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Drug-resistant Mycobacterium tuberculosis is a significant cause of infectious disease morbidity and mortality for which new antimicrobials are urgently needed. Inhibitors of mycobacterial respiratory energy metabolism have emerged as promising next-generation antimicrobials, but a number of targets remain unexplored. Succinate dehydrogenase (SDH), a focal point in mycobacterial central carbon metabolism and respiratory energy production, is required for growth and survival in M. tuberculosis under a number of conditions, highlighting the potential of inhibitors targeting mycobacterial SDH enzymes. To advance SDH as a novel drug target in M. tuberculosis , we utilized a combination of biochemical screening and in-silico deep learning technologies to identify multiple chemical scaffolds capable of inhibiting mycobacterial SDH activity. Antimicrobial susceptibility assays show that lead inhibitors are bacteriostatic agents with activity against wild-type and drug-resistant strains of M. tuberculosis . Mode of action studies on lead compounds demonstrate that the specific inhibition of SDH activity dysregulates mycobacterial metabolism and respiration and results in the secretion of intracellular succinate. Interaction assays demonstrate that the chemical inhibition of SDH activity potentiates the activity of other bioenergetic inhibitors and prevents the emergence of resistance to a variety of drugs. Overall, this study shows that SDH inhibitors are promising next-generation antimicrobials against M. tuberculosis .

Background: Succinate dehydrogenase inhibitor (SDHI) fungicides play important roles in the control of plant fungal diseases. However, they are facing serious challenges from issues with resistance and cross-resistance, primarily attributed to their frequent application and structural similarities. There is an urgent need to design and develop SDHI fungicides with novel structures., Results: Aiming to discover novel potent SDHI fungicides, 31 innovative pyrazole β-ketonitrile derivatives with diphenyl ether moiety were rationally designed and synthesized, which were guided by a 3D-QSAR model from our previous study. The optimal target compound A23 exhibited not only outstanding in vitro inhibitory activities against Rhizoctonia solani with a half-maximal effective concentration (EC 50 ) value of 0.0398 μg mL -1 comparable to that for fluxapyroxad (EC 50  = 0.0375 μg mL -1 ), but also a moderate protective efficacy in vivo against rice sheath blight. Porcine succinate dehydrogenase (SDH) enzymatic inhibitory assay revealed that A23 is a potent inhibitor of SDH, with a half-maximal inhibitory concentration of 0.0425 μm. Docking study within R. solani SDH indicated that A23 effectively binds into the ubiquinone site mainly through hydrogen-bonds, and cation-π and π-π interactions., Conclusion: The identified β-ketonitrile compound A23 containing diphenyl ether moiety is a potent SDH inhibitor, which might be a good lead for novel fungicide research and optimization. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: Plant fungal diseases present a major challenge to global agricultural production. Despite extensive efforts to develop fungicides, particularly succinate dehydrogenase inhibitors (SDHIs), their effectiveness is often limited by poor retention of fungicide droplets on hydrophobic leaves. The off-target losses and unintended release cause fungal resistance and severe environmental pollution., Results: To update the structure of existing SDHIs and synchronously realize the efficient utilization, we have employed a sophisticated supramolecular strategy to optimize a structurally novel SDH inhibitor (AoH25), creating an innovative supramolecular SDH fungicide (AoH25@β-CD), driven by the host-guest recognition principle between AoH25 and β-cyclodextrin (β-CD). Intriguingly, AoH25@β-CD self-assembles into biocompatible supramolecular nanovesicles, which reinforce the droplet/foliage (liquid-solid) interface interaction and the effective wetting and retention on leaf surfaces, setting the foundation for enhancing fungicide utilization. Mechanistic studies revealed that AoH25@β-CD exhibited significantly higher inhibition of SDH (IC 50  = 1.56 µM) compared to fluopyram (IC 50  = 244.41 µM) and AoH25 alone (IC 50  = 2.29 µM). Additionally, AoH25@β-CD increased the permeability of cell membranes in Botryosphaeria dothidea, facilitating better penetration of active ingredients into pathogenic cells. Further experimental outcomes confirmed that AoH25@β-CD was 88.5% effective against kiwifruit soft rot at a low-dose of 100 µg mL -1 , outperforming commercial fungicides such as fluopyram (52.4%) and azoxystrobin (65.4%). Moreover, AoH25@β-CD showed broad-spectrum bioactivity against oilseed rape sclerotinia, achieving an efficacy of 87.2%, outstripping those of fluopyram (48.7%) and azoxystrobin (76.7%)., Conclusion: This innovative approach addresses key challenges related to fungicide deposition and resistance, improving the bioavailability of agricultural chemicals. The findings highlight AoH25@β-CD as a novel supramolecular SDH inhibitor, demonstrating its potential as an efficient and sustainable solution for plant disease management., (© 2024. The Author(s).)

Dollar spot, a highly destructive turfgrasses disease worldwide, is caused by multiple species within the genus Clarireedia. Previous research indicated varying sensitivity to boscalid among Clarireedia populations not historically exposed to succinate dehydrogenase inhibitors (SDHIs). This study confirms that the differential sensitivity pattern is inherent among different Clarireedia spp., utilizing a combination of phylogenetic analyses, in vitro cross-resistance assays, and genetic transformation of target genes with different mutations. Furthermore, greenhouse inoculation experiments revealed that the differential boscalid sensitivity did not lead to pathogenicity issues or fitness penalties, thereby not resulting in control failure by boscalid. This research underscores the importance of continuous monitoring of fungicide sensitivity trends and highlights the complexity of chemical control of dollar spot due to the inherent variability in fungicide sensitivity among different Clarireedia spp., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study aimed to evaluate the effects of Euryale ferox Seed Shell Polyphenol Extract (EFSSPE) on a foodborne pathogenic bacterium. EFSSPE showed antimicrobial activity toward Salmonella Typhimurium CICC 22956; the minimum inhibitory concentration of EFSSPE was 1.25 mg/mL, the inhibition curve also reflected the inhibitory effect of EFSSPE on the growth of S . Typhimurium. Detection of alkaline phosphatase outside the cell revealed that EFSSPE treatment damaged the cell wall integrity of S. Typhimurium. EFSSPE also altered the membrane integrity, thereby causing leaching of 260-nm-absorbing material (bacterial proteins and DNA). Moreover, the activities of succinate dehydrogenase and malate dehydrogenase were inhibited by EFSSPE. The hydrophobicity and clustering ability of cells were affected by EFSSPE. Scanning electron microscopy showed that EFSSPE treatment damaged the morphology of the tested bacteria. These results indicate that EFSSPE can destroy the cell wall integrity and alter the permeability of the cell membrane of S. Typhimurium.

Consumers are exposed to succinate dehydrogenase inhibitor (SDHI) pesticides through their diet. A cumulative dietary risk assessment for the French population has been performed with French monitoring data (2017-2021) and consumption data from INCA3. The calculation followed a two-tiered approach, using deterministic then probabilistic methods. It was carried out, using European health based guidance values (HBGV) derived for each active substance to characterise their toxicity. In Tier I, the calculated hazard index of 0.12 was below the threshold of 1 and in Tier II, the total margin of exposure at percentile 99.9 remains above the trigger value of 100 (1798 [1631-2311]). In Tier II, the three main risk drivers identified at the upper tail of the distribution were strawberries-fluopyram (19.1%), peaches-fluopyram (14.1%) and table grapes-boscalid (10.5%). Finally, the impact of the major sources of uncertainties was qualitatively evaluated. All together, they were considered of low impact on the outcomes. This work demonstrates the absence of unacceptable chronic risk related to the cumulative exposure of SDHI for French consumers during the 2017-2021 period., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Understanding the stereoselective bioactivity of chiral pesticides is crucial for accurately evaluating their effectiveness and optimizing their use. Isopyrazam, a widely used chiral SDHI fungicide, has been studied for its antifungal activity only at the racemic level. Therefore, to clarify the highly bioactive isomers, the stereoselective bioactivity of isopyrazam isomers against four typical phytopathogens was studied for the first time. The bioactivity ranking of the isomers was trans- 1 S ,4 R ,9 R - (+)-isopyrazam > cis -1 R ,4 S ,9 R -(+)-isopyrazam > trans -1 R ,4 S ,9 S -(-)-isopyrazam > cis- 1 S ,4 R ,9 S -(-)-isopyrazam. SDH activity was assessed by molecular docking simulation and actual detection to confirm the reasons for stereoselective bioactivity. The results suggest that the stereoselective bioactivity of isopyrazam is largely dependent on the differential binding ability of each isomer to the SDH ubiquitin-binding site, located within a cavity formed by the iron-sulfur subunit, the cytochrome b560 subunit, and the cytochrome b small subunit. Moreover, to reveal the molecular mechanism of isopyrazam stereoselectively affecting mycelial growth, the contents of succinic acid, fumaric acid, and ATP were measured. Furthermore, by measuring exospore polysaccharides and oxalic acid content, it was determined that 1 S ,4 R ,9 R -(+)- and 1 R ,4 S ,9 R -(+)-isopyrazam more strongly inhibited the ability of Sclerotinia sclerotiorum to infect plants. The findings provided essential data for the development of high-efficiency isopyrazam fungicides and offered a methodological reference for analyzing the enantioselective activity mechanism of SDHI fungicides.

