First Investigation of Secondary Metabolites from Aspergillus xerophilus Reveals Compounds with Inhibitive Effects against Three Phytopathogenic Fungi of Agrarian Crops.

Fungal secondary metabolites play a highly significant role in crop protection, which is related to their antifungal activity against agriculturally important phytopathogens. In fact, plant diseases caused by fungi including species belonging to the genera of Alternaria, Botrytis, and Fusarium have become increasingly serious affecting crop yield and quality. Hence, there is increasing awareness by the scientific community of the importance of exploiting fungal products for finding new compounds able to inhibit phytopathogens. In this study several drimane-type sesquiterpenes have been detected for the first time as products of Aspergillus xerophilus by GC-MS analysis of the organic extracts obtained from the mycelia and culture filtrates of the fungus grown on two different substrates. Seven pure drimane-type sesquiterpenes were also isolated and identified by spectroscopic methods. The inhibitory effects of the pure compounds have been investigated against three phytopathogenic fungi of agrarian crops (i.e., Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum f. sp. pisi). Among the drimane-type sesquiterpenes isolated in this study, 9,11-dihydroxy-6-oxodrim-7-ene is the most active against the three phytopathogens. Our findings also reveal the high sensitivity of A. alternata to the isolated compounds. These results pave the way for future applications in agriculture of both A. xerophilus and its metabolites.

BODIPY-Based Analogue of the TREM2-Binding Molecular Adjuvant Sulfavant A, a Chemical Tool for Imaging and Tracking Biological Systems.

Recently, we described synthetic sulfolipids named Sulfavants as a novel class of molecular adjuvants based on the sulfoquinovosyl-diacylglycerol skeleton. The members of this family, Sulfavant A (1), Sulfavant R (2), and Sulfavant S (3), showed important effects on triggering receptor expressed on myeloid cells 2 (TREM2)-induced differentiation and maturation of human dendritic cells (hDC), through a novel cell mechanism underlying the regulation of the immune response. As these molecules are involved in biological TREM2-mediated processes crucial for cell survival, here, we report the synthesis and application of a fluorescent analogue of Sulfavant A bearing the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene moiety (Me4-BODIPY). The fluorescent derivative, named PB-SULF A (4), preserving the biological activity of Sulfavants, opens the way to chemical biology and cell biology experiments to better understand the interactions with cellular and in vivo organ targets and to improve our comprehension of complex molecular mechanisms underlying the not fully understood ligand-induced TREM2 activity.

In Vitro Evaluation of Antiviral Activities of Funicone-like Compounds Vermistatin and Penisimplicissin against Canine Coronavirus Infection.

Recent studies have demonstrated that 3-O-methylfunicone (OMF), a fungal secondary metabolite from Talaromyces pinophilus belonging to the class of funicone-like compounds, has antiviral activity against canine coronaviruses (CCoV), which causes enteritis in dogs. Herein, we selected two additional funicone-like compounds named vermistatin (VER) and penisimplicissin (PS) and investigated their inhibitory activity towards CCoV infection. Thus, both compounds have been tested for their cytotoxicity and for antiviral activity against CCoV in A72 cells, a fibrosarcoma cell line suitable for investigating CCoV. Our findings showed an increase in cell viability, with an improvement of morphological features in CCoV-infected cells at the non-toxic doses of 1 µM for VER and 0.5 µM for PS. In addition, we observed that these compounds caused a strong inhibition in the expression of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor which is activated during CCoV infection. Our results also showed the alkalinization of lysosomes in the presence of VER or PS, which may be involved in the observed antiviral activities.

First report of 6-methylpyridione analogues from Dothiorella sarmentorum, a botryosphaeriaceous fungus associated with grapevine trunk diseases.

Dothiorella species are fungal plant pathogens associated with Botryosphaeria dieback of grapevine. Symptoms caused by these fungi on grapevines suggest possible implication of phytotoxic metabolites in the infection mechanisms. However, few studies were conducted to investigate the secondary metabolism of these fungi. In this study, 6-methylpyridione analogues were isolated and identified for the first time in liquid cultures of Dothiorella sarmentorum isolated from symptomatic grapevine in Algeria.

Exploring the Binding of Natural Compounds to Cancer-Related G-Quadruplex Structures: From 9,10-Dihydrophenanthrenes to Their Dimeric and Glucoside Derivatives.

