The Interaction Mechanism of Picolinamide Fungicide Targeting on the Cytochrome bc1 Complex and Its Structural Modification.

Picolinamide fungicides, structurally related to UK-2A and antimycin-A, bind into the Qi-site in the bc1 complex. However, the detailed binding mode of picolinamide fungicides remains unknown. In the present study, antimycin-A and UK-2A were selected to study the binding mode of picolinamide inhibitors with four protonation states in the Qi-site by integrating molecular dynamics simulation, molecular docking, and molecular mechanics Generalized Born surface area (MM/GBSA) calculations. Subsequently, a series of new picolinamide derivatives were designed and synthesized to further understand the effects of substituents on the tail phenyl ring. The computational results indicated that the substituted aromatic rings in antimycin-A and UK-2A were the pharmacophore fragments and made the primary contribution when bound to a protein. Compound 9g-hydrolysis formed H-bonds with Hie201 and Ash228 and showed an IC50 value of 6.05 ± 0.24 µM against the porcine bc1 complex. Compound 9c, with a simpler chemical structure, showed higher control effects than florylpicoxamid against cucumber downy mildew and expanded the fungicidal spectrum of picolinamide fungicides. The structural and mechanistic insights obtained from the present study will provide a valuable clue for the future designing of new promising Qi-site inhibitors.

Modulation of Cellular Levels of Adenosine Phosphates and Creatine Phosphate in Cultured Primary Astrocytes.

Adenosine triphosphate (ATP) is the main energy currency of all cells, while creatine phosphate (CrP) is considered as a buffer of high energy-bond phosphate that facilitates rapid regeneration of ATP from adenosine diphosphate (ADP). Astrocyte-rich primary cultures contain ATP, ADP and adenosine monophosphate (AMP) in average specific contents of 36.0 ± 6.4 nmol/mg, 2.9 ± 2.1 nmol/mg and 1.7 ± 2.1 nmol/mg, respectively, which establish an adenylate energy charge of 0.92 ± 0.04. The average specific cellular CrP level was found to be 25.9 ± 10.8 nmol/mg and the CrP/ATP ratio was 0.74 ± 0.28. The specific cellular CrP content, but not the ATP content, declined with the age of the culture. Absence of fetal calf serum for 24 h caused a partial loss in the cellular contents of both CrP and ATP, while application of creatine for 24 h doubled the cellular CrP content and the CrP/ATP ratio, but did not affect ATP levels. In glucose-deprived astrocytes, the high cellular ATP and CrP contents were rapidly depleted within minutes after application of the glycolysis inhibitor 2-deoxyglucose and the respiratory chain inhibitor antimycin A. For those conditions, the decline in CrP levels always preceded that of ATP contents. In contrast, incubation of glucose-fed astrocytes for up to 30 min with antimycin A had little effect on the high cellular ATP content, while the CrP level was significantly lowered. These data demonstrate the importance of cellular CrP for maintaining a high cellular ATP content in astrocytes during episodes of impaired ATP regeneration.

AMP-activated protein kinase (AMPK) suppresses Ibaraki virus propagation.

The Ibaraki virus (IBAV) causes Ibaraki disease in cattle. Our previous studies have shown that IBAV uses macropinocytosis to enter the host cell and exit from the endosome to the cytosol in response to endosomal acidification. To further explore the mechanism of IBAV infection and replication, we examined the effect of inhibitors of mitochondrial oxidative phosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and antimycin A, on IBAV propagation. These inhibitors significantly suppressed IBAV propagation, with reduced cellular ATP levels resulting from suppression of ATP synthesis. Furthermore, we identified AMP-activated protein kinase (AMPK), which is activated by CCCP or antimycin A, as a key signaling molecule in IBAV suppression. We also observed that IBAV infection induces ATP depletion and increases AMPK activity. Our findings suggest that AMPK is a potential target in Ibaraki disease.

[Inactivation of Ras1 in Fission Yeast Aggravates the Oxidative Stress Response Induced by Tert Butyl Hydroperoxide (tBHP)].

