Electron Transport Chain Inhibitors Research Articles

N-acetylcysteine amide and di- N-acetylcysteine amide protect retinal cells in culture via an antioxidant action

Reactive oxygen species (ROS) play a significant role in toxicity to the retina in a variety of diseases. N-acetylcysteine (NAC), N-acetylcysteine amide (NACA) and the dimeric di-N-acetylcysteine amide (diNACA) were evaluated in terms of protecting retinal cells, in vitro, in a variety of stress models.Three types of rat retinal cell cultures were utilized in the study: macroglial-only cell cultures, neuron-only retinal ganglion cell (RGC) cultures, and mixed cultures containing retinal glia and neurons. Ability of test agents to attenuate oxidative stress in all cultures was ascertained. In addition, capability of agents to protect against a variety of alternate clinically-relevant stressors, including excitotoxins and mitochondrial electron transport chain inhibitors, was also evaluated. Capacity of test agents to elevate cellular levels of reduced glutathione under normal and compromised conditions was also determined.NAC, NACA and diNACA demonstrated concentration-dependent cytoprotection against oxidative stress in all cultures. These three compounds, however, had differing effects against a variety of alternate insults to retinal cells. The most protective agent was NACA, which was most potent against the most stressors (including oxidative stress, mitochondrial impairment by antimycin A and azide, and glutamate-induced excitotoxicity). Similar to NAC, NACA increased glutathione levels in non-injured cells, although diNACA did not, suggesting a different, unknown mechanism of antioxidant activity for the latter. In support of this, diNACA was the only agent to attenuate rotenone-induced toxicity in mitochondria.NAC, NACA and diNACA exhibited varying degrees of antioxidant activity, i.e., protected cultured rat retinal cells from a variety of stressors which were designed to mimic aspects of the pathology of different retinal diseases. A general rank order of activity was observed: NACA ≥ diNACA > NAC. These results warrant further exploration of NACA and diNACA as antioxidant therapeutics for the treatment of retinal diseases, particularly those involving oxidative stress. Furthermore, we have defined the battery of tests carried out as the “Wood, Chidlow, Wall and Casson (WCWC) Retinal Antioxidant Indices”; we believe that these are of great value for screening molecules for potential to reduce retinal oxidative stress in a range of retinal diseases.

Targeting Asparagine Metabolism in Well-Differentiated/Dedifferentiated Liposarcoma.

mTORC1 activity is dependent on the presence of micronutrients, including Asparagine (Asn), to promote anabolic cell signaling in many cancers. We hypothesized that targeting Asn metabolism would inhibit tumor growth by reducing mTORC1 activity in well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS). Human tumor metabolomic analysis was utilized to compare abundance of Asn in WD vs. DD LPS. Gene set enrichment analysis (GSEA) compared relative expression among metabolic pathways upregulated in DD vs. WD LPS. Proliferation assays were performed for LPS cell lines and organoid models by using the combination treatment of electron transport chain (ETC) inhibitors with Asn-free media. 13C-Glucose-labeling metabolomics evaluated the effects of combination treatment on nucleotide synthesis. Murine xenograft models were used to assess the effects of ETC inhibition combined with PEGylated L-Asparaginase (PEG-Asnase) on tumor growth and mTORC1 signaling. Asn was enriched in DD LPS compared to WD LPS. GSEA indicated that mTORC1 signaling was upregulated in DD LPS. Within available LPS cell lines and organoid models, the combination of ETC inhibition with Asn-free media resulted in reduced cell proliferation. Combination treatment inhibited nucleotide synthesis and promoted cell cycle arrest. In vivo, the combination of ETC inhibition with PEG-Asnase restricted tumor growth. Asn enrichment and mTORC1 upregulation are important factors contributing to WD/DD LPS tumor progression. Effective targeting strategies require limiting access to extracellular Asn and inhibition of de novo synthesis mechanisms. The combination of PEG-Asnase with ETC inhibition is an effective therapy to restrict tumor growth in WD/DD LPS.

New functions of pirin proteins and a 2-ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile.

