New cytotoxic ergosterols from a plant-associated fungus Colletotrichum magnisporum.

Three new ergosterols, colletosterols A-C (1-3), together with two known analogues 4 and 5, were isolated from the endophytic fungus Colletotrichum magnisporum associated with the leaves of Rauvolfia verticillata by a bioassay-guided fractionation method. The new structures were elucidated on the basis of extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. All the ergosterols were evaluated for their cytotoxic activities against A549 and HeLa cell lines. Compounds 1-3 exhibited notable cytotoxicity with the IC50 values of 3.76-11.18 µM.

Colletofragarone A2 and Colletoins A-C from a Fungus Colletotrichum sp. Decrease Mutant p53 Levels in Cells.

p53 is frequently mutated in tumor cells. Mutant p53 (mut p53) accumulates in cells to promote cancer progression, invasion, and metastasis, and it is attracting attention as a target for cancer therapies. In this study, we used immunofluorescence staining of Saos-2 cells harboring doxycycline-inducible p53R175H [Saos-2 (p53R175H) cells] to search for compounds from natural sources that can target mut p53 and found an extract of Colletotrichum sp. (13S020) that was active. Bioassay-guided fractionation of the extract afforded a known polyketide, colletofragarone A2 (1), and three new analogues, colletoins A-C (2-4). The relative and absolute configurations of 1 were determined by the spectroscopic method and DFT calculation. Compounds 1 and 2 inhibited the growth of Saos-2 (p53R175H) cells and decreased mut p53 in the cells.

Prodrugs for nitroreductase based cancer therapy-4: Towards prostate cancer targeting: Synthesis of N-heterocyclic nitro prodrugs, Ssap-NtrB enzymatic activation and anticancer evaluation.

In this study, various N-heterocyclic nitro prodrugs (NHN1-16) containing pyrimidine, triazine and piperazine rings were designed and synthesized. The final compounds were identified using FT-IR, 1H NMR, 13C NMR as well as elemental analyses. Enzymatic activities of compounds were conducted by using HPLC analysis to investigate the interaction of substrates with Ssap-NtrB nitroreductase enzyme. MTT assay was performed to evaluate the toxic effect of compounds against Hep3B and PC3 cancer cell lines and healthy HUVEC cell. It was observed that synthesized compounds NHN1-16 exhibited different cytotoxic profiles. Pyrimidine derivative NHN3 and triazine derivative NHN5 can be good drug candidates for prostate cancer with IC50 values of 54.75 µM and 48.9 µM, respectively. Compounds NHN6, NHN10, NHN12, NHN14 and NHN16 were selected as prodrug candidates because of non-toxic properties against three different cell models. The NHN prodrugs and Ssap-NtrB combinations were applied to SRB assay to reveal the prodrug capabilities of these selected compounds. SRB screening results showed that the metabolites of all selected non-toxic compounds showed remarkable cytotoxicity with IC50 values in the range of 1.71-4.72 nM on prostate cancer. Among the tested compounds, especially piperazine derivatives NHN12 and NHN14 showed significant toxic effect with IC50 values of 1.75 nM and 1.79 nM against PC3 cell compared with standart prodrug CB1954 (IC50: 1.71 nM). Novel compounds NHN12 and NHN14 can be considered as promising prodrug candidates for nitroreductase-prodrug based prostate cancer therapy.

Colletotrichalactones A-Ca, unusual 5/6/10-fused tricyclic polyketides produced by an endophytic fungus, Colletotrichum sp. JS-0361.

Three unusual polyketides with a 5/6/10-fused ring system, named colletotrichalactones A-Ca (1-3a), were isolated from cultures of the endophytic fungus, Colletotrichum sp. JS-0361, which was isolated from a leaf of Morus alba. Their structures, including their absolute stereochemistries, were completely established using extensive spectroscopic methods together with a chemical reaction utilizing competing enantioselective acylation coupled with LC/MS. Compounds possessing this ring skeleton were previously reported in three studies. Our rigorous chemical investigation revealed the complete configuration of this skeleton, which agreed with the results for glabramycin B with this ring skeleton established by computational chemistry and enantioselective synthesis in previous reports. 1 and 2 had unstable aldehyde groups that were easily converted to acetal groups in the presence of solvents. Meanwhile, compound 3a, with terminal acetal functionality, was deduced to be an artefact originating from compound 3 with a terminal aldehyde group. Compounds 1 and 3a displayed moderate-to-potent cytotoxic activities against MCF7 cells with IC50s of 35.06 and 25.20 µM, respectively.