Ras proteins are small GTPases and function as molecular switches to regulate cellular homeostasis. Ras-dependent signalling pathways regulate several essential processes such as cell cycle progression, growth, migration, apoptosis, and senescence. The dysregulation of Ras signaling pathway has been linked to several pathological outcomes. A potential role of RAS in regulating the redox signalling pathway has been established that includes the manipulation of ROS levels to provide a redox milieu that might be conducive to carcinogenesis. Reactive oxygen species (ROS) and mitochondrial impairment have been proposed as major factors affecting the physiology of cells and implicated in several pathologies. The present study was conducted to evaluate the role of Ras1, tert Butyl hydroperoxide (tBHP), and antimycin A in oxidative stress response in Schizosaccharomyces pombe cells. We observed decreased cell survival, higher levels of ROS, and mitochondrial dysfunctionality in ras1Δ cells and tBHP as well as respiratory inhibitor, antimycin A treated wild type cells. Furthermore, these defects were more profound in ras1Δ cells treated with tBHP or antimycin A. Additionally, Ras1 also has been shown to regulate the expression and activity of several antioxidant enzymes like glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), and catalase. Together, these results suggest the potential role of S. pombe Ras1 in mitigating oxidative stress response.

Identification of antimycin A as a c-Myc degradation accelerator via high-throughput screening.

c-Myc is a critical regulator of cell proliferation and growth. Elevated levels of c-Myc cause transcriptional amplification, leading to various types of cancers. Small molecules that specifically inhibit c-Myc-dependent regulation are potentially invaluable for anticancer therapy. Because c-Myc does not have enzymatic activity or targetable pockets, researchers have attempted to obtain small molecules that inhibit c-Myc cofactors, activate c-Myc repressors, or target epigenetic modifications to regulate the chromatin of c-Myc-addicted cancer without any clinical success. In this study, we screened for c-Myc inhibitors using a cell-dependent assay system in which the expression of c-Myc and its transcriptional activity can be inferred from monomeric Keima and enhanced GFP fluorescence, respectively. We identified one mitochondrial inhibitor, antimycin A, as a hit compound. The compound enhanced the c-Myc phosphorylation of threonine-58, consequently increasing the proteasome-mediated c-Myc degradation. The mechanistic analysis of antimycin A revealed that it enhanced the degradation of c-Myc protein through the activation of glycogen synthetic kinase 3 by reactive oxygen species (ROS) from damaged mitochondria. Furthermore, we found that the inhibition of cell growth by antimycin A was caused by both ROS-dependent and ROS-independent pathways. Interestingly, ROS-dependent growth inhibition occurred only in the presence of c-Myc, which may reflect the representative features of cancer cells. Consistently, the antimycin A sensitivity of cells was correlated to the endogenous c-Myc levels in various cancer cells. Overall, our study provides an effective strategy for identifying c-Myc inhibitors and proposes a novel concept for utilizing ROS inducers for cancer therapy.

Identification of In Vitro Inhibitors of Monkeypox Replication.

Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating their inhibitory activity in vitro against two orthopoxviruses. IMPORTANCE Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV.

Genome Mining and Molecular Networking Guided Isolation of Antimycin Analogs with Antifeedant Activities from the Deep-Sea-Derived Streptomyces sp. NA13.

Polyphagous insects could affect agricultural production, which leads to serious economic losses. Due to the negative effects of synthesized insecticides, finding eco-friendly and new biopesticides is emergent. To develop natural origin insecticides, an integrative approach combining antifeedant activity screening, genome mining, and molecular networking has been applied to discover antifeedant secondary metabolites from Streptomyces sp. NA13, which leads to the isolation of a novel antimycin Q (1) and six known antimycin analogs (antimycins A1a, A2a, A3a, A4a, A7a, and N-formylantimycic acid methyl ester, 2-7). Their structures were identified by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopic. The absolute configuration of 1 was elucidated by the comparison of coupling constant, electronic circular dichroism (ECD) analysis, and NMR calculations. 1-6 exhibited different levels of antifeedant activities against Helicoverpa armigera, especially 1-4. At the same time, the antifeedant activity of antimycin was reported firstly.

Pharmacological Progress of Mitophagy Regulation.