The understanding of how central metabolism and fermentation pathways regulate antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is still incomplete. Our study reveals that B. fragilis encodes two iron-dependent, redox-sensitive regulatory pirin protein genes, pir1 and pir2. The mRNA expression of these genes increases when exposed to oxygen and during growth in iron-limiting conditions. These proteins, Pir1 and Pir2, influence the production of short-chain fatty acids and modify the susceptibility to metronidazole and amixicile, a new inhibitor of pyruvate: ferredoxin oxidoreductase in anaerobes. We have demonstrated that Pir1 and Pir2 interact directly with this oxidoreductase, as confirmed by two-hybrid system assays. Furthermore, structural analysis using AlphaFold2 predicts that Pir1 and Pir2 interact stably with several central metabolism enzymes, including the 2-ketoglutarate:ferredoxin oxidoreductases Kor1AB and Kor2CDAEBG. We used a series of metabolic mutants and electron transport chain inhibitors to demonstrate the extensive impact of bacterial metabolism on metronidazole and amixicile susceptibility. We also show that amixicile is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. Our investigation led to the discovery that the kor2AEBG genes are essential for growth and have dual functions, including the formation of 2-ketoglutarate via the reverse TCA cycle. However, the metabolic activity that bypasses the function of Kor2AEBG following the addition of phospholipids or fatty acids remains undefined. Overall, our study provides new insights into the central metabolism of B. fragilis and its regulation by pirin proteins, which could be exploited for the development of new narrow-spectrum antimicrobials in the future.

Controlling Pericellular Oxygen Tension in Cell Culture Reveals Distinct Breast Cancer Responses to Low Oxygen Tensions.

In oxygen (O2)-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O2 tension experienced by cells (i.e., pericellular O2). However, it is discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O2 consumption. A reaction-diffusion model is developed to predict pericellular O2 tension a priori, demonstrating that the effect of cellular O2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O2 tension in cell culture incubators is insufficient to regulate O2 in cell culture, thus introducing the concept of pericellular O2-controlled cell culture.

Acute Metabolic Stress Induces Lymphatic Dysfunction Through KATP Channel Activation.

Lymphatic dysfunction is an underlying component of multiple metabolic diseases, including diabetes, obesity, and metabolic syndrome. We investigated the roles of KATP channels in lymphatic contractile dysfunction in response to acute metabolic stress induced by inhibition of the mitochondrial electron transport chain. Ex vivo popliteal lymphatic vessels from mice were exposed to the electron transport chain inhibitors antimycin A and rotenone, or the oxidative phosphorylation inhibitor/protonophore, CCCP. Each inhibitor led to a significant reduction in the frequency of spontaneous lymphatic contractions and calculated pump flow, without a significant change in contraction amplitude. Contraction frequency was restored by the KATP channel inhibitor, glibenclamide. Lymphatic vessels from mice with global Kir6.1 deficiency or expressing a smooth muscle-specific dominant negative Kir6.1 channel were resistant to inhibition. Antimycin A inhibited the spontaneous action potentials generated in lymphatic muscle and this effect was reversed by glibenclamide, confirming the role of KATP channels. Antimycin A, but not rotenone or CCCP, increased dihydrorhodamine fluorescence in lymphatic muscle, indicating ROS production. Pretreatment with tiron or catalase prevented the effect of antimycin A on wild-type lymphatic vessels, consistent with its action being mediated by ROS. Our results support the conclusion that KATP channels in lymphatic muscle can be directly activated by reduced mitochondrial ATP production or ROS generation, consequent to acute metabolic stress, leading to contractile dysfunction through inhibition of the ionic pacemaker controlling spontaneous lymphatic contractions. We propose that a similar activation of KATP channels contributes to lymphatic dysfunction in metabolic disease.

Electron transport chain inhibition increases cellular dependence on purine transport and salvage

Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.

Experimental and computational investigation of the kinetic evolution of the glutaminolysis pathway and its interplay with the glycolysis pathway.

