Inhibition Of Oxygen Consumption Research Articles

The fungicide cymoxanil impairs respiration in Saccharomyces cerevisiae via cytochrome c oxidase inhibition.

Cymoxanil (CYM) is a widely used synthetic acetamide fungicide, but its biochemical mode of action remains elusive. Since CYM inhibits cell growth, biomass production, and respiration in Saccharomyces cerevisiae, we used this model to characterize the effect of CYM on mitochondria. We found it inhibits oxygen consumption in both whole cells and isolated mitochondria, specifically inhibiting cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking, we propose that CYM blocks the interaction of cytochrome c with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that will be instrumental in driving informed management of the use of this fungicide.

Smart multi‐functional aggregates reoxygenate tumor microenvironment through a two‐pronged strategy to revitalize cancer immunotherapy

AbstractPD‐1/PD‐L1 inhibitors have emerged as standard treatments for advanced solid tumors; however, challenges such as a low overall response rate and systemic side effects impede their implementation. Hypoxia drives the remodeling of the tumor microenvironment, which is a leading reason for the failure of immunotherapies. Despite some reported strategies to alleviate hypoxia, their individual limitations constrain further improvements. Herein, a novel two‐pronged strategy is presented to efficiently address hypoxia by simultaneously adopting atovaquone (ATO, inhibiting oxygen consumption) and oxyhemoglobin (HbO2, directly supplementing oxygen) within a multifunctional aggregate termed NPs‐aPD‐1/HbO2/ATO. In addition to eliminating hypoxia with these two components, this smart aggregate also includes albumin and an ROS‐responsive cross‐linker as a controlled release scaffold, along with PD‐1 antibody (aPD‐1) for immunotherapy. Intriguingly, NPs‐aPD‐1/HbO2/ATO demonstrates exceptional tumor targeting in vivo, exhibiting ≈4.2 fold higher accumulation in tumors than in the liver. Consequently, this aggregate not only effectively mitigates hypoxia and significantly assists aPD‐1 immunotherapy but also simultaneously resolves the targeting and systemic toxicity issues associated with individual administration of each component. This study proposes substantial implications for drug‐targeted delivery, addressing tumor hypoxia and advancing immunotherapy, providing valuable insights for advancing cancer treatment strategies.

Interactive effects of cyanobacterial metabolites aeruginosin-98B, anabaenopeptin-B and cylindrospermopsin on physiological parameters and novel in vivo fluorescent indicators in Chironomus aprilinus larvae

We aimed to determine the effects of single cyanobacterial metabolites aeruginosin-B (AER-B), anabaenopeptin-B (ANA-B), cylindrospermopsin (CYL), their binary and ternary mixtures on biomarkers of Chironomus aprilinus larvae: oxygen consumption, fat body structure and two novel fluorescent indicators: imaging of nuclei in cells of body integument, and the catecholamine level. The obtained results showed that oxygen consumption was inhibited by single tested cyanobacterial metabolites except for ANA-B at the lowest concentration (250 μg/L). Although the mixtures of the metabolites inhibited oxygen consumption with antagonistic interactions between the components stimulation was noted in the group exposed to the lowest concentrations of AER-B + CYL (125 μg/L + 125 μg/L, respectively) and the ternary mixture of AER-B + ANA-B + CYL (83.3 μg/L + 83.3 μg/L + 83.3 μg/L, respectively). In vivo fluorescent staining with Hoechst 34580 showed that single AER-B had lower cytotoxic potential on body integument cells than ANA-B and CYL and most binary mixtures except for AER-B + CYL induced synergistic toxicity. Catecholamine level was decreased in animals exposed to single metabolites, their binary and ternary mixtures; however, the interactions between the components in the ternary mixture were antagonistic. Fat body was found to be disrupted in the larvae exposed to single metabolites and their combinations. Antagonistic toxic interactions between the oligopeptide components were found in most binary and the ternary mixtures; however, synergistic effect was noted in the binary mixture of AER-B + CYL. The results suggest that in natural conditions Chironomus larvae and possibly other benthic invertebrates may be affected by cyanobacterial metabolites, however various components and in mixtures and their concentrations may determine varied physiological effects and diverse interactions.

