Ophiobolin A derivatives with enhanced activities under tumor-relevant acidic conditions.

Glioblastoma (GBM) is the most common form of malignant primary brain tumor and is one of the most lethal cancers. The difficulty in treating GBM stems from its highly developed mechanisms of drug resistance. Our research team has recently identified the fungal secondary metabolite ophiobolin A (OpA) as an agent with significant activity against drug-resistant GBM cells. However, the OpA's mode of action is likely based on covalent modification of its intracellular target(s) and thus possible off-target reactivity needs to be addressed. This work involves the investigation of an acid-sensitive OpA analogue approach that exploits the elevated acidity of the GBM microenvironment to enhance the selectivity for tumor targeting. This project identified analogues that showed selectivity at killing GBM cells grown in cultures at reduced pH compared to those maintained under normal neutral conditions. These studies are expected to facilitate the development of OpA as an anti-GBM agent by investigating its potential use in an acid-sensitive analogue form with enhanced selectivity for tumor targeting.

Correction: Masi et al. Araufuranone: A New Phytotoxic Tetrasubstituted Dihydrofuro[3,2-b]furan-2(5H)-One Isolated from Ascochyta araujiae. Biomolecules 2022, 12, 1274.

In the original article [...].

(±)-3-Deoxyradicinin Induces Stomata Opening and Chloroplast Oxidative Stress in Tomato (Solanum lycopersicum L.).

Radicinin is a phytotoxic dihydropyranopyran-4,5-dione isolated from the culture filtrates of Cochliobolus australiensis, a phytopathogenic fungus of the invasive weed buffelgrass (Cenchrus ciliaris). Radicinin proved to have interesting potential as a natural herbicide. Being interested in elucidating the mechanism of action and considering radicinin is produced in small quantities by C. australiensis, we opted to use (±)-3-deoxyradicinin, a synthetic analogue of radicinin that is available in larger quantities and shows radicinin-like phytotoxic activities. To obtain information about subcellular targets and mechanism(s) of action of the toxin, the study was carried out by using tomato (Solanum lycopersicum L.), which, apart from its economic relevance, has become a model plant species for physiological and molecular studies. Results of biochemical assays showed that (±)-3-deoxyradicinin administration to leaves induced chlorosis, ion leakage, hydrogen peroxide production, and membrane lipid peroxidation. Remarkably, the compound determined the uncontrolled opening of stomata, which, in turn, resulted in plant wilting. Confocal microscopy analysis of protoplasts treated with (±)-3-deoxyradicinin ascertained that the toxin targeted chloroplasts, eliciting an overproduction of reactive singlet oxygen species. This oxidative stress status was related by qRT-PCR experiments to the activation of transcription of genes of a chloroplast-specific pathway of programmed cell death.

Biochemical Analyses of Bioactive Extracts from Plants Native to Lampedusa, Sicily Minor Island.

Major threats to the human lifespan include cancer, infectious diseases, diabetes, mental degenerative conditions and also reduced agricultural productivity due to climate changes, together with new and more devastating plant diseases. From all of this, the need arises to find new biopesticides and new medicines. Plants and microorganisms are the most important sources for isolating new metabolites. Lampedusa Island host a rich contingent of endemic species and subspecies. Seven plant species spontaneously growing in Lampedusa, i.e., Atriplex halimus L. (Ap), Daucus lopadusanus Tineo (Dl), Echinops spinosus Fiori (Es) Glaucium flavum Crantz (Gf) Hypericum aegypticum L: (Ha), Periploca angustifolia Labill (Pa), and Prasium majus L. (Pm) were collected, assessed for their metabolite content, and evaluated for potential applications in agriculture and medicine. The HPLC-MS analysis of n-hexane (HE) and CH2Cl2 (MC) extracts and the residual aqueous phases (WR) showed the presence of several metabolites in both organic extracts. Crude HE and MC extracts from Dl and He significantly inhibited butyrylcholinesterase, as did WR from the extraction of Dl and Pa. HE and MC extracts showed a significant toxicity towards hepatocarcinoma Huh7, while Dl, Ha and Er HE extracts were the most potently cytotoxic to ileocecal colorectal adenocarcinoma HCT-8 cell lines. Most extracts showed antiviral activity. At the lowest concentration tested (1.56 µg/mL), Dl, Gf and Ap MC extracts inhibited betacoronavirus HCoV-OC43 infection by> 2 fold, while the n-hexane extract of Pm was the most potent. In addition, at 1.56 µg/mL, potent inhibition (>10 fold) of dengue virus was detected for Dl, Er, and Pm HE extracts, while Pa and Ap MC extracts dampened infections to undetectable levels. Regarding to phytotoxicity, MC extracts from Er, Ap and Pm were more effective in inhibiting tomato rootlet elongation; the same first two extracts also inhibited seed cress germination while its radicle elongation, due to high sensitivity, was affected by all the extracts. Es and Gf MC extracts also inhibited seed germination of Phelipanche ramosa. Thus, we have uncovered that many of these Lampedusa plants displayed promising biopesticide, antiviral, and biological properties.