With the advancement in novel drug discovery, biologically active compounds are considered pharmacological tools to understand complex biological mechanisms and the identification of potent therapeutic agents. Mitochondria boast a central role in different integral biological processes and mitochondrial dysfunction is associated with multiple pathologies. It is, therefore, prudent to target mitochondrial quality control mechanisms by using pharmacological approaches. However, there is a scarcity of biologically active molecules, which can interact with mitochondria directly. Currently, the chemical compounds used to induce mitophagy include oligomycin and antimycin A for impaired respiration and acute dissipation of mitochondrial membrane potential by using CCCP/FCCP, the mitochondrial uncouplers. These chemical probes alter the homeostasis of the mitochondria and limit our understanding of the energy regulatory mechanisms. Efforts are underway to find molecules that can bring about selective removal of defective mitochondria without compromising normal mitochondrial respiration. In this report, we have tried to summarize and status of the recently reported modulators of mitophagy.

Endogenous Energy Stores Maintain a High ATP Concentration for Hours in Glucose-Depleted Cultured Primary Rat Astrocytes.

Adenosine triphosphate (ATP) is the central energy currency of all cells. Cultured primary rat astrocytes contain a specific cellular ATP content of 27.9 ± 4.7 nmol/mg. During incubation in a glucose- and amino acid-free incubation buffer, this high cellular ATP content was maintained for at least 6 h, while within 24 h the levels of ATP declined to around 30% of the initial value without compromising cell viability. In contrast, cells exposed to 1 mM and 5 mM glucose maintained the initial high cellular ATP content for 24 and 72 h, respectively. The loss in cellular ATP content observed during a 24 h glucose-deprivation was fully prevented by the presence of glucose, fructose or mannose as well as by the mitochondrial substrates lactate, pyruvate, ß-hydroxybutyrate or acetate. The high initial specific ATP content in glucose-starved astrocytes, was almost completely abolished within 30 min after application of the respiratory chain inhibitor antimycin A or the mitochondrial uncoupler BAM-15, while these inhibitors lowered in glucose-fed cells the ATP content only to 60% (BAM-15) and 40% (antimycin A) within 5 h. Inhibition of the mitochondrial pyruvate carrier by UK5099 alone or of mitochondrial fatty acid uptake by etomoxir alone hardly affected the high ATP content of glucose-deprived astrocytes during an incubation for 8 h, while the co-application of both inhibitors depleted cellular ATP levels almost completely within 5 h. These data underline the importance of mitochondrial metabolism for the ATP regeneration of astrocytes and demonstrate that the mitochondrial oxidation of pyruvate and fatty acids strongly contributes to the maintenance of a high ATP concentration in glucose-deprived astrocytes.

Total synthesis of isoneoantimycin.

Antimycins are one of the well-known antifungal metabolites produced by Streptomyces bacteria. Neoantimycin and its analogues, the ring-expanded antimycins featuring a 15-membered tetraester ring, have been shown to be effective regulators of the oncogenic proteins GRP78/BiP and K-Ras. Isoneoantimycin was isolated from Streptomyces fradiae IFO12773 (ISP 5063) as a minor metabolite during the fermentation of neoantimycin and is the first reported antibiotic of the antimycin family without the macrolide core. In this study, we explored the total synthesis and stereochemical assignment of isoneoantimycin as an approach to perform structure-activity studies on neoantimycins. Taking the neoantimycin biosynthesis pathway into account, we presumed that the stereochemistry of isoneoantimycin is the same as that of neoantimycin. The synthesis of our target molecule with the (1S,2R,5S,6S,14R,15R,17S) configuration has been achieved by using chiral-pool building blocks. A comparison of the spectroscopic data between the synthetic and natural samples verified our presumption of the stereochemistry of natural isoneoantimycin.

Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic ß-cell dysfunction.

