Additional Sarasinosides from the Marine Sponge Melophlus sarasinorum Collected from the Bismarck Sea.

In our continuing efforts to describe the biological and chemical diversity of sponges from Kimbe Bay, Papua New Guinea, the known 30-norlanostane saponin sarasinoside C1 (1) was identified along with six new analogues named sarasinosides C4, C5, C6, C7, C8, and C9 (2-7) from the sponge Melophlus sarasinorum. The structures of the new compounds were elucidated by analysis of 1D and 2D NMR and HRMS data, as well as comparison with literature data. All new compounds are characterized by the same tetraose moiety, ß-d-Xylp-(1→6)-ß-d-GlcNAcp-(1→2)-[ß-d-GalNAcp-(1→4)]-ß-d-Xylp, as described previously for sarasinoside C1, but differed in their aglycone moieties. When comparing NMR data of sarasinoside C8 with those of known analogues, a misassignment was identified in the configuration of the C-8/C-9 diol for the previously described sarasinoside R (8), and it has been corrected here using a combination of ROESY analysis and molecular modeling.

Bioactive Potential: A Pharmacognostic Definition through the Screening of Four Hypericum Species from the Canary Islands.

In this work, we propose a general methodology to assess the bioactive potential (BP) of extracts in the quest of vegetable-based drugs. To exemplify the method, we studied the anticancer potential (AP) of four endemic species of genus Hypericum (Hypericum canariense L, Hypericum glandulosum Aiton, Hypericum grandifolium Choisy and Hypericum reflexum L.f) from the Canary Islands. Microextracts were obtained from the aerial parts of these species and were tested against six human tumor cell lines, A549 (non-small-cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small-cell lung), T-47D (breast) and WiDr (colon). The methanol-water microextracts were evaluated further for cell migration, autophagy and cell death. The most promising bioactive polar microextracts were analyzed by UHPLC-DAD-MS. The extraction yield, the bioactivity evaluation and the chemical profiling by LC-MS suggested that H. grandifolium was the species with the highest AP. Label-free live-cell imaging studies on HeLa cells exposed to the methanol-water microextract of H. grandifolium enabled observing cell death and several apoptotic hallmarks. Overall, this study allows us to select Hypericum grandifolium Choisy as a source of new chemical entities with a potential interest for cancer treatment.

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool.

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, which are proposed to be introduced by YcaO-TfuA proteins. Using RiPPER, we show that previously undescribed RiPP gene clusters encoding YcaO and TfuA proteins are widespread in Actinobacteria and encode a highly diverse landscape of precursor peptides that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new structural class of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

Caniferolide A, a Macrolide from Streptomyces caniferus, Attenuates Neuroinflammation, Oxidative Stress, Amyloid-Beta, and Tau Pathology in Vitro.

The macrolide caniferolide A was isolated from extracts of a culture of the marine-derived actinomycete Streptomyces caniferus, and its ability to ameliorate Alzheimer's disease (AD) hallmarks was determined. The compound reduced neuroinflammatory markers in BV2 microglial cells activated with lipopolysaccharide (LPS), being able to block NFκB-p65 translocation to the nucleus and to activate the Nrf2 pathway. It also produced a decrease in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), reactive oxygen species (ROS) and nitric oxide release and inhibited iNOS, JNK, and p38 activities. Moreover, the compound blocked BACE1 activity and attenuated Aß-activation of microglia by drastically diminishing ROS levels. The phosphorylated state of the tau protein was evaluated in SH-SY5Y tau441 cells. Caniferolide A reduced Thr212 and Ser214 phosphorylation by targeting p38 and JNK MAPK kinases. On the other side, the antioxidant properties of the macrolide were determined in an oxidative stress model with SH-SY5Y cells treated with H2O2. The compound diminished ROS levels and increased cell viability and GSH content by activating the nuclear factor Nrf2. Finally, the neuroprotective ability of the compound was confirmed in two trans-well coculture systems with activated BV2 cells (both with LPS and Aß) and wild type and transfected SH-SY5Y cells. The addition of caniferolide A to microglial cells produced a significant increase in the survival of neuroblastoma in both cases. These results indicate that the compound is able to target many pathological markers of AD, suggesting that caniferolide A could be an interesting drug lead for a polypharmacological approach to the illness.