Gallic acid triphenylphosphonium derivatives TPP+-C10 and TPP+-C12 inhibit mitochondrial function in Candida albicans exerting antifungal and antibiofilm effects.

To evaluate the antifungal and antibiofilm activity of gallic acid derivatives TPP+-C10 and TPP+-C12 and their effects on mitochondrial function on two Candida albicans reference strains (ATCC 90029 and ATCC 10231). First, we determined minimal inhibitory concentration (MIC) using a microdilution assay. Both compounds exerted antifungal effects, and their MICs ranged from 3.9 to 13µM, with no statistically significant differences between them (P>0.05, t-test). These concentrations served as references for following assays. Subsequently, we measured oxygen consumption with a Clark electrode. Our observations revealed that both drugs inhibited oxygen consumption in both strains with TPP+-C12 exerting a more pronounced inhibitory effect. We then employed flow cytometry with TMRE as a probe to assess mitochondrial membrane potential. For each strain assayed, the compounds induced a decay in transmembrane potential by 75%-90% compared to the control condition (P<0.05, ANOVA). Then, we measured ATP levels using a commercial kit. TPP+-C12 showed a 50% decrease of ATP content (P<0.05 ANOVA), while TPP+-C10 exhibited a less pronounced effect. Finally, we assessed the antibiofilm effect using the MTT reduction assay. Both compounds were effective, but TPP+-C12 displayed a greater potency, requiring a lower concentration to inhibit 50% of biofilms viability (P<0.05, t-test). Derivatives of gallic acid linked to a TPP+ group exert antifungal and antibiofilm activity through impairment of mitochondrial function in C. albicans.

Synthesis and biological evaluation of novel bi-gold mitocans in lung cancer cells.

Mitochondria are promising drug target for cancer treatment. We previously demonstrated that a bi-gold compound BGC2a was more potent than the mono-gold drug auranofin in suppressing cancer cells due to increased gold atom number that led to higher drug accumulation in and thereby inhibition of mitochondria. To exploit the potential of this new strategy, we further designed and synthesized a series of bi-gold mitocans, the compounds targeting mitochondria. The results showed that most of the newly synthesized mitocans exhibited obviously lower IC50 than auranofin, an old drug that is repurposed in clinical trials for cancer treatment. The best mitocan C3P4 was nearly 2-fold more potent than BGC2a in human non-small cell lung cancer A549 cells and mantle cell lymphoma Jeko-1 cells, exhibiting substantial colony formation-suppressing and tumor-suppressing effects in A549 cells xenograft model. C3P4 induced apoptosis in a dose-dependent manner and arrested cell cycle at G0/G1 phase. The mechanistic study showed that C3P4 significantly increased the global reactive oxygen species and mitochondrial superoxide level, and reduced the mitochondrial membrane potential. C3P4 preferentially accumulated in mitochondria as measured by the gold content and substantially inhibited oxygen consumption rate and ATP production. These results further validated our hypothesis that targeting mitochondria would be promising to develop more potent anticancer agents. C3P4 may be further evaluated as a drug candidate for lung cancer treatment.

Effects of cadmium exposure on metabolism, antioxidant defense, immune function, and the hepatopancreas transcriptome of Cipangopaludina cathayensis

