Succinate Dehydrogenase Inhibition Research Articles

Novel Pyrazole-4-Carboxamide Derivatives Containing Oxime Ether Group as Potential SDHIs to Control Rhizoctonia solani.

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 EC50 value of 0.039 μg/mL, which was about 20-fold better than that of boscalid (EC50 = 0.799 μg/mL) and 4-fold more potent than fluxapyroxad (EC50 = 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 (IC50 = 2.04 μM), which was obviously more potent than those of boscalid (IC50 = 7.92 μM) and fluxapyroxad (IC50 = 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.

Long-Term Protective Effects of Succinate Dehydrogenase Inhibition during Reperfusion with Malonate on Post-Infarction Left Ventricular Scar and Remodeling in Mice.

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.

A universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle

A prevalent side-reaction of succinate dehydrogenase oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a strong inhibitor of succinate dehydrogenase. We purified from cow heart mitochondria an enzyme (OAT1) with OAA tautomerase (OAT) activity that converts enol-OAA to the physiological keto-OAA form, and determined that it belongs to the highly conserved and previously uncharacterized Fumarylacetoacetate_hydrolase_domain-containing protein family. From all three domains of life, heterologously expressed proteins were shown to have strong OAT activity, and ablating the OAT1 homolog caused significant growth defects. In Escherichia coli, expression of succinate dehydrogenase was necessary for OAT1-associated growth defects to occur, and ablating OAT1 caused a significant increase in acetate and other metabolites associated with anaerobic respiration. OAT1 increased the succinate dehydrogenase reaction rate by 35% in in vitro assays with physiological concentrations of both succinate and malate. Our results suggest that OAT1 is a universal metabolite repair enzyme that is required to maximize aerobic respiration efficiency by preventing succinate dehydrogenase inhibition.

Dimethyl malonate preserves renal and mitochondrial functions following ischemia-reperfusion via inhibition of succinate dehydrogenase

BackgroundAcute kidney injury (AKI), often experienced at the intensive care units, is associated with high morbidity/mortality where ischemia-reperfusion injury is a main causative factor. Succinate accumulation during ischemia contributes to the excessive generation of reactive oxygen species at reperfusion. Inhibition of succinate dehydrogenase has been associated with protective outcome in cardiac ischemia-reperfusion after 24h, but the effects on kidney and mitochondrial functions are less well studied. AimTo investigate the therapeutic potential of succinate dehydrogenase inhibition, by using dimethyl malonate (DMM), on kidney and mitochondria functions in a mouse model of AKI. MethodsMale C57BL/6J mice were pre-treated with DMM or placebo, i.p. 30min prior to bilateral renal ischemia (20min). After 3-days of reperfusion, glomerular filtration rate (GFR) was calculated from plasma clearance of FITC-inulin. Kidney mitochondria was isolated and mass specific and intrinsic mitochondrial function were evaluated by high resolution respirometry. Kidney sections were stained (i.e., hematoxylin-eosin and TUNEL) and analyzed for histopathological evaluation of injuries and apotosis, respectively. NADPH oxidase activity in kidney and human proximal tubular cell-line (HK2) were measured luminometrically. ResultsDMM treatment improved GFR (p < 0.05) and reduced levels of blood urea nitrogen (p < 0.01) compared to untreated animals, which was associated with lower degree of ischemia-reperfusion-induced tubular injuries (P < 0.001) and apoptosis (P < 0.01). These therapeutic renal effects were linked with improved mitochondrial function, both mass-specific and intrinsic. Finally, DMM treatment prevented ischemia-reperfusion-induced NADPH oxidase activity in the kidney (p < 0.001), which was showed also in HK2 cells exposed to hypoxia and reoxygenation (P < 0.01). ConclusionInhibition of succinate dehydrogenase with DMM, in conjunction with the ischemia-reperfusion phase, significantly improved both renal and mitochondrial functions. These findings may have clinical implications for future therapeutic strategies to prevent development of AKI and associated adverse complications, especially in high risk hospitalized patients.

