Expanding the Structural Diversity of Tubulin-Targeting Agents: Development of Highly Potent Benzimidazoles for Treating Fungal Diseases.

Benzimidazoles, the representative pharmacophore of fungicides, have excellent antifungal potency, but their simple structure and single site of action have hindered their wider application in agriculture. In order to extend the structural diversity of tubulin-targeted benzimidazoles, novel benzimidazole derivatives were prepared by introducing the attractive pyrimidine pharmacophore. 2-((6-(4-(trifluoromethyl)phenoxy)pyrimidin-4-yl)thio)-1H-benzo[d]imidazole (A25) exhibited optimal antifungal activity against Sclerotinia sclerotiorum (S. s.), affording an excellent half-maximal effective concentration (EC50) of 0.158 µg/mL, which was higher than that of the reference agent carbendazim (EC50 = 0.594 µg/mL). Pot experiments revealed that compound A25 (200 µg/mL) had acceptable protective activity (84.7%) and curative activity (78.1%), which were comparable with that of carbendazim (protective activity: 90.8%; curative activity: 69.9%). Molecular docking displayed that multiple hydrogen bonds and π-π interactions could be formed between A25 and ß-tubulin, resulting in a stronger bonding effect than carbendazim. Fluorescence imaging revealed that the structure of intracellular microtubules can be changed significantly after A25 treatment. Overall, these remarkable antifungal profiles of constructed novel benzimidazole derivatives could facilitate the application of novel microtubule-targeting agents.

Evaluation of Ointments with Daldinia eschscholtzii in Wound Healing in an In Vivo Model.

Fungi are a source of a variety of secondary metabolites of importance in different areas of biotechnology. Several compounds have been characterized with antioxidant, antimicrobial, and anti-inflammatory activity from fungi of the division of the Ascomycota, among which is the species Daldinia eschscholtzii, an endophyte fungus of pantropical distribution. In this study, we evaluated the effect of an ointment made with D. eschscholtzii on the wound healing of BALB/c mice. The species was corroborated using a molecular marker Internal Transcribed Spacer (ITS1 and ITS4). The extracts and dust of the fungus were considered nontoxic as they caused a mortality of <15% in the nematode Panagrellus redivivus, and experimental ointments had no adverse effects on the skin of BALB/c mice. Wounds treated with the D. eschscholtzii ointments had 99.9-100% wound contraction after 17 days, which was similar to commercial healing (positive control). As such, the ointment of D. eschscholtzii is a natural alternative to improve wound healing.

Formation of Biogenic Manganese Oxide Nodules on Hyphae of a New Fungal Isolate of Periconia That Immobilizes Aqueous Copper.

Mn(II)-oxidizing microorganisms are considered to play significant roles in the natural geochemical cycles of Mn and other heavy metals because the insoluble biogenic Mn oxides (BMOs) that are produced by these microorganisms adsorb other dissolved heavy metals and immobilize them as precipitates. In the present study, a new Mn(II)-oxidizing fungal strain belonging to the ascomycete genus Periconia, a well-studied plant-associating fungal genus with Mn(II)-oxidizing activity that has not yet been exami-ned in detail, was isolated from natural groundwater outflow sediment. This isolate, named strain TS-2, was confirmed to oxidize dissolved Mn(II) and produce insoluble BMOs that formed characteristic, separately-located nodules on their hyphae while leaving major areas of the hyphae free from encrustation. These BMO nodules also adsorbed and immobilized dissolved Cu(II), a model analyte of heavy metals, as evidenced by elemental mapping ana-lyses of fungal hyphae-BMO assemblages using a scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX). Analyses of functional genes within the whole genome of strain TS-2 further revealed the presence of multiple genes predicted to encode laccases/multicopper oxidases that were potentially responsible for Mn(II) oxidation by this strain. The formation of BMO nodules may have functioned to prevent the complete encrustation of fungal hyphae, thereby enabling the control of heavy metal concentrations in their local microenvironments while maintaining hyphal functionality. The present results will expand our knowledge of the physiological and morphological traits of Mn(II)-oxidizing Periconia, which may affect the natural cycle of heavy metals through their immobilization.

