Potent Fungicidal Activity Research Articles

An Eco-Friendly Method to Synthesize Potent Antimicrobial Tricyclic Flavonoids.

The rapid emergence and spread of multidrug-resistant microorganisms is threatening our ability to treat common infections, with serious medical, social, and economic consequences. Despite substantial progress in the global fight against antibiotic resistance, the number of effective antibiotics is rapidly decreasing, underlying the urgent need to develop novel antimicrobials. In the present study, the green synthesis of novel iodine-substituted tricyclic flavonoids has been accomplished using an eco-friendly reagent, HPW-SiO2, as a cyclization agent for the precursor 3-dithiocarmamic flavanones. In vitro antimicrobial activity of the new compounds was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentrations. All tested compounds displayed potent inhibitory activity against all tested microbial strains, with the lowest MIC values of 0.12 µg/mL and 0.48 µg/mL recorded for compound 5c against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. Higher MIC values (7.81 µg/mL) were registered for several flavonoids against Gram-negative bacteria Escherichia coli and Acinetobacter pittii. No inhibitory activity was evidenced against Pseudomonas aeruginosa strain. The highest antifungal activity was displayed by flavonoid 5d against Candida krusei (MIC = 3.9 µg/mL). The same compound also exhibited the most potent bactericidal and fungicidal activity against Bacillus subtilis (0.9 µg/mL) and Staphylococcus aureus (1.97 µg/mL), Candida albicans, and Candida krusei (7.81 µg/mL). Based on the reported results, we believe that the novel iodine-substituted tricyclic flavonoids have good potential to become new antimicrobial agents effective against bacterial and fungal strains, including WHO-priority pathogens.

A novel antifungal peptide, SP1.2, from Rhodopseudomonas palustris against the rice blast pathogen.

Rice blast has a significant detrimental impact on rice yields, so developing efficient biological control technologies is an effective means for rice blast prevention and control. The GroEL protein has proven to be effective at preventing and managing the pathogenicity of rice blast. Here, we analyzed the amino acid sequence of the GroEL protein and synthesized the '60 kDa chaperonin signature' (350-373 amino acids) peptide SP1.2, which has potent antifungal activity. Notably, the SP1.2 peptide exhibited potent fungicidal activity against Magnaporthe oryzae, effectively inhibiting appressorium germination. Electron microscopy revealed that SP1.2 disrupted the fungal plasma membrane and bound to multiple bioactive phosphoinositides in vitro, triggering the production of reactive oxygen species. Furthermore, it also caused an increase in the acetylation of M. oryzae and induced autophagy in cells. The spray application of SP1.2 significantly reduced the number of disease spots caused by the fungal pathogen M. oryzae in rice, enhancing the defense response of rice plants. Field trials showed that the control effect was 64.59% after spraying SP1.2. Our study illustrates the antifungal activity of the structurally unique SP1.2 peptide against plant fungal pathogens and paves the way for the future development of this class of peptides as antifungal agents. © 2024 Society of Chemical Industry.

In vitro activity of olorofim against 507 filamentous fungi including antifungal drug-resistant strains at a tertiary laboratory in Australia: 2020-2023.