Mitochondrial succinate transport as a novel therapeutic target for cardiac ischemia/reperfusion injury

Abstract Background The accumulation of the mitochondrial metabolite succinate during ischemia and its rapid oxidation by succinate dehydrogenase (SDH) upon reperfusion drives ischemia/reperfusion injury (IRI) in myocardial infarction. Succinate oxidation leads to the production of reactive oxygen species (ROS) by reverse electron transport (RET) at mitochondrial complex I, orchestrating a cascade of events leading to cardiomyocyte death. Modulating succinate metabolism, such as SDH inhibition, is a promising therapeutic strategy. During ischemia, the mitochondria produced succinate is transported into the cytosol, enabling the accumulation of a large succinate pool. On reperfusion, the succinate is transported back into mitochondria and oxidised by SDH, thus we hypothesised that targeting succinate mitochondrial transport could be a novel therapeutic target to prevent cardiac IRI. Purpose To assess the role of succinate mitochondrial transport as a novel therapeutic target to prevent cardiac IRI. Methods Isolated heart mitochondria were used to assess mitochondrial respiration by Oroboros, RET-ROS production by Amplex Red and butylmalonate delivery. Cultured H9C2 myoblasts and isolated primary adult cardiomyocytes from C57BL/6J mice were used to assess butylmalonate uptake and delivery from esters, as well as their impact on succinate accumulation and oxidation in vitro. LAD ligation and reperfusion in C57BL/6J mice was used as an in vivo MI model. Metabolite levels were measured by LC-MS/MS. Results Butylmalonate, an inhibitor of the mitochondrial dicarboxylate carrier (DIC), blocks succinate-dependent respiration and RET-ROS production in isolated mitochondria. However, butylmalonate entry into cells was inefficient and does not impact infarct size after IRI in vivo. We developed novel butylmalonate esters to improve butylmalonate delivery to cardiomyocytes in vivo. We found that diacetoxymethyl butylmalonate (DAB) effectively delivered butylmalonate and could prevent succinate accumulation and oxidation in vitro. DAB could also prevent succinate-mediated respiration and RET-ROS at lower concentrations than butylmalonate alone. Finally, DAB reduced acute infarct size in vivo when administered at reperfusion. Conclusions Blocking succinate transport back into mitochondria at reperfusion prevents the damage in IRI by preventing the oxidation of succinate by SDH and downstream RET-ROS production. Therefore, targeting the mitochondrial transport of succinate may be a promising novel strategy in preventing cardiac IRI.

Metabolic reprogramming in inflammatory microglia indicates a potential way of targeting inflammation in Alzheimer's disease

Microglia activation drives the pro-inflammatory activity in the early stages of Alzheimer's disease (AD). However, the mechanistic basis is elusive, and the hypothesis of targeting microglia to prevent AD onset is little explored. Here, we demonstrated that upon LPS exposure, microglia shift towards an energetic phenotype characterised by high glycolysis and high mitochondrial respiration with dysfunction. Although the activity of electron transport chain (ETC) complexes is boosted by LPS, this is mostly devoted to the generation of reactive oxygen species. We showed that by inhibiting succinate dehydrogenase (SDH) with dimethyl malonate (DMM), it is possible to modulate the LPS-induced metabolic rewiring, facilitating an anti-inflammatory phenotype. DMM improves mitochondrial function in a direct way and by reducing LPS-induced mitochondrial biogenesis. Moreover, the block of SDH with DMM inhibits the recruitment of hypoxia inducible-factor 1 α (HIF-1α), which mediates the induction of glycolysis and cytokine expression. Similar bioenergetic alterations were observed in the microglia isolated from AD mice (3xTg-AD), which present high levels of circulating LPS and brain toll-like receptor4 (TLR4). Moreover, this well-established model of AD was used to show a potential effect of SDH inhibition in vivo as DMM administration abrogated brain inflammation and modulated the microglia metabolic alterations of 3xTg-AD mice. The RNA-sequencing analysis from a public dataset confirmed the consistent transcription of genes encoding for ETC subunits in the microglia of AD mice (5xFAD). In conclusion, TLR4 activation promotes metabolic changes and the pro-inflammatory activity in microglia, and SDH might represent a promising therapeutic target to prevent AD development.

Design, Synthesis, and Antifungal Activities of Novel Pyrazole-4-carboxamide Derivatives Containing an Ether Group as Potential Succinate Dehydrogenase Inhibitors.