Macroautophagy/autophagy or mitophagy plays crucial roles in the maintenance of pancreatic ß-cell function. PPP3/calcineurin can modulate the activity of TFEB, a master regulator of lysosomal biogenesis and autophagy gene expression, through dephosphorylation. We studied whether PPP3/calcineurin inhibitors can affect the mitophagy of pancreatic ß-cells and pancreatic ß-cell function employing FK506, an immunosuppressive drug against graft rejection. FK506 suppressed rotenone- or oligomycin+antimycin-A-induced mitophagy measured by Mito-Keima localization in acidic lysosomes or RFP-LC3 puncta colocalized with TOMM20 in INS-1 insulinoma cells. FK506 diminished nuclear translocation of TFEB after treatment with rotenone or oligomycin+antimycin A. Forced TFEB nuclear translocation by a constitutively active TFEB mutant transfection restored impaired mitophagy by FK506, suggesting the role of decreased TFEB nuclear translocation in FK506-mediated mitophagy impairment. Probably due to reduced mitophagy, recovery of mitochondrial potential or quenching of mitochondrial ROS after removal of rotenone or oligomycin+antimycin A was delayed by FK506. Mitochondrial oxygen consumption was reduced by FK506, indicating reduced mitochondrial function by FK506. Likely due to mitochondrial dysfunction, insulin release from INS-1 cells was reduced by FK506 in vitro. FK506 treatment also reduced insulin release and impaired glucose tolerance in vivo, which was associated with decreased mitophagy and mitochondrial COX activity in pancreatic islets. FK506-induced mitochondrial dysfunction and glucose intolerance were ameliorated by an autophagy enhancer activating TFEB. These results suggest that diminished mitophagy and consequent mitochondrial dysfunction of pancreatic ß-cells contribute to FK506-induced ß-cell dysfunction or glucose intolerance, and autophagy enhancement could be a therapeutic modality against post-transplantation diabetes mellitus caused by PPP3/calcineurin inhibitors.

Antimycin A induced apoptosis in HCT-116 colorectal cancer cells through the up- and downregulation of multiple signaling pathways.

Colorectal cancer is the third most life-threatening cancer in the western countries. For the treatment, several chemotherapeutic drugs are using those that have severe side effects on the patient. So, finding alternative drugs is important. In the present research antimycin A was selected to evaluate the anticancer properties on the HCT-116 colorectal cancer cells. Antimycin A inhibited HCT-116 cells proliferation with the IC50 value of 29 µg/mL concentration. As a long-term effect, HCT-116 cells were incubated with 10-40 µg/mL concentration of antimycin A for 7 days. No colony was observed in the treated wells. Apoptotic features in HCT-116 cells were observed in antimycin A treated cells after being stained with Hoechst 33342 dye. Apoptosis was further confirmed by FITC-annexin V/PI. Role of caspase-3 protein in the apoptosis process was also confirmed by the caspase-3 inhibitor. After treatment of HCT-116 cells with antimycin A, apoptotic related gene expression was checked by reverse transcription polymerase chain reaction. p53 and caspase-9 genes were upregulated consequently mitogen-activated protein kinases (MAPK), poly(ADP-Ribose) polymerase (PARP), and nuclear factor kappa B (NF-κB) genes were downregulated. Molecular docking simulation indicated significant binding affinity of antimycin A with the five proteins. The results indicated antimycin A would be a promising anticancer agent for further anticancer research.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

Insecticidal characteristics and structural identification of the potential active compounds from Streptomyces sp. KR0006: Strain improvement through mutagenesis.

Pest control by biological means is an effective, eco-friendly, and promising method that typically involves compounds naturally derived from actinomycetes. Thus, the present study aimed to screen, characterize, and identify the structure of insecticidal compounds from Streptomyces sp. KR0006 and increase the activity through mutagenesis. In the examination of the insecticidal activity level of the isolates, Streptomyces sp. KR0006 metabolite showed significant activity against larvae and moths of Plutella xylostella. Taxonomic analyses of the 16S rRNA gene sequences revealed that the isolated KR0006 strain tended to be 99% consistent with Streptomyces cinereoruber strain NBRC 12756. Three active compounds isolated from the culture filtrate of KR0006 were purified by solvent partition, mid-pressure liquid chromatography (MPLC), Sephadex LH20 column chromatography, and high-performance liquid chromatography (HPLC). By performing 1H-NMR, 13C-NMR, and 2D-NMR experiments, and high-resolution electrospray ionization mass spectrometry analysis, the 316-HP2, 316-HP3, and 316-HP5 compounds were inferred as antimycin A3a (MW, 519.; C26H36N2O9), antimycin A8a (MW, 534; C27H38N2O9), and antimycin A1a (MW, 548; C28H40N2O9) respectively. Mutant U67 obtained from exposure to ultraviolet (UV) irradiation (254 nm, height 17 cm) for 70 seconds resulted in a 70% more larval mortality than that of the initial wild culture. The second mutation of the culture broth enhanced insecticidal activity by 80 and 100% compared with the first mutation and initial medium, respectively. Our study found that Streptomyces sp. KR0006 strain produces insecticidal active compounds and could be used for practical pest management.