Exploring cellular responses necessitates studying real-time metabolic pathway kinetics, considering the adaptable nature of cells. Glycolysis and glutaminolysis are interconnected pathways fundamental to driving cellular metabolism, generating both energy and essential biosynthetic molecules. While prior studies explored glycolysis tracking, this research focuses on monitoring the kinetics of the glutaminolysis pathway by evaluating the effect of glutamine availability on glycolytic kinetics and by investigating the impact of a stimulator (oligomycin) and inhibitor (2DG) on the glycolytic flux in the presence of glutamine. Additionally, we adapted a rate equation model to provide improved understanding of the pathway kinetics. The experimental and simulated results indicate a significant reduction in extracellular lactate production in the presence of glutamine, reflecting a shift from glycolysis towards oxidative phosphorylation, due to the additional contribution of glutamine to energy production through the ETC (electron transport chain), reducing the glycolytic load. Oligomycin, an ETC inhibitor, increases lactate production to the original glycolytic level, despite the presence of glutamine. Nevertheless, its mechanism is influenced by the presence of glutamine, as predicted by the model. Conversely, 2DG notably reduces lactate production, affirming its glycolytic origin. The gradual increase in lactate production under the influence of 2DG implies increased activation of glutaminolysis as an alternative energy source. The model also simulates the varying metabolic responses under varying carbon/modulator concentrations. In conclusion, the kinetic model described here contributes to the understanding of changes in intracellular metabolites and their interrelationships in a way which would be challenging to obtain solely through kinetic assays.

Heat stress upregulates arachidonic acid to trigger autophagy in sertoli cells via dysfunctional mitochondrial respiratory chain function

As a key factor in determining testis size and sperm number, sertoli cells (SCs) play a crucial role in male infertility. Heat stress (HS) reduces SCs counts, negatively impacting nutrient transport and supply to germ cells, and leading to spermatogenesis failure in humans and animals. However, how HS affects the number of SCs remains unclear. We hypothesized that changes in SC metabolism contribute to the adverse effects of HS. In this study, we first observed an upregulation of arachidonic acid (AA), an unsaturated fatty acid after HS exposure by LC-MS/MS metabolome detection. By increasing ROS levels, expression of KEAP1 and NRF2 proteins as well as LC3 and LAMP2, 100 µM AA induced autophagy in SCs by activating oxidative stress (OS). We observed adverse effects of AA on mitochondria under HS with a decrease of mitochondrial number and an increase of mitochondrial membrane potential (MMP). We also found that AA alternated the oxygen transport and absorption function of mitochondria by increasing glycolysis flux and decreasing oxygen consumption rate as well as the expression of mitochondrial electron transport chain (ETC) proteins Complex I, II, V. However, pretreatment with 5 mM NAC (ROS inhibitor) and 2 µM Rotenone (mitochondrial ETC inhibitor) reversed the autophagy induced by AA. In summary, AA modulates autophagy in SCs during HS by disrupting mitochondrial ETC function, inferring that the release of AA is a switch-like response, and providing insight into the underlying mechanism of high temperatures causing male infertility.

The differential effects of acute and prolonged L-NAME exposure on A23187-induced NO-dependent dilation in human arterioles