New antifungal agents are required to mitigate against azole-resistant Aspergillus and drug-resistant non-Aspergillus moulds. The novel orotomide, olorofim (F2G, Manchester, UK), has potent fungicidal activity against Aspergillus including azole-resistant Aspergillus fumigatus, Lomentospora prolificans and Scedosporium spp. Development of olorofim-specific clinical breakpoints/epidemiological cut-off values requires reliable MIC data. Determine the in vitro activity of olorofim compared with standard antifungals against mould pathogens at an Australian hospital. Olorofim MICs were determined for 507 clinical mould isolates using the CLSI M38-A3 standard. MICs of amphotericin B, anidulafungin, posaconazole, voriconazole and isavuconazole were obtained using Sensititre™ YeastOne YO10 and AUSNMRCI panels (Thermo-Fisher Scientific). A. fumigatus sensu stricto was the commonest species (33.3%) followed by L. prolificans (18.3%), Scedosporium (11.4%) and Fusarium (6%) species. Olorofim modal MICs were ≤0.25 mg/L (MIC90 0.25 mg/L) for all Aspergillus except Aspergillus Section Usti (1 mg/L); MICs for nine azole-resistant/non-wild-type A. fumigatus ranged from 0.008 to 0.125 mg/L. The MIC90 of olorofim for L. prolificans was 0.5 mg/L, 0.25-0.5 mg/L for Scedosporium spp. and 8 mg/L for the F. solani complex but with modal MICs of 0.25 and 0.008 mg/L for F. oxysporum and F. proliferatum complexes, respectively. For Verruconis gallopava (n = 10), the olorofim MIC90 was 0.06 mg/L (voriconazole MIC90 2 mg/L, isavuconazole MICs of 4->8 mg/L). Olorofim had little activity against other dematiaceous moulds including Exophiala species. Olorofim was highly active against Aspergillus spp. including azole-resistant A. fumigatus, L. prolificans, Scedosporium spp. and some Fusarium species with the new finding of potent activity against V. gallopava.

Discovery of Novel α,β-Unsaturated Amide Derivatives as Candidate Antifungals to Overcome Fungal Resistance.

In our previous study, coumarin-containing CYP51 inhibitor A32 demonstrated potent antiresistance activity. However, compound A32 demonstrated unsatisfied metabolic stability, necessitating modifications to overcome these limitations. In this study, α,β-unsaturated amides were used to replace the unstable coumarin ring, which increased metabolic stability by four times while maintaining antifungal activity, including activity against resistant strains. Subsequently, the sterol composition analysis and morphological observation experiments indicated that the target of these novel compounds is lanosterol 14α-demethylase (CYP51). Meanwhile, biofilm growth was inhibited and resistance genes (ERG11, CDR1, CDR2, and MDR1) expression was downregulated to find out how the antiresistance works. Importantly, compound C07 demonstrated the capacity to stimulate reactive oxygen species, thus displaying potent fungicidal activity. Moreover, C07 exhibited encouraging effectiveness in vivo following intraperitoneal administration. Additionally, the most potent compound C07 showed satisfactory pharmacokinetic properties and low toxicity. These α,β-unsaturated amide derivatives, particularly C07, are potential candidates for treating azole-resistant candidiasis.

Silver Nanoparticles as Antimicrobial Agents: Mechanisms, Challenges, and Applications

Silver nanoparticles (AgNPs) have garnered considerable attention for their potent antimicrobial properties and broad spectrum of applications in various fields. This review delves into the multifaceted realm of AgNPs as antimicrobial agents, aiming to provide a comprehensive understanding of their mechanisms of action, challenges, and diverse applications. The article begins by tracing the historical significance of silver as an antimicrobial agents and transitions into the contemporary role of AgNPs in modern applications. The synthesis of AgNPs is explored, encompassing diverse methods such as chemical, physical, and biological approaches. Particular emphasis is placed on green synthesis methods, which not only yield nanoparticles with controlled properties but also align with principles of environmental sustainability and biocompatibility. The review subsequently dissects the intricate mechanisms underpinning AgNPs antimicrobial process. The interaction of AgNPs with microbial cell membranes, generation of reactive oxygen species (ROS), and disruption of vital microbial processes collectively contribute to their potent bactericidal and fungicidal activities. However, the incorporation of AgNPs into various applications is not devoid of challenges. This article examines potential toxicity concerns, addressing issues related to stability, aggregation, and controlled release of nanoparticles, as well as the emergence of resistance mechanisms in microorganisms. Delving into the realm of applications, the review unveils AgNPs’ significance in biomedical and clinical settings, where they are employed in wound healing, medical devices, and infection control. Moreover, the environmental implications of AgNPs are explored, including their use in water and air purification, as well as potential roles in food and agricultural sectors. Looking forward, the review discusses emerging trends and suggests future research directions. Combinational therapies, integration with advanced materials, and exploration of AgNPs’ potential in addressing global antimicrobial resistance are outlined as promising avenues. In conclusion, this comprehensive review underscores the vital role of silver nanoparticles as versatile and potent antimicrobial agents, shedding light on their mechanisms, challenges, and multifarious applications across diverse sectors.