Preparations of Terminal Oxidase Cytochrome bd-II Isolated from Escherichia coli Reveal Significant Hydrogen Peroxide Scavenging Activity.

Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II oxidase isolated from the bacterium were shown to scavenge hydrogen peroxide (H2O2) with high rate producing molecular oxygen (O2). Addition of H2O2 to the same buffer that does not contain enzyme or contains thermally denatured cytochrome bd-II does not lead to any O2 production. The latter observation rules out involvement of adventitious transition metals bound to the protein. The H2O2-induced O2 production is not susceptible to inhibition by N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (the compound that binds specifically to a quinol binding site), and CO (diatomic gas that binds specifically to the reduced heme d). However, O2 formation is inhibited by cyanide (IC50 = 4.5 ± 0.5 µM) and azide. Addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate cytochrome bd-II and apparently does not affect the O2 reductase activity of the enzyme. The ability of cytochrome bd-II to detoxify H2O2 could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

MiR-6918-5p prevents renal tubular cell apoptosis by targeting MBD2 in ischemia/reperfusion-induced AKI.

AIMS: Although previous studies reported that miRNAs are involved in the progression of acute kidney injury (AKI), their exact function and mechanism in ischemic AKI remains largely unknown. This study aims to define the role of miR-6918-5p in ischemia-reperfusion AKI. Materials and methods The renal arteries of C57BL/6J mice were clamped to establish a model of ischemia-reperfusion renal injury. BUMPT cells were added with Antimycin A and calcium ionophore to establish a model of ATP depletion in vitro. Cell apoptosis was detected by CCK8, flow cytometry and western blot, while HE staining and TUNEL staining were used to assess the degree of kidney damage. KEY FINDINGS: We suppressed mmu_miR-6918-5p by ischemic injury in vitro and in vivo. We found that ischemia-reperfusion (I/R)-induced renal tubular cell apoptosis and the expression of cleaved caspase3 were enhanced by the inhibitor of mmu_miR-6918-5p; this effect was attenuated by an mmu_miR-6918-5p mimic. Mechanistically, mmu_miR-6918-5p binds to the 3' UTR region of MBD2 and represses its expression. The mmu_miR-6918-5p mimic alleviated the ischemic AKI by targeting MBD2. Conversely, the inhibitor of mmu_miR-6918-5p enhanced the ischemic AKI; this was diminished by MBD2-KO. SIGNIFICANCE: Mmu_miR-6918-5p protected against the development of ischemic AKI by targeting MBD2.

How to Use Respiratory Chain Inhibitors in Toxicology Studies-Whole-Cell Measurements.