The Ca2+ ionophore A23187 induces endothelium-dependent and non-receptor-mediated vasodilation in human adipose arterioles (HAA). Preliminary data from our group suggest that while A23187 dilation mainly depends on NO as a vasodilator factor in healthy HAAs, there is a component of non-NO and likely hydrogen peroxide (H2O2)-mediated dilation, especially in arterioles from patients with coronary artery disease (CAD). The mechanisms underlying the release of two different vasodilator factors remain largely unclear. There is evidence that NO-mediated S-nitrosylation inhibits the catalytic activity of mitochondrial electron transport chain (ETC) complexes or other reactive oxygen species (ROS)-producing enzymes such as NADPH oxidases (NOX). The objective of this study was to compare the effects of acute (30 minutes) and prolonged (90 minutes) nitric oxide (NO) synthase inhibitor nitro-L-arginine methyl ester (L-NAME) exposure on A23187 dilation to elucidate whether prolonged incubation with L-NAME results in transition from NO-dependent to H2O2-dependent vasodilation in HAAs. HAAs (~150 μm ID), isolated from fat tissues obtained from subjects with and without CAD, were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In non-CAD HAAs constricted with endothelin-1, A23187 (10−11 to 10−7 M) elicited concentration-dependent dilation (maximal dilation at 10−7 M: 68±5%, n=5), and this dilation was largely abolished after acute exposure to L-NAME (maximal dilation at 10−7 M: 21±7%, n=5). Prolonged L-NAME incubation restored A23187-dependent dilation (maximal dilation at 10−7 M: 74±2%, n=5), which was subsequently inhibited by hydrogen peroxide scavenger catalase (500 U/ml; maximal dilation at 10−7 M: 14±4%, n=5). HAAs obtained from CAD patients, responded to A23187 in a similar dose-dependent fashion (maximal dilation at 10−7 M: 68±3%, n=5), and this dilation was not affected by acute L-NAME exposure (maximal dilation at 10−7 M: 65±3%, n=5), but was reduced by catalase (maximal dilation at 10−7 M: 31±6%, n=6). To further determine the role of mitochondria-derived ROS in A23187-induced dilation, we treated arterioles with ETC inhibitor rotenone (1 mM). In non-CAD HAAs not pretreated with L-NAME, rotenone had no effect on A23187-induced dilation (maximal dilation at 10−7 M: 68±6%, n=5). In the presence of rotenone, however, prolonged L-NAME incubation no longer restored A23187-dependent dilation (maximal dilation at 10−7 M: 20±3%, n=5). Together, these data provide the first evidence that in healthy HAAs, prolonged L-NAME exposure changes the mechanism of vasodilation from NO to H2O2-mediated CAD-like vasodilation in response to A23187. In addition, the signaling process likely involves mitochondrial ROS. To elucidate the precise mechanisms underlying this process, future studies will be required to evaluate whether in healthy vasculature, basal NO inhibits mitochondrial ETC or other proteins such as NOXs through NO-mediated protein modification, which can be reversed by prolonged L-NAME incubation thereby promoting a switch from NO to H2O2 -mediated dilation. This work was supported by the National Heart, Lung, and Blood Institute [R01HL096647]. This is the full abstract presented at the American Physiology Summit 2024 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.

Cyclin-dependent kinase 12 deficiency reprogrammes cellular metabolism to alleviate ferroptosis potential and promote the progression of castration-resistant prostate cancer.

Cyclin-dependent kinase 12 (CDK12)-deficient prostate cancer defines a subtype of castration-resistant prostate cancer (CRPC) with a poor prognosis. Current therapy, including PARP inhibitors, shows minimal treatment efficacy for this subtype of CRPC, and the underlying mechanism remains elusive. Based on bioinformatics analysis, we evaluated the relationship between CDK12 deficiency and prostate cancer patient's prognosis and treatment resistance. Furthermore, we used CRISPR-Cas9 technology and mass spectrometry-based metabolomic profiling to reveal the metabolic characteristics of CDK12-deficient CRPC. To elucidate the specific mechanisms of CDK12 deficiency-mediated CRPC metabolic reprogramming, we utilized cell RNA-seq profiling and other molecular biology techniques, including cellular reactive oxygen species probes, mitochondrial function assays, ChIP-qPCR and RNA stability analyses, to clarify the role of CDK12 in regulating mitochondrial function and its contribution to ferroptosis. Finally, through in vitro drug sensitivity testing and in vivo experiments in mice, we identified the therapeutic effects of the electron transport chain (ETC) inhibitor IACS-010759 on CDK12-deficient CRPC. CDK12-deficient prostate cancers reprogramme cellular energy metabolism to support their aggressive progression. In particular, CDK12 deficiency enhanced the mitochondrial respiratory chain for electronic transfer and ATP synthesis to create a ferroptosis potential in CRPC cells. However, CDK12 deficiency downregulated ACSL4 expression, which counteracts the lipid oxidation stress, leading to the escape of CRPC cells from ferroptosis. Furthermore, targeting the ETC substantially inhibited the proliferation of CDK12-deficient CRPC cells in vitro and in vivo, suggesting a potential new target for the therapy of CDK12-deficient prostate cancer. Our findings show that energy and lipid metabolism in CDK12-deficient CRPC work together to drive CRPC progression and provide a metabolic insight into the worse prognosis of CDK12-deficient prostate cancer patients. CDK12 deficiency promotes castration-resistant prostate cancer (CRPC) progression by reprogramming cellular metabolism. CDK12 deficiency in CRPC leads to a more active mitochondrial electron transport chain (ETC), ensuring efficient cell energy supply. CDK12 phosphorylates RNA Pol II to ensure the transcription of ACSL4 to regulate ferroptosis. Mitochondrial ETC inhibitors exhibit better selectivity for CDK12-deficient CRPC cells, offering a promising new therapeutic approach for this subtype of CRPC patients.