Araufuranone: A New Phytotoxic Tetrasubstituted Dihydrofuro[3,2-b]furan-2(5H)-One Isolated from Ascochyta araujiae.

Araujia hortorum is a perennial vining plant species native to South America. It was introduced into many countries for ornamental and medicinal purposes as well as for its edible fruits, but it has become highly invasive, generating severe environmental problems. Biological control using bioherbicides and natural compounds is an interesting control option. The pathogenic fungus Ascochyta araujiae, isolated from infected leaves of A. hortorum, could be considered as a potential biocontrol agent. Its ability to produce bioactive metabolites was studied. The organic extract of the fungal culture filtrates showed interesting phytotoxic activities consisting of clearly visible necrotic symptoms (0.5-1 cm in diameter) in the punctured leaves. Thus, it was purified; this afforded three main metabolites. These were chemically and biologically characterised: one proved to be a new pentasubstituted dihydrofuro[3,2-b]furan-2(5H)-one, named araufuranone (1). The others were the already known fungal metabolites neovasinin and 2,4-dihydroxy-6-hydoxymethylbenzaldehyde (2 and 3). The structure of araufuranone was determined using spectroscopic methods (essentially 1D and 2D 1H and 13C NMR and HR ESIMS spectra); its relative configuration was assigned by a NOESY spectrum. To the best of our knowledge, araufuranone is the first example of a naturally occurring compound showing that carbon skeleton. Assayed by a puncture, araufuranone proved to be weakly active on the leaves of Diplotaxis sp. and Sonchus sp.; the other two metabolites were even less toxic. Tested on cress, compounds 2 and 3 were able to partially inhibit rootlet elongation whereas araufuranone was almost inactive.

Augmented phytotoxic effect of nanoencapsulated ophiobolin A.

Ophiobolin A is a secondary phytotoxic metabolite produced by some pathogenic fungal species responsible for severe plant diseases, considered to play a role in disease development and symptom appearance. Herein we investigated whether the phytotoxic activities of ophiobolin A against weed species could be improved by nanoencapsulation. Given the rapid natural degradation of the compound, it was hoped that nanoencapsulation would prolong the phytotoxic effects or enhance the bioactivity, thus leading to improved weed control capabilities. This article presents an assessment of the effectiveness of encapsulated ophiobolin A on 11 commonly found weed species, compared to the pure ophiobolin, to the particle alone, and a combination of mixed particles and ophiobolin A, by applying the solution droplets to both intact or injured leaf surface, on the adaxial or abaxial side. The bioassays showed the improved efficacy of the encapsulated ophiobolin, and the need for leaf lesions to diffuse the particles into the tissues.[Formula: see text].

Terpestacin, a toxin produced by Phoma exigua var. heteromorpha, the causal agent of a severe foliar disease of oleander (Nerium oleander L.).

