Inhibition Of Mitochondrial Electron Transport Research Articles

Hypoxia Enhances Endothelial Intercellular Adhesion Molecule 1 Protein Level Through Upregulation of Arginase Type II and Mitochondrial Oxidative Stress.

Hypoxia plays a crucial role in the pathogenesis of cardiovascular diseases. Mitochondrial enzyme arginase type II (Arg-II) is reported to lead to endothelial dysfunction and enhance the expression of endothelial inflammatory adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). In this study, we investigate the role of Arg-II in hypoxia-induced endothelial activation and the potential underlying mechanisms. Exposure of the human endothelial cells to hypoxia induced a time-dependent increase in Arg-II, HIF1α, HIF2α, and ICAM-1 protein level, whereas no change in the protein level of VCAM-1 and E-selectin was observed. Similar effects were obtained in cells treated with a hypoxia mimetic Dimethyloxaloylglycine (DMOG). Silencing HIF1α, but not HIF2α, reversed hypoxia-induced upregulation of Arg-II. Moreover, silencing Arg-II prevented the ICAM-1 upregulation induced by hypoxia or DMOG. Furthermore, the endothelial cells incubated under hypoxic condition or treated with DMOG or hypoxia enhanced monocyte adhesion, which was inhibited by silencing Arg-II. Lastly, silencing Arg-II prevented hypoxia-induced mitochondrial superoxide production in endothelial cells, and hypoxia-induced ICAM-1 upregulation was reversed by mitochondrial electron transport inhibitor rotenone. These data demonstrate that hypoxia enhances ICAM-1 protein level and monocyte-endothelial interaction through HIF1α-mediated increase in Arg-II protein level on leading to increased mitochondrial reactive oxygen species production. These effects of hypoxia on endothelial cells may play a key role in cardiovascular diseases. Our results suggest that Arg-II could be a promising therapeutic target to prevent hypoxia-induced vascular damage/dysfunction.

Synthesis and biological activity of analogs of the antifungal antibiotic UK-2A. III. Impact of modifications to the macrocycle isobutyryl ester position.

Fenpicoxamid (Inatreq™ active), a new fungicide under development by Corteva Agriscience™, Agriculture Division of DowDuPont, is an isobutyryl acetal derivative of the antifungal antibiotic UK-2A. SAR studies around the picolinamide ring and benzyl substituents attached at positions 3 and 8, respectively, of the UK-2A bislactone macrocycle have recently been documented. This study focuses on replacement of the isobutyryl ester group in the 7 position. Thirty analogs, predominantly esters and ethers, were prepared and evaluated for inhibition of mitochondrial electron transport and in vitro growth of Zymoseptoria tritici, Leptosphaeria nodorum, Pyricularia oryzae and Ustilago maydis. Aliphatic substituents containing four to six carbon atoms deliver strong intrinsic activity, the pivaloate ester (IC50 1.44 nM) and the n-butyl, 1-Me-propyl, 3,3-diMe-propyl and 2-c-propyl propyl ethers (IC50 values = 1.08, 1.14, 1.15 & 1.32 nM, respectively) being the most active derivatives. QSAR modelling identified solvation energy (Esolv ) and critical packing parameters (vsurf_CP) as highly significant molecular descriptors for explaining relative intrinsic activity of analogs. Activity translation to fungal growth inhibition and disease control testing was significantly influenced by intrinsic activity and physical properties, the cyclopropanecarboxylate ester (log D 3.67, IC50 3.36 nM, Z. tritici EC50 12 μg L-1 ) showing the strongest Z. tritici activity in protectant tests. Substitution of the isobutyryl ester group of UK-2A generates analogs that retain strong antifungal activity against Z. tritici and other fungi. © 2019 Society of Chemical Industry.

Intervention of mitochondrial activity attenuates cisplatin-induced acute kidney injury.