Phytochemicals demonstrate potential fungicidal activity against Fusarium commune, a newly identified pathogenic fungus causing rhizome rot in Polygonatum cyrtonema (giant Chinese Solomon's seal)

AbstractPolygonatum cyrtonema (giant Chinese Solomon's seal) is a perennial herb belonging to the family Liliaceae, with significant medicinal and dietary value. However, a severe rhizome rot has led to a decline in the yield and quality of this species. In this study, a fungus was isolated from symptomatic rhizome samples and identified as Fusarium commune through molecular and morphological analyses. In pathogenicity tests, the identification of F. commune as the causative agent of rhizome rot was confirmed by fulfilling Koch's postulates. Biological characteristics indicated that the optimal growth conditions for F. commune were a temperature of 25°C, pH of 8, carbon source of soluble starch, nitrogen source of peptone and a photoperiod of 24 h light. In order to reduce the use of chemical fungicides and explore natural substances to control the disease, the sensitivity of F. commune to seven phytochemicals was studied. Four phytochemicals (honokiol, 2‐allylphenol, magnolol and carvacrol) showed inhibitory activity, with honokiol having a 50% of the maximal effect concentration (EC50) of 9.99 ± 0.14 mg/L. The results of pot experiment showed that the four phytochemicals had significant control efficacy on the rhizome rot of P. cyrtonema, and the highest control efficacy was observed with honokiol. These findings provide a scientific basis for the control of rhizome rot in P. cyrtonema.

Efficacy of rhizobacteria Paenibacillus polymyxa SY42 for the biological control of Atractylodes chinensis root rot

Atractylodes chinensis is one of the most commonly used bulk herbs in East Asia; however, root rot can seriously affect its quality and yields. In contrast to chemical pesticides, biological control strategies are environmentally compatible and safe. For this study, 68 antagonistic bacterial strains were isolated from the rhizospheres of healthy Atractylodes chinensis. Strain SY42 exhibited the most potent fungicidal activities, with inhibition rates against F. oxysporum, F. solani, and F. redolens of 67.07 %, 63.40 % and 68.45 %, respectively. Through morphological observation and molecular characterization, strain SY42 was identified as Paenibacillus polymyxa. The volatile organic components (VOCs) produced by SY42 effectively inhibited the mycelial growth of pathogenic fungi through diffusion. SY42 significantly inhibited the germination of pathogenic fungal spores. Following co-culturing with SY42, the mycelium of the pathogenic fungus was deformed, folded, and even ruptured. SY42 could produce cellulases and proteases to degrade fungal cell walls. Pot experiments demonstrated the excellent biocontrol efficacy of SY42. This study revealed that P. polymyxa SY42 inhibited pathogenic fungi through multiple mechanisms, which verified its utility as a biocontrol agent for the control of A. chinensis root rot.

Assessing the efficacy of gutiferone E in photodynamic therapy for oral candidiasis

The rise in antifungal resistance and side effects of conventional treatments drive the search for innovative therapies like Photodynamic Therapy (PDT). This study explored the efficacy of PDT mediated by gutiferone, an isolated compound from red propolis, for candidiasis treatment. Multiple evaluation methods were employed, including determining the minimum inhibitory concentration (MIC) via broth microdilution, quantifying biomass using crystal violet detachment, and cell counting through total plate count. PDT mediated by gutiferone was also assessed in five groups of mice, followed by histopathological examination and agar plating of lingual tissue samples. Among the seven Candida species tested, gutiferone displayed efficacy against C. albicans, C. glabrata, and C. tropicalis, with MIC values of 1000 μg/mL. In C. tropicalis biofilms, exposure to gutiferone led to a reduction of 1.61 Log10 CFU/mL. PDT mediated by gutiferone achieved an average reduction of 3.68 Log10 CFU/mL in C. tropicalis biofilm cells, underscoring its potent fungicidal activity. Histopathological analysis revealed fungal structures, such as pseudohyphae and hyphae, in infected groups (G2) and irradiated mice. In contrast, groups treated with gutiferone or subjected to gutiferone-assisted PDT (G5) exhibited only few blastoconidia. Furthermore, CFU/mL assessments in lingual tissue post-treatment demonstrated a significantly lower count (0.30 Log10 CFU/mL) in the G5 group compared to G2 (2.43 Log10 CFU/mL). These findings highlight the potential of PDT mediated by gutiferone as a promising alternative for managing denture stomatitis. Future research and clinical investigations offer the promise of validating its clinical applicability and improving outcomes in the treatment of oral candidiasis.

Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography

The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

Synthesis, crystal structure, biological evaluation, in silico ADME properties, enzymatic target prediction and molecular docking studies of pyrazolone-azomethine analogs

A series of twelve azomethine analogs (3a-l) of 4-aminoantipyrine (4-AA) were prepared and characterized using FT-IR, 1HNMR and EI mass spectrometric analysis. Analog 3f was obtained as a single crystal, and X-ray diffraction (XRD) data indicated its adoption of a trans configuration around the central azomethine (CN) group, forming monoclinic P2(1)/n symmetry. The unit cell of 3f contains four molecules, and its crystal packing is stabilized through significant intermolecular host-guest CH⋯O, CH⋯π, and π⋯π interactions, resulting in a stacked supramolecular structure. In vitro bactericidal and fungicidal screening of analogs 3a-l was performed against twelve bacterial and five fungal strains, respectively. Most of the analogs demonstrated weak to moderate antimicrobial activities, specifically 3a, 3b, 3e and 3g exhibited highest activity against S. boydii, B. megaterium; S. sonnei and E. coli, respectively and 3c, 3d and 3l showed potent fungicidal activity against A. flavus, C. albicans, and N. crassa, respectively. The results indicate that the antimicrobial potency is largely dependent on the nature of the aromatic ring linked pyrazolone moiety via azomethine (CN) group. DPPH free radical scavenging assay revealed analogs 3e, 3l, 3g, 3k, 3h and 3d had IC50 values of 0.07, 1.19, 2.31, 2.57, 4.04 and 4.07 µg mL−1, respectively, comparable to that of the standard ascorbic acid (IC50 = 4.10 µg mL−1). Docking simulations were conducted to elucidate the binding mode of potent antibacterial analogs (3e and 3g) and antifungal analogs (3d and 3l) within the active sites of KAS (E. coli) and CYP 51 (C. albicans) receptors, respectively. The results revealed strong binding affinities between the studied molecules and the respective proteins. All the tested analogs fulfill the condition of Lipinski's rule of five (ROF) and Veber's rule, and the calculated ADMET profile indicated favorable drug-likeness, pharmacokinetic properties, and drug scores. Ligand-based enzymatic target predictions suggest that analogs 3a-l have potential as inhibitors of kinases, enzymes, proteases, or ligands for Family A GPCR. Finally, DFT calculations using the B3LYP/6-311G(d,p) level of theory provide insights into the FMO energies, global molecular reactivity, and biological properties of the azomethine analogs of 4-AA.

Structural Optimization, Fungicidal Activities Evaluation, DFT Study and Structure-Activity Relationship of Dopamine Derivatives with Benzothiazole Fragment from Polyrhachis dives.

In our previous work, two dopamine derivatives with benzothiazole fragment were isolated and identified from Polyrhachis dives (P. dives). Based on their characteristic structure, we used them as lead compound to carry out structural optimization and subsequent fungicidal evaluation. Here 20 dopamine derivatives with benzothiazole fragment were designed and synthesized by a facile method, and their structures were characterized by 1 H-NMR, 13 CNMR and HMRS. In bioassays, most of the title compounds possess potential fungicidal activities against Altenaia alternala (A. alternala) and Botrytis cinerea (B. cinerea). Especially, (E)-N-(2-(benzo[d]thiazol-6-yl)ethyl)-3-(p-tolyl)acrylamide and (E)-N-(2-(benzo[d]thiazol-6-yl)ethyl)-3-(4-(trifluoromethyl)phenyl)acrylamide displayed 29.3 mg/L and 10.7 mg/L EC50 value against A. alternala, respectively, which possessed equivalent fungicidal activities level to hymexazol.