Since 1987, several cytochalasins were isolated from Phoma exigua var. heteromorpha, the causal agent of foliar blight disease of oleander (Nerium oleander L.), and chemically and biologically characterised. During the purification process of a large-scale production of cytochalasins A and B, necessary to continue the study on their anticancer activity, a metabolite having a different carbon skeleton compared to that of cytochalasans, was isolated. It was identified as terpestacin, a well-known toxic fungal stestertepenoid, isolated for the first time from P. exigua var. heteromorpha, by spectroscopic investigation (essentially 1D and 2D 1H and 13C-NMR and ESI MS) and optical methods in comparison with the literature data. Terpestacin and some its derivatives (including a natural one, fusaproliferin) were prepared and tested for their biological activity. Terpestacin and fusaproliferin had some inhibitory effects on seed germination of Phelipanche ramosa, whereas none of the compounds caused phytotoxic effects on weed leaves.[Formula: see text].

Epithelial-mesenchymal transition sensitizes breast cancer cells to cell death via the fungus-derived sesterterpenoid ophiobolin A.

The epithelial-mesenchymal transition (EMT) imparts properties of cancer stem-like cells, including resistance to frequently used chemotherapies, necessitating the identification of molecules that induce cell death specifically in stem-like cells with EMT properties. Herein, we demonstrate that breast cancer cells enriched for EMT features are more sensitive to cytotoxicity induced by ophiobolin A (OpA), a sesterterpenoid natural product. Using a model of experimentally induced EMT in human mammary epithelial (HMLE) cells, we show that EMT is both necessary and sufficient for OpA sensitivity. Moreover prolonged, sub-cytotoxic exposure to OpA is sufficient to suppress EMT-imparted CSC features including sphere formation and resistance to doxorubicin. In vivo growth of CSC-rich mammary cell tumors, is suppressed by OpA treatment. These data identify a driver of EMT-driven cytotoxicity with significant potential for use either in combination with standard chemotherapy or for tumors enriched for EMT features.

Arabidopsis Defense against the Pathogenic Fungus Drechslera gigantea Is Dependent on the Integrity of the Unfolded Protein Response.

Drechslera gigantea Heald & Wolf is a worldwide-spread necrotrophic fungus closely related to the Bipolaris genus, well-known because many member species provoke severe diseases in cereal crops and studied because they produce sesterpenoid phytoxins named ophiobolins which possess interesting biological properties. The unfolded protein response (UPR) is a conserved mechanism protecting eukaryotic cells from the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER). In plants, consolidated evidence supports the role of UPR in the tolerance to abiotic stress, whereas much less information is available concerning the induction of ER stress by pathogen infection and consequent UPR elicitation as part of the defense response. In this study, the infection process of D. gigantea in Arabidopsis thaliana wild type and UPR-defective bzip28 bzip60 double mutant plants was comparatively investigated, with the aim to address the role of UPR in the expression of resistance to the fungal pathogen. The results of confocal microscopy, as well as of qRT-PCR transcript level analysis of UPR genes, proteomics, microRNAs expression profile and HPLC-based hormone analyses demonstrated that ophiobolin produced by the fungus during infection compromised ER integrity and that impairment of the IRE1/bZIP60 pathway of UPR hampered the full expression of resistance, thereby enhancing plant susceptibility to the pathogen.

Evaluation of Dittrichia viscosa (L.) Greuter Dried Biomass for Weed Management.

Dittrichia viscosa (L.) Greuter, a plant species common in the Mediterranean basin, produces several bioactive compounds, some of which have herbicidal effects. A number of greenhouse and field experiments were carried out in order to evaluate if these effects could be obtained also by using the whole plant biomass, to identify the efficacious doses, determine their effects on seed germination and weed emergence, and to evaluate influence of soil characteristics on biomass efficacy. The experiments carried out evidenced that: (i) the dried biomass completely hampers plant emergence when high doses (30-40 kg biomass m-3 of soil) are mixed into the soil, or delays it at a lower dose (10 kg m-3); (ii) the detrimental effects are not affected by soil type. The exploitation of the D. viscosa dried biomass appears to be a feasible option in weed management practices and its potential is discussed.

Biodegradable polymers as carriers for tuning the release and improve the herbicidal effectiveness of Dittrichia viscosa plant organic extracts.