The dysfunction of mitochondrial respiratory chain induced by cisplatin results in overproduction of reactive oxygen species (ROS) which contributes to kidney injury. The current study aimed to evaluate the effect of a mitochondrial electron transport inhibitors of rotenone (mitochondrial complex I inhibitor) and azoxystrobin (mitochondrial complex III inhibitor), in cisplatin-induced kidney injury. In vivo, cisplatin was administered to male C57BL/6J mice by a single intraperitoneal (i.p.) injection (20mg/kg). Then the mice were treated with or without 200ppm rotenone in food. Mice were sacrificed after cisplatin administration for 72h. The serum and the kidney tissues were collected for further analysis. In vitro, mouse proximal tubular cells (mPTCs) were treated with cisplatin (5µg/mL) and rotenone/azoxystrobin for 24h. Flow cytometry, Western blotting, and TUNEL staining were used to evaluate the cell injury. In vivo, rotenone treatment obviously ameliorated cisplatin-induced renal tubular injury evidenced by the improved histology and blocked NGAL upregulation. Meanwhile, cisplatin-induced renal dysfunction shown by the increased levels of serum creatinine (Scr), blood urea nitrogen (BUN), and cystatin C were significantly reduced by rotenone treatment. Moreover, the increments of cleaved caspase-3 and transferase dUTP nick-end labeling (TUNEL)-positive cells were markedly decreased in line with the attenuated mitochondrial dysfunction and oxidative stress after rotenone administration. In vitro, rotenone and azoxystrobin protected against mitochondrial dysfunction, oxidative stress, and renal tubular cell apoptosis induced by cisplatin. Our results demonstrated that inhibition of mitochondrial activity significantly attenuated cisplatin nephrotoxicity possibly by inhibiting mitochondrial oxidative stress.

High-resolution QTL mapping in Tetranychus urticae reveals acaricide-specific responses and common target-site resistance after selection by different METI-I acaricides

Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance.To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a non-canonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific.

Mitochondrial dysfunction reduces the activity of KIR2.1 K+ channel in myoblasts via impaired oxidative phosphorylation.

Myoblast fusion depends on mitochondrial integrity and intracellular Ca2+ signaling regulated by various ion channels. In this study, we investigated the ionic currents associated with [Ca2+]i regulation in normal and mitochondrial DNA-depleted (ρ0) L6 myoblasts. The ρ0 myoblasts showed impaired myotube formation. The inwardly rectifying K+ current (IKir) was largely decreased with reduced expression of KIR2.1, whereas the voltage-operated Ca2+ channel and Ca2+-activated K+ channel currents were intact. Sustained inhibition of mitochondrial electron transport by antimycin A treatment (24 h) also decreased the IKir. The ρ0 myoblasts showed depolarized resting membrane potential and higher basal [Ca2+]i. Our results demonstrated the specific downregulation of IKir by dysfunctional mitochondria. The resultant depolarization and altered Ca2+ signaling might be associated with impaired myoblast fusion in ρ0 myoblasts.

Roles of NADPH oxidase and mitochondria in flow-induced vasodilation of human adipose arterioles: ROS-induced ROS release in coronary artery disease.

H2 O2 contributes to FID of human arterioles. This study is designed to examine the roles of mitochondria and NADPH oxidase in modulating the release of ROS and in mediating FID. We tested whether NADPH oxidase contributes to mitochondrial ROS generation in arterioles during CAD. Visceral adipose arterioles obtained from patients with or without CAD were cannulated and pressurized for videomicroscopic measurement of arteriolar diameters. Dilator responses and ROS production during flow were determined in the presence and absence of the NADPH oxidase inhibitor gp91ds-tat and the mitochondrial electron transport inhibitor rotenone. Both dilation and H2 O2 generation during flow were reduced in the presence of rotenone (13.5±8% vs 97±% without rotenone) or gp91ds-tat in patients with CAD, while patients without CAD exhibited H2 O2 -independent dilations. Mitochondrial superoxide production during flow was attenuated by gp91ds-tat in arterioles from CAD patients. These findings indicate that ROS produced by NADPH oxidase are an upstream component of the mitochondria-dependent pathway contributing to flow-dependent H2 O2 generation and dilation in peripheral microvessels from patients with CAD. We conclude that in CAD, both mitochondria and NADPH oxidase contribute to FID through a redox mechanism in visceral arterioles.