Structure elucidation and biosynthetic gene cluster analysis of caniferolides A-D, new bioactive 36-membered macrolides from the marine-derived Streptomyces caniferus CA-271066.

Bioassay-guided isolation based on the antifungal activity of a culture broth of the marine-derived actinomycete Streptomyces caniferus CA-271066 led to the discovery of new 36-membered polyol macrolides, caniferolides A-D (1-4). Their connectivity was determined by spectroscopic methods including ESITOF-MS and 1D/2D NMR. The relative stereochemistry of each stereocluster in these compounds was established using NOE analysis, the universal database method and J-based configuration analysis, further assisted by comparisons with NMR data of structurally related macrolides. Genome sequencing followed by detailed bioinformatics analysis led to the identification of the corresponding biosynthetic gene cluster and allowed the prediction of the stereochemical outcome of their biosynthesis, confirming the relative stereochemistry of each stereocluster already determined by NMR and establishing their stereochemical relationship, ultimately rendering the absolute configuration of all chiral centers. Furthermore, based on our results and already published data, it has been possible to derive the complete absolute configuration of the related macrolides PM100117 and PM100118, astolides A and B, and deplelides A and B. Caniferolides A-D have shown pronounced antifungal activity against Candida albicans and Aspergillus fumigatus alongside antiproliferative activity against five human tumoral cell lines.

Discovery and Biosynthesis of the Antibiotic Bicyclomycin in Distantly Related Bacterial Classes.

Bicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesized by Streptomyces cinnamoneus DSM 41675. BCM is structurally characterized by a core cyclo(l-Ile-l-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesized by a cyclodipeptide synthase, and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded by the genomes of hundreds of Pseudomonas aeruginosa isolates distributed globally, and heterologous expression of the pathway from P. aeruginosa SCV20265 demonstrated that the product is chemically identical to BCM produced by S. cinnamoneus Overall, putative BCM gene clusters have been found in at least seven genera spanning Actinobacteria and Proteobacteria (Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.IMPORTANCE Bicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known bicyclomycin-producing organism Streptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including in the opportunistic pathogen Pseudomonas aeruginosa This wide distribution of a complex biosynthetic pathway is very unusual and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

MDN-0170, a New Napyradiomycin from Streptomyces sp. Strain CA-271078.

A new napyradiomycin, MDN-0170 (1), was isolated from the culture broth of the marine-derived actinomycete strain CA-271078, together with three known related compounds identified as 4-dehydro-4a-dechloronapyradiomycin A1 (2), napyradiomycin A1 (3) and 3-chloro-6,8-dihydroxy-8-α-lapachone (4). The structure of the new compound was determined using a combination of spectroscopic techniques, including 1D and 2D NMR and electrospray-time of flight mass spectrometry (ESI-TOF MS). The relative configuration of compound 1, which contains two independent stereoclusters, has been established by molecular modelling in combination with nOe and coupling constant analyses. Biosynthetic arguments also allowed us to propose its absolute stereochemistry. The antimicrobial properties of the compounds isolated were evaluated against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Aspergillus fumigatus, and Candida albicans. The potent bioactivity previously reported for compounds 2 and 3 against methicillin-sensitive S. aureus has been extended to methicillin-resistant strains in this report.

New ikarugamycin derivatives with antifungal and antibacterial properties from Streptomyces zhaozhouensis.

A bioassay guided fractionation of the ethyl acetate extract from culture broths of the strain Streptomyces zhaozhouensis CA-185989 led to the isolation of three new polycyclic tetramic acid macrolactams (1-3) and four known compounds. All the new compounds were structurally related to the known Streptomyces metabolite ikarugamycin (4). Their structural elucidation was accomplished using a combination of electrospray-time of flight mass spectrometry (ESI-TOF MS) and 1D and 2D NMR analyses. Compounds 1-3 showed antifungal activity against Aspergillus fumigatus, Candida albicans and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).