Collacyclumines A-D from the endophytic fungus Colletotrichum salsolae SCSIO 41021 isolated from the mangrove Kandelia candel.

Four undescribed alkaloids, namely collacyclumines A-D, along with a known analogue, agrocybenine, were isolated from the endophytic fungus Colletotrichum salsolae SCSIO 41021 derived from the mangrove plant Kandelia candel (L.) Druce. Collacyclumine A represents the first case of dimeric pyrrolidine alkaloid in nature. The structures of these compounds were elucidated by a combination of NMR spectra, HRESIMS data, and X-ray diffraction experiment. A proposed biosynthetic pathway of these isolated compounds were also discussed. None of compounds showed cytotoxic effects against ten cell lines.

Four New Chromone Derivatives from Colletotrichum gloeosporioides.

Four previously unreported chromones, 5-hydroxy-2-(hydroxymethyl)-8-methoxy-4H-chromen-4-one (1), (5R,7S)-5,7-dihydroxy-2-propyl-5,6,7,8-tetrahydro-4H-chromen-4-one (2), (5R,7S)-5,7-dihydroxy-2-methyl-5,6,7,8-tetrahydro-4H-chromen-4-one (3), and (5R,7S)-5,7-dihydroxy-2-[(E)-prop-1-en-1-yl]-5,6,7,8-tetrahydro-4H-chromen-4-one (4), as well as one known analogue 5-hydroxy-2-methyl-4H-chromen-4-one (5) were isolated from the fermentation broth of the endophytic fungus Colletotrichum gloeosporioides derived from the mangrove Ceriops tagal. Their structures were elucidated based on extensive spectroscopic analyses. The absolute configurations of 2-4 were determined by comparison the experimental and calculated electronic circular dichroism (ECD) spectra. Compound 2 showed cytotoxic activity against A549 cell line with the IC50 value of 0.094 mm.

Colletoindole A from the Mangrove Plant Endophytic Fungus Colletotrichum tropicale SCSIO 41022.

A new indole derivative colletoindole A (1), along with two new indole derivatives (2 and 3) and one known compound acropyrone (4) were isolated from cultures of Colletotrichum tropicale SCSIO 41022 derived from a mangrove plant Kandelia candel. The structures of 1-4 were determined by analysis of NMR and MS data. The cytotoxicity of 1, 2 and 4, and the COX-2 inhibitory activity of 1 and 2 were evaluated.

Colletopeptides A-D, Anti-inflammatory Cyclic Tridepsipeptides from the Plant Endophytic Fungus Colletotrichum sp. S8.

Four new hybrid peptide-polyketide cyclic tridepsipeptides, colletopeptides A-D (1-4), were isolated and characterized from the endophytic fungus Colletotrichum sp. S8 derived from the stems of Rubia podantha with the guidance of LC-UV-MS detection. Their structures were elucidated by extensive spectroscopic analysis and X-ray crystallography. Compounds 1-4 are rare natural 12-membered cyclic tridepsipeptides containing a 3,5,11-trihydroxy-2-methyl dodecanoic acid unit in their structures. 1-4 inhibited lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages with the IC50 values of 8.3, 38.7, 13.5, and 22.2 µM, respectively. 1 also inhibited the production of inflammatory factors IL-6 and TNF-α, and decreased the phosphorylation of NF-κB-associated proteins IκBα and p65.

CfPDIP1, a novel secreted protein of Colletotrichum falcatum, elicits defense responses in sugarcane and triggers hypersensitive response in tobacco.