Bactericidal and sterilizing activity of sudapyridine-clofazimine-TB47 combined with linezolid or pyrazinamide in a murine model of tuberculosis.

As an obligate aerobe, Mycobacterium tuberculosis relies on its branched electron transport chain (ETC) for energy production through oxidative phosphorylation. Regimens targeting ETC exhibit promising potential to enhance bactericidal activity against M. tuberculosis and hold the prospect of shortening treatment duration. Our previous research demonstrated that the bacteriostatic drug candidate TB47 (T) inhibited the growth of M. tuberculosis by targeting the cytochrome bc1 complex and exhibited synergistic activity with clofazimine (C). Here, we found synergistic activities between C and sudapyridine (S), a structural analog of bedaquiline (B). S has shown similar anti-tuberculosis efficacy and may share a mechanism of action with B, which inhibits ATP synthesis and the energy metabolism of bacteria. We evaluated the efficacy of SCT in combination with linezolid (L) or pyrazinamide (Z) using a well-established murine model of tuberculosis. Compared to the BPa(pretomanid)L regimen, SCT and SCTL demonstrated similar bactericidal and sterilizing activities. There was no significant difference in activity between SCT and SCTL. In contrast, SCZ and SCTZ showed much higher activities, with none of the 15 mice experiencing relapse after 2 months of treatment with either SCZ or SCTZ. However, T did not contribute to the activity of the SCZ. Our findings emphasize the efficacy and the potential clinical significance of combination therapy with ETC inhibitors. Additionally, cross-resistance exists not only between S and B but also between S/B and C. This is supported by our findings, as spontaneous S-resistant mutants exhibited mutations in Rv0678, which are associated with cross-resistance to B and C.

Investigating impacts of the mycothiazole chemotype as a chemical probe for the study of mitochondrial function and aging.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), MTZ, a newly employed ETC complex I inhibitor, exhibited higher cytotoxicity against cancer cell lines compared to certain non-cancer cell lines. Interestingly, 8-OAc demonstrated greater selectivity for cancer cells when compared to both MTZ and Rote, which has promising potential for anticancer therapeutic development. Furthermore, in vivo experiments with these small molecules utilizing a C. elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. We also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilizes the transcription factors ATFS-1 and HSF1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

Inducing vulnerability to InhA inhibition restores isoniazid susceptibility in drug-resistant Mycobacterium tuberculosis.

Of the approximately 10 million cases of Mycobacterium tuberculosis (Mtb) infections each year, over 10% are resistant to the frontline antibiotic isoniazid (INH). INH resistance is predominantly caused by mutations that decrease the activity of the bacterial enzyme KatG, which mediates the conversion of the pro-drug INH to its active form INH-NAD. We previously discovered an inhibitor of Mtb respiration, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a collection of INH-resistant mutants to INH through an unknown mechanism. To investigate the mechanism of action of C10, we exploited the toxicity of high concentrations of C10 to select for resistant mutants. We discovered two mutations that confer resistance to the disruption of energy metabolism and allow for the growth of Mtb in high C10 concentrations, indicating that growth inhibition by C10 is associated with inhibition of respiration. Using these mutants as well as direct inhibitors of the Mtb electron transport chain, we provide evidence that inhibition of energy metabolism by C10 is neither sufficient nor necessary to potentiate killing by INH. Instead, we find that C10 acts downstream of INH-NAD synthesis, causing Mtb to become particularly sensitive to inhibition of the INH-NAD target, InhA, without changing the concentration of INH-NAD or the activity of InhA, the two predominant mechanisms of potentiating INH. Our studies revealed that there exists a vulnerability in Mtb that can be exploited to render Mtb sensitive to otherwise subinhibitory concentrations of InhA inhibitor.IMPORTANCEIsoniazid (INH) is a critical frontline antibiotic to treat Mycobacterium tuberculosis (Mtb) infections. INH efficacy is limited by its suboptimal penetration of the Mtb-containing lesion and by the prevalence of clinical INH resistance. We previously discovered a compound, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a set of INH-resistant mutants to INH. Resistance is typically mediated by katG mutations that decrease the activation of INH, which is required for INH to inhibit the essential enzyme InhA. Our current work demonstrates that C10 re-sensitizes INH-resistant katG-hypomorphs without enhancing the activation of INH. We furthermore show that C10 causes Mtb to become particularly vulnerable to InhA inhibition without compromising InhA activity on its own. Therefore, C10 represents a novel strategy to curtail the development of INH resistance and to sensitize Mtb to sub-lethal doses of INH, such as those achieved at the infection site.