Cadmium (Cd) is a common contaminant in aquatic environments. However, little is known about the mechanisms underlying Cd toxicity in the freshwater snail Cipangopaludina cathayensis (Heude, 1890). This study to investigate the toxic effects of Cd on the standard metabolism, antioxidant activities, immune function, and hepatopancreas transcriptome profiles of C. cathayensis. C. cathayensis was exposed to 0.25, 0.5, 1.0, or 1.5 mg/L Cd for 3 h, with results showing that Cd significantly inhibited oxygen consumption and ammonia excretion and disrupted the respiratory metabolism of C. cathayensis. In addition, the O:N ratio dropped below 7, indicating that C. cathayensis may rely exclusively on proteins as an energy source under Cd stress. To understand how Cd impacts the antioxidant activities, immune function, and transcriptional profiles, C. cathayensis were exposed to 0.5 (low exposure, L14) or 1.5 (high exposure, H14) mg/L Cd for 14 days. Our results indicate that Cd exposure leads to oxidative stress and immunosuppression, with the latter effect being larger for exposure to higher Cd concentrations. A total of 2172 differentially expressed genes (DEGs) were identified by transcriptome analysis of the hepatopancreas, of which 885 were upregulated and 1287 were downregulated. Gene ontology and KEGG analyses revealed that the DEGs in the H14 group are enriched for energy generation terms and the “oxidative phosphorylation” pathway, respectively. Therefore, up-regulation of energy metabolism may be an adaptive strategy under Cd stress. Moreover, several genes involved in antioxidant activity were downregulated, whereas genes related to reactive oxygen species generation were upregulated. In addition, many immunity-related genes were identified within the DEGs, indicating that Cd toxicity may affect immune defense. Further, DEGs in the H14 group were enriched for disease-associated pathways. Taken together, our results indicate that Cd exposure leads to metabolic disorders, oxidative stress, and immunosuppression and thus may potentially contribute to disease outbreaks.

Mitochondria-targeted melatonin photorelease supports the presence of melatonin MT1 receptors in mitochondria inhibiting respiration

The presence of signaling-competent G protein-coupled receptors in intracellular compartments is increasingly recognized. Recently, the presence of Gi/o protein-coupled melatonin MT1 receptors in mitochondria has been revealed, in addition to the plasma membrane. Melatonin is highly cell permeant, activating plasma membrane and mitochondrial receptors equally. Here, we present MCS-1145, a melatonin derivative bearing a triphenylphosphonium cation for specific mitochondrial targeting and a photocleavable o-nitrobenzyl group releasing melatonin upon illumination. MCS-1145 displayed low affinity for MT1 and MT2 but spontaneously accumulated in mitochondria, where it was resistant to washout. Uncaged MCS-1145 and exogenous melatonin recruited β-arrestin 2 to MT1 in mitochondria and inhibited oxygen consumption in mitochondria isolated from HEK293 cells only when expressing MT1 and from mouse cerebellum of WT mice but not from MT1-knockout mice. Overall, we developed the first mitochondria-targeted photoactivatable melatonin ligand and demonstrate that melatonin inhibits mitochondrial respiration through mitochondrial MT1 receptors.

Oxidative stress and mitochondrial damage induced by a novel pesticide fluopimomide in Caenorhabditis elegans.

Fluopimomide is a novel pesticide intensively used in agricultural pest control; however, its excessive use may have toxicological effects on non-target organisms. In this study, Caenorhabditis elegans was used to evaluate the toxic effects of fluopimomide and its possible mechanisms. The effects of fluopimomide on the growth, pharyngeal pumping, and antioxidant systems of C. elegans were determined. Furthermore, the gene expression levels associated with mitochondria in the nematodes were also investigated. Results indicated that fluopimomide at 0.2, 1.0, and 5.0 mg/L notably (p < 0.001) decreased body length, pharyngeal pumping, and body bends in the nematodes compared to the untreated control. Additionally, fluopimomide at 0.2, 1.0, and 5.0 mg/L notably (p < 0.05) increased the content of malondialdehyde by 3.30-, 21.24-, and 33.57-fold, respectively, while fluopimomide at 1.0 and 5.0 mg/L significantly (p < 0.001) increased the levels of reactive oxygen species (ROS) by 49.14% and 77.06% compared to the untreated control. In contrast, fluopimomide at 1.0 and 5.0 mg/L notably reduced the activities of target enzyme succinate dehydrogenase and at 5.0 mg/L reduced the activities of antioxidant enzyme superoxide dismutase. Further evidence revealed that fluopimomide at 1.0 and 5.0 mg/L significantly inhibited oxygen consumption and at 0.2, 1.0, and 5.0 mg/L significantly inhibited ATP level in comparison to the untreated control. The expression of genes related to the mitochondrial electron transport chain mev-1 and isp-1 was significantly downregulated. ROS levels in the mev-1 and isp-1 mutants after fluopimomide treatments did not change significantly compared with the untreated mutants, suggesting that mev-1 and isp-1 may play critical roles in the toxicity induced by fluopimomide. Overall, the results demonstrate that oxidative stress and mitochondrial damage may be involved in toxicity of fluopimomide in C. elegans.