Succinate dehydrogenase (SDH) has been considered an ideal target for discovering fungicides. To develop novel SDH inhibitors, in this work, 31 novel benzothiazol-2-ylthiophenylpyrazole-4-carboxamides were designed and synthesized using active fragment exchange and a link approach as promising SDH inhibitors. The findings from the tests on antifungal activity indicated that most of the synthesized compounds displayed remarkable inhibition against the fungi tested. Compound Ig N -(2-(((5-chlorobenzo[ d ]thiazol-2-yl)thio)methyl)phenyl)-3-(difluoromethyl)-1-methyl-1 H -yrazole-4-carboxamide, with EC 50 values against four kinds of fungi tested below 10 μg/mL and against Cercospora arachidicola even below 2 μg/mL, showed superior antifungal activity than that of commercial fungicide thifluzamide, and specifically compounds Ig and Im were found to show preventative potency of 90.6% and 81.3% against Rhizoctonia solani Kühn , respectively, similar to the positive fungicide thifluzamide. The molecular simulation studies suggested that hydrophobic interactions were the main driving forces between ligands and SDH. Encouragingly, we found that compound Ig can effectively promote the wheat seedlings and the growth of Arabidopsis thaliana . Our further studies indicated that compound Ig could stimulate nitrate reductase activity in planta and increase the biomass of plants.

In this study, a series of novel hydrazide-containing flavonol derivatives was designed, synthesized, and evaluated for antifungal activity. In the in vitro antifungal assay, most of the target compounds exhibited potent antifungal activity against seven tested phytopathogenic fungi. In particular, compound C32 showed the best antifungal activity against Rhizoctonia solani (EC 50 = 0.170 μg/mL), outperforming carbendazim (EC 50 = 0.360 μg/mL) and boscalid (EC 50 = 1.36 μg/mL). Compound C24 exhibited excellent antifungal activity against Valsa mali , Botrytis cinerea , and Alternaria alternata with EC 50 values of 0.590, 0.870, and 1.71 μg/mL, respectively. The in vivo experiments revealed that compounds C32 and C24 were potential novel agricultural antifungals. 3D quantitative structure-activity relationship (3D-QSAR) models were used to analyze the structure-activity relationships of these compounds. The analysis results indicated that introducing appropriate electronegative groups at position 4 of a benzene ring could effectively improve the anti- R. solani activity. In the antifungal mechanism study, scanning electron microscopy and transmission electron microscopy analyses revealed that C32 disrupted the normal growth of hyphae by affecting the structural integrity of the cell membrane and cellular respiration. Furthermore, compound C32 exhibited potent succinate dehydrogenase (SDH) inhibitory activity (IC 50 = 8.42 μM), surpassing that of the SDH fungicide boscalid (IC 50 = 15.6 μM). The molecular dynamics simulations and docking experiments suggested that compound C32 can occupy the active site and form strong interactions with the key residues of SDH. Our findings have great potential for aiding future research on plant disease control in agriculture.

The diphenyl ether molecular pharmacophore has played a significant role in the development of fungicidal compounds. In this study, a variety of pyrazol-5-yl-phenoxybenzamide derivatives were synthesized and evaluated for their potential to act as succinate dehydrogenase inhibitors (SDHIs). The bioassay results indicate certain compounds to display a remarkable and broad-spectrum in their antifungal activities. Notably, compound 12x exhibited significant in vitro activities against Valsa mali , Gaeumannomyces graminis , and Botrytis cinerea , with EC 50 values of 0.52, 1.46, and 3.42 mg/L, respectively. These values were lower or comparable to those of Fluxapyroxad (EC 50 = 12.5, 1.93, and 8.33 mg/L, respectively). Additionally, compound 12x showed promising antifungal activities against Sclerotinia sclerotiorum (EC 50 = 0.82 mg/L) and Rhizoctonia solani (EC 50 = 1.86 mg/L), albeit lower than Fluxapyroxad (EC 50 = 0.23 and 0.62 mg/L). Further in vivo experiments demonstrated compound 12x to possess effective protective antifungal activities against V. mali and S. sclerotiorum at a concentration of 100 mg/L, with inhibition rates of 66.7 and 89.3%, respectively. In comparison, Fluxapyroxad showed inhibition rates of 29.2 and 96.4% against V. mali and S. sclerotiorum , respectively. Molecular docking analysis revealed that compound 12x interacts with SDH through hydrogen bonding, π-cation, and π-π interactions, providing insights into the probable mechanism of action. Furthermore, compound 12x exhibited greater binding energy and SDH enzyme inhibitory activity than Fluxapyroxad (Δ G cal = -46.8 kcal/mol, IC 50 = 1.22 mg/L, compared to Δ G cal = -41.1 kcal/mol, IC 50 = 8.32 mg/L). Collectively, our results suggest that compound 12x could serve as a promising fungicidal lead compound for the development of more potent SDHIs for crop protection.

A series of new piperidine-4-carbohydrazide derivatives bearing a quinazolinyl moiety were prepared and evaluated for their fungicidal activities against agriculturally important fungi. Among these derivatives, the chemical structure of compound A45 was clearly verified by X-ray crystallographic analysis. The antifungal bioassays revealed that many compounds in this series possessed good to excellent inhibition effects toward the tested fungi. For example, compounds A13 and A41 had EC 50 values of 0.83 and 0.88 μg/mL against Rhizoctonia solani in vitro , respectively, superior to those of positive controls Chlorothalonil and Boscalid (1.64 and 0.96 μg/mL, respectively). Additionally, the above two compounds also exhibited notable inhibitory activities against Verticillium dahliae (with EC 50 values of 1.12 and 3.20 μg/mL, respectively), far better than the positive controls Carbendazim and Chlorothalonil (19.3 and 11.0 μg/mL, respectively). More importantly, compound A13 could potently inhibit the proliferation of R. solani in the potted rice plants, showing good in vivo curative and protective efficiencies of 76.9% and 76.6% at 200 μg/mL, respectively. Furthermore, compound A13 demonstrated an effective inhibition of succinate dehydrogenase (SDH) activity in vitro with an IC 50 value of 6.07 μM. Finally, the molecular docking study revealed that this compound could be well embedded into the active pocket of SDH via multiple noncovalent interactions, involving residues like SER39, ARG43, and GLY46.

Bioisosteric silicon replacement has proven to be a valuable strategy in the design of bioactive molecules for crop protection and drug development. Twenty-one novel carboxamides possessing a silicon-containing biphenyl moiety were synthesized and tested for their antifungal activity and succinate dehydrogenase (SDH) enzymatic inhibitory activity. Among these novel succinate dehydrogenase inhibitors (SDHIs), compounds 3a , 3e , 4l, and 4o possessing appropriate c log P and topological polar surface area values showed excellent inhibitory effects against Rhizoctonia solani , Sclerotinia sclerotiorum , Botrytis cinerea , and Fusarium graminearum at 10 mg/L in vitro, and the EC 50 values of 4l and 4o were 0.52 and 0.16 mg/L against R. solani and 0.066 and 0.054 mg/L against S. sclerotiorum , respectively, which were superior to those of Boscalid. Moreover, compound 3a demonstrated superior SDH enzymatic inhibitory activity (IC 50 = 8.70 mg/L), exhibiting 2.54-fold the potency of Boscalid (IC 50 = 22.09 mg/L). Docking results and scanning electron microscope experiments revealed similar mode of action between compound 3a and Boscalid. The new silicon-containing carboxamide 3a is a promising SDHI candidate that deserves further investigation.