A series of novel pyrazole-4-carboxamides bearing an ether group were designed and synthesized on the basis of the structure of commercial succinate dehydrogenase inhibitor (SDHI) fungicide flubeneteram via scaffold hopping and evaluated for their antifungal activities against five fungi. The bioassay results showed that most of the target compounds exhibited excellent in vitro antifungal activity against Rhizoctonia solani and some compounds exerted remarkable antifungal activities against Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, and Alternaria alternate. Particularly, compounds 7d and 12b displayed outstanding antifungal activity against R. solani, with an EC50 value of 0.046 μg/mL, far superior to that of boscalid (EC50 = 0.741 μg/mL) and fluxapyroxad (EC50 = 0.103 μg/mL). Meanwhile, compound 12b also presented a broader fungicidal spectrum than other compounds. Moreover, in vivo anti-R. solani results showed that compounds 7d and 12b could significantly inhibit the growth of R. solani in rice leaves with excellent protective and curative efficacies. In addition, the results of the succinate dehydrogenase (SDH) enzymatic inhibition assay showed that compound 7d generated significant SDH inhibition, with an IC50 value of 3.293 μM, which was about 2 times better than that of boscalid (IC50 = 7.507 μM) and fluxapyroxad (IC50 = 5.991 μM). Furthermore, scanning electron microscopy (SEM) analysis indicated that compounds 7d and 12b significantly destroyed the typical structure and morphology of R. solani hyphae. The molecular docking study revealed that compounds 7d and 12b could embed into the binding pocket of SDH and form hydrogen bond interactions with TRP173 and TRY58 at the activity site of SDH, which was in line with fluxapyroxad, indicating that they had a similar mechanism of action. These results demonstrated that compounds 7d and 12b could be promising candidates of SDHI fungicides, which deserved further investigation.

Hemocytes in blue mussel Mytilus edulis adopt different energy supply modes to cope with different BDE-47 exposures

The energetic response of blue mussel Mytilus edulis when coping with tetrabromodiphenyl ether (BDE-47) exposure was evaluated from the perspective of alterations in energy supply mode, and the possible regulating mechanism was discussed based on a 21-day bioassay. The results showed that the energy supply mode changed with concentration: 0.1 μg/L BDE-47 decreased the activity of isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), malate dehydrogenase and oxidative phosphorylation, suggesting inhibition of the tricarboxylic (TCA) acid cycle and aerobic respiration. The coincident increase in phosphofructokinase and the decrease in lactate dehydrogenase (LDH) indicated that glycolysis and anaerobic respiration were increased. When exposed to 1.0 μg/L BDE-47, M. edulis mainly utilized aerobic respiration, but lowered glucose metabolism as indicated by the decrease in glutamine and l-leucine was suggested to be involved in this process, which was differed from that in the control. The reoccurrence of IDH and SDH inhibition as well as LDH elevation indicated attenuation of aerobic and anaerobic respiration when the concentration increased to 10 μg/L, but severe protein damage was evidenced based on the elevation of amino acids and glutamine. Under the 0.1 μg/L BDE-47, activation of the AMPK-Hif-1a signaling pathway promoted the expression of glut1, which was the potential mechanism for the improvement of anaerobic respiration, and further activated glycolysis and anaerobic respiration. This study shows that the energy supply mode experienced a conversion from aerobic respiration under normal conditions to anaerobic mode in the low BDE-47 treatment and back to aerobic respiration with increasing BDE-47 concentrations, which may represent a potential mechanism for mussel physiological responses when faced with different levels of BDE-47 stress.

Succinate dehydrogenase inhibition decreases mitochondrial respiratory capacity in human airway smooth muscle cells