12-OR: Urine Lactate Is a Disease Biomarker for Diabetic Kidney Disease

As lactate (LA) elevation is associated with mitochondrial dysfunction, we examined the role of LA in diabetic kidney disease (DKD) progression. Urine LA (ULA) was measured in patients with type 2 diabetes (T2D) in three independent cohort studies (HUNT3, SMART2D, and CRIC). ULA and plasma LA (PLA) were also measured in patients with type 1 diabetes (T1D) following eu- or hyperglycemic clamp, in mouse kidney sections, and sodium-glucose cotransporter-2 (SGLT2)-deficient mice. Mitochondrial functional changes in human kidney proximal tubule (HK2) cells were assayed in response to exogenous LA. Subjects in HUNT3 with DKD (n=39) had elevated ULA levels compared to healthy controls (n=53, p=0.023) (Fig. 1). In subjects with T2D from SMART2D (n=230) and CRIC (n=889), the third tertile of ULA/creatinine ratio was associated with a greater estimated glomerular filtration rate (eGFR) decline [-1.63 mL/min/1.73m2/year, p=0.007, and -0.63 mL/min/1.73m2, p=0.042], relative to the first tertile. Acute hyperglycemia stimulated PLA (p&amp;lt;0.0001) and ULA (p=0.001) in patients with T1D (n=39), and in kidney sections incubated with high glucose. Compared to wild-type, SGLT2-deficient mice had lower ULA levels. LA above 2.5 mM inhibited oxygen consumption rate in HK2 cells. Elevated ULA/creatinine ratio inhibits mitochondrial function in diabetes and identifies subjects at risk of rapid renal decline independent of chronic glycemic control. Disclosure L.Kugathasan: None. L.Natarajan: None. S.Lim: None. S.Waikar: None. Y.M.Osman: None. J.He: None. V.Vallon: Research Support; NIH - National Institutes of Health, Gilead Sciences, Inc., Merck Sharp &amp; Dohme Corp., Novo Nordisk, Maze Therapeutics, Boehringer-Ingelheim, Speaker's Bureau; AstraZeneca. S.Hallan: None. D.Cherney: Other Relationship; Boehringer Ingelheim-Lilly, Merck, AstraZeneca, Sanofi, Mitsubishi-Tanabe, Abbvie, Janssen, Bayer, Prometic, BMS, Maze, Gilead, CSL-Behring, Otsuka, Novartis, Youngene, Lexicon and Novo-Nordisk, Research Support; Boehringer Ingelheim-Lilly, Merck, Janssen, Sanofi, AstraZeneca, CSL-Behring and Novo-Nordisk. K.Sharma: Advisory Panel; Reata Pharmaceuticals, Inc., Otsuka America Pharmaceutical, Inc. Cric study investigators: n/a. M.Darshi: Employee; Janssen Research &amp; Development, LLC. V.Srinivasan sridhar: Other Relationship; Merck &amp; Co., Inc. V.Drel: None. J.Tumova: None. J.Liu: None. J.Wang: None. B.A.Perkins: Advisory Panel; Dexcom, Inc., Insulet Corporation, Novo Nordisk, Sanofi, Vertex Pharmaceuticals Incorporated, Other Relationship; Abbott, Medtronic, Sanofi, Research Support; Novo Nordisk, Bank of Montreal (BMO). Y.Lytvyn: None.

Actin-Cytoskeleton Drives Caveolae Signaling to Mitochondria during Postconditioning.