In-depth studies on the interaction of natural compounds with cancer-related G-quadruplex structures have been undertaken only recently, despite their high potential as anticancer agents, especially due to their well-known and various bioactivities. In this frame, aiming at expanding the repertoire of natural compounds able to selectively recognize G-quadruplexes, and particularly focusing on phenanthrenoids, a mini-library including dimeric (1-3) and glucoside (4-5) analogues of 9,10-dihydrophenanthrenes, a related tetrahydropyrene glucoside (6) along with 9,10-dihydrophenanthrene 7 were investigated here by several biophysical techniques and molecular docking. Compounds 3 and 6 emerged as the most selective G-quadruplex ligands within the investigated series. These compounds proved to mainly target the grooves/flanking residues of the hybrid telomeric and parallel oncogenic G-quadruplex models exploiting hydrophobic, hydrogen bond and π-π interactions, without perturbing the main folds of the G-quadruplex structures. Notably, a binding preference was found for both ligands towards the hybrid telomeric G-quadruplex. Moreover, compounds 3 and 6 proved to be active on different human cancer cells in the low micromolar range. Overall, these compounds emerged as useful ligands able to target G-quadruplex structures, which are of interest as promising starting scaffolds for the design of analogues endowed with high and selective anticancer activity.

Hands-on synthesis of furanamides and evaluation of their antimicrobial activity.

Diverse natural and synthetic furan derivatives have shown biological activity. Here, we describe the preparation of benzyl and arylethyl ß-furanamides with OH or OMe aryl substituents by an adapted sustainable method from a furoic acid using methyl chloroformate. Symmetric and asymmetric ß,ß'-furanamides have instead been prepared using azabenzotriazole based catalyst (HATU). The products have been evaluated for their antimicrobial properties on Gram positive and Gram negative bacteria. Just a minimal not-significant activity has been observed in some derivatives.

Mitidjospirone, a new spirodioxynaphthalene and GC-MS screening of secondary metabolites produced by strains of Lasiodiplodia mitidjana associated to Citrus sinensis dieback.

Mitidjospirone, a new spiridioxynaphthalene, was isolated from the mycelial extract of a strain of Lasiodiplodia mitidjana, a recently described species belonging to the family Botryosphaeriaceae. Its structure was elucidated by extensive spectroscopic analysis and the absolute configuration was determined by electronic circular dichroism (ECD) experiment. Furthermore, several known compounds were identified during the screening of secondary metabolites produced by four strains of L. mitidjana.

Effectiveness of the Fungal Metabolite 3-O-Methylfunicone towards Canine Coronavirus in a Canine Fibrosarcoma Cell Line (A72).

Canine coronavirus (CCoV), an alphacoronavirus, may cause self-limiting enteric disease in dogs, especially in puppies. The noteworthy plasticity of coronaviruses (CoVs) occurs through mutation and recombination processes, which sometimes generate new dangerous variants. The ongoing SARS-CoV-2 pandemic and the isolation of a novel canine-feline recombinant alphacoronavirus from humans emphasizes the cross-species transmission ability of CoVs. In this context, exploring antiviral compounds is essential to find new tools for fighting against CoVs infections. Fungi produce secondary metabolites, which are often developed as antibiotics, fungicides, hormones, and plant growth regulators. Previous examinations of benzo-γ-pyrone 3-O-methylfunicone (OMF), obtained from Talaromyces pinophilus, showed that it reduces the infectivity of hepatitis C virus and bovine herpesvirus 1. Based on this evidence, this study evaluated the antiviral ability of OMF against CCoV infection in a canine fibrosarcoma (A72) cell line. During CCoV infection, a non-toxic dose of OMF markedly increased features of cell viability. Moreover, OMF induced a significant reduction in virus yield in the presence of an intense downregulation of the viral nucleocapsid protein (NP). These findings occurred in the presence of a marked reduction in the aryl hydrocarbon receptor (AhR) expression. Taken together, preliminary findings suggest that OMF inhibiting AhR shows promising activity against CCoV infection.

Interaction of the Fungal Metabolite Harzianic Acid with Rare-Earth Cations (Pr3+, Eu3+, Ho3+, Tm3+).