Magnaporthe oryzae is a rice blast pathogen that seriously threatens rice yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor (SDHI) fungicide that effectively controls many crop diseases. Benzovindiflupyr has a strong inhibitory effect on M. oryzae; however, control of rice blast by benzovindiflupyr and risk of resistance to benzovindiflupyr are not well studied in this pathogen. In this study, six benzovindiflupyr-resistant strains were obtained by domestication induced in the laboratory. The MoSdhB H245D mutation was the cause of M. oryzae resistance to benzovindiflupyr, which was verified through succinate dehydrogenase (SDH) activity assays, molecular docking, and site-specific mutations. Survival fitness analysis showed no significant difference between the benzovindiflupyr-resistant and parent strains. Positive cross-resistance to benzovindiflupyr and other SDHIs and negative cross-resistance to azoxystrobin were observed. Therefore, the risk of benzovindiflupyr resistance in M. oryzae might be medium to high. It should be combined with other classes of fungicides (tebuconazole and azoxystrobin) to slow the development of resistance., (Copyright © 2024. Published by Elsevier Inc.)

Rice blast, caused by Magnaporthe oryzae, is a devastating fungal disease worldwide. Pydiflumetofen (Pyd) is a new succinate dehydrogenase inhibitor (SDHI) that exhibited anti-fungal activity against M. oryzae. However, control of rice blast by Pyd and risk of resistance to Pyd are not well studied in this pathogen. The baseline sensitivity of 109 M. oryzae strains to Pyd was determined using mycelial growth rate assay, with EC 50 values ranging from 0.291 to 2.1313 μg/mL, and an average EC 50 value of 1.1005 ± 0.3727 μg/mL. Totally 28 Pyd-resistant (PydR) mutants with 15 genotypes of point mutations in succinate dehydrogenase (SDH) complex were obtained, and the resistance level could be divided into three categories of very high resistance (VHR), high resistance (HR) and moderate resistance (MR) with the resistance factors (RFs) of >1000, 105.74-986.13 and 81.92-99.48, respectively. Molecular docking revealed that all 15 mutations decreased the binding-force score for the affinity between Pyd and target subunits, which further confirmed that these 15 genotypes of point mutations were responsible for the resistance to Pyd in M. oryzae. There was positive cross resistance between Pyd and other SDHIs, such as fluxapyroxad, penflufen or carboxin, while there was no cross-resistance between Pyd and carbendazim, prochloraz or azoxystrobin in M. oryzae, however, PydR mutants with SdhB P198Q , SdhC L66F or SdhC L66R genotype were still sensitive to the other 3 SDHIs, indicating lack of cross resistance. The results of fitness study revealed that the point mutations in MoSdhB/C/D genes might reduce the hyphae growth and sporulation, but could improve the pathogenicity in M. oryzae. Taken together, the risk of resistance to Pyd might be moderate to high, and it should be used as tank-mixtures with other classes of fungicides to delay resistance development when it is used for the control of rice blast in the field., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Succinate dehydrogenase inhibitors are essential fungicides used in agriculture. To explore new pyrazole-carboxamides with high fungicidal activity, a series of N-substitutedphenyl-3-di/trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamides bearing a branched alkyl ether moiety were designed and synthesized. The in vitro bioassay indicated that some target compounds displayed appreciable fungicidal activity. For example, compounds 5d and 5e showed high efficacy against S. sclerotiorum with EC 50 values of 3.26 and 1.52 μg/mL respectively, and also exhibited excellent efficacy against R. solani with EC 50 values of 0.27 and 0.06 μg/mL respectively, which were comparable or superior to penflufen. The further in vivo bioassay on cucumber leaves demonstrated that 5e provided strong protective activity of 94.3 % against S. sclerotiorum at 100 μg/mL, comparable to penflufen (99.1 %). Cytotoxicity assessment against human renal cell lines (239A cell) revealed that 5e had low cytotoxicity within the median effective concentrations. Docking study of 5e with succinate dehydrogenase illustrated that R-5e formed one hydrogen bond and two π-π stacking interactions with amino acid residues of target enzyme, while S-5e formed only one π-π stacking interaction with amino acid residue. This study provides a valuable reference for the design of new succinate dehydrogenase inhibitor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Sclerotium rolfsii is a destructive soil-borne fungal pathogen which is distributed worldwide. In previous study, the succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr has been identified for its great antifungal activity against Sclerotium rolfsii. This study is aimed to investigate the resistance risk and mechanism of benzovindiflupyr in Sclerotium rolfsii., Results: Eight stable benzovindiflupyr-resistant isolates were generated by fungicide adaptation. Although the obtained eight resistant isolates have a stronger pathogenicity than the parental sensitive isolate, they have a fitness penalty in the mycelial growth and sclerotia formation compared to the parental isolate. A positive cross-resistance existed in the resistant isolates between benzovindiflupyr and thifluzamide, carboxin, boscalid and isopyrazam. Three-point mutations, including SdhB N180D , SdhC Q68E and SdhD H103Y , were identified in the benzovindiflupyr-resistant isolates. However, molecular docking analysis indicated that only SdhD H103Y could influence the sensitivity of Sclerotium rolfsii to benzovindiflupyr. After mycelial co-incubation of resistant isolates and the sensitive isolate, resistance genes may be transmitted to the sensitive isolate. The in vivo efficacy of benzovindiflupyr and thifluzamide against benzovindiflupyr-resistant isolates was a little lower than that against the sensitive isolate but with no significant difference., Conclusion: The results suggested a low to medium resistance risk of Sclerotium rolfsii to benzovindiflupyr. However, once resistance occurs, it is possible to spread in the population of Sclerotium rolfsii. This study is helpful to understanding the risk and mechanism of resistance to benzovindiflupyr in multinucleate pathogens such as Sclerotium rolfsii. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

The development of new fungicide molecules is a crucial task for agricultural chemists to enhance the effectiveness of fungicides in agricultural production. In this study, a series of novel fluoroalkenyl modified succinate dehydrogenase inhibitors were synthesized and evaluated for their antifungal activities against eight fungi. The results from the in vitro antifungal assay demonstrated that compound 34 exhibited superior activity against Rhizoctonia solani with an EC 50 value of 0.04 μM, outperforming commercial fluxapyroxad (EC 50 = 0.18 μM) and boscalid (EC 50 = 3.07 μM). Furthermore, compound 34 showed similar effects to fluxapyroxad on other pathogenic fungi such as Sclerotinia sclerotiorum (EC 50 = 1.13 μM), Monilinia fructicola (EC 50 = 1.61 μM), Botrytis cinerea (EC 50 = 1.21 μM), and also demonstrated protective and curative efficacies in vivo on rapeseed leaves and tomato fruits. Enzyme activity experiments and protein-ligand interaction analysis by surface plasmon resonance revealed that compound 34 had a stronger inhibitory effect on succinate dehydrogenase compared to fluxapyroxad. Additionally, molecular docking and DFT calculation confirmed that the fluoroalkenyl unit in compound 34 could enhance its binding capacity with the target protein through p-π conjugation and hydrogen bond interactions.