A mutation in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Tetranychus urticae is associated with resistance to METI acaricides

The acaricidal compounds pyridaben, tebufenpyrad and fenpyroximate are frequently used in the control of phytophagous mites such as Tetranychus urticae, and are referred to as Mitochondrial Electron Transport Inhibitors, acting at the quinone binding pocket of complex I (METI-I acaricides). Because of their very frequent use, resistance evolved fast more than 20 years ago, and is currently wide-spread. Increased activity of P450 monooxygenases has been often associated with resistance, but target-site based resistance mechanisms were never reported.Here, we report the discovery of a mutation (H92R) in the PSST homologue of complex I in METI-I resistant T. urticae strains. The position of the mutation was studied using the high-resolution crystal structure of Thermus thermophilus, and was located in a stretch of amino acids previously photo-affinity labeled by fenpyroximate. Selection experiments with a strain segregating for the mutant allele, together with marker-assisted back-crossing of the mutation in a susceptible background, confirmed the involvement of the mutation in METI-I resistance. Additionally, an independent genetic mapping approach; QTL analysis identified the genomic region of pyridaben resistance, which included the PSST gene. Last, we used CRISPR-Cas9 genome editing tools to introduce the mutation in the Drosophila PSST homologue.

Quercetin Protects Rat L6 Myocytes from Antimycin A-Induced Mitochondrial Dysfunction

Objective: Mitochondrial dysfunction is often associated with various disorders such as diabetes, Alzheimer’s etc. Reactive oxygen species (ROS), aging, and reduction of mitochondrial biogenesis contribute to mitochondrial dysfunction. Antimycin-A (AMA) damages the mitochondria through inhibition of mitochondrial electron transport. The present study sought to investigate effects of quercetin on rat L6 cells and whether quercetin protects mitochondria against oxidative damage caused by AMA. Methods: Rat L6 myocytes were used for the study. Effects of quercetin on Antimycin-A induced mitochondrial dysfunction was studied using cytotoxicity, ATP levels, mitochondrial superoxide production and NDUFB8 mRNA expression. Results: In this study, exposure of L6 myocytes to AMA induced an increase cell death, decreased ATP content, followed by a decrease in mitochondrial superoxide, and decreased expression of NDUFB8. We found that quercetin protected myocytes from antimycin-A (AMA) induced L6 cell death as evidenced from increased lactate dehydrogenase (LDH) leakage into extracellular medium, protected ATP production, prevented increase in oxidative stress and restored levels of NDUFB8 mRNA expression implying improved mitochondrial function. Conclusion: These results suggest that the quercetin showed protective effect against AMA-induced mitochondrial dysfunction by increasing ATP production, decreasing oxidative stress, and restoring mitochondrial function.

Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (kcat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (kcat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance.The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.

Abstract 4451: Novel small molecule AG311 induces tumor cell death through inhibition of mitochondrial electron transport

Abstract Mitochondria are emerging as an attractive target for new anticancer therapy. The inhibition of the mitochondrial electron transport chain (ETC) provides an alternate and selective mechanism for killing cancer cells, bypassing upstream apoptosis-inducing pathways. The mechanism is based on the fact that cancer cells have higher mitochondrial membrane potential rendering them inherently susceptible to increased reactive oxygen species (ROS) generation such as superoxide through the respiratory chain. In addition pharmacological inhibition of complex I and III at the ubiquinone-binding site could further increase superoxide production. Taken together, this mitochondrial sensitivity represents a promising approach to selectively kill cancer cells. The purpose of this study was to investigate the mitochondria-associated cell death of AG311, a novel anticancer compound. AG311 (5-[(4-Methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine) is a small molecular weight compound designed by our group. AG311 has been shown to induce rapid mitochondrial depolarization resulting in necrotic cell death in a cancer cell-selective manner. In two mouse orthotopic breast cancer models (MDA-MB-435 and 4T1), AG311 significantly reduced tumor volume by 85% and 81%, respectively (n = 4 - 6), with no apparent systemic toxicity. In this study, we aimed to identify the mitochondrial protein target(s) of AG311 in cancer cells. First, upregulation of the mitochondrial ETC by culturing MDA-MB-435 cells in galactose media sensitized cells to AG311-induced cell death. Mitochondrial oxygen consumption (XFe96 extracellular flux analyzer) drastically decreased by 62.9% (± 12.9, n = 8) in response to AG311 (7.5 μM). The effect of AG311 on mitochondrial ETC complexes was determined by spectrophotometrically measuring NADH oxidation in the presence of antimycin A (complex I), ubiquinol reduction (complex III-IV) or cytochrome c oxidation (complex IV). It was found that AG311 inhibited complex I and III activity, but not complex IV. Further kinetic assays suggested that AG311 competitively inhibited ubiquinone binding and significantly induced generation of mitochondrial superoxide (estimated with MitoSOX Red). Finally, treatment with antioxidants (e.g. lipoic acid) partially prevented AG311-induced cell death. In summary, the present results indicate mitochondrial ubiquinone as a likely target for AG311-induced selective cancer cell death through generation of mitochondrial superoxide. This together with the previously demonstrated efficacy further substantiates the use of AG311 as an anticancer agent. Citation Format: Anja Bastian, Lora C. Bailey-Downs, Jessica E. Thorpe, Ravi Kumar Vyas Devambatla, Aleem Gangjee, Kenneth M. Humphries, Michael A. Ihnat. Novel small molecule AG311 induces tumor cell death through inhibition of mitochondrial electron transport. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4451. doi:10.1158/1538-7445.AM2015-4451