Antifungal effects of volatile organic compounds produced by Trichoderma hamatum against Neocosmospora solani.

Neocosmospora solani causes Fusarium wilt disease and root rot, which are serious problems worldwide. To determine the growth inhibition of Neocosmospora solani by Trichoderma hamatum volatile organic compounds (VOCs), the major chemical components of Trichoderma hamatum VOCs and the differences in their contents at different times were analysed, and the activity of these components was evaluated. The antifungal activity of Trichoderma hamatum was measured by a screening test, as Trichoderma hamatum exhibited strong antagonism against Neocosmospora solani in vitro. The double plate technique was used to verify the activity of Trichoderma hamatum VOCs, and the inhibition rate was 63.77%. Neocosmospora solani mycelia were uneven and expanded, the contents of the cells leaked, and the mycelia shrank and presented a diaphragm in the hyphae upon Trichoderma hamatum VOCs treatment. Trichoderma hamatum VOCs and their contents at different times were analysed by using gas chromatography-mass spectrometry. 6-Pentyl-2H-pyran-2-one clearly presented in greater amounts than the other components on day 3, 4, 5, and 6. VOCs from Trichoderma hamatum exhibited evident effects on the percentage of healthy fruit after day 3. Moreover, Trichoderma hamatum can improve the biological control of diseases caused by soilborne pathogens, and can be applied in biocontrol fields.

Escuzarmycins A-D, Potent Biofungicides to Control Septoria tritici Blotch.

A study targeting novel antifungal metabolites identified potent in vitro antifungal activity against key plant pathogens in acetone extracts of Streptomyces sp. strain CA-296093. Feature-based molecular networking revealed the presence in this extract of antimycin-related compounds, leading to the isolation of four new compounds: escuzarmycins A-D (1-4). Extensive structural elucidation, employing 1D and 2D NMR, high-resolution mass spectrometry, Marfey's analysis, and NOESY correlations, confirmed their structures. The bioactivity of these compounds was tested against six fungal phytopathogens, and compounds 3 and 4 demonstrated strong efficacy, particularly against Zymoseptoria tritici, with compound 3 exhibiting the highest potency (EC50: 11 nM). Both compounds also displayed significant antifungal activity against Botrytis cinerea and Colletotrichum acutatum, with compound 4 proving to be the most potent. Despite moderate cytotoxicity against the human cancer cell line HepG2, compounds 3 and 4 emerge as promising fungicides for combating Septoria tritici blotch, anthracnose, and gray mold.

Six undescribed 23-norursane triterpenoids from the biotransformation of ilexgenin a by endophytic fungi and their vascular protective activity.

Biotransformation of ursane-type triterpenoid ilexgenin A by endophytic fungi Lasiodiplodia sp. MQD-4 and Pestalotiopsis sp. ZZ-1, isolated from Ilex pubescences and Callicarpa kwangtungensis respectively, was investigated for the first time. Six previously undescribed metabolites (1-6) with 23-norursane triterpenoids skeleton were isolated and their structures were unambiguously established by the analysis of spectroscopic data and single-crystal X-ray crystallographic experiments. Decarboxylation, oxidation, and hydroxylation reactions were observed on the triterpenoid skeleton. Especially, the decarboxylation of C-23 provided definite evidence to understand the biogenetic process of 23-norursane triterpenoids. Moreover, the qualitative analysis of the extract of I. pubescences showed metabolites 1, 3, 4, and 6 could be detected in the originated plant, indicating biotransformation by endophytic fungi is a practical strategy for the isolation of novel natural products. Finally, all isolates were evaluated for the protective activities against H2O2-induced HUVECs dysfunction in vitro. Compound 5 could improve the viability of endothelial cells and decrease the level of intracellular ROS.

In vitro Antibacterial Activity and Secondary Metabolite Profiling of Endolichenic Fungi Isolated from Genus Parmotrema.