Colletotrichum falcatum, a hemibiotrophic fungal pathogen, causes one of the major devastating diseases of sugarcane-red rot. C. falcatum secretes a plethora of molecular signatures that might play a crucial role during its interaction with sugarcane. Here, we report the purification and characterization of a novel secreted protein of C. falcatum that elicits defense responses in sugarcane and triggers hypersensitive response (HR) in tobacco. The novel protein purified from the culture filtrate of C. falcatum was identified by MALDI TOF/TOF MS and designated as C. falcatum plant defense-inducing protein 1 (CfPDIP1). Temporal transcriptional profiling showed that the level of CfPDIP1 expression was greater in incompatible interaction than the compatible interaction until 120 h post-inoculation (hpi). EffectorP, an in silico tool, has predicted CfPDIP1 as a potential effector. Functional characterization of full length and two other domain deletional variants (CfPDIP1ΔN1-21 and CfPDIP1ΔN1-45) of recombinant CfPDIP1 proteins has indicated that CfPDIP1ΔN1-21 variant elicited rapid alkalinization and induced a relatively higher production of hydrogen peroxide (H2O2) in sugarcane suspension culture. However, in Nicotiana tabacum, all the three forms of recombinant CfPDIP1 proteins triggered HR along with the induction of H2O2 production and callose deposition. Further characterization using detached leaf bioassay in sugarcane revealed that foliar priming with CfPDIP1∆1-21 has suppressed the extent of lesion development, even though the co-infiltration of CfPDIP1∆1-21 with C. falcatum on unprimed leaves increased the extent of lesion development than control. Besides, the foliar priming has induced systemic expression of major defense-related genes with the concomitant reduction of pathogen biomass and thereby suppression of red rot severity in sugarcane. Comprehensively, the results have suggested that the novel protein, CfPDIP1, has the potential to trigger a multitude of defense responses in sugarcane and tobacco upon priming and might play a potential role during plant-pathogen interactions.

Secondary metabolites from the Colletotrichum gloeosporioides A12, an endophytic fungus derived from Aquilaria sinensis.

Two new cyclohexene derivatives colletotricones A and B (1 and 2) and a new thiazole derivative colletotricole A (5), along with six known natural metabolites were isolated from the extract of Colletotrichum gloeosporioides A12, an endophytic fungus derived from Aquilaria sinensis. Among them, the colletotricones A and B possess a cyclohexenone skeleton, whereas the colletotricole A is a thiazole derivative. Their structures were fully assigned with the aid of extensive spectroscopic analysis and data from the literature. Moreover, cytotoxic activity in vitro of compounds 1 and 3-9 were evaluated against MCF-7, NCI-H460, HepG-2 and SF-268 tumour cell lines. The new compound 1 exhibited growth inhibitory activity against all the four tumour cell lines with IC50 values ranging from 15.7 to 46.8 µM.

Citrinal B, natural 11 beta-hydroxysteroid dehydrogennase type 1 inhibitor identified from structure-based virtual screening.

Citrinal B, a tricyclic compound from endophytic fungus Colletotrichum capsici in our previous studies, exhibited significant inhibitory activity against 11ß-hydroxysteroid dehydrogenase type 1 (11 ß-HSD1) in vitro and showed strong binding affinity to 11ß-HSD1. Moreover, citrinal B treatments decreased the lipid droplet accumulation associate with the inhibition of 11ß-HSD1 expression in differentiate induced 3T3-L1 preadipocytes. Furthermore, the molecular docking demonstrated that citrinal B coordinated in the active site of 11ß-HSD1 is essential for the ability of diminishing the enzyme activity.

Comparative secretome analysis of Colletotrichum falcatum identifies a cerato-platanin protein (EPL1) as a potential pathogen-associated molecular pattern (PAMP) inducing systemic resistance in sugarcane.