BACKGROUND: The organic extracts (OEs) of Dittrichia viscosa, a ruderal plant common in the Mediterranean regions, proved to have herbicidal properties. In order to improve OE effectiveness and to develop novel eco-friendly bioherbicidal products, different amounts of OE were included in poly(butylene succinate)- and polycaprolactone-based films (PBS and PCL, respectively). Particular attention was given to the study of interactions between the polymers and OEs, with a deep spotlight concerning the influence of OEs on structural, morphological and thermal properties of both polymers, in order to assess the OE releasing kinetics from the matrices and its tuned herbicidal action against seeds. RESULTS: The bioassays carried out on Lepidium sativum and Phelipanche ramosa seeds evidenced a more controlled and effective OE release by PBS than PCL, and a longer lasting efficacy by the polymers with a higher OE content. The chemical-physical analyses were performed on films before and after biological assays. The thermogravimetric analysis confirmed that OE was a thermal stabilizer of the polymer; the presence of OE and polymer separated degradative kinetics suggested that only a partial and functional miscibility between polymers and OE occurred. The morphological analysis confirmed the good OE dispersion between PBS and PCL molecular chains. Infrared spectroscopy highlighted the enhanced hydrolysed structure of the doped polymers after the bioassays. These outcomes well matched the quantitative information outlined by release kinetics. DISCUSSION: The use of biodegradable polymers allows the effectiveness and tuning of the release of the formulated bioactive compounds to be improved. The easy-to-obtain and easy-to-formulate OE could become a suitable and environmentally friendly instrument in weed management programmes.

Secondary metabolites produced by Colletotrichum lupini, the causal agent of anthachnose of lupin (Lupinus spp.).

COLLETOTRICHUM LUPINI: is the causal agent of lupin (Lupinus albus L.) anthracnose, a destructive seed-borne disease affecting stems and pods. Despite that several biological studies have been carried out on this pathogen, the production of secondary metabolites has not yet been investigated. Thus, a strain of C. lupini, obtained from symptomatic stems of L. albus, has been grown in vitro to evaluate its ability to produce bioactive compounds. From its culture filtrates, a 3-substituted indolinone, named lupindolinone, and a 5,6-disubstituted tetrahydro-α-pyrone, named lupinlactone, were isolated together with the known (3R)-mevalonolactone and tyrosol. Lupindolinone and lupinlactone were characterized as 3-ethylindolin-2-one and 5-hydroxy-6-methyltetrahydropyran-2-one by spectroscopic methods (essentially nuclear magnetic resonance [NMR] and high-resolution electrospray ionization mass spectrometry [HR ESI-MS]). The R absolute configuration (AC) at C-5 of lupinlactone was determined by applying the modified Mosher's method. Thus, considering its relative stereochemistry assigned by NMR spectroscopy, the AC of lupinlactone could be formulated as 5R,6S. Lupindolinone was isolated as racemic mixture as shown by investigation using chiroptical methods. The metabolites were assayed in different biological tests and proved to have some activities at the used concentration.

Inuloxin E, a New Seco-Eudesmanolide Isolated from Dittrichia viscosa, Stimulating Orobanche cumana Seed Germination.

A new sesquiterpenoid belonging to the subgroup seco-eudesmanolides and named inuloxin E was isolated from Dittrichia viscosa, together with the already known sesquiterpenoids inuloxins A-D and α-costic acid. Inuloxin E was characterized by spectroscopic data (essentially NMR and ESI MS) as 3-methylene-6-(1-methyl-4-oxo-pentyl)-3a,4,7,7a-tetrahydro-3H-benzofuran-2-one. Its relative configuration was determined by comparison with the closely related inuloxin D and chemical conversion of inuloxin E into inuloxin D and by the observed significant correlation in the NOESY spectrum. Both inuloxins D and E induced germination of the parasitic weed Orobanche cumana, but were inactive on the seeds of Orobanche minor and Phelipanche ramosa. The germination activity of some hemisynthetic esters of inuloxin D was also investigated.

Encapsulation of inuloxin A, a plant germacrane sesquiterpene with potential herbicidal activity, in ß-cyclodextrins.