The endolichenic fungi are an unexplored group of organisms for the production of bioactive secondary metabolites. The aim of the present study is to determine the antibacterial potential of endolichenic fungi isolated from genus Parmotrema. The study is continuation of our previous work, wherein a total of 73 endolichenic fungi were isolated from the lichenized fungi, which resulted in 47 species under 23 genera. All the isolated endolichenic fungi were screened for preliminary antibacterial activity. Five endolichenic fungi-Daldinia eschscholtzii, Nemania diffusa, Preussia sp., Trichoderma sp. and Xylaria feejeensis, were selected for further antibacterial activity by disc diffusion method. The zone of inhibition ranged from 14.3 ± 0.1 to 23.2 ± 0.1. The chemical composition of the selected endolichenic fungi was analysed through GC-MS, which yielded a total of 108 compounds from all the selected five endolichenic fungi. Diethyl phthalate, 1-hexadecanol, dibutyl phthalate, n-tetracosanol-1, 1-nonadecene, pyrrol[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methyl) and tetratetracontane were found to be common compounds among one or the other endolichenic fungi, which possibly were responsible for antibacterial activity. GC-MS data were further analysed through Principal Component Analysis which showed D. eschscholtzii to be with unique pattern of expression of metabolites. Compound confirmation test revealed coumaric acid to be responsible for antibacterial activity in D. eschscholtzii. So, the study proves that endolichenic fungi that inhabit lichenized fungal thalli could be a source of potential antibacterial compounds.

Bioactivity-Based Molecular Networking-Guided Isolation of Epicolidines A-C from the Endophytic Fungus Epicoccum sp. 1-042.

Bioactivity-based molecular networking-guided fractionation enabled the isolation of three new polycyclic tetramic acids bearing cis-decalin, epicolidines A-C (1-3), along with one known compound, PF 1052 (4), from the endophytic fungus Epicoccum sp. 1-042 collected in Tibet, China. Their structures were assigned on the basis of extensive spectroscopic data, partial hydrolysis, advanced Marfey's method, quantum chemistry calculations, and X-ray diffraction analysis. Compounds 2-4 displayed promising activities against Gram-positive bacteria in vitro. Particularly, compound 4 displayed remarkable potential against vancomycin-resistant Enterococcus faecium (VRE) with an MIC value of 0.25 µg/mL, lower than the MIC (0.5 µg/mL) of the antibiotic combination quinupristin/dalfopristin (Q/D). In a further in vivo study, compound 4 increased the survival rate to 100% in the VRE-G. mellonella infection model at a concentration of 10 mg/kg.

Three new nonenes from culture broth of marine-derived fungus Albifimbria verrucaria and their cytotoxic and anti-viral activities.

Three new nonenes, verrucanonenes A‒C (1‒3), were isolated from culture broth of marine-derived fungus Albifimbria verrucaria. These compounds were isolated using silica gel column chromatography, reversed-phase medium pressure liquid chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. Their structures were determined using a spectroscopic method. Cytotoxicities of these isolated compounds to A549, DU145, HCT116, and HT1080 cancer cell lines were assessed. Compounds 1‒3 exhibited cytotoxicities to DU145 cancer cell line, with IC50 values of 23.4, 28.6, and 20.1 µM, respectively. Compound 2 decreased H1N1-induced cytopathic effects on MDCK cells in a dose-dependent manner.

The cryptic metabolites and anti-phytopathogenic activities from Nigrospora lacticolonia and Penicillium rubens uncovered by the synergism with host Paris polyphylla, monoculture, and co-culture.

