-shift calculations followed by DP4+ calculations, and modified Mosher's method. In addition, our method was applied to five known naturally occurring compounds, which led to the successful verification of their absolute configurations. Our newly developed method using the HBTM catalyst provides a highly sensitive, simple, and cost- and time-effective approach and an applicable and convenient analytical method for determining the absolute configuration of one secondary alcohol in natural products.

Chemical characterization of cytotoxic indole acetic acid derivative from mulberry fruit (Morus alba L.) against human cervical cancer.

The fruit of the white mulberry tree (Morus alba L.) is a multiple fruit with a sweet flavor commonly consumed around the world. Chemical investigation of the fruits led to the isolation of two indole acetic acid derivatives (1 -2) including a new compound, which turned out to be an isolation artifact, 3S-(ß-D-glucopyranosyloxy)-2,3-dihydro-2-oxo-1H-indole-3-acetic acid butyl ester (1), along with five known compounds (3 -7). Compounds 2 and 7 were newly identified from mulberry fruit. The new isolation artifact (1) exhibited cytotoxic effect on human cervical cancer Hela cells in a dose-dependent manner. Compound 1 activated caspase-8, caspase-9, and caspase-3, followed by cleavage of PARP, a substrate of caspase-3, in a dose-dependent manner. Simultaneous alterations in protein expression of mitochondrial factors Bax, BID and Bcl-2 were also observed. A comparison between compounds 1 and 2 led to a structure-activity relationship analysis of the cytotoxic effect. These results suggest that compound 1 could be beneficial in human cervical cancer treatment, and provide a theoretical basis for further application of compound 1.

Beneficial Effects of Bioactive Compounds in Mulberry Fruits against Cisplatin-Induced Nephrotoxicity.

Mulberry, the fruit of white mulberry tree (Morus alba L., Moraceae), is commonly used in traditional Chinese medicines as a sedative, tonic, laxative, and emetic. In our continuing research of the bioactive metabolites from mulberry, chemical analysis of the fruits led to the isolation of five compounds, 1-5. The compounds were identified as butyl pyroglutamate (1), quercetin 3-O-ß-d-glucoside (2), kaempferol 3-O-ß-d-rutinoside (3), rutin (4), and 2-phenylethyl d-rutinoside (5) by spectroscopic data analysis, comparing their nuclear magnetic resonance (NMR) data with those in published literature, and liquid chromatography-mass spectrometry analysis. The isolated compounds 1-5 were evaluated for their effects on anticancer drug-induced side effects by cell-based assays. Compound 1 exerted the highest protective effect against cisplatin-induced kidney cell damage. This effect was found to be mediated through the attenuation of phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, p38, mitogen-activated protein kinase, and caspase-3 in cisplatin-induced kidney cell damage.