Discovery of pyridachlometyl: A new class of pyridazine fungicides

Pyridachlometyl is a unique pyridazine fungicide with a novel mode of action. Herein, we describe the pathway for the invention of pyridachlometyl. First, we identified a diphenyl-imidazo[1,2-a]pyrimidine as our proprietary lead with potent fungicidal activity. Then, aiming to simplify the chemical structure, we applied judicious estimations to explore monocyclic heterocycles as pharmacophores. This enabled the identification of a novel class of tetrasubstituted pyridazine compounds with potent fungicidal activity, likely retaining the same mode of action as the aforementioned compounds. The findings indicated bioisosteric similarity between diphenyl-imidazo[1,2-a]pyrimidine and pyridazine. Further structure–activity and mammalian safety investigations of pyridazine compounds resulted in the discovery of pyridachlometyl as a candidate for commercial development.

Transcriptomic Profile of Penicillium digitatum Reveals Novel Aspects of the Mode of Action of the Antifungal Protein AfpB.

Antifungal proteins (AFPs) from filamentous fungi are promising biomolecules to control fungal pathogens. Understanding their biological role and mode of action is essential for their future application. AfpB from the citrus fruit pathogen Penicillium digitatum is highly active against fungal phytopathogens, including its native fungus. Our previous data showed that AfpB acts through a multitargeted three-stage process: interaction with the outer mannosylated cell wall, energy-dependent cell internalization, and intracellular actions that result in cell death. Here, we extend these findings by characterizing the functional role of AfpB and its interaction with P. digitatum through transcriptomic studies. For this, we compared the transcriptomic response of AfpB-treated P. digitatum wild type, a ΔafpB mutant, and an AfpB-overproducing strain. Transcriptomic data suggest a multifaceted role for AfpB. Data from the ΔafpB mutant suggested that the afpB gene contributes to the overall homeostasis of the cell. Additionally, these data showed that AfpB represses toxin-encoding genes, and they suggest a link to apoptotic processes. Gene expression and knockout mutants confirmed that genes coding for acetolactate synthase (ALS) and acetolactate decarboxylase (ALD), which belong to the acetoin biosynthetic pathway, contribute to the inhibitory activity of AfpB. Moreover, a gene encoding a previously uncharacterized extracellular tandem repeat peptide (TRP) protein showed high induction in the presence of AfpB, whereas its TRP monomer enhanced AfpB activity. Overall, our study offers a rich source of information to further advance in the characterization of the multifaceted mode of action of AFPs. IMPORTANCE Fungal infections threaten human health worldwide and have a negative impact on food security, damaging crop production and causing animal diseases. At present, only a few classes of fungicides are available due to the complexity of targeting fungi without affecting plant, animal, or human hosts. Moreover, the intensive use of fungicides in agriculture has led to the development of resistance. Therefore, there is an urgent need to develop antifungal biomolecules with new modes of action to fight human-, animal-, and plant-pathogenic fungi. Fungal antifungal proteins (AFPs) offer great potential as new biofungicides to control deleterious fungi. However, current knowledge about their killing mechanism is still limited, which hampers their potential applicability. AfpB from P. digitatum is a promising molecule with potent and specific fungicidal activity. This study further characterizes its mode of action, opening avenues for the development of new antifungals.