Succinate dehydrogenase (SDH) is a mitochondrial enzyme that participates in both the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) as Complex II. In the TCA cycle, SDH catalyzes the oxidation of succinate to fumarate, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. Previously we developed and validated a quantitative histochemical technique to determine the maximum velocity of the SDH (SDHmax) reaction as a direct measurement of mitochondrial respiratory capacity in individual human airway smooth muscle (hASM) cells. The measurement of SDHmax was shown to provide a reliable estimate of the maximum respiratory capacity of an individual cell within a heterogeneous population of cells when compared to respirometry-based OCR measurements. In the present study we further assessed the specificity of this method by performing a targeted knockdown of the SDHB subunit in hASM cells. As a part of the catalytic core of the SDH complex, the SDHB subunit mediates electron transfer to ubiquinone and thereby regulates electron transfer by SDH. Therefore, we hypothesize that Sdhb-knockdown decreases SDHmax in hASM cells. This study was performed in hASM cells dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors with no current history of smoking or respiratory diseases. Targeted Sdhb-knockdown was generated by siRNA mediated knockdown. hASM cells were also treated with malonate (1 mM for 24 h), which is a competitive inhibitor of SDH. To measure SDHmax, the cells were exposed to a solution containing 1-methoxyphenazine methosulphate (mPMS; exogenous electron carrier), azide (cytochrome oxidase inhibitor), 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT; reaction indicator). As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. In the quantitative histochemical procedure, the change in optical density (OD), reflecting the progressive accumulation of NBT diformazan (NBTdfz; peak absorbance of 570 nm) in the cell, was measured every 15 s over an 8-min period (a linear reaction). SDHmax was determined from the OD using the Beer-Lambert equation and expressed as mM fumarate/L cell/min. In addition, we used MitoTracker Green (200 nM) to label and image mitochondria in hASM cells to determine mitochondrial volume density and mitochondrial complexity index (MCI). Our results show that SDHmax was markedly decreased in both Sdhb-knockdown and malonate treated hASM cells indicative of loss of function of SDH. Sdhb-knockdown increased mitochondrial volume density and decreased MCI in hASM cells, indicative of increased mitochondrial fragmentation and a role of SDH in the maintenance of mitochondrial morphology. The results confirm that the measurement of SDHmax is specific to SDH. Supported by NIH grant HL157984 (GCS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial redox adaptations enable alternative aspartate synthesis in SDH-deficient cells.

The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD +to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss-of-function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC), but mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports human cell proliferation through aspartate synthesis but, unlike other ETC impairments, the effects of SDH inhibition are not ameliorated by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH-impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context depend on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production through pyruvate carboxylation and reductive carboxylation of glutamine. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. These data therefore identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal how compartmentalized redox changes can impact cellular fitness.

Abstract P3-11-11: Impaired Succinate Metabolism Supports Endocrine Therapy Resistance in ER Positive Breast Cancers

Abstract Estrogen receptor positive (ER+) breast cancer is the most common subtype among all breast cancers and is responsible for most breast cancer related deaths. Endocrine therapies, such as selective estrogen receptor modulators (SERM) and aromatase inhibitors (AI), often given in combination with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, are initially effective but the majority of advanced ER+ breast cancers eventually become refractory to endocrine therapies. Therefore, understanding the underlying molecular mechanisms that enable or promote resistance to endocrine therapies may lead to novel therapeutic targets. Cancer cells consume greater quantities of glucose to meet their energy and anabolic demands to support cell growth and proliferation. In this study, we used breast cancer cell models that are endocrine therapy sensitive and resistant and investigated the metabolites by growing the cells in the presence of the stable glucose isotope, UC13-Glucose. We measured the C13-labelled metabolites of glycolysis and the TCA cycle. Glucose consumption was higher in endocrine therapy resistant MCF7/LCC9 (LCC9) cells compared with parental MCF7 (MCF7) and estrogen independent, endocrine therapy sensitive MCF7/LCC1 (LCC1) cells. Notably, in LCC9 cells the m+4 and m+6 isotopomer of C-13 labelled citrate was absent with a concurrent high succinate:fumarate ratio. Furthermore, in LCC9 cells, most of the fumarate was unlabeled suggesting that fumarate was not generated from glucose. These results suggest that the TCA cycle is impaired in LCC9 cells and the conversion of succinate to fumarate is dysregulated. Succinate dehydrogenase (SDH) enzyme is responsible for reversible catalytic conversion of succinate to fumarate. SDH is a multimeric protein comprised of four different subunits and SDH assembly factor 2 (SDHAF2), which is a tumor suppressor gene. Comparable SDH enzyme activity was observed in LCC9 and LCC1 cells. Intriguingly, when SDH activity was inhibited using dimethylmalonate, the LCC9 cells were re-sensitized to both Fulvestrant and 4-hydroxytamoxifen. Succinate accumulation was accompanied with HIF 1-alpha stabilization and SDH inhibition led to lower levels of HIF1-alpha. This indicated that SDH may function in the conversion of fumarate to succinate in LCC9 cells. Our study further investigates the source of fumarate in LCC9 cells. Overall, this study suggests that targeting succinate metabolism may help to restore sensitivity to Fulvestrant and tamoxifen in endocrine therapy resistance in ER+ breast cancer cells. Citation Format: Anil Yadav, Karla Andrade de Oliveira, Lu Jin, Robert Clarke, Surojeet Sengupta. Impaired Succinate Metabolism Supports Endocrine Therapy Resistance in ER Positive Breast Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-11.