Aims: Azoles have been widely employed for the treatment of invasive fungal diseases; however, their efficacy is diminished as pathogenic fungi tolerate them due to their fungistatic properties. Geldanamycin (GdA) can render azoles fungicidal by inhibiting the ATPase and molecular chaperone activities of heat shock protein 90 (Hsp90). Nonetheless, the clinical applicability of GdA is restricted due to its cytotoxic ansamycin scaffold structure, its induction of cytoprotective heat shock responses, and the conservative nature of Hsp90. Hence, it is imperative to elucidate the mechanism of action of GdA to confer fungicidal properties to azoles and mitigate the toxic adverse effects associated with GdA., Materials and Methods: Through various experimental methods, including the construction of gene-deleted Candida albicans mutants, in vitro drug sensitivity experiments, Western blot analysis, reactive oxygen species (ROS) assays, and succinate dehydrogenase activity assays, we identified Hsp90 client proteins associated with the tolerance of C. albicans to azoles., Key Findings: It was observed that GdA effectively hindered the entry of Hsp90 into mitochondria, resulting in the alleviation of inhibitory effect of Hsp90 on succinate dehydrogenase. Consequently, the activation of succinate dehydrogenase led to an increased production of ROS. within the mitochondria, thereby facilitating the antifungal effects of azoles against C. albicans., Significance: This research presents a novel approach for conferring fungicidal properties to azoles, which involves specifically disrupting the interaction of between Hsp90 and succinate dehydrogenase rather than employing a non-specific inhibition of ATPase activity of Hsp90., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Mitochondrial electron transfer inhibitors at complex II (METI-II), also referred to as succinate dehydrogenase inhibitors (SDHI), represent a recently developed class of acaricides encompassing cyflumetofen, cyenopyrafen, pyflubumide and cyetpyrafen. Despite their novelty, resistance has already developed in the target pest, Tetranychus urticae. In this study a new mutation, H146Q in a highly conserved region of subunit B of complex II, was identified in a T. urticae population resistant to all METI-IIs. In contrast to previously described mutations, H146Q is located outside the ubiquinone binding site of complex II. Marker-assisted backcrossing of this mutation in a susceptible genetic background validated its association with resistance to cyflumetofen and pyflubumide, but not cyenopyrafen or cyetpyrafen. Biochemical assays and the construction of inhibition curves with isolated mitochondria corroborated this selectivity. In addition, phenotypic effects of H146Q, together with the previously described H258L, were further examined via CRISPR/Cas9 gene editing. Although both mutations were successfully introduced into a susceptible T. urticae population, the H146Q gene editing event was only recovered in individuals already harboring the I260V mutation, known to confer resistance towards cyflumetofen. The combination of H146Q + I260V conferred high resistance levels to all METI-II acaricides with LC 50 values over 5000 mg a.i./L for cyflumetofen and pyflubumide. Similarly, the introduction of H258L via gene editing resulted in high resistance levels to all tested acaricides, with extreme LC 50 values (>5000 mg a.i./L) for cyenopyrafen and cyetpyrafen, but lower resistance levels for pyflubumide and cyflumetofen. Together, these findings indicate that different mutations result in a different cross-resistance spectrum, probably also reflecting subtle differences in the binding mode of complex II acaricides., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bursaphelenchus xylophilus is a dangerous quarantine pest that causes extensive damage to pine ecosystems worldwide. Cyclobutrifluram, a succinate dehydrogenase inhibitor (SDHI), is a novel nematicide introduced by Syngenta in 2013. However, the nematocidal effect of cyclobutrifluram against plant-parasitic nematodes remains underexplored. Therefore, here, we aim to address this knowledge gap by evaluating the toxicity, effects, and mode of action of cyclobutrifluram on B. xylophilus . The result shows that cyclobutrifluram is the most effective agent, with an LC 50 value of 0.1078 mg·L -1 . At an LC 20 dose, it significantly reduced the population size to 10.40 × 10 3 ± 737.56-approximately 1/23 that of the control group. This notable impact may stem from the agent's ability to diminish egg-laying and hatching rates, as well as to impede the nematodes' development. In addition, it has also performed well in the prevention of pine wilt disease, significantly reducing the incidence in greenhouses and in the field. SDH consists of a transmembrane assembly composed of four protein subunits (SDHA to SDHD). Four sdh genes were characterized and proved by RNAi to regulate the spawning capacity, locomotion ability, and body size of B. xylophilus. The mortality of nematodes treated with sdhc -dsRNA significantly decreased upon cyclobutrifluram application. Molecular docking further confirmed that SDHC, a cytochrome-binding protein, is the target. In conclusion, cyclobutrifluram has a good potential for trunk injection against B. xylophilus. This study provides valuable information for the screening and application of effective agents in controlling and preventing PWD in forests.

Plant pathogenic fungi pose a significant threat to agricultural production, necessitating the development of new and more effective fungicides. The ring replacement strategy has emerged as a highly successful approach in molecular design. In this study, we employed the ring replacement strategy to successfully design and synthesize 32 novel hydrazide derivatives containing diverse heterocycles, such as thiazole, isoxazole, pyrazole, thiadiazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, thiophene, pyridine, and pyrazine. Their antifungal activities were evaluated in vitro and in vivo . Bioassay results revealed that most of the title compounds displayed remarkable antifungal activities in vitro against four tested phytopathogenic fungi, including Fusarium graminearum , Botrytis cinerea , Sclerotinia sclerotiorum , and Rhizoctonia solani . Especially, compound 5aa displayed a broad spectrum of antifungal activity against F. graminearum , B. cinerea , S. sclerotiorum , and R. solani , with the corresponding EC 50 values of 0.12, 4.48, 0.33, and 0.15 μg/mL, respectively. In the antifungal growth assay, compound 5aa displayed a protection efficacy of 75.5% against Fusarium head blight (FHB) at a concentration of 200 μg/mL. In another in vivo antifungal activity evaluation, compound 5aa exhibited a noteworthy protective efficacy of 92.0% against rape Sclerotinia rot (RSR) at a concentration of 100 μg/mL, which was comparable to the positive control tebuconazole (97.5%). The existing results suggest that compound 5aa has a broad-spectrum antifungal activity. Electron microscopy observations showed that compound 5aa might cause mycelial abnormalities and organelle damage in F. graminearum . Moreover, in the in vitro enzyme assay, we found that the target compounds 5aa , 5ab , and 5ca displayed significant inhibitory effects toward succinate dehydrogenase, with the corresponding IC 50 values of 1.62, 1.74, and 1.96 μM, respectively, which were superior to that of boscalid (IC 50 = 2.38 μM). Additionally, molecular docking and molecular dynamics simulation results revealed that compounds 5aa , 5ab , and 5ca have the capacity to bind in the active pocket of succinate dehydrogenase (SDH), establishing hydrogen-bonding interactions with neighboring amino acid residues.

Background: Resistance to succinate dehydrogenase inhibitor (SDHI) fungicides has been reported in some rust fungi within Pucciniales. However, measuring the resistance factors conferred by a specific substitution at the target site is difficult for most species because of the difficulty in performing in vitro experiments and the complexity of the binuclear state in these obligate parasites. We focused on Puccinia horiana because it easily forms homozygous basidiospores that are sensitive to SDHIs during in vitro germination, whereas the uredospores of other rust fungi are less sensitive., Results: We identified two substitutions, SdhC-I88F and SdhD-C125Y, that drive SDHI resistance in Pu. horiana. Using basidiospore germination inhibition tests, we measured the resistance factors for six SDHI fungicides in Pu. horiana isolates harboring SdhC-I88F substitutions, wherein orthologous substitutions were most frequently observed in SDHI-resistant Pucciniales, such as soybean rust (Phakopsora pachyrhizi). The resistance factors were high for penthiopyrad and benzovindiflupyr (>150), moderate for oxycarboxin and inpyrfluxam (10-30), and low for mepronil and fluxapyroxad (3-10). The most potent SDHI against SdhC-I88F-harboring isolates was inpyrfluxam, with a half-maximal effective concentration (EC 50 ) of 0.0082 mg L -1 owing to its high intrinsic activity. SdhD-C125Y played a minor, but significant role in increasing the resistance factors (one- to tenfold increases), depending on the individual SDHIs., Conclusion: This study is the first to use basidiospore germination inhibitory tests to quantify the resistance factors for SDHI-resistant Pucciniales. Owing to its homozygous binucleate nature and the high availability of basidiospores, Pu. horiana is useful for investigating SDHI resistance in Pucciniales. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Ischaemia-reperfusion (IR) injury is the paradoxical consequence of the rapid restoration of blood flow to an ischaemic organ. Although reperfusion is essential for tissue survival in conditions such as myocardial infarction and stroke, the excessive production of mitochondrial reactive oxygen species (ROS) upon reperfusion initiates the oxidative damage that underlies IR injury, by causing cell death and inflammation. This ROS production is caused by an accumulation of the mitochondrial metabolite succinate during ischaemia, followed by its rapid oxidation upon reperfusion by succinate dehydrogenase (SDH), driving superoxide production at complex I by reverse electron transport. Inhibitors of SDH, such as malonate, show therapeutic potential by decreasing succinate oxidation and superoxide production upon reperfusion. To better understand the mechanism of mitochondrial ROS production upon reperfusion and to assess potential therapies, we set up an in vitro model of IR injury. For this, isolated mitochondria were incubated anoxically with succinate to mimic ischaemia and then rapidly reoxygenated to replicate reperfusion, driving a burst of ROS formation. Using this system, we assess the factors that contribute to the magnitude of mitochondrial ROS production in heart, brain, and kidney mitochondria, as well as screening for inhibitors of succinate oxidation with therapeutic potential., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michael P. Murphy is on the Scientific Advisory Board of MitoQ, Inc. and holds stock in the company, is CSO of Camoxis Therapeutics Inc., which is developing malonate as a therapy and he holds patents in therapeutic applications of malonate. Thomas Krieg is CMO of Camoxis Therapeutics Inc., which is developing malonate as a therapy and he holds patents in therapeutic applications of malonate. Hiran A. Prag holds a patent in therapeutic applications of malonate. All other authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