Antifeedant, antifungal and nematicidal compounds from the endophyte Stemphylium solani isolated from wormwood.

The continuous search for natural product-based biopesticides from fungi isolated from untapped sources is an effective tool. In this study, we studied a pre-selected fungal endophyte, isolate Aa22, from the medicinal plant Artemisia absinthium, along with the antifungal, insect antifeedant and nematicidal compounds present in the extract. The endophyte Aa22 was identified as Stemphylium solani by molecular analysis. The antifungal activity was tested by broth microdilution against Fusarium solani, F. oxysporum, F. moniliforme and Botrytis cinerea, the insect antifeedant by choice bioassays against Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi and the in vitro mortality against the root-knot nematode Meloiydogyne javanica. The structures of bioactive compounds were determined on the basis of 1D and 2D NMR spectroscopy and mass spectrometry. The ethyl acetate extract obtained from the solid rice fermentation showed mycelial growth inhibition of fungal pathogens (EC50 0.08-0.31 mg/mL), was antifeedant to M. persicae (99%) and nematicidal (68% mortality). A bioguided fractionation led to the isolation of the new compound stempholone A (1), and the known stempholone B (2) and stemphol (3). These compounds exhibited antifeedant (EC50 0.50 mg/mL), antifungal (EC50 0.02-0.43 mg/L) and nematicidal (MLD 0.5 mg/mL) activities. The extract activities can be explained by 3 (antifungal), 1-3 (antifeedant) and 1 (nematicidal). Phytotoxicity tests on Lolium perenne and Lactuca sativa showed that the extract and 1 increased L. sativa root growth (121-130%) and 1 reduced L. perenne growth (48-49%). These results highlight the potential of the endophytic fungi Aa22 as biotechnological source of natural product-based biopesticides.

Allelopathic potential of Rapanea umbellata leaf extracts.

The stressful conditions associated with the Brazilian savanna (Cerrado) environment were supposed to favor higher levels of allelochemicals in Rapanea umbellata from this ecosystem. The allelopathic potential of R. umbellata leaf extracts was studied using the etiolated wheat coleoptile and standard phytotoxicity bioassays. The most active extract was selected to perform a bioassay-guided isolation, which allowed identifying lutein (1) and (-)-catechin (2) as potential allelochemicals. Finally, the general bioactivity of the two compounds was studied, which indicated that the presence of 1 might be part of the defense mechanisms of this plant.

Antileishmanial, antitrypanosomal, and cytotoxic screening of ethnopharmacologically selected Peruvian plants.

Extracts (34) from eight plant species of the Peruvian Amazonia currently used in traditional Peruvian medicine, mostly as antileishmanial remedies and also as painkiller, antiseptic, antipyretic, anti-inflamatory, antiflu, astringent, diuretic, antipoison, anticancerous, antiparasitic, insecticidal, or healing agents, have been tested for their antileishmanial, antitrypanosomal, and cytotoxic activity. Plant species were selected based on interviews conducted with residents of rural areas. The different plant parts were dried, powdered, and extracted by maceration with different solvents (hexane, chloroform, and 70% ethanol-water). These extracts were tested on promastigote forms of Leishmania infantum strain PB75, epimastigote forms of Trypanosoma cruzi strain Y, and the mammalian CHO cell line. Parasite viability and nonspecific cytotoxicity were analyzed by a modified MTT colorimetric assay method. The isolation and identification of pure compounds from selected extracts were performed by column chromatography, gas chromatography mass spectrometry (GC-MS; mixtures), spectroscopic techniques [MS, infrared (IR), ultraviolet (UV)], and mono and two-dimensional (1)H and (13)C nuclear magnetic resonance (NMR; COSY, HSQC, NOESY) experiments. Chondodendron tomentosum bark and Cedrela odorata were the most active extracts against Leishmania, while C. odorata and Aristoloquia pilosa were the most active against Trypanosoma, followed by Tabebuia serratifolia, Tradescantia zebrina, and Zamia ulei. Six compounds and two mixtures were isolated from Z. ulei [cycasin (1)], T. serratifolia {mixtures 1-2, and naphthoquinones 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione (2) and 2-(1-hydroxyethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione (3)}, and C. tomentosum [chondrocurine (4); (S,S')-12-O-methyl(+)-curine (5); and cycleanine (6)]. Four compounds and the two mixtures exhibited significant activity.