Caveolae-associated signaling toward mitochondria contributes to the cardioprotective mechanisms against ischemia-reperfusion (I/R) injury induced by ischemic postconditioning. In this work, we evaluated the role that the actin-cytoskeleton network exerts on caveolae-mitochondria communication during postconditioning. Isolated rat hearts subjected to I/R and to postconditioning were treated with latrunculin A, a cytoskeleton disruptor. Cardiac function was compared between these hearts and those exposed only to I/R and to the cardioprotective maneuver. Caveolae and mitochondria structures were determined by electron microscopy and maintenance of the actin-cytoskeleton was evaluated by phalloidin staining. Caveolin-3 and other putative caveolae-conforming proteins were detected by immunoblot analysis. Co-expression of caveolin-3 and actin was evaluated both in lipid raft fractions and in heart tissue from the different groups. Mitochondrial function was assessed by respirometry and correlated with cholesterol levels. Treatment with latrunculin A abolishes the cardioprotective postconditioning effect, inducing morphological and structural changes in cardiac tissue, reducing F-actin staining and diminishing caveolae formation. Latrunculin A administration to post-conditioned hearts decreases the interaction between caveolae-forming proteins, the co-localization of caveolin with actin and inhibits oxygen consumption rates in both subsarcolemmal and interfibrillar mitochondria. We conclude that actin-cytoskeleton drives caveolae signaling to mitochondria during postconditioning, supporting their functional integrity and contributing to cardiac adaption against reperfusion injury.

The Omega-3 Docosahexaenoyl Ethanolamide Reduces CCL5 Secretion in Triple Negative Breast Cancer Cells Affecting Tumor Progression and Macrophage Recruitment.

Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype lacking effective targeted therapies, is considered to feature a unique cellular microenvironment with high infiltration of tumor-associated macrophages (TAM), which contribute to worsening breast cancer patient outcomes. Previous studies have shown the antitumoral actions of the dietary omega-3 docosahexaenoic acid (DHA) in both tumor epithelial and stromal components of the breast cancer microenvironment. Particularly in breast cancer cells, DHA can be converted into its conjugate with ethanolamine, DHEA, leading to a more effective anti-oncogenic activity of the parent compound in estrogen receptor-positive breast cancer cells. Here, we investigated the ability of DHEA to attenuate the malignant phenotype of MDA-MB-231 and MDA-MB-436 TNBC cell lines, which in turn influenced TAM behaviors. Our findings revealed that DHEA reduced the viability of TNBC cells in a concentration-dependent manner and compromised cell migration and invasion. Interestingly, DHEA inhibited oxygen consumption and extracellular acidification rates, reducing respiration and the glycolytic reserve in both cell lines. In a co-culture system, TNBC cells exposed to DHEA suppressed recruitment of human THP-1 cells, reduced their viability, and the expression of genes associated with TAM phenotype. Interestingly, we unraveled that the effects of DHEA in TNCB cells were mediated by reduced C-C motif chemokine ligand 5 (CCL5) expression and secretion affecting macrophage recruitment. Overall, our data, shedding new light on the antitumoral effects of DHA ethanolamine-conjugated, address this compound as a promising option in the treatment of TNBC patients.

Metabolic reprogramming mediated PD-L1 depression and hypoxia reversion to reactivate tumor therapy.