Risk Assessment of Psychotropic Drugs on Mitochondrial Function Using In Vitro Assays.

Mitochondria are potential targets responsible for some drug- and xenobiotic-induced organ toxicities. However, molecular mechanisms of drug-induced mitochondrial toxicities are mostly unknown. Here, multiple in vitro assays were used to investigate the effects of 22 psychotropic drugs on mitochondrial function. The acute extracellular flux assay identified inhibitors of the electron transport chain (ETC), i.e., aripiprazole, phenytoin, and fluoxetine, an uncoupler (reserpine), substrate inhibitors (quetiapine, carbamazepine, buspirone, and tianeptine), and cytotoxic compounds (chlorpromazine and valproic acid) in HepG2 cells. Using permeabilized HepG2 cells revealed minimum effective concentrations of 66.3, 6730, 44.5, and 72.1 µM for the inhibition of complex-I-linked respiration for quetiapine, valproic acid, buspirone, and fluoxetine, respectively. Assessing complex-II-linked respiration in isolated rat liver mitochondria revealed haloperidol is an ETC inhibitor, chlorpromazine is an uncoupler in basal respiration and an ETC inhibitor under uncoupled respiration (IC50 = 135 µM), while olanzapine causes a mild dissipation of the membrane potential at 50 µM. This research elucidates some mechanisms of drug toxicity and provides some insight into their safety profile for clinical drug decisions.

GaMF1.39's antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876.

New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

A patatin-like phospholipase is important for mitochondrial function in malaria parasites.

For their proliferation within red blood cells, malaria parasites depend on a functional electron transport chain (ETC) within their mitochondrion, which is the target of several antimalarial drugs. Here, we have used gene disruption to identify a patatin-like phospholipase, PfPNPLA2, as important for parasite replication and mitochondrial function in Plasmodium falciparum. Parasites lacking PfPNPLA2 show defects in their ETC and become hypersensitive to mitochondrion-targeting drugs. Furthermore, PfPNPLA2-deficient parasites show differences in the composition of their cardiolipins, a unique class of phospholipids with key roles in mitochondrial functions. Finally, we demonstrate that parasites devoid of PfPNPLA2 have a defect in gametocyte maturation, underlining the importance of a functional ETC for parasite transmission to the mosquito vector.

The role of mitochondrial reactive oxygen species in chondrocyte mechanotransduction.

Chondrocytes are mechanosensitive cells able to sense and respond to external mechanical stimuli through the process of mechanotransduction. Previous studies have demonstrated that mechanical stimulation causes mitochondrial deformation leading to mitochondrial reactive oxygen species (ROS) release in a dose-dependent manner. For this reason, we focused on elucidating the role of mitochondrial ROS as anabolic signaling molecules in chondrocyte mechanotransduction. Chondrocyte-seeded agarose gels were subjected to mechanical stimuli and the effect on matrix synthesis, ROS production, and mitogen-activated protein kinases (MAPK) signaling was evaluated. Through the use of ROS-specific staining, superoxide anion was the primary ROS released in response to mechanical stimuli. The anabolic effect of mechanical stimulation was abolished in the presence of electron transport chain inhibitors (complexes I, III, and V) and superoxide anion scavengers. Subsequent studies were centered on the involvement of MAPK pathways (ERK1/2, p38, and JNK) in the mechanotransduction cascade. While disruption of the ERK1/2 pathway had no apparent effect, the anabolic effect of mechanical stimulation was abolished in the presence of p38 and JNK pathway inhibitors. This suggest the involvement of apoptosis stimulating kinase 1 (ASK1), an upstream redox-sensitive MAP3K shared by both the JNK and p38 pathways. Future experiments will focus on the involvement of the thioredoxin-ASK1 complex which disassociates in the presence of oxidative stress, allowing ASK1 to phosphorylate several MAP2Ks. Overall, these findings indicate superoxide anion as the primary ROS released in response to mechanical stimuli and that the resulting anabolic effect on chondrogenic matrix biosynthesis arises from the ROS-dependent activation of the p38 and JNK MAPKs.