Anthratectone and naphthotectone, two quinones from bioactive extracts of Tectona grandis.

Two new quinones, (an isoprenoid quinone, and a dimeric anthraquinone) named naphthotectone and anthratectone, respectively, were isolated from bioactive leaf extracts from Tectona grandis. Their structures were determined by a combination of 1D and 2D NMR techniques. The bioactivity profile of naphthotectone was assessed using the etiolated wheat coleoptiles bioassay in aqueous solutions at concentrations ranging from 10(-3) to 10(-5)M, as well as the standard target species lettuce, cress, tomato, and onion. Naphthotectone showed high level of activities in both bioassays. This fact, along with the presence of this compound as the major component in Tectona grandis, suggests that it may be involved in the allelopathic activity previously described for this species, and probably in other defense mechanisms.

Understanding thioamitide biosynthesis using pathway engineering and untargeted metabolomics.

Thiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very few thioamitide biosynthetic steps have been elucidated, even though the biosynthetic gene clusters (BGCs) of multiple thioamitides have been identified. We hypothesised that engineering the thiostreptamide S4 BGC in a heterologous host could provide insights into its biosynthesis when coupled with untargeted metabolomics and targeted mutations of the precursor peptide. Modified BGCs were constructed, and in-depth metabolomics enabled a detailed understanding of the biosynthetic pathway to thiostreptamide S4, including the identification of a protein critical for amino acid dehydration that has homology to HopA1, an effector protein used by a plant pathogen to aid infection. We use this biosynthetic understanding to bioinformatically identify diverse RiPP-like BGCs, paving the way for future RiPP discovery and engineering.

Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature.

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a wide range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor annotation of the short precursor peptides that are ultimately modified into the final molecule. Here, we utilise a previously described genome mining tool, RiPPER, to identify novel RiPP precursor peptides near YcaO-domain proteins, enzymes that catalyse various RiPP post-translational modifications including heterocyclisation and thioamidation. Using this dataset, we identified a novel and diverse family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albidoflavus J1074 (formerly known as Streptomyces albus) was characterised, leading to the discovery of streptamidine, a novel amidine-containing RiPP. This new BGC family highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.

Isolation and phytotoxicity of terpenes from Tectona grandis.

A study was carried out on the allelopathic potential of four forest species, Tectona grandis, Aleurites fordii, Gliricidia sepium, and Maytenus buxifolia. The most active species, T. grandis, was selected to perform a phytochemical study. A new compound, abeograndinoic acid, was isolated, and elucidation of its structure showed that this compound has an unusual carbon skeleton. A further 21 known terpenoids-including 4 sesquiterpenoids, 8 diterpenes and 9 triterpenes-also were isolated. A biosynthetic scheme for the presence of the new compound is proposed. Bioactivity profiles that used etiolated wheat coleoptiles and phytotoxicity bioassays on the isolated compounds were conducted. The compounds that presented the highest phytotoxic activity are the diterpenes 9 (2-oxokovalenic acid) and 12 (19-hydroxyferruginol).

Strasseriolides A-D, A Family of Antiplasmodial Macrolides Isolated from the Fungus Strasseria geniculata CF-247251.

A novel family of four potent antimalarial macrolides, strasseriolides A-D (1-4), has been isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. The structures of these compounds, including their absolute configurations, were elucidated by HRMS, NMR spectroscopy, and X-ray single-crystal diffraction. The four compounds gave respective IC50 values of 9.810, 0.013, 0.123, and 0.128 µM against Plasmodium falciparum 3D7 parasites with no significant cytotoxicity against the HepG2 cell line.

MDN-0171, a new medermycin analogue from Streptomyces albolongus CA-186053.