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.

Potential Fungicide Candidates: A Dual Action Mode Study of Novel Pyrazole-4-carboxamides against Gibberella zeae

Pyrazole carboxamides are a class of traditional succinate dehydrogenase inhibitors (SDHIs) that have developed into a variety of commercialized fungicides. In the present work, a series of novel 1,5-disubstituted-1H-pyrazole-4-carboxamide derivatives were designed and synthesized based on the active backbone of 5-trifluoromethyl-1H-4-pyrazole carboxamide. Bioassay results indicated that some target compounds exhibited excellent in vitro antifungal activities against six phytopathogenic fungi. Notably, the EC50 values of Y47 against Gibberella zeae, Nigrospora oryzae, Thanatephorus cucumeris, and Verticillium dahliae were 5.2, 9.2, 12.8, and 17.6 mg/L, respectively. The in vivo protective and curative activities of Y47 at 100 mg/L against G. zeae on maize were 50.7 and 44.2%, respectively. Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis revealed that the large steric hindrance and electronegative groups on the 5-position of the pyrazole ring were important for the activity. The IC50 value of Y47 against succinate dehydrogenase (SDH) was 7.7 mg/L, superior to fluopyram (24.7 mg/L), which was consistent with the docking results. Morphological studies with fluorescence microscopy (FM) and scanning electron microscopy (SEM) found that Y47 could affect the membrane integrity of mycelium by inducing endogenous reactive oxygen species (ROS) production and causing peroxidation of cellular lipids, which was further verified by the malondialdehyde (MDA) content. Antifungal mechanism analysis demonstrated that the target compound Y47 not only had significant SDH inhibition activity but could also affect the membrane integrity of mycelium, exhibiting obvious dual action modes. This research provides a novel approach to the development of traditional SDHIs and their derivatives.

Itaconate controls its own synthesis via feedback-inhibition of reverse TCA cycle activity at IDH2

Macrophages undergo extensive metabolic reprogramming during classical pro-inflammatory polarization (M1-like). The accumulation of itaconate has been recognized as both a consequence and mediator of the inflammatory response. In this study we first examined the specific functions of itaconate inside fractionated mitochondria. We show that M1 macrophages produce itaconate de novo via aconitase decarboxylase 1 (ACOD1) inside mitochondria. The carbon for this reaction is not only supplied by oxidative TCA cycling, but also through the reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase (IDH). While macrophages are capable of sustaining a certain degree of itaconate production during hypoxia by augmenting the activity of IDH-dependent reductive carboxylation, we demonstrate that sufficient itaconate synthesis requires a balance of reductive and oxidative TCA cycle metabolism in mouse macrophages. In comparison, human macrophages increase itaconate accumulation under hypoxic conditions by augmenting reductive carboxylation activity. We further demonstrated that itaconate attenuates reductive carboxylation at IDH2, restricting its own production and the accumulation of the immunomodulatory metabolites citrate and 2-hydroxyglutarate. In line with this, reductive carboxylation is enhanced in ACOD1-depleted macrophages. Mechanistically, the inhibition of IDH2 by itaconate is linked to the alteration of the mitochondrial NADP+/NADPH ratio and competitive succinate dehydrogenase inhibition. Taken together, our findings extend the current model of TCA cycle reprogramming during pro-inflammatory macrophage activation and identified novel regulatory properties of itaconate.

Ischemia-Selective Cardioprotection by Malonate for Ischemia/Reperfusion Injury.

Background:Inhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored.Methods:C57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining.Results:Malonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate.Conclusions:Cardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate’s preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury.

Mode of action of fluopyram in plant-parasitic nematodes

Plant-parasitic nematodes (PPN) are responsible for severe yield losses in crop production. Management is challenging as effective and safe means are rare. Recently, it has been discovered that the succinate dehydrogenase (SDH) inhibitor fluopyram is highly effective against PPN while accompanying an excellent safety profile. Here we show that fluopyram is a potent inhibitor of SDH in nematodes but not in mammals, insects and earthworm, explaining the selectivity on molecular level. As a consequence of SDH inhibition, fluopyram impairs ATP generation and causes paralysis in PPN and Caenorhabditis elegans. Interestingly, efficacy differences of fluopyram amongst PPN species can be observed. Permanent exposure to micromolar to nanomolar amounts of fluopyram prevents Meloidogyne spp. and Heterodera schachtii infection and their development at the root. Preincubation of Meloidogyneincognita J2 with fluopyram followed by a recovery period effectively reduces gall formation. However, the same procedure does not inhibit H.schachtii infection and development. Sequence comparison of sites relevant for ligand binding identified amino acid differences in SDHC which likely mediate selectivity, coincidently revealing a unique amino acid difference within SDHC conserved among Heterodera spp. Docking and C.elegans mutant studies suggest that this minute difference mediates altered sensitivity of H.schachtii towards fluopyram.