In silico and in vitro investigation of the antifungal activity of trimetallic Cu–Zn-magnetic nanoparticles against Fusarium oxysporum with stimulation of the tomato plant's drought stress tolerance response

Fusarium oxysporum is the fungus responsible for Fusarium wilt. Tomatoes and other plants acquire Fusarium wilt through their root systems. Occasionally, fungicides applied to the soil are used to combat the disease; however, some strains have developed resistance. Carboxymethyl cellulose (CMC) trimetallic magnetic zinc and copper nanoparticles CMC-Cu-Zn-FeMNPs are one of the most promising antifungal agents against a wide range of fungi. One of the most important aspects of using magnetic nanoparticles is their ability to target cells, which confirms the drug's potent fungicidal activity. Using a UV-spectrophotometer, the characterization of synthesized CMC-Cu-Zn-FeMNPs revealed four peaks at226,271, 321 and 335 nm, as well as spherical nanoparticles with a mean size of 5.905 nm and a surface potential of −61.7 mv. In this study, CMC-Cu-Zn-FeMNPs were used to inhibit the growth of F. oxysporum by interfering with the ergosterol production metabolic pathway. Molecular docking experiments demonstrated that the nanoparticles were able to bind to sterol 14-alpha demethylase responsible for inhibiting ergosterol biosynthesis. Real-time PCR analysis showed that the nanoparticles upregulated tomato plants and other assessed parameters under drought stress and downregulated the velvet complex and virulence factors of F. oxysporum on plants. The study results indicate that CMC-Cu-Zn-FeMNPs may be a promising and eco-friendly solution with low potential of accumulation and easy to collected alternative to conventional chemical pesticides that can have negative impacts on the environment and human health. Furthermore, it could provide a sustainable solution for managing Fusarium wilt disease, which can significantly reduce tomato yield and quality.

Anti-phytopathogenic activity and the mechanisms of phthalides from Angelica sinensis (Oliv.) Diels.

The resistance of traditional chemical fungicides to plant pathogenic fungi and the threats to the safety of humans and the environment highlight an urgent need to find safe and efficient alternatives to chemical fungicides. Owing to the wide spectrum of antifungal activities, low persistence and nontoxicity to mammals and aquatic life, essential oils have considerable potential as low-risk pesticides. In this study, the essential oil and the main components of Angelica sinensis (Oliv.) Diels (Danggui) were extracted, analyzed by GC-MS, and evaluated for their antifungal activities against six plant pathogenic fungi. 3-butylidenephthalide (3-BPH) showed the best antifungal activity against Fusarium graminearum with an EC50 value of 14.35 μg mL-1 . The antifungal mechanistic studies revealed that 3-BPH induced the generation of endogenous ROS to cause lipid peroxidation of the cell membrane and inhibited the biosynthesis of ergosterol, thereby causing the cell membranedamaged to exert its fungicidal activity. Significantly, 3-BPH could reduce deoxynivalenol production compared to the control. This study demonstrated the potent fungicidal activity of natural phthalide compound 3-BPH and highlighted its potential as an alternative agent to control F. graminearum. © 2023 Society of Chemical Industry.

Design, synthesis, and antifungal activity of novel dithiin tetracarboximide derivatives as potential succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitor (SDHI) fungicides are an important class of agricultural fungicides with the advantages of high efficiency and a broad bactericidal spectrum. To pursue novel SDHIs, a series of N-substituted dithiin tetracarboximide derivatives were designed, synthesized, and characterized by 1 H NMR, 13 C NMR, and high resolution mass spectrum (HRMS). These engineered compounds displayed potent fungicidal activity against phytopathogens, including Sclerotinia sclerotiorum, Botrytis cinerea, and Rhizoctonia solani, comparable with that of the commercial SDHI fungicide boscalid. In particular, compound 18 stood out with prominent activity against S. sclerotiorum with a half-maximal effective concentration (EC50 ) value of 1.37 μg ml-1 . Compound 1 exhibited the most potent antifungal activity against B. cinerea with EC50 values of 5.02 μg ml-1 . As for R. solani, 12 and 13 exhibited remarkably inhibitory activity with EC50 values of 4.26 and 5.76 μg ml-1 , respectively. In the succinate dehydrogenase (SDH) inhibition assay, 13 presented significant inhibitory activity with a half-maximal inhibitory concentration (IC50 ) value of 15.3μm, which was approximately equivalent to that of boscalid (14.2μm). Furthermore, molecular docking studies revealed that 13 could anchor in the binding site of SDH. Taken together, results suggested that the dithiin tetracarboximide scaffold possessed a huge potential to be developed as novel fungicides and SDHIs. © 2023 Society of Chemical Industry.