To promote the development and exploitation of novel antifungal agents, a series of thiazol-2-ylbenzamide derivatives (3A-3V) and thiazole-2-ylbenzimidoyl chloride derivatives (4A-4V) were designed and selective synthesis. The bioassay results showed that most of the target compounds exhibited excellent in vitro antifungal activities against five plant pathogenic fungi (Valsa mali, Sclerotinia scleotiorum, Botrytis cinerea, Rhizoctonia solani and Trichoderma viride). The antifungal effects of compounds 3B (EC 50  = 0.72 mg/L) and 4B (EC 50  = 0.65 mg/L) against S. scleotiorum were comparable to succinate dehydrogenase inhibitors (SDHIs) thifluzamide (EC 50  = 1.08 mg/L) and boscalid (EC 50  = 0.78 mg/L). Especially, compounds 3B (EC 50  = 0.87 mg/L) and 4B (EC 50  = 1.08 mg/L) showed higher activity against R. solani than boscalid (EC 50  = 2.25 mg/L). In vivo experiments in rice leaves revealed that compounds 3B (86.8 %) and 4B (85.3 %) exhibited excellent protective activities against R. solani comparable to thifluzamide (88.5 %). Scanning electron microscopy (SEM) results exhibited that compounds 3B and 4B dramatically disrupted the typical structure and morphology of R. solani mycelium. Molecular docking demonstrated that compounds 3B and 4B had significant interactions with succinate dehydrogenase (SDH). Meanwhile, SDH inhibition assay results further proved their potential as SDHIs. In addition, acute oral toxicity tests on A. mellifera L. showed only low toxicity for compounds 3B and 4B to A. mellifera L. populations. These results suggested that these two series of compounds had merit for further investigation as potential low-risk agricultural SDHI fungicides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Watermelon is affected by diseases such as Fusarium wilt, gummy stem blight, and root-knot nematode (RKN). Succinate dehydrogenase inhibitors (SDHIs) with potential fungicide and nematicide activity provide the opportunity to control multiple diseases with one compound. In this study, we aimed to determine the sensitivity of Meloidogyne incognita race 4 (MI4), Fusarium oxysporum f. sp. niveum (FON), and Stagonosporopsis citrulli (SCIT) to existing SDHIs: benzovindiflupyr, fluopyram, cyclobutrifluram, and pydiflumetofen. All SDHIs had fungicidal activity against 19 SCIT isolates in mycelial growth assays, but isolates were most sensitive to pydiflumetofen (median EC 50 = 0.41 μg/ml). Most of the 50 FON isolates tested were sensitive to cyclobutrifluram for mycelial growth (median EC 50 = 4.04 μg/ml) and conidial germination (median EC 50 = 0.2 μg/ml) assays but were not sensitive to fluopyram. MI4 was most sensitive to cyclobutrifluram for egg hatch (mean EC 50 = 0.0019 μg/ml) and J2 motility (mean EC 50 = 1.16 μg/ml) assays but was not sensitive to pydiflumetofen. Significant positive correlations between the sensitivity of SCIT (mycelial growth) and FON (mycelial growth and conidial germination) for cyclobutrifluram and benzovindiflupyr (SCIT r = 0.88; FON r = 0.7; P < 0.0001) and cyclobutrifluram and pydiflumetofen (SCIT r = 0.83; FON r = 0.67 and 0.77; P < 0.0001) indicate a potential for cross-resistance between these SDHIs for these fungal pathogens. Overall, results suggest that cyclobutrifluram may be used for managing RKN, whereas it should be used judiciously for Fusarium wilt of watermelon and gummy stem blight due to the existence of insensitive isolates to the fungicide., Competing Interests: The author(s) declare no conflict of interest.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC 50 values in the 1.0-3.9 μM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC 50  = 2.0 μM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC 50  = 3.9 μM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The pursuit of new succinate dehydrogenase (SDH) inhibitors is a leading edge in fungicide research and development. The use of 3D quantitative structure-activity relationship (3D-QSAR) models significantly enhances the development of compounds with potent antifungal properties. In this study, we leveraged the natural product coumarin as a molecular scaffold to synthesize 74 novel 3-coumarin hydrazide derivatives. Notably, compounds 4ap (0.28 μg/mL), 6ae (0.32 μg/mL), and 6ah (0.48 μg/mL) exhibited exceptional in vitro effectiveness against Rhizoctonia solani , outperforming the commonly used fungicide boscalid (0.52 μg/mL). Furthermore, compounds 4ak (0.88 μg/mL), 6ae (0.61 μg/mL), 6ah (0.65 μg/mL), and 6ak (1.11 μg/mL) showed significant activity against Colletotrichum orbiculare , surpassing both the SDHI fungicide boscalid (43.45 μg/mL) and the broad-spectrum fungicide carbendazim (2.15 μg/mL). Molecular docking studies and SDH enzyme assays indicate that compound 4ah may serve as a promising SDHI fungicide. Our ongoing research aims to refine this 3D-QSAR model further, enhance molecular design, and conduct additional bioactivity assays.

The dearomatization at the hydrophobic tail of the boscalid was carried out to construct a series of novel pyrazole-4-carboxamide derivatives containing an oxime ether fragment. By using fungicide-likeness analyses and virtual screening, 24 target compounds with theoretical strong inhibitory effects against fungal succinate dehydrogenase (SDH) were designed and synthesized. Antifungal bioassays showed that the target compound E1 could selectively inhibit the in vitro growth of R. solani , with the EC 50 value of 1.1 μg/mL that was superior to that of the agricultural fungicide boscalid (2.2 μg/mL). The observations by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that E1 could reduce mycelial density and significantly increase the mitochondrial number in mycelia cytoplasm, which was similar to the phenomenon treated with boscalid. Enzyme activity assay showed that the E1 had the significant inhibitory effect against the SDH from R. solani , with the IC 50 value of 3.3 μM that was superior to that of boscalid (7.9 μM). The mode of action of the target compound E1 with SDH was further analyzed by molecular docking and molecular dynamics simulation studies. Among them, the number of hydrogen bonds was significantly more in the SDH- E1 complex than that in the SDH-boscalid complex. This research on the dearomatization strategy of the benzene ring for constructing pyrazole-4-carboxamides containing an oxime ether fragment provides a unique thought to design new antifungal drugs targeting SDH.