The synergism of host Paris polyphylla medium, the monoculture, and the coculture led to seventeen new metabolites, including eight sesquiterpenes, 1-7 having uncommon structural motifs compared to similar caryophyllene derivatives, 8 with an unprecedented bicyclic framework, and three xyloketals (13-15) with unprecedented frameworks from Nigrospora lacticolonia; one polyketide, 17 with novel bicyclo [2.2.2] undecane skeleton, and five polyketide-terpenoid hybrids, 20 (one novel sulfated), 21-24 from Penicillium rubens. The structures were determined mainly by the NMR, HRESIMS, ECD calculation, and single-crystal X-ray diffraction. Nine cryptic compounds (2-4, 5, 12-15, 17) were produced by the inductions of host medium and the coculture. The compounds 13 from N. lacticolonia, 24-26, 28, 29, and 31 from P. rubens indicated significant antiphytopathogenic activities against N. lacticolonia with MICs at 2-4 µg/mL. Moreover, compounds 22-26, 28, 29, and 31 from P. rubens showed antifungal activities against P. rubens with MICs at 2-4 µg/mL. The synergistic effects of host medium and the coculture can induce the structural diversity of metabolites.

Xylarkarynone A and B: Two Bioactive Eremophilane Sesquiterpenes from Xylaria sp. HHY-2.

A new compound xylarkarynone A (1), a first reported natural product compound xylarkarynone B (2) and eight known compounds (3-10) were isolated from Xylaria sp. HHY-2. Their structures were elucidated by spectroscopic methods, DP4+ probability analyses and electronic circular dichroism (ECD) calculations. The bioactivities of isolated compounds were assayed. Compound 1 exhibited obvious activity against A549 cells with an IC50 value of 6.12±0.28 µM. Additionally, compound 1 showed moderate antifungal activities against Plectosphaerella cucumerina and Aspergillus niger with minimum inhibitory concentrations (MICs) of both 16 µg/mL, which was at the same grade with positive control nystatin. Most compounds exhibited varying degrees of inhibitory activity against P. cucumerina, indicating that Xylaria sp. has potential as inhibitors against P. cucumerina.

Bioactive Alkaloids from the Mangrove-Derived Fungus Nigrospora oryzae SYSU-MS0024.

Chemical investigation of marine fungus Nigrospora oryzae SYSU-MS0024 cultured on solid-rice medium led to the isolation of three new alkaloids, including a pair of epimers, nigrosporines A (1) and B (2), and a pair of enantiomers, (+)-nigrosporine C (+)-3, and (-)-nigrosporine C (-)-3, together with eight known compounds (4-11). Their structures were elucidated based on extensive mass spectrometry (MS) and 1D/2D nuclear magnetic resonance (NMR) spectroscopic analyses and compared with data in the literature. The absolute configurations of compounds 1-3 were determined by a combination of electronic circular dichroism (ECD) calculations, Mosher's method, and X-ray single-crystal diffraction technique using Cu Kα radiation. In bioassays, compound 2 exhibited moderate inhibition on NO accumulation induced by lipopolysaccharide (LPS) on BV-2 cells in a dose-dependent manner at 20, 50, and 100 µmol/L and without cytotoxicity in a concentration of 100.0 µmol/L. Moreover, compound 2 also showed moderate acetylcholinesterase (AChE) inhibitory activities with IC50 values of 103.7 µmol/L. Compound 5 exhibited moderate antioxidant activity with EC50 values of 167.0 µmol/L.

Chemical Composition and Antimicrobial Properties of Morchella crassipes (Ascomycota) from Kashmir Valley (India).

In the current era, wild macrofungi are being focused for developing and overing novel bioactive compounds for the management of agricultural, horticultural, and other infectious diseases. In that view, current research work was designed to evaluate the biochemical composition and medicinal properties of Morchella crassipes mushroom. The mycochemical screening of aqueous extract exposed the incidence of glycosides, free amino acids and proteins, alkaloids, carbs, flavonoids, terpenoids, phenolic compounds and tannins, except volatile oils, resins, steroids, and anthraquinones. However, hexane extract exhibited the occurrence of glycosides, alkaloids, volatile oils, steroids and terpenoids while as all other phytochemicals were not detected. The gas chromatography mass spectrometry profiling has disclosed the identification of three predominant naturally occurring bioactive volatile monoterpenoids, namely neral, citral, and epoxy-linalool oxide with well-known biological activities. The methanolic extract resulted in strong antifungal efficacy against the tested fungal strains such as Penicillium chrysogenum (20.33 ± 0.57 mm) followed by Pythium ultimum (15.33 ± 0.76 mm) and Aspergillus niger (12.50 ± 0.50 mm) at highest concentrations. Likewise, marked antibacterial effects were reported in case of Staphylococcus aureus (15.16 ± 0.76 mm), followed by Salmonella gallinarum (14.33 ± 0.57 mm) and Escherichia coli (13.66 ± 0.57 mm), respectively. This data may offer baseline information regarding the bioactive metabolites and opening new ways for conducting trails to find natural management strategies to combat multi drug resistant pathogens in horticulture, agriculture, and aquaculture.