Inuloxin A is a promising plant phytotoxic sesquiterpene that deserves further studies to evaluate its potential as a bioherbicide. However, its low solubility in water and its bioavailability could hamper its practical applications. For this reason, inuloxin A was complexed with ß-cyclodextrins by using three different methods, i.e., kneading, co-precipitation and grinding. The resulted complexes were fully characterized by different techniques such as 1H NMR, UV-vis, XRD, DSC and SEM, and they were biologically assayed in comparison with the pure compound in several biological systems. The efficacy of the kneading and grinding complexes was similar to that of inuloxin A and these complexes almost completely inhibit Phelipanche ramosa seed germination. The complete solubility in water and the preservation of the biological properties of these two complexes could allow further studies to develop a novel natural herbicide for parasitic plant management based on these formulations.

Lentiquinones A, B, and C, Phytotoxic Anthraquinone Derivatives Isolated from Ascochyta lentis, a Pathogen of Lentil.

A strain of the pathogenic fungus Ascochyta lentis isolated from lentil ( Lens culinaris) was studied to ascertain its capability to produce bioactive metabolites. From the culture filtrates were found three new anthraquinone derivatives, named lentiquinones A (1), B (2), and C (3), and the known lentisone. From the mycelium, four known analogues were identified, namely pachybasin (in larger amount), ω-hydroxypachybasin, 1,7-dihydroxy-3-methylanthracene-9,10-dione, and phomarin. Lentiquinones A-C were characterized by spectroscopic methods as 3,4,6-trihydroxy-8-methyl-2 H-benzo[ g]chromene-5,10-dione, 2,3,4,5,10-pentahydroxy-7-methyl-3,4,4a,10-tetrahydroanthracen-9(2 H)-one, and its 2-epimer, respectively, and the relative configuration of the two latter compounds was deduced by X-ray diffraction data analysis. The absolute configuration of lentiquinones B and C was determined as (2 R,3 S,4 S,4a S,10 R) and (2 S,3 S,4 S,4a S,10 R), respectively, by electronic circular dichroism (ECD) in solution and solid state, and TDDFT calculations. When tested by using different bioassays, the novel compounds showed interesting activities. In particular, applied to punctured leaves of host and nonhost plants, the three new compounds and lentisone caused severe necrosis, with lentiquinone A being the most active among the new metabolites. On cress ( Lepidium sativum), this latter compound proved to be particularly active in inhibiting root elongation. On Lemna minor all the compounds reduced the content of chlorophyll, with 1,7-dihyroxy-3-methylanthracene-9,10-dione being the most active. The new compounds, together with lentisone, proved to have antibiotic properties.

Lathyroxins A and B, Phytotoxic Monosubstituted Phenols Isolated from Ascochyta lentis var. lathyri, a Fungal Pathogen of Grass Pea ( Lathyrus sativus).

Ascochyta lentis var. lathyri has recently been reported to be the causal agent of Ascochyta blight of grass pea ( Lathyrus sativus), a disease characterized by the appearance of necrotic lesions of leaves and stems. Considering the novelty of the pathogen and the possible involvement of secondary metabolites in symptom appearance, a study was carried out to ascertain the capability of this fungus to produce bioactive metabolites. Some phytotoxic phenols were isolated from the culture filtrates of the fungus. In particular, two new phytotoxic metabolites, named lathyroxins A and B, were characterized by spectroscopic methods as 4-(2-hydroxy-3,3-dimethoxypropyl)phenol and 3-(4-hydroxyphenyl)propane-1,2-diol, respectively, and the R absolute configuration of C-2 of their 2-dimethoxy- and 2,3-diol-propyl side chain was assigned. Moreover, other well-known fungal metabolites, namely, p-hydroxybenzaldehyde, p-methoxyphenol, and tyrosol, were also identified. Lathyroxins A and B showed interesting phytotoxic properties, being able to cause necrosis on leaves and to inhibit seed germination and rootlet elongation. Moreover, both of the new metabolites had no effect against bacteria, arthropods, and nematodes.