Abstract 2327: Dysregulated TCA cycle is associated with endocrine therapy resistance in ER+ breast cancer cells

Abstract Estrogen receptor positive (ER+) breast cancer is the most common breast cancer subtype. Endocrine therapies, such as selective estrogen receptor modulators (SERM) and aromatase inhibitors (AI), along with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, are the current mainstays of treatment. Despite being initially effective, the majority of advanced ER+ breast cancers become resistant to the endocrine therapies. Therefore, it is important to understand the underlying molecular mechanisms that enable or promote resistance to endocrine therapies. Rapidly proliferating cells have a high energy demand and consequently consume greater quantities of glucose to fulfill their energy and anabolic demands to support cell growth. Using endocrine therapy sensitive and resistant breast cancer cell models we investigated the metabolic flux of glucose by growing the cells in the presence of the stable glucose isotope, UC13-Glucose and measured the C13-labelled metabolites of glycolysis and the TCA cycle. Glucose consumption was higher in endocrine therapy resistant MCF7/LCC9 (LCC9) cells compared with parental MCF7 (MCF7) and estrogen independent, endocrine therapy sensitive MCF7/LCC1 (LCC1) cells. The TCA cycle was noticeably impaired in LCC9 cells as the C-13 labelled citrate (m+4 and m+6) isotopomer was absent in these cells. In addition, C-13 labelled fumarate was very low in LCC9 cells with a concurrent increase in the succinate:fumarate ratio. These observations show that in LCC9 cells the TCA cycle is dysregulated at the succinate to fumarate conversion step, which is catalyzed by the succinate dehydrogenase (SDH) enzyme. SDH is a multimeric protein comprised of four different subunits and SDH assembly factor 2 (SDHAF2), which is a tumor suppressor gene. Further investigation revealed that SDHAF2 was under expressed in LCC9 cells compared with LCC1 cells, likely contributing to lower SDH activity. Intriguingly, SDH inhibition by dimethyl-malonate re-sensitized LCC9 cells to both Fulvestrant and 4-hydroxytamoxifen. Clinically, lower expression levels of SDHAF2 correlates with poor relapse free survival in ER+ breast cancer patients. In summary, our study suggests that accumulation of succinate driven by deficient SDH activity is functionally associated with endocrine therapy resistance in ER+ breast cancer cells. Citation Format: Surojeet Sengupta, Karla Andrade de Oliviera, Lu Jin, Robert Clarke. Dysregulated TCA cycle is associated with endocrine therapy resistance in ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2327.

Reverse Electron Transfer is a More Dominant Source of Mitochondrial ROS Production in the Heart and Kidney Outer Medulla than in the Kidney Cortex