Colletotrichum falcatum, an intriguing hemibiotrophic fungal pathogen causes red rot, a devastating disease of sugarcane. Repeated in vitro subculturing of C. falcatum under dark condition alters morphology and reduces virulence of the culture. Hitherto, no information is available on this phenomenon at molecular level. In this study, the in vitro secretome of C. falcatum cultured under light and dark conditions was analyzed using 2-DE coupled with MALDI TOF/TOF MS. Comparative analysis identified nine differentially abundant proteins. Among them, seven proteins were less abundant in the dark-cultured C. falcatum, wherein only two protein species of a cerato-platanin protein called EPL1 (eliciting plant response-like protein) were found to be highly abundant. Transcriptional expression of candidate high abundant proteins was profiled during host-pathogen interaction using qRT-PCR. Comprehensively, this comparative secretome analysis identified five putative effectors, two pathogenicity-related proteins and one pathogen-associated molecular pattern (PAMP) of C. falcatum. Functional characterization of three distinct domains of the PAMP (EPL1) showed that the major cerato-platanin domain (EPL1∆N1-92) is exclusively essential for inducing defense and hypersensitive response (HR) in sugarcane and tobacco, respectively. Further, priming with EPL1∆N1-92 protein induced systemic resistance and significantly suppressed the red rot severity in sugarcane. BIOLOGICAL SIGNIFICANCE: Being the first secretomic investigation of C. falcatum, this study has identified five potential effectors, two pathogenicity-related proteins and a PAMP. Although many reports have highlighted the influence of light on pathogenicity, this study has established a direct link between light and expression of effectors, for the first time. This study has presented the influence of a novel N-terminal domain of EPL1 in physical and biological properties and established the functional role of major cerato-platanin domain of EPL1 as a potential elicitor inducing systemic resistance in sugarcane. Comprehensively, the study has identified proteins that putatively contribute to virulence of C. falcatum and for the first time, demonstrated the potential role of EPL1 in inducing PAMP-triggered immunity (PTI) in sugarcane.

Microbial Natural Product Alternariol 5-O-Methyl Ether Inhibits HIV-1 Integration by Blocking Nuclear Import of the Pre-Integration Complex.

While Highly Active Antiretroviral Therapy (HAART) has significantly decreased the mortality of human immunodeficiency virus (HIV)-infected patients, emerging drug resistance to approved HIV-1 integrase inhibitors highlights the need to develop new antivirals with novel mechanisms of action. In this study, we screened a library of microbial natural compounds from endophytic fungus Colletotrichum sp. and identified alternariol 5-O-methyl ether (AME) as a compound that inhibits HIV-1 pre-integration steps. Time-of addition analysis, quantitative real-time PCR, confocal microscopy, and WT viral replication assay were used to elucidate the mechanism. As opposed to the approved integrase inhibitor Raltegravir, AME reduced both the integrated viral DNA and the 2-long terminal repeat (2-LTR) circular DNA, which suggests that AME impairs the nuclear import of viral DNA. Further confocal microscopy studies showed that AME specifically blocks the nuclear import of HIV-1 integrase and pre-integration complex without any adverse effects on the importin α/ß and importin ß-mediated nuclear import pathway in general. Importantly, AME inhibited Raltegravir-resistant HIV-1 strains and exhibited a broad anti-HIV-1 activity in diverse cell lines. These data collectively demonstrate the potential of AME for further development into a new HIV inhibitor, and suggest the utility of viral DNA nuclear import as a target for anti-HIV drug discovery.

Fungal Endophytes: an Alternative Source for Production of Volatile Compounds from Agarwood Oil of Aquilaria subintegra.

Fungal endophytes are microorganisms that are well-known for producing a diverse array of secondary metabolites. Recent studies have uncovered the bioprospecting potential of several plant endophytic fungi. Here, we demonstrate the presence of highly bioactive fungal endophytic species in Aquilaria subintegra, a fragrant wood plant collected from Thailand. Thirty-three fungal endophytic strains were isolated and further identified to genus level based on morphological characteristics. These genera included Colletotrichum, Pestalotiopsis, Fusarium, Russula, Arthrinium, Diaporthe and Cladosporium. All strains were cultured on potato dextrose broth for 30 days prior to partitioning with ethyl acetate. The volatile compounds of all extracts were investigated by gas chromatography-mass spectrometry (GC-MS). Four strains-Arthrinium sp. MFLUCC16-0042, Colletotrichum sp. MFLUCC16-0047, Colletotrichum sp. MFLUCC16-0048 and Diaporthe sp. MFLUCC16-0051-produced a broad spectrum of volatile compounds, including ß-agarofuran, α-agarofuran, δ-eudesmol, oxo-agarospirol, and ß-dihydro agarofuran. These compounds are especially important, because they greatly resemble those originating from the host-produced agarwood oil. Our findings demonstrate the potential of endophytic fungi to produce bioactive compounds with applications in perfumery and cosmetic industries. Antioxidant activity of all extracts was also evaluated by using 2,2-diphenyl-2-picrylhydrazyl radical scavenging assays. The ethyl acetate extract of Diaporthe sp. MFLUCC16-0051 demonstrated superior antioxidant capacity, which was comparable to that of the gallic acid standard. Our results indicate that the MFLUCC16-0051 strain is a resource of natural antioxidant with potential medicinal applications.