Discovering antimalarials with a differential mode of action

Malaria is a global health problem that causes significant mortality and morbidity, with more than 1 million deaths per year caused by P. falciparum. Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites. Therefore, the efforts to improve current treatments are focused on the identification of new antimalarial drugs displaying novel mechanisms of action. To increase the probability of success to identify compounds active against all relevant parasite drug targets, phenotypic assays have been used to screen large compound libraries against P. falciparum blood stages. At GSK, 2M compounds were screened and 13500 confirmed hits (TCAMS-Tres Cantos Antimalarial Set) were selected which inhibited P. falciparum growth with IC50s below 2 μM. In order to select chemotypes with a presumptive novel mechanism of action, an early profiling is carried out which involves several phases: 1. In the first phase, compounds are tested against a wide panel of P. falciparum strains with well characterized genetic linkage to drug resistance to establish which series had no cross-resistance with already known antimalarial compounds. Panel includes laboratory strains as well as clinical isolates. 2. In a second phase, compounds are tested using functional assays to identify those molecules interfering with parasite pathways that are targets of already known antimalarials (electron transport, folate and isoprenoid). Both mitochondrial electron transport and pyrimidine biosynthesis inhibitors are identified using transgenic parasites overexpressing Saccharomyces cerevisiae DHODH. Addition of proguanil to cultures restores cytochrome bc1 blockage but not DHODH inhibition. Antifolates are identified by supplementing the growth medium with folic acid and, similarly, inhibitors of the isoprenoid biosynthesis pathway by adding geranyl geranyol or isopentenyl pyrophosphate, which shifts the IC50 of inhibitors of these pathways. 3. Finally compounds are subjected to hematin polymerization assays to establish the interference with this essential pharmacological target of malaria parasites. New scaffolds with novel modes of action can be selected using these approaches avoiding the interference with pathways which are known to develop resistance. This strategy has already been succesful and our current antimalarial pipeline is based on scaffolds displaying new mechanisms of action. Chemoproteomics and chemical genomics efforts are being used to decipher new MoA.

P22: Mitochondira-targeted hydrogen sulfide donors AP39 and AP123 attenuate hyperglycaemia-induced oxidative stress and loss of bioenergetics in microvascular endothelial cells