RationaleReactive oxygen species (ROS; e.g. O2·– and H2O2) play important roles in both physiological and pathophysiological processes. ROS in low concentrations contribute to physiological processes, such as cellular redox signaling and phagocytosis, whereas ROS in high concentrations are toxic to the cell causing tissue injury contributing to the pathogenesis of cardiovascular and chronic renal diseases, including salt‐sensitive hypertension. Mitochondria, which produce ROS as byproducts of aerobic respiration via both forward electron transfer (FET) and reverse electron transfer (RET) are known to be one of the major cellular sources of ROS. Although it is recognized that the RET mechanism in which electrons flow back from complex II to complex I contribute significantly to ROS production in cardiac mitochondria, the mechanisms of ROS production and the role of RET in kidney mitochondria has remained poorly understood.MethodWe evaluated the relative contributions of FET and RET towards overall ROS production in mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague‐Dawley rats. H2O2 emission was measured by a spectrofluorometer in isolated mitochondria in the presence of either Succinate (Suc) simulating RET or succinate+rotenone (Suc+Rot) simulating FET. Furthermore, we measured mitochondrial rates of H2O2 production along with respiration and membrane potential under three respiratory states namely (i) leak state (state 2; after substrate addition), (ii) oxidative phosphorylation (OxPhos) state (state 3; after ADP addition), and (iii) maximum respiratory state (state 5; after the addition of the uncoupler FCCP).ResultsIt was found that mitochondria isolated from the heart and kidney cortex produced the least and the most ROS, respectively. The rate of ROS production in the presence of Suc+Rot compared to Suc alone decreased significantly in the heart and to a lesser extent in OM, indicating significant contribution of RET to overall ROS production in the heart and slightly in the OM. In contrast, there was not significant difference in ROS production rates in the presence of Suc and Suc +Rot in mitochondria from kidney cortex, showing that RET is not predominant in the kidney cortex. Also, we observed significant reduction in the ROS production rate in state 4 compared to state 2 in the heart mitochondria compared to kidney cortex and OM. A possible explanation for these differential results is that oxaloacetate (OAA), produced by the tricarboxylic acid cycle, accumulates resulting in succinate dehydrogenase (SDH) inhibition more rapidly in the heart than in the kidney affecting mitochondrial ROS production, respiration, and bioenergetics.ConclusionRET mechanism contributes to mitochondrial ROS production significantly in the heart and slightly in the kidney OM, but not in the kidney cortex. OAA accumulation contributes to SDH inhibition significantly in the heart than in the kidney.

Functional deficiency of succinate dehydrogenase promotes tumorigenesis and development of clear cell renal cell carcinoma through weakening of ferroptosis

ABSTRACT Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinomas, with high mortality and poor prognoses worldwide. Succinate dehydrogenase (SDH) consists of four nuclear-encoded subunits and it is the only complex involved in both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Previous studies have shown decreased SDH activity in ccRCC. However, the role and underlying molecular mechanisms of SDH in ccRCC initiation and development remain unclear. In the present study, pan-cancer analysis of SDH gene expression was analyzed and the relationship between SDH gene expression and clinicopathological parameters was assessed using different databases. cBioPortal, UACLAN, and Tumor Immune Estimation Resource (TIMER) were subsequently utilized to analyze genetic alterations, methylation, and immune cell infiltration of SDH genes in ccRCC patients. We found SDHs were significantly downregulated in ccRCC tissues and correlated with ccRCC progression. Increased methylation and high SDH promoter mutation rates may be the cause of reduced expression of SDHs in ccRCC. Moreover, the interaction network showed that SDH genes were correlated with ferroptosis-related genes. We further demonstrated that SDH inhibition dampened oxidative phosphorylation, reduced ferroptotic events, and restored ferroptotic cell death, characterized by eliminated mitochondrial ROS levels, decreased cellular ROS and diminished peroxide accumulation. Collectively, this study provides new insights into the regulatory role of SDH in the carcinogenesis and progression of ccRCC, introducing a potential target for advanced antitumor therapy through ferroptosis.

Synthesis and Biological Activity of Novel Antifungal Leads: 3,5-Dichlorobenzyl Ester Derivatives.

Succinate dehydrogenase (SDH) is one of the most important molecular targets for the development of new fungicides. Carboxamide fungicides are a class of SDH inhibitors widely used to inhibit highly destructive plant pathogens. Although cases of resistance have been found in fungal pathogens due to the unrestricted use in recent years, there is still demand for new compounds with improved fungicidal activity. Therefore, a series of ester compounds were designed to investigate potential novel antifungal molecules. First, the antifungal activity of different benzyl alcohol compounds (A1-A21) was tested, and a highly active fragment (3,5-dichlorobenzyl alcohol) was found. Subsequently, various compounds were synthesized by esterification between different acids and 3,5-dichlorobenzyl alcohol, among which compound 5 exhibited remarkable antifungal activity against Botrytis cinerea and Rhizoctonia solani with EC50 values of 6.60 and 1.61 mg/L, respectively, which were comparable to those of commercial fungicide boscalid (EC50 = 1.24 and 1.01 mg/L). In vivo testing further demonstrated that compound 5 was effective in suppressing B. cinerea (200 mg/L, 50.9%). Moreover, SDH inhibition assays, fluorescence quenching analysis, and determination of mitochondrial membrane potential revealed that compound 5 has similar effects to boscalid. Furthermore, the fungicidal activity of target compounds can be maintained by modifying the amide bond to an ester bond. These results will provide basis for the development of novel fungicides.