Mitochondrial electron transport chain (ETC) inhibition is a phenomenon interesting in itself and serves as a tool for studying various cellular processes. Despite the fact that searching the term "rotenone" in PubMed returns more than 6900 results, there are many discrepancies regarding the directions of changes reported to be caused by this RTC inhibitor in the delicate redox balance of the cell. Here, we performed a multifaceted study of the popular ETC inhibitors rotenone and antimycin A, involving assessment of mitochondrial membrane potential and the production of hydrogen peroxide and superoxide anions at cellular and mitochondrial levels over a wide range of inhibitor concentrations (1 nmol/dm3-100 µmol/dm3). All measurements were performed with whole cells, with accompanying control of ATP levels. Antimycin A was more potent in hindering HepG2 cells' abilities to produce ATP, decreasing ATP levels even at a 1 nmol/dm3 concentration, while in the case of rotenone, a 10,000-times greater concentration was needed to produce a statistically significant decrease. The amount of hydrogen peroxide produced in the course of antimycin A biological activity increased rapidly at low concentrations and decreased below control level at a high concentration of 100 µmol/dm3. While both inhibitors influenced cellular superoxide anion production in a comparable manner, rotenone caused a greater increase in mitochondrial superoxide anions compared to a modest impact for antimycin A. IC50 values for rotenone and antimycin A with respect to HepG2 cell survival were of the same order of magnitude, but the survival curve of cells treated with rotenone was clearly biphasic, suggesting a concentration-dependent mode of biological action. We propose a clear experimental setup allowing for complete and credible analysis of the redox state of cells under stress conditions which allows for better understanding of the effects of ETC inhibition.

Reprint of: Ubisemiquinone Is the Electron Donor for Superoxide Formation by Complex III of Heart Mitochondria.

Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.

Novel Antimycin Analogues with Agricultural Antifungal Activities from the Sponge-Associated Actinomycete Streptomyces sp. NBU3104.

Phytopathogenic fungi could affect the growth of agricultural products and result in serious economic losses. To develop novel and potent fungicides, secondary metabolites of an oceanic mesophotic zone Streptomyces sp. NBU3104 was isolated by metabolomics and genomics, which led to the discovery of eight novel antimycins I-P (1-8), including antimycin I (1), six rare acetylated actimycins J-N (2-6), P (8), and an unusual deformylated antimycin O (7). The chemical structures of these metabolites were identified using nuclear magnetic resonance (NMR) spectroscopic analysis, high-resolution electrospray ionization mass spectrometry (HRESIMS) data, and the known reported metabolites in the literature. Their absolute configurations were elucidated by comparison of coupling constant and experimental electronic circular dichroism (ECD) spectra. Among them, compound 1 exhibited excellent inhibitory activities against phytopathogenic fungi, such as Candida albicans, Penicillium expansum, Penicillium citrinum, and Botrytis cinerea. Furthermore, compound 1 could effectively control gray mold of apple in vivo (minimum inhibitory concentration (MIC) = 8 µg/mL). The structure-activity relations of antimycins I-P (1-8) suggested that the aldehyde group in 3-formamidosalicylate unit moiety should be the key factor in their antifungal activities.

An evaluation of a new high-sensitivity PrestoBlue assay for measuring cell viability and drug cytotoxicity using EA.hy926 endothelial cells.

INTRODUCTION: Commercially-available resazurin-based reagents used for cell viability assessment contain varying amounts of resorufin; these may contribute to differences in autofluorescence, signal-to-background (S/B) ratio and the dynamic range of the assay. OBJECTIVES: This in vitro study compares the sensitivity of a new, high-sensitivity PrestoBlue (hs-PB) assay with standard PrestoBlue (PB) in assessing the efficacy of valinomycin and antimycin A in human vascular endothelial EA.hy926 cells, as well as cell viability. METHODS: The metabolic activity of EA.hy926 was evaluated based on resorufin fluorescence (PB assays) or formazan absorbance (MTT assay). RESULTS: The hs-PB assay demonstrated lower resorufin autofluorescence than the PB, resulting in a ≥ 1.4-fold increase in S/B ratio in hs-PB compared to PB. Valinomycin was more potent cytotoxic agent than antimycin A. The hs-PB, PB and MTT produced similar IC50 values for valinomycin. Antimycin A showed significantly higher potency in the MTT than in the resazurin-based assays. The EA.hy926 cells demonstrated higher metabolic activity in the presence of the antimycin A solvent - DMSO. CONCLUSION: All the examined methods may be used interchangeably to analyze drug cytotoxicity. Any differences in drug cytotoxicity observed between the assays may be due to relatively low drug potency and/or the influence of solvent on metabolism of assay reagent. The hs-PB assay appears to more effectively detect cell viability and produce a stronger signal than its conventional counterpart.