Succinate dehydrogenase (SDH) is an integral component of the tricarboxylic acid cycle (TCA) and respiratory electron transport chain (ETC), targeted by succinate dehydrogenase inhibitors (SDHIs). Fusarium asiaticum is a prominent phytopathogen causing Fusarium head blight (FHB) on wheat. Here, we characterized the functions of the FaSdhA, FaSdhB, FaSdhC 1 , FaSdhC 2 , and FaSdhD subunits. Deletion of FaSdhA , FaSdhB , or FaSdhD resulted in significant growth defects in F. asiaticum . The FaSdhC 1 or FaSdhC 2 deletion mutants exhibited substantial reductions in fungal growth, conidiation, virulence, and reactive oxygen species (ROS). The FaSdhC 1 expression was significantly induced by pydiflumetofen (PYD). The Δ FaSdhC 1 mutant displayed hypersensitivity to SDHIs, whereas the Δ FaSdhC 2 mutant exhibited resistance against most SDHIs. The transmembrane domains of FaSdhC 1 are essential for regulating mycelial growth, virulence, and sensitivity to SDHIs. These findings provided valuable insights into how the two SdhC paralogues regulated the functional integrity of SDH, ROS homeostasis, and the sensitivity to SDHIs in phytopathogenic fungi.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC 50 =22.5 µM), fluopyram (EC 50 =4.8 µM) and flutolanil (EC 50 =53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In the search for novel succinate dehydrogenase inhibitor (SDHI) fungicides to control Rhizoctonia solani , thirty-five novel pyrazole-4-carboxamides bearing either an oxime ether or an oxime ester group were designed and prepared based on the strategy of molecular hybridization, and their antifungal activities against five plant pathogenic fungi were also investigated. The results indicated that the majority of the compounds containing oxime ether demonstrated outstanding in vitro antifungal activity against R. solani, and some compounds also displayed pronounced antifungal activities against Sclerotinia sclerotiorum and Botrytis cinerea . Particularly, compound 5e exhibited the most promising antifungal activity against R. solani with an EC 50 value of 0.039 μg/mL, which was about 20-fold better than that of boscalid (EC 50 = 0.799 μg/mL) and 4-fold more potent than fluxapyroxad (EC 50 = 0.131 μg/mL). Moreover, the results of the detached leaf assay showed that compound 5e could suppress the growth of R. solani in rice leaves with significant protective efficacies (86.8%) at 100 μg/mL, superior to boscalid (68.1%) and fluxapyroxad (80.6%), indicating promising application prospects. In addition, the succinate dehydrogenase (SDH) enzymatic inhibition assay revealed that compound 5e generated remarkable SDH inhibition (IC 50 = 2.04 μM), which was obviously more potent than those of boscalid (IC 50 = 7.92 μM) and fluxapyroxad (IC 50 = 6.15 μM). Furthermore, SEM analysis showed that compound 5e caused a remarkable disruption to the characteristic structure and morphology of R. solani hyphae, resulting in significant damage. The molecular docking analysis demonstrated that compound 5e could fit into the identical binding pocket of SDH through hydrogen bond interactions as well as fluxapyroxad, indicating that they had a similar antifungal mechanism. The density functional theory and electrostatic potential calculations provided useful information regarding electron distribution and electron transfer, which contributed to understanding the structural features and antifungal mechanism of the lead compound. These findings suggested that compound 5e could be a promising candidate for SDHI fungicides to control R. solani , warranting further investigation.

Succinate dehydrogenase inhibitor (SDHI) fungicides are the most commonly and effectively used class of fungicides for controlling gray mold. Among them, only boscalid has been registered in China for controlling grape gray mold, whereas isofetamid and pydiflumetofen are two new SDHI fungicides that have demonstrated high efficacy against various fungal diseases. However, the sensitivity of Botrytis cinerea isolates from vineyards in China to these three fungicides is currently unknown. In this study, the sensitivity of 55 B. cinerea isolates from vineyards to boscalid, isofetamid, and pydiflumetofen was determined, with the effective concentrations for inhibiting 50% of spore germination (EC 50 ) values ranging from 1.10 to 393, 0.0300 to 42.0, and 0.0990 to 25.5 μg ml -1 , respectively. The resistance frequencies for boscalid, isofetamid, and pydiflumetofen were 60.0, 7.2, and 12.8%, respectively. Three mutations (H272R, H272Y, and P225F) were detected in the SdhB subunit, with H272R being the most prevalent (75.7%), followed by H272Y (16.2%) and P225F (8.1%). All three mutations are associated with resistance to boscalid, and of them, H272R mutants exhibited high resistance. Only P225F and H272Y mutants exhibited resistance to isofetamid and pydiflumetofen, respectively. A weakly positive cross-resistance relationship was observed between boscalid and pydiflumetofen ( r = 0.38, P < 0.05). Additionally, the H272R mutants showed no significant fitness costs, whereas the remaining mutants exhibited reduced mycelial growth (P225F) and sporulation (H272Y and P225F). These results suggest that isofetamid and pydiflumetofen are effective fungicides against B. cinerea in vineyards, but appropriate rotation strategies must be implemented to reduce the selection of existing SDHI-resistant isolates., Competing Interests: The author(s) declare no conflict of interest.

Succinate dehydrogenase (SDH) is an important inner mitochondrial membrane-bound enzyme involved in redox reactions during the tricarboxylic acid cycle. Therefore, a series of novel chitosan derivatives were designed and synthesized as potential microbicides targeting SDH and precisely characterized by FTIR, 1 H NMR and SEM. Their antifungal and antibacterial activities were evaluated against Botrytis cinerea, Fusarium graminearum, Staphylococcus aureus and Escherichia coli. The bioassays revealed that these chitosan derivatives exerted significant antifungal effects, with four of the compounds achieving 100 % inhibition of Fusarium graminearum merely at a concentration of 0.5 mg/mL. Additionally, CSGDCH showed 79.34 % inhibition of Botrytis cinerea at a concentration of 0.1 mg/mL. In vitro antibacterial tests revealed that CSGDCH and CSGDBH have excellent Staphylococcus aureus and Escherichia coli inhibition with MICs of 0.0156 mg/mL and 0.03125 mg/mL, respectively. Molecular docking studies have been carried out to explore the binding energy and binding mode of chitosan and chitosan derivatives with SDH. The analyses indicated that chitosan derivatives targeted the active site of the SDH protein more precisely, disrupting its normal function and ultimately repressing the growth of microbial cells. Furthermore, the chitosan derivatives were also evaluated biologically for antioxidation, and all of these compounds had a greater degree of reducing power, superoxide radical, hydroxyl radical and DPPH-radical scavenging activity than chitosan. This research has the potential for the development of agricultural antimicrobial agents., Competing Interests: Declaration of competing interest The authors declare that there is no competing financial interests or conflict relationships between them., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Succinate dehydrogenase inhibition with malonate during initial reperfusion reduces myocardial infarct size in both isolated mouse hearts subjected to global ischemia and in in situ pig hearts subjected to transient coronary ligature. However, the long-term effects of acute malonate treatment are unknown. Here, we investigated whether the protective effects of succinate dehydrogenase inhibition extend to a reduction in scar size and adverse left ventricular remodeling 28 days after myocardial infarction. Initially, ten wild-type mice were subjected to 45 min of left anterior descending coronary artery (LAD) occlusion, followed by 24 h of reperfusion, and were infused during the first 15 min of reperfusion with saline with or without disodium malonate (10 mg/kg/min, 120 μL/kg/min). Malonate-treated mice depicted a significant reduction in infarct size (15.47 ± 3.40% of area at risk vs. 29.34 ± 4.44% in control animals, p < 0.05), assessed using triphenyltetrazolium chloride. Additional animals were then subjected to a 45 min LAD ligature, followed by 28 days of reperfusion. Treatment with a single dose of malonate during the first 15 min of reperfusion induced a significant reduction in scar area, measured using Picrosirius Red staining (11.94 ± 1.70% of left ventricular area (n = 5) vs. 23.25 ± 2.67% (n = 9), p < 0.05), an effect associated with improved ejection fraction 28 days after infarction, as determined using echocardiography, and an attenuated enhancement in expression of the pro-inflammatory and fibrotic markers NF-κB and Smad2/3 in remote myocardium. In conclusion, a reversible inhibition of succinate dehydrogenase with a single dose of malonate at the onset of reperfusion has long-term protective effects in mice subjected to transient coronary occlusion., Competing Interests: The authors declare no conflicts of interest.

