New insights into the anti-inflammatory and anti-melanoma mechanisms of action of azelaic acid and other Fusarium solani metabolites via in vitro and in silico studies.

Metabolites exploration of the ethyl acetate extract of Fusarium solani culture broth that was isolated from Euphorbia tirucalli root afforded five compounds; 4-hydroxybenzaldehyde (1), 4-hydroxybenzoic acid (2), tyrosol (3), azelaic acid (4), malic acid (5), and fusaric acid (6). Fungal extract as well as its metabolites were evaluated for their anti-inflammatory and anti-hyperpigmentation potential via in vitro cyclooxygenases and tyrosinase inhibition assays, respectively. Azelaic acid (4) exhibited powerful and selective COX-2 inhibition followed by fusaric acid (6) with IC50 values (2.21 ± 0.06 and 4.81 ± 0.14 µM, respectively). As well, azelaic acid (4) had the most impressive tyrosinase inhibitory effect with IC50 value of 8.75 ± 0.18 µM compared to kojic acid (IC50 = 9.27 ± 0.19 µM). Exclusive computational studies of azelaic acid and fusaric acid with COX-2 were in good accord with the in vitro results. Interestingly, this is the first time to investigate and report the potential of compounds 3-6 to inhibit cyclooxygenase enzymes. One of the most invasive forms of skin cancer is melanoma, a molecular docking study using a set of enzymes related to melanoma suggested pirin to be therapeutic target for azelaic acid and fusaric acid as a plausible mechanism for their anti-melanoma activity.

A target fishing study to spot possible biological targets of fusaric acid: Inhibition of protein kinase-A and insights on the underpinning mechanisms.

Fusaric acid is a secondary metabolite produced by various Fusarium fungi, present with relatively high incidence in Fusarium-contaminated foods. It was already described as phytotoxic and cytotoxic. However, the understanding of its molecular mechanisms is still fragmentary and further data are needed to ensure an informed assessment of the risk related to its presence in food. This work applied an integrated in silico/in vitro approach to reveal novel potential biological activities of fusaric acid and to investigate the underpinning mechanisms. An in silico reverse screening was used to identify novel biological targets for fusaric acid. Computational results indicated as target protein kinase-A, which was confirmed with biochemical cell-free assays providing evidence of its actual inhibitory potential. Cell-based experiments on intestinal cells (HCEC-1CT cells) identified the mitochondrial network and cell membranes as potentially affected organelles, possibly resulting from PKA inhibition. The integration of 3D molecular modeling supported the plausibility of fusaric acid-dependent inhibition. From the hazard identification perspective, considering the Low Observed Adverse Effect Level described here (0.1 mM) and the possible level of contamination in food, fusaric acid might raise concern from a food safety standpoint and the gastrointestinal tract was described as a meaningful system to investigate with priority.

Concise Total Synthesis and Antifungal Activities of Fusaric Acid, a Natural Product.

The total synthesis of a natural product alkaloid fusaric acid (FA), which exhibits herbicide, fungicide, insecticide and even diverse notable pharmacological activities, was accomplished in four steps using commercially available materials. The synthesis, based on a unified and flexible strategy using 6-bromonicotinaldehyde as a common intermediate, is concise, convergent, practical and can be carried out on a two-gram scale. This approach could be readily applicable to the synthesis of its analogues. In addition, FA had a wide range of inhibitory activities against 14 plant pathogenic fungi in this study, which demonstrated that as a leading compound, and it has great potential to be further developed as an agricultural fungicide.

Hypothermia induced by central injection of sucralose potentially occurs via monoaminergic pathways in the hypothalamus of chicks.

Oral administration of sucralose has been reported to stimulate food intake through inducing hypothalamic neuropeptide Y (NPY) in mice and fruit flies. However, the underlying mechanisms of action of sucralose in hypothermia and NPY and monoamine regulation remain unknown. The aim of the present study was to investigate central effects of sucralose on body temperature, NPY, and monoamine regulation, as well as its peripheral effects, in chicks. In Experiment 1, 5-day-old chicks were centrally injected with 1 µmol of sucralose, other sweeteners (erythritol and glucose), or saline. In Experiment 2, chicks were centrally injected with 0.2, 0.4, and 1.6 µmol of sucralose or saline. In Experiment 3, chicks were centrally injected with 0.8 µmol of sucralose or saline, with a co-injection of 100 µg fusaric acid (FA), an inhibitor of dopamine-ß-hydroxylase, to examine the role dopamine in sucralose induced hypothermia. In Experiment 4, 7-16-day-old chicks were orally administered with 75, 150, and 300 mg/2 ml distilled water or sucralose, daily. We observed that the central injection of sucralose, but not other sweeteners, decreased body temperature (P < .05) in chicks; however, the oral injection did not influence body temperature, food intake, and body weight gain. Central sucralose administration decreased dopamine and serotonin and stimulated dopamine turnover rate in the hypothalamus significantly (P < .05). Notably, sucralose co-injection with FA impeded sucralose-induced hypothermia. Sucralose decreases body temperature potentially via central monoaminergic pathways in the hypothalamus.