Eudistomins Y-Inspired Design and Divergent Optimization of Heteroaryl Ketones for New Antifungal Leads.

The discovery of structurally distinct leads is imperative in modern agrochemical science. Inspired by eudistomins Y and the framework-related pharmaceuticals, aryl heteroaryl ketone was drawn as a common model intriguing the design and divergent synthesis of 14 kinds of heteroaryl ketones aligned with their oxime derivatives. Antifungal function-oriented phenotypical screen protruded benzothiazolyl-phenyl oxime 5a as a promising model, and the concomitant modification led to benzothiazolyl oxime 5am (EC50 = 5.17 µM) as a superior lead than fluoxastrobin (EC50 = 7.54 µM) against Sclerotinia sclerotiorum. Scaffold hopping of the phenyl subunit identified benzothiazolyl-pyridyl oxime as a novel antifungal scaffold accompanied by acquiring oxime 5bm with remarkable activity (EC50 = 3.57 µM) against Pyricularia oryzae. Molecular docking showed that candidate 5am could form more hydrogen bonds with the amino acid residues of actin than metrafenone. This compound also demonstrated better curative efficacy than that of fluoxastrobin and metrafenone in controlling the plant disease caused by S. sclerotiorum. These results rationalize the discovery of antifungal candidates based on aryl heteroaryl ketone.

A new resorcylic acid lactone from the endophytic fungus Chaetosphaeronema sp.

A new 14-membered resorcylic acid lactone (RAL14), chaetolactone A (1), along with three known ones (2-4), was obtained from the fermentation of the soil-derived fungus Chaetosphaeronema sp. SSJZ001. Their structures were established based on extensive spectroscopic data analyses (UV, IR, HRESIMS, 1D, and 2D NMR),13C NMR chemical shifts calculations coupled with the DP4+ probability method, theoretical calculations of ECD spectra, as well as X-ray diffraction analysis. All compounds were evaluated for their cytotoxic effects against A549, HO-8910, and MCF-7 cell lines.

Diaporchalasins A-E, New Cytochalasins from the Endophytic Fungus Diaporthe sp. BMX12 Isolated from Aquilaria sinensis.

Five new cytochalasins, diaporchalasins A-E (1-5), together with 14 known congeners (6-19) were isolated from the endophytic fungus Diaporthe sp. BMX12, which was isolated from the branches of Aquilaria sinensis. The structures of the new compounds were elucidated by extensive spectroscopic analyses including high-resolution electron spray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR). Their absolute configurations were assigned by theoretical electronic circular dichroism (ECD) calculations. Compounds 11 and 12 featuring a keto carbonyl at C-21 displayed cytotoxicity toward K562, BEL-7402, SGC-7901, A549, and HeLa cell lines with IC50 values ranging from 4.4 to 47.4 µM.

Identification of Bis(methylsulfanyl)methane and Furan-2(5H)-one as Volatile Marker Compounds for the Differentiation of the White Truffle Species Tuber magnatum and Tuber borchii.

Some truffles are expensive and, therefore, are prone to food fraud. A particular problem is the differentiation of high-priced Tuber magnatum truffles from cheaper Tuber borchii truffles, both of which are white truffles with similar morphological characteristics. Using an untargeted approach, the volatiles isolated from samples of both species were screened for potential marker compounds by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and statistical analysis of the obtained semiquantitative data. Results suggested bis(methylsulfanyl)methane and furan-2(5H)-one as compounds characterizing T. magnatum and T. borchii, respectively. Exact quantitation of both volatiles by conventional one-dimensional gas chromatography-mass spectrometry in combination with stable isotopologues of the target compounds as internal standards confirmed both as marker compounds. The method is suitable to be used in the routine analysis for the objective species differentiation of T. magnatum and T. borchii.