Mitochondria are key mediators in determining cell fate and some mitochondrial functions are regulated by endogenous H2S. 3-MST is constitutively expressed in mitochondria and CSE/CBS translocate to mitochondria during oxidative/hypoxic insult and mitochondrial H2S synthesis is cytoprotective. With this in mind, we have designed a series of novel compounds to generate H2S within the mitochondria containing triphenylphosphonium (mitochondria-targeting) and either anethole dithiolethione (AP39) or hydroxythiobenzamide (AP123). We have evaluated the effectiveness of AP39, AP123 (and respective their control compounds) at preventing/reversing hyperglycaemia (HG)-induced oxidative stress, and metabolic changes in brain microvascular endothelial cells in vitro. Incubation of endothelial cells with AP39 and AP123 (30–300 nM) increased intracellular H2S levels, with a preferential response seen in the mitochondrial regions confirming mitochondrial H2S delivery. HG (10 days, 25 mM), induced significant mitochondrial ‘oxidant’ production (MitoSox and CM-DCF), hyperpolarisation of the mitochondrial membrane (JC-1) and inhibition of mitochondrial electron transport, ATP production and cellular bioenergetics (Seahorse, XF24 Extracellular Flux Analyzer). These metabolic changes were significantly attenuated by treatment of cells with 200 microM of the non-targeted H2S donor GYY4137. This study suggests that very low concentrations of mitochondrial-targeted H2S (sub nM) induce positive bioenergetic effects, preserve mitochondrial function and cell integrity. Moreover, by targeting mitochondria, the mitochondria- and cytoprotective potency of H2S (e.g with AP39 and AP123) is increased by >2000 fold. Mitochondria-targeted H2S donor molecules may be useful in the treatment/prevention in pathologies associated with mitochondrial dysfunction (e.g. diabetes, sepsis, hypertension, stroke etc).

Actein Isolated from Black Cohosh Promotes the Function of Osteoblastic MC3T3-E1 Cells

Actein, isolated from black cohosh, was subjected to in vitro experiments to investigate its functional bioactivities in osteoblastic MC3T3-E1 cells. Actein caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, mineralization, and glutathione content in the cells, suggesting that actein has a stimulatory effect on osteoblastic bone formation or has potential activity against osteoporosis. We investigated the protective effects of actein on mitochondrial electron transport inhibitor, antimycin A induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant decrease in cell viability and mineralization. However, pretreatment with actein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, cardiolipin oxidation, ROS release, and nitrotyrosine increase, suggesting that actein may be useful for protecting mitochondria against a burst of oxidative stress. In addition, actein increased the phosphorylation of CREB (cAMP-response element-binding protein) inhibited by antimycin A and decreased the production of TNF-α induced by antimycin A. These findings suggest that actein could prevent oxidative damage to osteoblasts in osteoporotic patients.

Odporność przędziorka chmielowca (Tetranychus urticae, Koch) na METI akarycydy analizowana w wybranych sadach jabłoniowych w Polsce

Summary Two spotted spider mite ( Tetranychus urticae , Koch) is one of important apple orchards pest. Some of the commonly used acaricides against motil stages of this pest are those from the METI group (mitochondrial electron transport inhibitors). The aim of the study was to evaluate the prevalence of the phenomenon of resistance in apple orchards in Poland. The mites collected in orchards located in different regions of country were tested in laboratory with registered compounds belonging to the METI acaricides (Magus 200 SC, Ortus 05 SC, Sanmite 20 WP). Some of the tested populations were characterized by a high resistance, comparable with the resistance of individuals from laboratory breeding resistant strain Akita.

The protective effects of Achyranthes bidentata root extract on the antimycin A induced damage of osteoblastic MC3T3-E1 cells

Achyranthes bidentata (A. bidentata) Blume is a medicinal herb with the property of strengthening bones and muscles and ensuring proper downward flow of blood in terms of the therapeutic theory of traditional medicine. In the present study, the effect of A. bidentata root extract (AE) on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. AE caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, and mineralization in the cells (P<0.05). AE also decreased the production of TNF-α, IL-6, and RANKL induced by antimycin A, mitochondrial electron transport inhibitor. Exposure of MC3T3-E1 cells to antimycin A caused significant reduction of cell viability and mineralization. However, pretreatment with AE prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, ATP loss, ROS release, and nitrotyrosine increase, suggesting that AE may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, AE increased the phosphorylation of cAMP-response element-binding protein inhibited by antimycin A. Our study demonstrates that A. bidentata could significantly prevent osteoblast damage in aged patients.

Biological evaluation of hydroxynaphthoquinones as anti-malarials.