Fusaricates H-K and fusolanones A-B from a mangrove endophytic fungus Fusarium solani HDN15-410.

Seven compounds including four undescribed fusaric acid derivatives, namely fusaricates H-K, and two undescribed γ-pyrone derivatives, named fusolanones A-B, as well as a known compound fusaric acid, were isolated from a mangrove endophytic fungus Fusarium solani. Fusaricates H-K represent the first cases of fusaric acid butanediol esters and are diastereoisomers. Their structures including absolute configurations were elucidated based on NMR, MS, chemical synthesis, chiral HPLC analysis and ECD calculations. The antibacterial activity of all undescribed compounds were tested and fusolanone B showed the best activity with MIC value 6.25 µg/mL on Vibrio parahaemolyticus.

Methyl Fumarate-Derived Iron Carbonyl Complexes (FumET-CORMs) as Powerful Anti-inflammatory Agents.

Autoimmune diseases are characterized by dendritic cell (DC)-driven activation of pro-inflammatory T cell responses. Therapeutic options for these severe diseases comprise small molecules such as dimethyl fumarate, or "gasotransmitters" such as CO. Herein we describe the synthesis of bifunctional enzyme-triggered CO-releasing molecules (ET-CORMs) that allow the simultaneous intracellular release of both CO and methyl fumarate. Using bone-marrow-derived DCs the impressive therapeutic potential of these methyl fumarate-derived compounds (FumET-CORMs) is demonstrated by strong inhibition of lipopolysaccharide-induced pro-inflammatory signaling pathways and blockade of downstream interleukin-12 or -23 production. The data also show that FumET-CORMs are able to transform DCs into an anti-inflammatory phenotype. Thus, these novel compounds have great clinical potential, for example, for the treatment of psoriasis or other inflammatory conditions of the skin.

Role of fusaric acid in the development of 'Fusarium wilt' symptoms in tomato: Physiological, biochemical and proteomic perspectives.

Fusarium wilt is one of the most prevalent and damaging diseases of tomato. Among various toxins secreted by the Fusarium oxysporum f. sp. lycopersici (causal agent of Fusarium wilt of tomato), fusaric acid (FA) is suspected to be a potent pathogenicity factor in tomato wilt disease development. With this rationale the present study was carried out with physiological, biochemical and proteomic perspectives. Treatment of FA was given to the leaves of tomato directly through infiltration to show the characteristic features of Fusarium wilt of tomato. The phytotoxic effect of FA was assessed in the form of cell death in tomato leaves which was observed by increased uptake of Evans blue stain. The measurement of electrolyte leakage was used as an indicator of the extent of cell death. The influence of FA on the leaf photosynthesis of tomato plant was investigated and it was found that FA strongly reduced the photosynthetic pigment contents of tomato leaves resulting to heavy suppression of leaf photosynthesis processes, which therefore affected leaf physiology finally leading to leaf wilting and necrosis. This cell death inducer (FA) produced an enormous oxidative burst during which large quantities of reactive oxygen species (ROS) like H2O2 was generated in the treated leaf tissues of tomato plants which was evident from enhancement in lipid peroxidation. To assess the involvement of proteolysis in the cell death cascade induced by FA treatment, total protease activity was measured in the leaf tissues and it was found that the total protease activity increased with the treatment and leading to cell death. Furthermore, proteomic study was used as a powerful tool to understand the alterations in cellular protein expression in response to FA exposure. Differential expression in several proteins was observed in the present study. Proteomic analyses, thus, clearly indicate that proteins belonging to different functional classes are significantly affected in the plant leaf tissues after FA exposure leading to deterioration of structure and metabolism of cells. Thus, it is concluded that FA plays an important role in fungal pathogenicity by decreasing cell viability.

Detoxification of Fusaric Acid by the Soil Microbe Mucor rouxii.

Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114), which causes root rot and wilt of cotton (Gossypium hirsutum and G. barbadense), has been identified recently for the first time in the western hemisphere in certain fields in the San Joaquin Valley of California. This pathotype produces copious quantities of the plant toxin fusaric acid (5-butyl-2-pyridinecarboxylic acid) compared to other isolates of F. oxysporum f. sp. vasinfectum (Fov) that are indigenous to the United States. Fusaric acid is toxic to cotton plants and may help the pathogen compete with other microbes in the soil. We found that a laboratory strain of the fungus Mucor rouxii converts fusaric acid into a newly identified compound, 8-hydroxyfusaric acid. The latter compound is significantly less phytotoxic to cotton than the parent compound. On the basis of bioassays of hydroxylated analogues of fusaric acid, hydroxylation of the butyl side chain of fusaric acid may affect a general detoxification of fusaric acid. Genes that control this hydroxylation may be useful in developing biocontrol agents to manage Fov.

Fusaric acid and analogues as Gram-negative bacterial quorum sensing inhibitors.

Taking advantage of microwave-assisted synthesis, efficient and expedite procedures for preparation of a library of fusaric acid and 39 analogues are reported. The fusaric acid analogues were tested in cell-based screening assays for inhibition of the las and rhl quorum sensing system in Pseudomonas aeruginosa and the lux quorum sensing system in Vibrio fischeri. Eight of the 40 compounds in the library including fusaric acid inhibited lux quorum sensing and one compound inhibited activity of the las quorum sensing system. To our delight, none of the compounds showed growth inhibitory effects in the tested concentration ranges.

Mycotoxigenic Potentials of Fusarium Species in Various Culture Matrices Revealed by Mycotoxin Profiling.

In this study, twenty of the most common Fusarium species were molecularly characterized and inoculated on potato dextrose agar (PDA), rice and maize medium, where thirty three targeted mycotoxins, which might be the secondary metabolites of the identified fungal species, were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Statistical analysis was performed with principal component analysis (PCA) to characterize the mycotoxin profiles for the twenty fungi, suggesting that these fungi species could be discriminated and divided into three groups as follows. Group I, the fusaric acid producers, were defined into two subgroups, namely subgroup I as producers of fusaric acid and fumonisins, comprising of F. proliferatum, F. verticillioides, F. fujikuroi and F. solani, and subgroup II considered to only produce fusaric acid, including F. temperatum, F. subglutinans, F. musae, F. tricinctum, F. oxysporum, F. equiseti, F. sacchari, F. concentricum, F. andiyazi. Group II, as type A trichothecenes producers, included F. langsethiae, F. sporotrichioides, F. polyphialidicum, while Group III were found to mainly produce type B trichothecenes, comprising of F. culmorum, F. poae, F. meridionale and F. graminearum. A comprehensive picture, which presents the mycotoxin-producing patterns by the selected fungal species in various matrices, is obtained for the first time, and thus from an application point of view, provides key information to explore mycotoxigenic potentials of Fusarium species and forecast the Fusarium infestation/mycotoxins contamination.

Fusaric acid induces a notochord malformation in zebrafish via copper chelation.

Over a thousand extracts were tested for phenotypic effects in developing zebrafish embryos to identify bioactive molecules produced by endophytic fungi. One extract isolated from Fusarium sp., a widely distributed fungal genus found in soil and often associated with plants, induced an undulated notochord in developing zebrafish embryos. The active compound was isolated and identified as fusaric acid. Previous literature has shown this phenotype to be associated with copper chelation from the active site of lysyl oxidase, but the ability of fusaric acid to bind copper ions has not been well described. Isothermal titration calorimetry revealed that fusaric acid is a modest copper chelator with a binding constant of 4.4 × 10(5) M(-1). These results shed light on the toxicity of fusaric acid and the potential teratogenic effects of consuming plants infected with Fusarium sp.

Identification of toxin inhibitors using a magnetic nanosensor-based assay.

A magnetic nanosensor-based method is described to screen a library of drugs for potential binding to toxins. Screening is performed by measuring changes in the magnetic relaxation signal of the nanosensors (bMR nanosensors) in aqueous suspension upon addition of the toxin. The Anthrax lethal factor (ALF) is selected as a model toxin to test the ability of our bMR nanosensor-based screening method to identify potential inhibitors of the toxin. Out of 30 molecules screened, sulindac, naproxen and fusaric acid are found to bind LF, with dissociation constants in the low micromolar range. Further biological analysis of the free molecules in solution indicate that sulindac and its metabolic products inhibited LF cytotoxicity to macrophages with IC50 values in the micromolar range. Meanwhile, fusaric acid is found to be less effective at inhibiting LF cytotoxicity, while naproxen does not inhibit LF toxicity. Most importantly, when the sulindac and fusaric acid-bMR nanosensors themselves are tested as LF inhibitors, as opposed to the corresponding free molecules, they are stronger inhibitors of LF with IC50 values in the nanomolar range. Taken together, these studies show that a bMR nanosensors-based assay can be used to screen known drugs and other small molecules for inhibitor of toxins. The method can be easily modified to screen for inhibitors of other molecular interactions and not only the selected free molecule can be study as potential inhibitors but also the bMR nanosensors themselves achieving greater inhibitory potential.