Rare-earth elements (REEs) are in all respect a class of new contaminants that may have toxic effects on organisms and microorganisms and information on their interactions with natural ligands should be of value to predict and control their diffusion in natural environments. In the current study, we investigate interactions of tripositive cations of praseodymium, europium, holmium, and thulium with harzianic acid (H2L), a secondary metabolite produced by selected strains of fungi belonging to the Trichoderma genus. We applied the same techniques and workflow previously employed in an analogous study concerning lanthanum, neodymium, samarium, and gadolinium tripositive cations. Therefore, in the current study, HPLC-ESI-HRMS experiments, circular dichroism (CD), and UV-Vis spectrophotometric absorption data, as well as accurate pH measurements, were applied to characterize bonding interactions between harzianic acid and Pr3+, Eu3+, Ho3+, and Tm3+ cations. Problems connected to the low solubility of harzianic acid in water were overcome by employing a 0.1 M NaClO4/(CH3OH + H2O 50/50 w/w) mixed solvent. For Pr3+, Ho3+, and Tm3+, only the mono complexes PrL+, HoL+, and TmL+ were detected and their formation constant determined. Eu3+ forms almost exclusively the bis complex EuL2- for which the corresponding formation constant is reported; under our experimental conditions, the mono complex EuL+ is irrelevant. Combining the results of the present and previous studies, a picture of interactions of harzianic acid with rare-earth cations extending over 8 of the 17 REEs can be composed. In order to complement chemical information with toxicological information, a battery of bioassays was applied to evaluate the effects of praseodymium, europium, holmium, and thulium tripositive cations on a suite of bioindicators including Aliivibrio fischeri (Gram-negative bacterium), Raphidocelis subcapitata (green alga), and Daphnia magna (microcrustacean), and median effective concentration (EC50) values of Pr3+, Eu3+, Ho3+, and Tm3+ for the tested species were assessed.

New Insights into Chemical and Biological Properties of Funicone-like Compounds.

Funicone-like compounds are a homogeneous group of polyketides that, so far, have only been reported as fungal secondary metabolites. In particular, species in the genus Talaromyces seem to be the most typical producers of this group of secondary metabolites. The molecular structure of funicone, the archetype of these products, is characterized by a γ-pyrone ring linked through a ketone group to a α-resorcylic acid nucleus. This review provides an update on the current knowledge on the chemistry of funicone-like compounds, with special emphasis on their classification, occurrence, and diverse biological activities. In addition, their potential relevance as mycotoxins is discussed.

Anti-Inflammatory Potential of Compounds Isolated from Tunisian Lichens Species.

The lichen's special symbiotic structure enables it to produce bioactive substances. They have historically been recognized for their aesthetic and medicinal benefits. Furthermore, in recent years, they have performed in various fields, including perfumery, dyeing, and pharmacology due to their rich secondary metabolites. From our study, four compounds were isolated from organic extracts of Parmotrema hypoleucinum, Roccella phycopsis, and Xanthoria parietina and identified by spectroscopic investigation as atranorin, (+)-iso-usnic acid, methyl orsellinate, and parietin, respectively. The anti-inflammatory effects of lichens extracts, and pure compounds were evaluated on RAW 264.7 macrophages cells at different concentrations. At 25 µg/mL all treated samples did not show any effect on cell viability. Atranorin and (+)-iso-usnic acid showed an inhibitory effect on nitric oxide (NO) levels in lipopolysaccharide (LPS)-stimulated macrophages. Nitric oxide (NO) production was measured using Griess reagent, atranorin and (+)-iso-usnic acid showed a high anti-inflammatory potential (75.99 % and 57.27 % at 25 µg/mL). On the other hand, methyl orsellinate and the organic extracts of three lichens showed good anti-inflammatory activity ranging from 29.16 % at 25 µg/mL to 86.91 % at 100 µg/mL.

Secondary Metabolites, including a New 5,6-Dihydropyran-2-One, Produced by the Fungus Diplodia corticola. Aphicidal Activity of the Main Metabolite, Sphaeropsidin A.

An undescribed 5,6-dihydropyran-2-one, namely diplopyrone C, was isolated and characterized from the cultures of an isolate of the fungus Diplodia corticola recovered from Quercus suber in Algeria. The structure and relative stereostructure of (5S,6S,7Z,9S,10S)-5-hydroxy-6-(2-(3-methyloxiran-2-yl)vinyl)-5,6-dihydro-2H-pyran-2-one were assigned essentially based on NMR and MS data. Furthermore, ten known compounds were isolated and identified in the same cultures. The most abundant product, the tetracyclic pimarane diterpene sphaeropsidin A, was tested for insecticidal effects against the model sucking aphid, Acyrthosiphon pisum. Results showed a toxic dose-dependent oral activity of sphaeropsidin A, with an LC50 of 9.64 mM.

Interaction of the Fungal Metabolite Harzianic Acid with Rare-Earth Cations (La3+, Nd3+, Sm3+, Gd3+).