Adjuvant Pam3CSk4 does not improve the immunization against Cryptococcus gattii infection in C57BL/6 mice.

Cryptococcosis is a relevant invasive fungal infection that affects immunocompromised and immunocompetent individuals when caused by Cryptococcus gattii. Host innate and adaptive immune responses can be subverted by C. gattii, that blocks the differentiation of T helper (Th) 1 and Th17 cells, which are involved in the protection against cryptococcosis. Moreover, the macrophage polarization is modulated by C. gattii infection that requires a balance in the macrophage subsets to control the C. gattii infection. Toll-like receptor (TLR) 2 agonists are important immunomodulators favoring a pro-inflammatory response with potential fungicidal activity, and TLR2 agonists have been used as adjuvants in vaccines against infections caused by bacteria or viruses. Therefore, this work aimed to evaluate the immunomodulatory effect of the tripalmitoyl lipopeptide S-glycerol cysteine (Pam3CSK4 or P3C4), a TLR2 agonist, as an adjuvant in the vaccination against C. gattii infection. C57BL/6 mice were immunized with 2×107 inactivated yeasts of C. gattii via intranasal route on day 1, 14 and 28 (Immunized group). Immunization was associated with 1µg or 10µg of adjuvant P3C4 (Immunized+P3C4-1µg or Immunized+P3C4-10µg), followed by C. gattii infection on day 42 after the immunization protocol. Immunized+P3C4-1µg group had reduced levels of IgG1, IgG2a and IgA and no significant difference in the IgG and IgM anti-GXM antibody titer was detected, compared to the Immunized group. High levels of IL-17 and IL-1β in lung tissue of mice from the Immunized+P3C4-1µg group did not promote a predominance of Th17 cells, in contrast, the frequency of TLR2+ cells was increased in immunized mice that received 1 µg of P3C4. The reduction in the relative expression of T-bet and high levels of Foxp3 detected in the lungs of the Immunized+P3C4-1µg group suggest a prevalence of regulatory T cells in the tissue, which did not contribute to the control of C. gattii infection. The immunization protocol associated with 10 µg of adjuvant P3C4 induced high levels of IL-17 in the lung tissue, whereas the levels of pro-inflammatory cytokines were downregulated. To evaluate the effect of adjuvant P3C4 in the control of C. gattii infection, quantification of the fungal burden in the lungs was performed by the CFU assay, and the groups with adjuvant P3C4 showed a pulmonary C. gattii burden that was not significantly altered when compared with the immunized group. The mice that received 1 µg of adjuvant P3C4 had a lower percentage of inflammatory infiltrate in the lungs. The immunomodulatory effect of P3C4, associated with the immunization protocol, plays an imbalance between pro- and anti-inflammatory response in the lungs that did not favor a protection against C. gattii infection, which is related to the immune response characterized by a suppressive/regulatory profile in the pulmonary microenvironment after C. gattii infection.

Inhibition of Fusarium Graminearum Growth and Deoxynivalenol Biosynthesis by Phenolic Compounds

AbstractPhytopathogenic fungi infections have become a major threat to reduce crop yields and dampen human health globally. The mycelium growth inhibition method was adopted to evaluate the antifungal activity of commercially available natural and natural‐like phenols, bioassay results showed that most phenolic compounds exhibited excellent fungicidal activity against Fusarium graminearum, Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum f. sp. Vasinfectum, Sclerotinia sclerotiorum, and Magnaporthe oryzae. Particularly, compound Z‐5 exhibited an excellent inhibition rate of 100.00 % at 100 μg/mL and EC50 value of 3.20 μg/mL against Fusarium graminearum. In addition, compound Z‐5 displayed the best efficacy and superior fungicidal activity compared to azoxystrobin (EC50=9.78 μg/mL) based on mycelial growth and spore germination. Significantly, compound Z‐5 at 200 μg/mL reduced 4‐deoxynivalenol production by over 98 % compared to the control, through down‐regulation of the gene expression of Tri5 and Tri6. Preliminary mechanism studies revealed that compound Z‐5 might exert its fungicidal activity by inducing accumulation of reactive oxygen species and lipid peroxidation, and causing cell membrane destruction and DNA synthesis interference. This study demonstrated the potent fungicidal activity of the natural‐like phenolic compound Z‐5 and highlighted its potential as an alternative agent to control F. graminearum.