To discover new succinate dehydrogenase inhibitors (SDHI) fungicides, a series of amide derivatives containing a pyrrolidine moiety were designed and synthesized, and their antifungal activities were evaluated against Monilinia fructicola (M. fructicola), Rhizoctonia solani (R. solani), Fusarium graminearum schw (F. graminearum), Fusarium oxysporum (F. oxysporum), and Phytophthora infestans (P. infestans). Some compounds showed excellent antifungal activities against the five fungi. Among them, compound 6 showed broad-spectrum inhibitory activities. The EC 50 of compound 6 against M. fructicola, R. solani, F. graminearum, F. oxysporum, and P. infestans were 2.13, 14.42, 1.69, 27.79, and 27.12 mg/L, respectively. In addition, compound 6 can effectively inhibit the spore germination of M. fructicola and has moderate damage to the cell membrane. Compound 6 can effectively inhibit succinate dehydrogenase (SDH) of M. fructicola, and can significantly increase the expression levels of SDHC and SDHD. Compound 6 can be used as a lead structure for developing new SDH inhibitors., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Succinate dehydrogenase inhibitors (SDHIs) have been widely used to manage plant diseases caused by phytopathogenic fungi. Although attention to and use of SDHI fungicides has recently increased, molecular responses of fungal pathogens to SDHIs have often not been investigated. A SDHI fungicide, fluopyram, has been used as a soybean seed treatment and has displayed effective control of Fusarium virguliforme, one of the causal agents of soybean sudden death syndrome. To examine genome-wide gene expression of F. virguliforme to fluopyram, RNA-seq analysis was conducted on two field strains of F. virguliforme with differing SDHI fungicide sensitivity in the absence and presence of fluopyram., Results: The analysis indicated that several xenobiotic detoxification-related genes, such as those of deoxygenase, transferases and transporters, were highly induced by fluopyram. Among the genes, four ATP-binding cassette (ABC) transporters were characterized by the yeast expression system. The results revealed that expression of three ABCG transporters was associated with reduced sensitivity to multiple fungicides including fluopyram. In addition, heterologous expression of a major facilitator superfamily (MFS) transporter that was highly expressed in the fluopyram-insensitive F. virguliforme strain in the yeast system conferred decreased sensitivity to fluopyram., Conclusion: This study demonstrated that xenobiotic detoxification-related genes were highly upregulated in response to fluopyram, and expression of ABC or MFS transporter genes was associated with reduced sensitivity to the SDHI fungicide. This is the first transcriptomic analysis of the fungal species response to fluopyram and the finding will help elucidate the molecular mechanisms of SDHI resistance. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Succinate dehydrogenase inhibitor (SDHI) fungicides are increasingly used in agriculture to combat molds and fungi, two major threats to both food supply and public health. However, the essential requirement for the succinate dehydrogenase (SDH) complex-the molecular target of SDHIs-in energy metabolism for almost all extant eukaryotes and the lack of species specificity of these fungicides raise concerns about their toxicity toward off-target organisms and, more generally, toward the environment. Herein we review the current knowledge on the toxicity toward zebrafish ( Brachydanio rerio ) of nine commonly used SDHI fungicides: bixafen, boscalid, fluxapyroxad, flutolanil, isoflucypram, isopyrazam, penthiopyrad, sedaxane, and thifluzamide. The results indicate that these SDHIs cause multiple adverse effects in embryos, larvae/juveniles, and/or adults, sometimes at developmentally relevant concentrations. Adverse effects include developmental toxicity, cardiovascular abnormalities, liver and kidney damage, oxidative stress, energy deficits, changes in metabolism, microcephaly, axon growth defects, apoptosis, and transcriptome changes, suggesting that glycometabolism deficit, oxidative stress, and apoptosis are critical in the toxicity of most of these SDHIs. However, other adverse outcome pathways, possibly involving unsuspected molecular targets, are also suggested. Lastly, we note that because of their recent arrival on the market, the number of studies addressing the toxicity of these compounds is still scant, emphasizing the need to further investigate the toxicity of all SDHIs currently used and to identify their adverse effects and associated modes of action, both alone and in combination with other pesticides.

Succinate dehydrogenase inhibitors (SDHIs) have become one of the fastest growing classes of new fungicides since entering the market, and have attracted increasing attention as a result of their unique structure, high activity and broad fungicidal spectrum. The mechanism of SDHIs is to inhibit the activity of succinate dehydrogenase, thereby affecting mitochondrial respiration and ultimately killing pathogenic fungi. At present, they have become popular varieties researched and developed by major pesticide companies in the world. In the review, we focused on the mechanism, the history, the representative varieties, structure-activity relationship and resistance of SDHIs. Finally, the potential directions for the development of SDHIs were discussed. It is hoped that this review can strengthen the individuals' understanding of SDHIs and provide some inspiration for the development of new fungicides., (Copyright © 2021. Published by Elsevier Ltd.)

Botrytis cinerea is a ubiquitous fungus that affects hundreds of plants, resulting in economic losses to the horticulture and fruit industry. The search for new antifungal agents is a matter of current interest. Thus, in this work a series of geranylated phenols in which the side alkyl chain has been hydrated have been synthesized, and their activity against B. cinerea has been evaluated. The coupling of phenol and geraniol has been accomplished under microwave irradiation obtaining the highest reaction yields in the shortest reaction times. Hydration of the side chain was carried out in dioxane with p -toluenesulfonic acid polymer-bound as the catalyst. All synthesized compounds were tested against B. cinerea using the growth inhibition assay and EC 50 values were determined. The results show that activity depends on the number and nature of functional groups in the phenol ring and hydration degree of the geranyl chain. The most active compound is 1,4-dihydroquinone with one hydroxyl group attached at the end of the alkyl chain. Results from a molecular docking study suggest that hydroxyl groups in the phenol ring and alkyl chain are important in the binding of compounds to the active site, and that the experimental antifungal activity correlates with the number of H-bond that can be formed in the binding site.

Inflammation is a critical component of cardiovascular disease (CVD), encompassing coronary artery disease (CAD), cerebrovascular events and heart failure and is the leading cause of mortality worldwide. In recent years, metabolism has been placed centrally in the governance of the immune response. Termed immunometabolism, immune cells adapt cellular metabolic pathways to meet demands of activation and thus function. This rewiring influences not only the bioenergetics of the cell but altered metabolites act as signalling molecules to regulate cellular response. In this review, we focus on the TCA cycle derivative, itaconate, as one such metabolite with promising immunomodulatory and therapeutic potential in inflammatory cardiovascular disease., (© 2021 The Author(s).)

Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.

Inhibition of succinate dehydrogenase (SDH) by Dimethyl Malonate (DiMal) reduces cardiac ischemia-reperfusion (IR) injury. We investigated the cardioprotective effect of DiMal in a rat model during advancing type 2 diabetes. Zucker Diabetic Fatty rats and lean controls were investigated corresponding to prediabetes, onset and mature diabetes. Hearts were mounted in an isolated perfused model, and subjected to IR for investigation of infarct size (IS) and mitochondrial respiratory control ratio (RCR). DiMal was administered for 10 min before ischemia. Compared with age-matched non-diabetic rats, prediabetic rats had larger IS (49 ± 4% vs. 36 ± 2%, p = 0.007), rats with onset diabetes smaller IS (51 ± 3% vs. 62 ± 3%, p = 0.05) and rats with mature diabetes had larger IS (79 ± 3% vs. 69 ± 2%, p = 0.06). At the prediabetic stage DiMal did not alter IS. At onset of diabetes DiMal 0.6 mM increased IS in diabetic but not in non-diabetic control rats (72 ± 4% vs. 51 ± 3%, p = 0.003). At mature diabetes DiMal 0.1 and 0.6 mM reduced IS (68 ± 3% vs. 79 ± 3% and 64 ± 5% vs. 79 ± 3%, p  = 0.1 and p = 0.01), respectively. DiMal 0.1 mM alone reduced IS in age-matched non-diabetic animals (55 ± 3% vs. 69 ± 2% p = 0.01). RCR was reduced at mature diabetes but not modulated by DiMal. Modulation of SDH activity results in variable infarct size reduction depending on presence and the stage of diabetes. Modulation of SDH activity may be an unpredictable cardioprotective approach.

Metabolic reprogramming is associated with NLRP3 inflammasome activation in activated macrophages, contributing to inflammatory responses. Tanshinone IIA (Tan-IIA) is a major constituent from Salvia miltiorrhiza Bunge, which exhibits anti-inflammatory activity. In this study, we investigated the effects of Tan-IIA on inflammation in macrophages in focus on its regulation of metabolism and redox state. In lipopolysaccharides (LPS)-stimulated mouse bone marrow-derived macrophages (BMDMs), Tan-IIA (10 μM) significantly decreased succinate-boosted IL-1β and IL-6 production, accompanied by upregulation of IL-1RA and IL-10 release via inhibiting succinate dehydrogenase (SDH). Tan-IIA concentration dependently inhibited SDH activity with an estimated IC 50 of 4.47 μM in LPS-activated BMDMs. Tan-IIA decreased succinate accumulation, suppressed mitochondrial reactive oxygen species production, thus preventing hypoxia-inducible factor-1α (HIF-1α) induction. Consequently, Tan-IIA reduced glycolysis and protected the activity of Sirtuin2 (Sirt2), an NAD + -dependent protein deacetylase, by raising the ratio of NAD + /NADH in activated macrophages. The acetylation of α-tubulin was required for the assembly of NLRP3 inflammasome; Tan-IIA increased the binding of Sirt2 to α-tubulin, and thus reduced the acetylation of α-tubulin, thus impairing this process. Sirt2 knockdown or application of Sirt2 inhibitor AGK-2 (10 μM) neutralized the effects of Tan-IIA, suggesting that Tan-IIA inactivated NLRP3 inflammasome in a manner dependent on Sirt2 regulation. The anti-inflammatory effects of Tan-IIA were observed in mice subjected to LPS challenge: pre-administration of Tan-IIA (20 mg/kg, ip) significantly attenuated LPS-induced acute inflammatory responses, characterized by elevated IL-1β but reduced IL-10 levels in serum. The peritoneal macrophages isolated from the mice displayed similar metabolic regulation. In conclusion, Tan-IIA reduces HIF-1α induction via SDH inactivation, and preserves Sirt2 activity via downregulation of glycolysis, contributing to suppression of NLRP3 inflammasome activation. This study provides a new insight into the anti-inflammatory action of Tan-IIA from the respect of metabolic and redox regulation.