Phytosterols isolated from endophytic fungus Colletotrichum gloeosporioides (Melanconiaceae) / Fotoesteróis isolados do fungo endofítico Colletotrichum gloeosporioides (Melanconiaceae)

Endophytic fungi are fungi that colonize internal tissues of plants. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, the aim this study was the isolation and structural identification of sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6) from fungus Colletotrichum gloeosporioidesisolated as endophytic from Virola michelli, a typical Amazon plant, used in folk medicine against skin infection. Compounds were isolated by chromatography column on silica and identified by 1H and 13C NMR and MS. The presence of phytosterols in fungi is rare and this is the first report of the isolation of the phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum.

Fungos endofíticos são fungos que colonizam os tecidos internos das plantas. Existem poucos estudos de compostos isolados de fungos endofíticos de plantas da Amazônia. Assim, o objetivo deste estudo foi o isolamento e identificação estrutural de sitosterol (1), estigmasterol (2), sitostenona (3), esqualeno (4), ergosterol (5) e peroxido de ergosterol (6) do fungo Colletotrichum gloeosporioidesisolado como endofítico de Virola michelli, uma planta típica da Amazônia, usada na medicina popular no combate a infecções de pele. Os compostos foram isolados por cromatografia em coluna de sílica e identificados por RMN 1H e 13C e EM. A presença de fitoesteróis em fungos é rara e este é o primeiro relato do isolamento dos fitoesteróides sitosterol, estigmasterol e sitostenona do gêreno Colletotrichum.

Antibacterial Azaphilones from an Endophytic Fungus, Colletotrichum sp. BS4.

Three new compounds, colletotrichones A-C (1-3), and one known compound, chermesinone B (4a), were isolated from an endophytic fungus, Colletotrichum sp. BS4, harbored in the leaves of Buxus sinica, a well-known boxwood plant used in traditional Chinese medicine (TCM). Their structures were determined by extensive spectroscopic analyses including 1D and 2D NMR, HRMS, ECD spectra, UV, and IR, as well as single-crystal X-ray diffraction, and shown to be azaphilones sharing a 3,6a-dimethyl-9-(2-methylbutanoyl)-9H-furo[2,3-h]isochromene-6,8-dione scaffold. Owing to the remarkable antibacterial potency of known azaphilones coupled to the usage of the host plant in TCM, we evaluated the antibacterial efficacy of the isolated compounds against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis, as well as against two human pathogenic clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa. Compound 1 exhibited marked antibacterial potencies against the environmental strains that were comparable to the standard antibiotics. Compound 3 was also active against E. coli. Finally, compound 2a exhibited the same efficacy as streptomycin against the clinically relevant bacterium S. aureus. The in vitro cytotoxicity of these compounds on a human acute monocytic leukemia cell line (THP-1) was also assessed. Our results provide a scientific rationale for further investigations into endophyte-mediated host chemical defense against specialist and generalist pathogens.

Higginsianins A and B, Two Diterpenoid α-Pyrones Produced by Colletotrichum higginsianum, with in Vitro Cytostatic Activity.