Rare-earth elements are emerging contaminants of soil and water bodies which destiny in the environment and effects on organisms is modulated by their interactions with natural ligands produced by bacteria, fungi and plants. Within this framework, coordination by harzianic acid (H2L), a Trichoderma secondary metabolite, of a selection of tripositive rare-earth cations Ln3+ (Ln3+ = La3+, Nd3+, Sm3+, and Gd3+) was investigated at 25 °C, and in a CH3OH/0.1 M NaClO4 (50/50 w/w) solvent, using mass spectrometry, circular dichroism, UV-Vis spectrophotometry, and pH measurements. Experimental data can be satisfactorily explained by assuming, for all investigated cations, the formation of a mono-complex (LnL+) and a bis-complex (LnL2-). Differences were found between the formation constants of complexes of different Ln3+ cations, which can be correlated with ionic radius. Since gadolinium is the element that raises the most concern among lanthanide elements, its effects on organisms at different levels of biological organization were explored, in the presence and absence of harzianic acid. Results of ecotoxicological tests suggest that harzianic acid can decrease gadolinium biotoxicity, presumably because of complex formation with Gd3+.

Direct evidence of the impact of aqueous self-assembly on biological behavior of amphiphilic molecules: The case study of molecular immunomodulators Sulfavants.

Sulfavant A and Sulfavant R, sulfoquinovoside-glycerol lipids under study as vaccine adjuvants, structurally differ only for the configuration of glyceridic carbon, R/S and R respectively. The in vitro activity of these substances follows a bell-shaped dose-response curve, but Sulfavant A gave the best response around 20 µM, while Sulfavant R at 10 nM. Characterization of aqueous self-assembly of these molecules by a multi-technique approach clarified the divergent and controversial biological outcome. Supramolecular structures were present at concentrations much lower than critical aggregation concentration for both products. The kind and size of these aggregates varied as a function of the concentration differently for Sulfavant A and Sulfavant R. At nanomolar range, Sulfavant A formed cohesive vesicles, while Sulfavant R arranged in spherical micellar particles whose reduced stability was probably responsible for an increase of monomer concentration in accordance with immunomodulatory profile. Instead, at micromolar concentrations transition from micellar to vesicular state of Sulfavant R occurred and thermodynamic stability of the aggregates, assessed by surface tensiometry, correlated with the bioactivity of Sulfavant A at 20 µM and the complete loss of efficacy of Sulfavant R. The study of Sulfavants provides clear evidence of how self-aggregation, often neglected, and the equilibria between monomers and aqueous supramolecular forms of lipophilic molecules deeply determine the overall bio-response.

Natural compounds from Juncus plants interacting with telomeric and oncogene G-quadruplex structures as potential anticancer agents.

Aiming at discovering novel, putative anticancer drugs featuring low-to-null side effects, natural compounds isolated from Juncaceae were studied here for their ability to target G-quadruplex structures originating from cancer-related telomeric and oncogene DNA sequences. Particularly, various dihydrophenanthrene, benzocoumarin and dihydrodibenzoxepin derivatives were firstly screened by the affinity chromatography-based G4-CPG assay, and the compound with the highest affinity and selectivity for G-quadruplexes (named J10) was selected for further studies. Fluorescence spectroscopy and circular dichroism experiments corroborated its capability to selectively recognize and stabilize G-quadruplexes over duplex DNA, also showing a preference for parallel G-quadruplexes. Molecular docking proved that the selective G-quadruplex interactions over duplex interactions could be due to the ability of J10 to bind to the grooves of the telomeric and oncogene G-quadruplex structures. Finally, biological assays demonstrated that J10 induces significant antiproliferative effects on human leukemia cells, with no relevant effects on healthy human fibroblasts. Interestingly, J10 exerts its antiproliferative action on tumor cells by activating the apoptotic pathway.

Ecotoxicity and photodegradation of Montelukast (a drug to treat asthma) in water.

The present work focuses on the ecotoxicological effects of montelukast sodium (MTL) and its photoproducts, obtained under environmentally-like conditions. Despite of the potential presence in surface waters and the common use of MTL as asthma drug, limited data has been published for its photodegradation, while no information is available for its ecotoxicity. Light-induced degradation is an effective way for drugs to degrade in aquatic environments, and MTL is highly photosensitive, even by exposure to sunlight. In this study, solar-simulated irradiation of the drug in water was investigated. The drug was quickly converted into a series of photoproducts that were spectroscopically characterized. The possible photoreaction pathways were proposed. Ecotoxicity tests were performed on parent compound and mixture of photoproducts towards two bioindicators (Raphidocelis subcapitata and Daphnia magna). Results evidenced that effects of MTL on D. magna (EC50 = 16.4 mg/L) were greater than effects on R. subcapitata (EC50 = 195.7 mg/L). Microscopy observations revealed that MTL had mainly accumulated in the gut of daphnia. Toxicity data on photolysed solutions highlighted the presence of residual toxicity in all samples, evidencing that no complete mineralization occurred. Future research should focus on monitoring of MTL concentrations in the environment and study its effects in bioaccumulation tests.