Determination of fusaric acid in maize using molecularly imprinted SPE clean-up.

A new LC method to detect fusaric acid (FA) in maize is reported based on a molecularly imprinted SPE clean-up using mimic-templated molecularly imprinted polymers. Picolinic acid was used as a toxin analog for imprinting polymers during a thermolytic synthesis. Both acidic and basic functional monomers were predicted to have favorable binding interactions by MP2 ab initio calculations. Imprinted polymers synthesized with methacrylic acid or 2-dimethylaminoethyl methacrylate exhibited imprinting effects in SPE analysis. FA levels were determined using RP ion-pairing chromatography with diode-array UV detection and tetrabutylammonium hydrogen sulfate in the mobile phase. A method was developed to detect FA in maize using molecularly imprinted SPE analysis within the range of 1-100 µg/g with recoveries between 83.9 and 92.1%.

Fusaric acid is a crucial factor in the disturbance of leaf water imbalance in Fusarium-infected banana plants.

Fusarium wilt of banana is caused by Fusarium oxysporum f. sp. cubense infection. The initial chlorosis symptoms occur progressively from lower to upper leaves, with wilt symptoms subsequently occurring in the whole plant. To determine the effect of the pathogen infection on the gas exchange characteristics and water content in banana leaves, hydroponic experiments with pathogen inoculation were conducted in a greenhouse. Compared with control plants, infected banana seedlings showed a higher leaf temperature as determined by thermal imaging. Reduced stomatal conductance (g(s)) and transpiration rate (E) in infected plants resulted in lower levels of water loss than in control plants. Water potential in heavily diseased plants (II) was significantly reduced and the E/g(s) ratio was higher than in noninfected plants, indicating the occurrence of uncontrolled water loss not regulated by stomata in diseased plants. As no pathogen colonies were detected from the infected plant leaves, the crude toxin was extracted from the pathogen culture and evaluated about the effect on banana plant to further investigate the probable reason of these physiological changes in Fusarium-infected banana leaf. The phytotoxin fusaric acid (FA) was found in the crude toxin, and both crude toxin and pure FA had similar effects as the pathogen infection on the physiological changes in banana leaf. Additionally, FA was present at all positions in diseased plants and its concentration was positively correlated with the incidence of disease symptoms. Taken together, these observations indicated that FA secreted by the pathogen is an important factor involved in the disturbance of leaf temperature, resulting in uncontrolled leaf water loss and electrolyte leakage due to damaging the cell membrane. In conclusion, FA plays a critical role in accelerating the development of Fusarium wilt in banana plants by acting as a phytotoxin.

In vitro acanthamoebicidal activity of fusaric acid and dehydrofusaric acid from an endophytic fungus Fusarium sp. Tlau3.

Acanthamoeba is a genus of free-living protozoa that can cause sight- and life-threatening diseases in man. Its control is still problematic due to the lack of effective and nontoxic acanthamoebicidal agents. Herein, we report the first finding of an in vitro killing effect of fusaric acid and dehydrofusaric acid, isolated from metabolites of the Fusarium fujikuroi species complex Tlau3, on Acanthamoeba trophozoites isolated from two clinical (AS, AR) and two soil (S3, S5) samples. AS, AR, and S3 were classified as members of the T4 genotype, whereas S5 belongs to T5. The fungal extract was found to exhibit acanthamoebicidal activity, and activity-guided fractionation led to the isolation and identification of active principles, fusaric acid and dehydrofusaric acid. Their effects were in concentration- and time-dependent manners. Fusaric acid and dehydrofusaric acid showed IC50 values against AS trophozoites of 0.31 and 0.34 µM, respectively. Commercial fusaric acid displayed the same acanthamoebicidal activity as that of the isolated fusaric acid, and therefore, commercial fusaric acid was used throughout this study. IC50 values of commercial fusaric acid against AR, S3, and S5 trophozoites were 0.33, 0.33, and 0.66 µM, respectively. Fusaric acid calcium salt has a history of usage as a hypotensive agent in humans with no observed toxicity. The present study suggests that fusaric acid may serve as a starting point for the development towards therapeutic and environmental acanthamoebicides with low toxicity to humans.