A new medermycin derivative, MDN-0171 (1), and two known structurally related compounds, medermycin (2) and antibiotic G15-F (3) were isolated from the acetone extract of culture broths of the marine-derived Streptomyces albolongus strain CA-186053. Their structures were determined using a combination of spectroscopic techniques, including 1D and 2D NMR and electrospray-time of flight mass spectrometry (ESI-TOF MS). Compounds 2 and 3 accounted for the antimicrobial activity (against methicillin-resistant Staphylococcus aureus and Escherichia coli) previously detected in the crude extract of this actinomycete.

Thioalbamide, A Thioamidated Peptide from Amycolatopsis alba, Affects Tumor Growth and Stemness by Inducing Metabolic Dysfunction and Oxidative Stress.

Thioalbamide, a thioamidated peptide biosynthesized by Amycolatopsis alba, is a thioviridamide-like molecule, and is part of a family of natural products representing a focus of biotechnological and pharmaceutical research in recent years due to their potent anti-proliferative and cytotoxic activities on malignant cells. Despite the high antitumor potential observed at nanomolar concentrations, the mechanisms underlying thioalbamide activity are still not known. In this work, the cellular effects induced by thioalbamide treatment on breast cancer cell lines were evaluated for the first time, highlighting the ability of this microbial natural peptide to induce mitochondrial dysfunction, oxidative stress, and apoptotic cell death. Furthermore, we demonstrate that thioalbamide can inhibit the propagation of cancer stem-like cells, which are strongly dependent on mitochondrial function and are responsible for chemotherapy resistance, metastasis, and tumor recurrence.

Bioactive apocarotenoids from Tectona grandis.

The bioactive fractions of Tectona grandis have yielded seven apocarotenoids, two of which have been isolated for the first time as natural products (tectoionols A and B). The chemical structures were determined through 1D and 2D nuclear magnetic resonance experiments. The absolute configuration of tectoionol A was determined using a modified Mosher methodology. Some NMR assignments for the compounds 9(S)-4-oxo-7,8-dihydro-beta-ionol and 3beta-hydroxy-7,8-dihydro-beta-ionone have been corrected on the basis of g-HSQC and g-HMBC experiments. The general bioactivities of isolated compounds have been studied using etiolated wheat coleoptiles. Those compounds that presented higher levels of activity were assayed on standard target species (Lactuca sativa, Lycopersicum esculentum, Lepidium sativum and Allium cepa).

Streptocyclinones A and B ameliorate Alzheimer's disease pathological processes in vitro.

Alzheimer's disease (AD) is a pathology characterized by the abnormal accumulation of amyloid-beta (Aß) and hyperphosphorylated tau. Oxidative stress and neuroinflammation are also strongly related to this disease. The ability of two new glycosylated angucyclinones, streptocyclinones A and B (1 and 2), isolated from Streptomyces sp to improve AD hallmarks was evaluated. Compounds were able to protect SH-SY5Y neuroblastoma cells from H2O2-induced oxidative injury by activating the nuclear factor E2-related factor (Nrf2). Their capacity to modulate neuroinflammation was tested in lipopolysaccharide-activated BV2 microglial cells. Compounds reduced the release of pro-inflammatory factors, inhibited the activation of NFκB and mitogen activated kinases (MAPK), and induced the translocation of Nrf2 to the nucleus of microglial cells. A trans-well co-culture was established to determine the effect of microglia treated with streptocyclinones on the survival of SH-SY5Y cells. The cell viability of neuroblastoma cells increased when the compounds were added to BV2 cells. SH-SY5Y-TMHT441 cells were used to determine the effect of compounds on tau phosphorylation. Both compounds reduced tau hyperphophorylation by targeting MAPK kinases. Moreover, streptocyclinone B (2) was able to inhibit the activity of ß-secretase 1 and decrease the release of reactive oxygen species in BV2 cells stimulated with Aß. With the same co-culture trans-well system, the treatment of Aß-stimulated microglia with compound 2 augmented the viability of SH-SY5Y-TMHT441 cells. The results presented in this work provide evidences of the multitarget activities displayed by these new Streptomyces compounds, making them good candidates for further studies in the treatment of AD.