Succinate dehydrogenase (SDH) inhibition with malonate during reperfusion reduced myocardial infarction in animals, whereas its endogenous substrate, succinate, is detected in plasma from STEMI patients. We investigated whether protection by SDH inhibition is additive to that of remote ischemic perconditioning (RIC) in pigs submitted to transient coronary artery occlusion, and whether protective maneuvers influence plasma levels of citric acid cycle metabolites. Forty pigs were submitted to 40 min coronary occlusion and reperfusion, and allocated to four groups (controls, sodium malonate 10 mmol/L, RIC, and malonate + RIC). Plasma was obtained from femoral and great cardiac veins and analyzed by LC-MS/MS. Malonate, RIC, and malonate + RIC reduced infarct size (24.67 ± 5.98, 25.29 ± 3.92 and 29.83 ± 4.62% vs. 46.47 ± 4.49% in controls, p < 0.05), but no additive effects were detected. Enhanced concentrations of succinate, fumarate, malate and citrate were observed in controls during initial reperfusion in the great cardiac vein, and most were reduced by cardioprotective maneuvers. Concentrations of succinate, fumarate, and malate significantly correlated with infarct size. In conclusion, despite the combination of SDH inhibition during reperfusion and RIC did not result in additive protection, plasma concentrations of selected citric acid cycle metabolites are attenuated by protective maneuvers, correlate with irreversible injury, and might become a prognosis tool in STEMI patients.

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae , combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.

Succinate dehydrogenase complex II inhibitors (SDHIs) are widely used fungicides since the 1960s. Recently, based on published in vitro cell viability data, potential health effects via disruption of the mitochondrial respiratory chain and tricarboxylic acid cycle have been postulated in mammalian species. As primary metabolic impact of SDH inhibition, an increase in succinate, and compensatory ATP production via glycolysis resulting in excess lactate levels was hypothesized. To investigate these hypotheses, genome-scale metabolic models of Rattus norvegicus and Homo sapiens were used for an in silico analysis of mammalian metabolism. Moreover, plasma samples from 28-day studies with the SDHIs boscalid and fluxapyroxad were subjected to metabolome analyses, to assess in vivo metabolite changes induced by SDHIs. The outcome of in silico analyses indicated that mammalian metabolic networks are robust and able to compensate different types of metabolic perturbation, e.g., partial or complete SDH inhibition. Additionally, the in silico comparison of rat and human responses suggested no noticeable differences between both species, evidencing that the rat is an appropriate testing organism for toxicity of SDHIs. Since no succinate or lactate accumulation were found in rats, such an accumulation is also not expected in humans as a result of SDHI exposure., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Thirty-three new 2, 3-dihydroquinolin-4(1H)-one analogues were designed, synthesized and characterized by IR, 1 H NMR, 13 C NMR and HRMS. The crystal structures of compounds 2g and 4l were characterized by single crystal X-ray diffraction. Their antifungal activities were determined against five plant pathogenic fungi namely Rhizoctonia solani, Fusarum graminearum, Helminthosporium maydis, Sclerotinia sclerotiorum and Botrytis cinerea. The results indicated that most of them revealed significant antifungal activity at 20 mg/L. Compound 4e showed the strongest antifungal activity against Botrytis cinerea and had better effects than the commercial fungicide fluopyram. Meanwhile, the active compounds were evaluated for their inhibitory activities against succinate dehydrogenase (SDH). The results displayed that they exhibited excellent activity. Compound 4e had better inhibitory activity than fluopyram. The molecular modeling results demonstrated that compound 4e could strongly bind to and interact with the binding sites of SDH. The inhibitory activity of 2, 3-dihydroquinolin-4(1H)-one derivatives against SDH has been reported for the first time., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

To continue our ongoing studies on discovery of new potent antifungal leads, 43 novel pyrazole-aromatic containing carboxamides were rationally designed and synthesized. Bioassays indicated that most target compounds displayed good in vitro antifungal activities against Botrytis cinerea, Rhizoctonia cerealis and Sclerotinia sclerotiorum and in vivo antifungal activity against R. solani. Compound 11ea exhibited the most significant in vitro activity against R. cerealis (EC 50  = 0.93 μg/mL) with about 2-fold more potent than a previously reported lead compound A1 (EC 50  = 2.01 μg/mL), and about 11-fold more potent than the positive control/commercial succinate dehydrogenase inhibitor thifluzamide (EC 50  = 23.09 μg/mL). Structure-activity relationship analysis and molecular docking simulations indicated that the presence of difluoromethyl pyrazole-(m-benzene) carboxamide scaffold obviously increased the antifungal activity. The further enzymatic bioassay showed that both thifluzamide and compound 11ea displayed excellent SDH inhibitory effects, and fluorescence quenching analysis suggested that they may share the same target SDH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

A series of new fungicides that can inhibit the succinate dehydrogenase (SDH) was classified and named as SDH inhibitors by the Fungicide Resistance Action Committee in 2009. To develop more potential SDH inhibitors, we designed and synthesized a novel series of N -(substituted pyridine-4-yl)-1-(substituted phenyl)-5-trifluoromethyl-1 H -pyrazole-4-carboxamide derivatives, 4a - 4i , namely, 5a - 5h , 6a - 6h , and 7a - 7j . The bioassay results demonstrated that some title compounds exhibited excellent antifungal activity against four tested phytopathogenic fungi ( Gibberella zea , Fusarium oxysporum , Cytospora mandshurica , and Phytophthora infestans ). The EC 50 values were 1.8 μg/mL for 7a against G. zeae , 1.5 and 3.6 μg/mL for 7c against F. oxysporum and C. mandshurica , respectively, and 6.8 μg/mL for 7f against P. infestans . The SDH enzymatic activity testing revealed that the IC 50 values of 4c , 5f , 7f , and penthiopyrad were 12.5, 135.3, 6.9, and 223.9 μg/mL, respectively. The molecular docking results of this series of title compounds with SDH model demonstrated that the compounds could completely locate inside of the pocket, the body fragment formed H bonds, and the phenyl ring showed a π-π interaction with Arg59, suggesting that these novel 5-trifluoromethyl-pyrazole-4-carboxamide derivatives might target SDH. These results could provide a benchmark for understanding the antifungal activity against the phytopathogenic fungus P. infestans and prompt us to discover more potent SDH inhibitors.

T cell activation is intimately linked to metabolism, as distinct metabolic requirements support the functional and phenotypical differences between quiescent and activated T cells. Metabolic transition from mitochondrial oxidative phosphorylation to aerobic glycolysis is crucial for a proper T cell activation. However, the role of tricarboxylic acid cycle (TCA), and in particular succinate dehydrogenase (SDH) in activated T cells needs further elucidation. Here we show that inhibition of SDH during activation of T cells results in strong impairment of proliferation, expression of activation markers, and production of key inflammatory cytokines, despite a concomitant increase in glycolytic metabolic activity. Similar effect of SDH inhibition were demonstrated in pre-activated T cell. Interestingly, itaconic acid, an endogenous SDH inhibitor released from activated macrophages and dendritic cells, had no immunomodulator effect. Taken together, our findings demonstrate that SDH enzyme fitness is critical for mounting and maintaining appropriate activation and function of human T cells.