Discovery of N-Methoxy-(biphenyl-ethyl)-pyrazole-carboxamides as Novel Succinate Dehydrogenase Inhibitors.

Succinate dehydrogenase (SDH) inhibitor is one of the research hotspots for the development of fungicides. Herein, we describe the design and synthesis of N-methoxy-(biphenyl-ethyl)-pyrazole-carboxamide derivatives with enhanced fungicidal activity by employing fragment combination strategy. The SDH enzymatic activity was evaluated for 24 title compounds, and compound 7s was identified as the highest activity against porcine SDH with an IC50 value of 0.014 μM, 205-fold greater than that of fluxapyroxad. Furthermore, the greenhouse experiments showed that compound 7u exhibited potent fungicidal activity against wheat powdery mildew with an EC50 value of 0.633 mg/L, higher activity than fluxapyroxad and benzovindiflupyr. The computational results showed that the fluorine atom substituted on the pyrazole ring formed an extra dipolar-dipolar interaction with C_S42 and then increased the van der Waals interaction between the compound and SDH. The structural and mechanistic insights obtained from the present work will provide a valuable clue to developing novel SDH inhibitors.

The Trisubstituted Isoxazole MMV688766 Exerts Broad-Spectrum Activity against Drug-Resistant Fungal Pathogens through Inhibition of Lipid Homeostasis.

Candida species are among the most prevalent causes of systemic fungal infection, posing a growing threat to public health. While Candida albicans is the most common etiological agent of systemic candidiasis, the frequency of infections caused by non-albicans Candida species is rising. Among these is Candida auris, which has emerged as a particular concern. Since its initial discovery in 2009, it has been identified worldwide and exhibits resistance to all three principal antifungal classes. Here, we endeavored to identify compounds with novel bioactivity against C. auris from the Medicines for Malaria Venture's Pathogen Box library. Of the five hits identified, the trisubstituted isoxazole MMV688766 emerged as the only compound displaying potent fungicidal activity against C. auris, as well as other evolutionarily divergent fungal pathogens. Chemogenomic profiling, as well as subsequent metabolomic and phenotypic analyses, revealed that MMV688766 disrupts cellular lipid homeostasis, driving a decrease in levels of early sphingolipid intermediates and fatty acids and a concomitant increase in lysophospholipids. Experimental evolution to further probe MMV688766's mode of action in the model fungus Saccharomyces cerevisiae revealed that loss of function of the transcriptional regulator HAL9 confers resistance to MMV688766, in part through the upregulation of the lipid-binding chaperone HSP12, a response that appears to assist in tolerating MMV688766-induced stress. The novel mode of action we have uncovered for MMV688766 against drug-resistant fungal pathogens highlights the broad utility of targeting lipid homeostasis to disrupt fungal growth and how screening structurally-diverse chemical libraries can provide new insights into resistance-conferring stress responses of fungi. IMPORTANCE As widespread antimicrobial resistance threatens to propel the world into a postantibiotic era, there is a pressing need to identify mechanistically distinct antimicrobial agents. This is of particular concern when considering the limited arsenal of drugs available to treat fungal infections, coupled with the emergence of highly drug-resistant fungal pathogens, including Candida auris. In this work, we demonstrate that existing libraries of drug-like chemical matter can be rich resources for antifungal molecular scaffolds. We discovered that the small molecule MMV688766, from the Pathogen Box library, displays previously undescribed broad-spectrum fungicidal activity through perturbation of lipid homeostasis. Characterization of the mode of action of MMV688766 provided new insight into the protective mechanisms fungi use to cope with the disruption of lipid homeostasis. Our findings highlight that elucidating the genetic circuitry required to survive in the presence of cellular stress offers powerful insights into the biological pathways that govern this important phenotype.