BackgroundThe hydroxynaphthoquinones have been extensively investigated over the past 50 years for their anti-malarial activity. One member of this class, atovaquone, is combined with proguanil in Malarone®, an important drug for the treatment and prevention of malaria.MethodsAnti-malarial activity was assessed in vitro for a series of 3-alkyl-2-hydroxy-1,4-naphthoquinones (N1-N5) evaluating the parasitaemia after 48 hours of incubation. Potential cytotoxicity in HEK293T cells was assessed using the MTT assay. Changes in mitochondrial membrane potential of Plasmodium were measured using the fluorescent dye Mitrotracker Red CMXROS.ResultsFour compounds demonstrated IC50s in the mid-micromolar range, and the most active compound, N3, had an IC50 of 443 nM. N3 disrupted mitochondrial membrane potential, and after 1 hour presented an IC50ΔΨmit of 16 μM. In an in vitro cytotoxicity assay using HEK 293T cells N3 demonstrated no cytotoxicity at concentrations up to 16 μM.ConclusionsN3 was a potent inhibitor of mitochondrial electron transport, had nanomolar activity against cultured Plasmodium falciparum and showed minimal cytotoxicity. N3 may serve as a starting point for the design of new hydroxynaphthoquinone anti-malarials.

Tumor cell death induced by the inhibition of mitochondrial electron transport: The effect of 3-hydroxybakuchiol

Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells.

Sciadopitysin protects osteoblast function via its antioxidant activity in MC3T3-E1 cells

Age-related osteoblast dysfunction is the main cause of age-related bone loss in both men and women. In the present study, the effect of sciadopitysin, a type of biflavonoids, on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. Sciadopitysin caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, mineralization, and glutathione content in the cells (P<0.05). Sciadopitysin also decreased the production of tumor necrosis factor-a (TNF-α) induced by antimycin A, a mitochondrial electron transport inhibitor. We investigated the protective effects of sciadopitysin on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused a significant reduction in osteoblast dysfunction. However, pretreatment with sciadopitysin prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, adenosine triphosphate (ATP) loss, reactive oxygen species (ROS) release, and nitrotyrosine increase, suggesting that sciadopitysin may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, sciadopitysin increased phosphorylation of cAMP-response element-binding protein (CREB) inhibited by antimycin A. Our results demonstrate that sciadopitysin may reduce or prevent osteoblasts degeneration.

Biogenesis of photosynthetic apparatus under the inhibition of energy processes in de-etiolated seedlings of barley (Hordeum vulgare L.)

Transformation of protochlorophyllide forms in etiolated barley seedlings and biogenesis of photosynthetic apparatus in greening leaves of 7-day-old etiolated barley seedlings (Hordeum vulgare L.) were studied under the inhibition of energy processes during illumination. Repression of electron transport between photosystem 2 and 1 (PS2 and PS1, respectively) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) inhibited the photochemical activity of PS2 but did not affect chlorophyll biosynthesis and ATP content in leaves compared to the control. Inhibition of mitochondrial electron transport with sodium azide increased relative content of nonphotoactive protochlorophyllide in etiolated leaves, decreased the content of ATP, chlorophylls, and carotenoids and completely suppressed the functional activity of PS 2. The inhibitor of glycolysis sodium fluoride affected all the parameters even more strongly. We observed synchronism in the accumulation of chlorophylls and carotenoids during greening for all inhibitor variants other than fluoride (correlation coefficient, r, equal to 0.98, 0.97, 0.97, and 0.47 with the significance level of 0.01; 0.015; 0.015, and 0.27 for control, diuron, azide, and sodium fluoride, respectively). The change in chlorophyll content under the influence of inhibitors positively correlated with the amount of ATP in the leaf tissue (for 24 h greening, r = 0.97 with significance level of 0.015). We suggest that sources of ATP involved in the synthesis of chlorophyll during greening of etiolated barley seedlings are mostly of non-plastid origin.

Vers la thrombolyse en Afrique Noire

Antimycin A (AMA) damages the mitochondria through inhibition of mitochondrial electron transport. In this study, exposure of L6 rat skeletal muscle cells to AMA induced a decrease in ATP content, followed by a decrease in mitochondrial membrane potential, leading to apoptosis. We evaluated the protective effects of water and ethanol extracts of Nelumbo nucifera seeds on L6 cells with AMA-induced oxidative stress. We found that the extracts reduced cellular apoptosis; preserved the mitochondrial membrane potential; protected mitochondrial ATP production; inhibited p53, Bax, and caspase 3 activities; and induced Bcl-2 production. Our results suggested that AMA induced apoptosis in L6 cells via impairment of mitochondrial function. N. nucifera extracts protected the cells from this mitochondria-mediated cell death.