Development of a rapid and sensitive HPLC method for the identification and quantification of cavoxin and cavoxone in Phoma cava culture filtrates.

Cavoxin is a tetrasubstituted phytotoxic chalcone and cavoxone is the corresponding chroman-4-one, both produced in vitro by Phoma cava, a fungus isolated from chestnut. Cavoxin showed biofungicide potential against fungal species responsible for food moulding. Therefore, cavoxin has potential to be incorporated into biopolymer to generate 'intelligent food packaging'. To reach this objective, large-scale production of cavoxin by P. cava fermentation needs to be optimized. A rapid and efficient method for cavoxin analysis, as well as of cavoxone, in the fungal culture filtrates and the corresponding organic extracts is the first experimental step. Thus, a HPLC method was developed and applied to quantify cavoxin and cavoxone production in two different fungal culture conditions. The analysis proved that cavoxin production in stirred culture filtrates is significantly higher than in static ones.

On the metabolites produced by Colletotrichum gloeosporioides a fungus proposed for the Ambrosia artemisiifolia biocontrol; spectroscopic data and absolute configuration assignment of colletochlorin A.

Ambrosia artemisiifolia L. is responsible for serious allergies induced on humans. Different approaches for its control were proposed during the COST Action FA1203 "Sustainable management of Ambrosia artemisiifolia in Europe" (SMARTER). Fungal secondary metabolites often show potential herbicidal activity. Three phytotoxins were purified from the fungal culture filtrates of Colletotrichum gloeosporioides, isolated from infected leaves of A. artemisiifolia. They were identified by spectroscopic and chemical methods as colletochlorin A, orcinol and tyrosol (1, 2 and 3). The absolute configuration 6'R to colletochlorin A was assigned for the first time applying the advanced Mosher's method. When assayed by leaf-puncture on A. artemisiifolia only 1 caused the appearance of large necrosis. The same symptoms were also induced by 1 on ambrosia plantlets associated with plant wilting. On Lemna minor, colletochlorin A caused a clear fronds browning, with a total reduction in chlorophyll content.

Fungal Phytotoxins in Sustainable Weed Management.

Fungal phytotoxins are natural secondary metabolites produced by plant pathogenic fungi during host-pathogen interactions. They have received considerable particular attention for elucidating disease etiology, and consequently to design strategies for disease control. Due to wide differences in their chemical structures, these toxic metabolites have different ecological and environmental roles and mechanisms of action. This review aims at summarizing the studies on the possible use of these metabolites as tools in biological and integrated weed management, e.g. as: novel and environmentally friendly herbicides; lead for novel compounds; sources of novel mechanisms of action. Moreover, the limiting factors for utilizing those metabolites in practice will also be briefly discussed.

Investigation of Amino Acids As Herbicides for Control of Orobanche minor Parasitism in Red Clover.

Certain amino acids induce inhibitory effects in plant growth due to feedback inhibition of metabolic pathways. The inhibition patterns depend on plant species and the plant developmental stage. Those amino acids with inhibitory action on specific weeds could be utilized as herbicides, however, their use for weed control has not been put into practice. Orobanche minor is a weed that parasitizes red clover. O. minor germination is stimulated by clover root exudates. The subsequent seedling is an obligated parasite that must attach quickly to the clover root to withdraw its nutrients. Early development of O. minor is vulnerable to amino acid inhibition and therefore, a series of in vitro, rhizotron, and field experiments were conducted to investigate the potential of amino acids to inhibit O. minor parasitism. In in vitro experiments it was found that among a collection of 20 protein amino acids, lysine, methionine and tryptophan strongly interfere with O. minor early development. Field research confirmed their inhibitory effect but revealed that methionine was more effective than lysine and tryptophan, and that two successive methionine applications at 308 and 543 growing degree days inhibited O. minor emergence in red clover up to 67%. We investigated additional effects with potential to influence the practical use of amino acids against broomrape weeds, whether the herbicidal effect may be reversible by other amino acids exuded by host plants or may be amplified by inducing host resistance barriers against O. minor penetration. This paper suggests that amino acids may have the potential to be integrated into biorational programs of broomrape management.