Antimicrobial properties of unusual eremophilanes from the endophytic Diaporthe sp. BCC69512.

Six undescribed infrequent eremophilane derivatives including diaportheremopholins A - F and its previously undescribed side chain (E)-2-methyloct-2-enoic acid, together with three known compounds (testacein, xestodecalactones B and C), were isolated from the endophytic fungus Diaporthe sp. BCC69512. The chemical structures were determined based on NMR spectroscopic information in conjunction with the evidence from NOESY spectrum, Mosher's application, and chemical reactions for corroborating the absolute configurations. The isolated compounds were evaluated for biological properties such as antimalarial, anti-TB, anti-phytopathogenic fungal, antibacterial activities and for cytotoxicity against malignant (MCF-7 and NCI-H187) and non-malignant (Vero) cells. Diaportheremopholins B (2) and E (5) possessed broad antimicrobial activity against Mycobacterium tuberculosis, Bacillus cereus, Alternaria brassicicola and Colletotrichum acutatum with MICs in a range of 25.0-50.0 µg/mL. Testacein (7) exhibited strong anti-A. brassicicola and anti-C. acutatum activities with equal MIC values of 3.13 µg/mL. Moreover, diaportheremopholin F (6) and compound 8 displayed antitubercular activity with equal MIC values of 50.0 µg/mL. All tested compounds were non-cytotoxic against MCF-7, NCI-H187, and Vero cells, except those compounds 2 and 5-7 exhibited weak cytotoxicity against both malignant and non-malignant cells with IC50 values in a range of 15.5-115.5 µM.

Structure-Activity Relationships of Natural C-9-Methyl-Substituted 10-Membered Lactones and Their Semisynthetic Derivatives.

Fungal 10-membered lactones (TMLs), such as stagonolide A, herbarumin I, pinolidoxin, and putaminoxin, are promising candidates for the development of nature-derived herbicides. The aim of this study was to analyze the structure-activity relationships (SAR) of C-9-methyl-substituted TMLs with a multitarget bioassay approach to reveal compounds with useful (phytotoxic, entomotoxic, antimicrobial) or undesirable (cytotoxic) bioactivities. A new TML, stagonolide L (1), along with five known compounds (stagonolides D (2) and E (3), curvulides A (4) and B1/B2 (5a,b), and pyrenolide C (6)), were purified from cultures of the phytopathogenic fungus Stagonospora cirsii, and five semisynthetic derivatives of 3 and 4 (7-11) were obtained. The absolute configuration of 4 was revised to 2Z, 4S, 5S, 6R, and 9R. The identity of 5a,b and stagonolide H is discussed. The phytotoxicity of compound 4, the entomotoxicity of 5a,b, and nonselective toxicity of compound 6 are demonstrated. The latter confirms the hypothesis that the α,ß-unsaturated carbonyl group is associated with the high general toxicity of TML, regardless of its position in the ring and other substituents. The epoxide in compound 4 is important for phytotoxicity. The revealed SAR patterns will be useful for further rational design of TML-based herbicides including curvulide A analogs with a 4,5-epoxy group.

New pimarane diterpenoids with antibacterial activity from fungus Arthrinium sp. ZS03.

A comprehensive chemical study of the endophytic fungus Arthrinium sp. ZS03, associated with Acorus tatarinowii Schott, yielded eleven pimarane diterpenoids (compounds 1-11), including seven novel compounds designated arthrinoids A-G (1-7). The determination of their structures and absolute configurations was achieved through extensive spectroscopic techniques, quantum chemical calculations of electronic circular dichroism (ECD), and single-crystal X-ray diffraction analysis. Furthermore, 7 demonstrated inhibitory activity against Klebsiella pneumoniae, comparable to the reference antibiotic amikacin, with a minimum inhibitory concentration (MIC) of 8 µg·mL-1.