A One-Pot Approach to Novel Pyridazine C-Nucleosides.

The synthesis of glycosides and modified nucleosides represents a wide research field in organic chemistry. The classical methodology is based on coupling reactions between a glycosyl donor and an acceptor. An alternative strategy for new C-nucleosides is used in this approach, which consists of modifying a pre-existent furyl aglycone. This approach is applied to obtain novel pyridazine C-nucleosides starting with 2- and 3-(ribofuranosyl)furans. It is based on singlet oxygen [4+2] cycloaddition followed by reduction and hydrazine cyclization under neutral conditions. The mild three-step one-pot procedure leads stereoselectively to novel pyridazine C-nucleosides of pharmacological interest. The use of acetyls as protecting groups provides an elegant direct route to a deprotected new pyridazine C-nucleoside.

A new cyanine from oxidative coupling of chlorogenic acid with tryptophan: Assessment of the potential as red dye for food coloring.

A red pigment was prepared by reaction of chlorogenic acid (CGA) with tryptophan (TRP) in air at pH 9 (37% w/w yield) and evaluated as food dye. The main component of pigment was formulated as an unusual benzochromeno[2,3-b]indole linked to a TRP unit, featuring a cyanine type chromophore (λmax 542, 546 nm, 1% extinction coefficient of the sodium salt = 244 ± 2). The chromophore showed a minimal pH dependence and proved stable for at least 3 h at 90 °C, both at pH 3.6 or 7.0, whereas red wine anthocyanins showed a substantial (30%) and betanin a complete abatement after 1 h at the acidic pHs. An intense coloring of different food matrices was obtained with the pigment at 0.01 % w/w. No toxicity was observed up to 0.2 mg/mL on hepatic and colonic cell lines. These data make this dye a promising alternative for red coloring of food.

Coordination Properties of the Fungal Metabolite Harzianic Acid toward Toxic Heavy Metals.

Some Trichoderma strains are known for their capacity to produce harzianic acid, a metabolite belonging to the tetramic acid derivatives. Harzianic acid has interesting biological properties, such as antimicrobial activities against phytopathogenic fungi and promotion of plant growth. It also possesses remarkable chemical properties, including the chelating properties toward essential transition metals, which might be related to the biological activities. Increasing knowledge on chelating properties might be relevant for understanding the various beneficial effects of harzianic acid in the interaction between the producer fungi and plants. In this work, the coordination capacity of harzianic acid was studied to evaluate the formation and stability of complexes formed with toxic heavy metals (i.e., Cd2+, Co2+, Ni2+, and Pb2+), which might have a crucial role in the tolerance of plants growing in metal-contaminated soils and in abiotic stress.

Synergistic Effect of Abietic Acid with Oxacillin against Methicillin-Resistant Staphylococcus pseudintermedius.

Resin acids are valued in traditional medicine for their antiseptic properties. Among these, abietic acid has been reported to be active against methicillin-resistant Staphylococcus aureus (MRSA) strains. In veterinary healthcare, the methicillin-resistant Staphylococcus pseudintermedius (MRSP) strain is an important reservoir of antibiotic resistance genes including mecA. The incidence of MRSP has been increasing, and treatment options in veterinary medicine are partial. Here, we investigated the antimicrobial and antibiofilm properties of abietic acid against three MRSP and two methicillin-susceptible Staphylococcus pseudintermedius (MSSP) strains, isolated from diseased pet animals and human wound samples. Abietic acid showed a significant minimal inhibitory concentration (MIC) value ranging from 32 to 64 µg/mL (MRSPs) and 8 µg/mL (MSSP). By checkerboard method we demonstrated that abietic acid increased oxacillin susceptibility of MRSP strains, thus showing a synergistic interaction with oxacillin. Abietic acid was also able to contrast the vitality of treated MSSP and MRSP1 biofilms at 20 µg/mL and 40 µg/mL, respectively. Finally, the compound moderately reduced mecA, mecR1 and mec1 gene expression. In conclusion, the results here reported demonstrate the antimicrobial activity of abietic acid against MRSP and support the use of this compound as a potential therapeutic agent to be used in combinatorial antibiotic therapy.