Two new diterpenoid α-pyrones, named higginsianins A (1) and B (2), were isolated from the mycelium of the fungus Colletotrichum higginsianum grown in liquid culture. They were characterized as 3-[5a,9b-dimethyl-7-methylene-2-(2-methylpropenyl)dodecahydronaphtho[2,1-b]furan-6-ylmethyl]-4-hydroxy-5,6-dimethylpyran-2-one and 4-hydroxy-3-[6-hydroxy-5,8a-dimethyl-2-methylene-5-(4-methylpent-3-enyl)decahydronaphthalen-1-ylmethyl]-5,6-dimethylpyran-2-one, respectively, by using NMR, HRESIMS, and chemical methods. The structure and relative configuration of higginsianin A (1) were confirmed by X-ray diffractometric analysis, while its absolute configuration was assigned by electronic circular dichroism (ECD) experiments and calculations using a solid-state ECD/TDDFT method. The relative and absolute configuration of higginsianin B (2), which did not afford crystals suitable for X-ray analysis, were determined by NMR analysis and by ECD in comparison with higginsianin A. 1 and 2 were the C-8 epimers of subglutinol A and diterpenoid BR-050, respectively. The evaluation of 1 and 2 for antiproliferative activity against a panel of six cancer cell lines revealed that the IC50 values, obtained with cells reported to be sensitive to pro-apoptotic stimuli, are by more than 1 order of magnitude lower than their apoptosis-resistant counterparts (1 vs >80 µM). Finally, three hemisynthetic derivatives of 1 were prepared and evaluated for antiproliferative activity. Two of these possessed IC50 values and differential sensitivity profiles similar to those of 1.

Antifungal compounds produced by Colletotrichum gloeosporioides, an endophytic fungus from Michelia champaca.

In this study, eight endophytic fungi were isolated from the leaves, stems and roots of Michelia champaca. The isolates were screened and evaluated for their antifungal, anticancer and acetylcholinesterase (AChE) inhibitory activities. All of the extracts exhibited potent activity against two evaluated phytopathogenic fungi. Chemical investigation of EtOAc extracts of the endophytic fungus Colletotrichum gloeosporioides resulted in the isolation of one new compound, 2-phenylethyl 1H-indol-3-yl-acetate (1), and seven known compounds: uracil (2), cyclo-(S*-Pro-S*-Tyr) (3), cyclo-(S*-Pro-S*-Val) (4), 2(2-aminophenyl)acetic acid (5), 2(4-hydroxyphenyl)acetic acid (6), 4-hydroxy- benzamide (7) and 2(2-hydroxyphenyl)acetic acid (8). All of the compound structures were elucidated using 1D and 2D NMR and MS analyses. The antifungal and AChE inhibitory activities of compounds 1-8 were evaluated in vitro. Compound 1 exhibited promising activity against Cladosporium cladosporioides and C. sphaerospermum that was comparable to that of the positive control nystatin.

[A new sesquiterpenoid from fungus Colletotrichum sp. and its cytotoxicity].

A novel sesquiterpenoid (1) and three known compounds identified as isoaltenuene (2), altenuene (3), and alternariol 4, 10-O-dimethyl ether (4), were isolated in our investigation of the cytotoxic constituents from solid cultures of the endophytic fungus Colletotrichum sp. The structures of these compounds were elucidated through spectroscopic data analysis. All compounds exhibited cytotoxic activity against lung cancer cell line A549, breast cancer cell line MDA-MB-231 and pancreatic cancer cell line PANC-1. Compound 4 could induce the PANC-1 cells inflation or death, but couldn't induce apoptosis at the IC50 of 60.2 microg x mL(-1).

Characteristics of the toxin extracted from liquid culture of Colletotrichum capsici f. nicotianae.

Colletotrichum capsici f. nicotianae is an important plant pathogen in tobacco-grown area of Weifang region of Shangdong Province, China. In this study, the toxicity of liquid culture media from different isolates was characterized, and some properties of the toxic ingredient were identified. The results indicated that the optimal toxin-producing conditions for C. capsici f. nicotianae were in potato dextrose broth under pH 6.0, at 25~30 °C for 13 days. The liquid culture media from all isolates were toxic to tobacco plants and induced the wilting symptoms. The toxin from the liquid culture media has thermal, acid-base stability and a broad spectrum of toxicity to the plants. Furthermore, the direct bioassay for two components of the liquid filtrates precipitated by ethanol showed that the active ingredient of the toxin is a kind of nonprotein substance, which was further supported by the papain hydrolysis test.