Antimycobacterial activity of fusaric acid from a mangrove endophyte and its metal complexes.

Due to the increasing prevalence of multidrug-resistant Mycobacterium tuberculosis, there is an urgent need for new antituberculosis drugs that have novel mechanisms of action. As part of our ongoing search for antimycobacterial metabolites from mangrove endophytes, chemical analysis of the active extract of a strain of Fusarium sp. was performed, which led to the isolation of fusaric acid as the predominant constituent. A variety of metal complexes of fusaric acid were prepared. Antimycobacterial assays showed that Cadmium (II) and Copper (II) complexes exhibited potent inhibitory activity against the M. bovis BCG strain [minimum inhibitory concentration (MIC) = 4 µg/mL] and the M. tuberculosis H37Rv strain (MIC = 10 µg/mL), respectively. This is the first report of the antimycobacterial activity of the mangrove Fusarium metabolite and its coordinating metal complexes.

Phytotoxicity of fusaric acid and analogs to cotton.

We developed a cotton cotyledonary leaf bioassay to test the phytotoxicity of fusaric acid (5-butylpicolinic acid), picolinic acid and related analogs. The compounds were dissolved in aqueous Tween 80, and 20 µL of the test solution was placed at three positions on the leaf, and a needle was used to puncture the leaf through each drop; the results were evaluated after 48 h. In contrast to previous studies, we found the carboxylic acid group is essential for phytotoxicity. Nicotinic acid was considerably less phytotoxic than picolinic acid and conversion of picolinic acid to the amide or N-oxide decreased phytotoxicity. Increasing the alkyl chain length at the 5-position on picolinic acid from two up to five carbons atoms increased phytotoxicity. Fusaric acid methyl ester, the most phytotoxic compound tested, is a naturally occurring compound; as such it has potential as a herbicide in organic farming.

Identification, mycotoxin risk and pathogenicity of Fusarium species associated with fig endosepsis in Apulia, Italy.

In a survey carried out on 87 rotted fig fruits samples collected in the Apulia region of Italy, the authors isolated 126 Fusarium strains identified as F. ramigenum (69 strains), F. solani (49), F. proliferatum (five) and three not identified. Investigation on the fertility of the strains belonging to F. proliferatum and F. ramigenum revealed that only strains of F. proliferatum were fertile. The identity of F. ramigenum strains was confirmed by sequencing a portion of the translation elongation factor-1alpha gene. When Fusarium species were analysed for their toxigenicity, 37/69 strains of F. ramigenum produced fusaric acid (FA) up to 525 mg kg(-1); 30 strains produced beauvericin (BEA) up to 190 mg kg(-1); 60 strains produced fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) up to 1575 mg kg(-1) of total FBs; and two strains produced fusaproliferin (FUP) up to 345 mg kg(-1); all five strains of F. proliferatum produced FA at low levels; two strains produced BEA up to 205 mg kg(-1); one strain produced FB(1) and FB(2), 1100 and 470 mg kg(-1), respectively; and one strain produced FUP, 820 mg kg(-1); F. solani (30 strains) produced FA, 13 strains up to 215 mg kg(-1). Few fungal extracts showed high toxicity toward brine shrimp larvae and in some cases in relation to BEA and FA content. A pathogenic assay on fig fruits showed that all three species were pathogenic, with higher virulence of F. ramigenum. These data report for the first time the production of BEA and FB(1)/FB(2) by F. ramigenum and show that it is a main agent of fig endosepsis in Apulia and can contribute to fumonisin contamination of fresh and dried figs.

Fusaric acid as a novel proton-affinitive derivatizing reagent for highly sensitive quantification of hydroxysteroids by LC-ESI-MS/MS.

A highly sensitive derivatization method for liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry of dehydroepiandrosterone (DHEA), testosterone (T), pregnenolone (P5), and 17alpha-OH-pregnenolone (17-OHP5) was developed based on the use of fusaric acid as a reagent. DHEA, P5, and 17-OHP5 were rapidly and quantitatively converted to the 3-fusarate esters by treatment with fusaric acid and 2-methyl-6-nitrobenzoic anhydride. The positive ESI-mass spectra of the fusarate esters of each steroid were dominated by the appearance of [M + H](+) as base peaks. The fusarate derivatization of these steroids showed 17.6-fold (DHEA), 11.9-fold (P5), 3.3-fold (17-OHP5), and 1.8-fold (T) higher sensitivity to those of the corresponding picolinate derivatives in LC-selected reaction monitoring.

Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans.

AIMS: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. METHODS AND RESULTS: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 microg ml(-1) suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 microg ml(-1). CONCLUSIONS: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.