Non-chemical signalling between mitochondria.

A wide variety of studies have reported some form of non-chemical or non-aqueous communication between physically isolated organisms, eliciting changes in cellular proliferation, morphology, and/or metabolism. The sources and mechanisms of such signalling pathways are still unknown, but have been postulated to involve vibration, volatile transmission, or light through the phenomenon of ultraweak photon emission. Here, we report non-chemical communication between isolated mitochondria from MCF7 (cancer) and MCF10A (non-cancer) cell lines. We found that mitochondria in one cuvette stressed by an electron transport chain inhibitor, antimycin, alters the respiration of mitochondria in an adjacent, but chemically and physically separate cuvette, significantly decreasing the rate of oxygen consumption compared to a control (p = <0.0001 in MCF7 and MCF10A mitochondria). Moreover, the changes in O2-consumption were dependent on the origin of mitochondria (cancer vs. non-cancer) as well as the presence of "ambient" light. Our results support the existence of non-chemical signalling between isolated mitochondria. The experimental design suggests that the non-chemical communication is light-based, although further work is needed to fully elucidate its nature.

NLG1, encoding a mitochondrial membrane protein, controls leaf and grain development in rice

BackgroundMitochondrion is the key respiratory organ and participate in multiple anabolism and catabolism pathways in eukaryote. However, the underlying mechanism of how mitochondrial membrane proteins regulate leaf and grain development remains to be further elucidated.ResultsHere, a mitochondria-defective mutant narrow leaf and slender grain 1 (nlg1) was identified from an EMS-treated mutant population, which exhibits narrow leaves and slender grains. Moreover, nlg1 also presents abnormal mitochondria structure and was sensitive to the inhibitors of mitochondrial electron transport chain. Map-based cloning and transgenic functional confirmation revealed that NLG1 encodes a mitochondrial import inner membrane translocase containing a subunit Tim21. GUS staining assay and RT-qPCR suggested that NLG1 was mainly expressed in leaves and panicles. The expression level of respiratory function and auxin response related genes were significantly down-regulated in nlg1, which may be responsible for the declination of ATP production and auxin content.ConclusionsThese results suggested that NLG1 plays an important role in the regulation of leaf and grain size development by maintaining mitochondrial homeostasis. Our finding provides a novel insight into the effects of mitochondria development on leaf and grain morphogenesis in rice.

Ether lipid biosynthesis promotes lifespan extension and enables diverse pro-longevity paradigms in Caenorhabditis elegans.

Biguanides, including the world's most prescribed drug for type 2 diabetes, metformin, not only lower blood sugar, but also promote longevity in preclinical models. Epidemiologic studies in humans parallel these findings, indicating favorable effects of metformin on longevity and on reducing the incidence and morbidity associated with aging-related diseases. Despite this promise, the full spectrum of molecular effectors responsible for these health benefits remains elusive. Through unbiased screening in Caenorhabditis elegans, we uncovered a role for genes necessary for ether lipid biosynthesis in the favorable effects of biguanides. We demonstrate that biguanides prompt lifespan extension by stimulating ether lipid biogenesis. Loss of the ether lipid biosynthetic machinery also mitigates lifespan extension attributable to dietary restriction, target of rapamycin (TOR) inhibition, and mitochondrial electron transport chain inhibition. A possible mechanistic explanation for this finding is that ether lipids are required for activation of longevity-promoting, metabolic stress defenses downstream of the conserved transcription factor skn-1/Nrf. In alignment with these findings, overexpression of a single, key, ether lipid biosynthetic enzyme, fard-1/FAR1, is sufficient to promote lifespan extension. These findings illuminate the ether lipid biosynthetic machinery as a novel therapeutic target to promote healthy aging.