As a promising cancer treatment, photodynamic therapy (PDT) still achieved limited clinical success due to the severe hypoxia and programmed death ligand-1 (PD-L1) over-expressed immunosuppression tumor microenvironment. At present, few methods have been proven to solve these two defects simply and effectively by a single drug or nano-system simultaneously. To ameliorate this situation, we designed and constructed MB@Bu@MnO2 nanoparticles with two-step oxygen regulation ability and PD-1/PD-L1 axis cascade-disruption capacity via a biomineralization method. In such a nanosystem, manganese dioxide albumin (MnO2@Alb) was used as the drug carrier, Butformin (Bu) as mitochondria-associated oxidative phosphorylation (OXPHOS) disruption agent with PD-L1 depression and oxygen reversion ability, and methylene blue (MB) as PDT drug with programmed cell death protein 1 (PD-1) inhibition capacity. Owing to the tumor-responsive capacity of MB@Bu@MnO2 nanoparticles, Bu and MB were selectively delivered and released in tumors. Then, the tumor hypoxia was dramatically reversed by Bu inhibited oxygen consumption, and MnO2 improved oxygen generation. Following this, the reactive oxygen species (ROS) generation was enhanced by MB@Bu@MnO2 nanoparticles mediated PDT owing to the reversed tumor hypoxia. Furthermore, the immunosuppression microenvironment was also obviously reversed by MB@Bu@MnO2 nanoparticles enhanced immunogenic cell death (ICD) and PD-1/PD-L1 axis cascade-disruption, which then enhanced T cell infiltration and improved its tumor cell killing ability. Finally, the growth of solid tumors was significantly depressed by MB@Bu@MnO2 nanoparticles mediated PDT. All in all, this well-designed nanosystem could solve the defects of traditional PDT via PD-1/PD-L1 axis dual disruption and reversing tumor hypoxia by two-step oxygen regulation.

Metabolic Adaptation to Tyrosine Kinase Inhibition in Chronic Myelogenous Leukemia Stem Cells

Objectives: BCR-ABL tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myelogenous leukemia (CML) but fail to eliminate leukemia stem cells, which persist as a source of disease recurrence. Previous studies have suggested that primitive CML cells rely on upregulated oxidative metabolism for survival. However, there is limited knowledge about the effects of TKI treatment on CML stem cell metabolism and the role of metabolic adaptations in stem cell persistence after TKI treatment. Here we evaluated the metabolic response to TKI treatment in CML stem and progenitor cells using a representative SCL-tTA/BCR-ABL mouse model of CML. Methods: CML mice were treated with the TKI Nilotinib or vehicle for 2 days, to evaluate initial effects of treatment, and for 2 weeks, to evaluate response to continued treatment. Bioenergetics, metabolite profiling, and stable isotope metabolic labeling was performed on BM c-Kit+ progenitor cells obtained from vehicle and TKI treated mice. The effects of TKI treatment on metabolism-related gene expression in CML hematopoietic stem cells (HSC) was studied using single cell RNA-Seq (scRNA) analysis of BM Lin- c-Kit+ Sca-1+ (LSK) cells from CML mice treated with vehicle or TKI for 2 days or 2 weeks. Results: Extracellular flux analysis revealed that TKI treatment initially inhibited oxygen consumption rate (OCR), representing mitochondrial respiration, and extracellular acidification rate (ECAR), representing glycolysis, in CML progenitor cells, but that these were restored with continued TKI treatment. Metabolite profiling using high-resolution mass spectrometry (HRMS) identified significant initial reduction in glycolytic, glutaminolytic and tricarboxylic acid cycle (TCA) intermediates after 2 days of TKI treatment. However, metabolite levels were restored to levels similar to controls with continued TKI treatment. Steady-state metabolic labeling with [U-13C6]-glucose or [U-13C5]-Glutamine showed reduced glycolytic flux, glutaminolysis and TCA intermediates after treatment with TKI for 2 days, but restoration of glycolytic activity and increased glucose-mediated lactate generation, and increased glutaminolytic and TCA cycle activity with continued treatment. The above studies indicate initial inhibition of energy metabolism in CML progenitors with TKI treatment, but metabolic adaptation to continued TKI exposure. scRNA analysis of CML HSC populations showed that TKI exposure led to selective enrichment of primitive HSC subsets characterized by reduced OXPHOS, glycolysis, nucleotide metabolism, and MYC gene signatures. Treatment with TKI for 2 days further reduced metabolic gene signatures, but continued treatment led to restoration of MYC and OXPHOS gene signature expression coupled with enrichment of HSC and quiescence signatures in persistent HSC. TKI-treated stem cells showed increased levels of Hif1a protein and enhanced activity of HIF-1, a key transcriptional regulator of energy metabolism. Treatment of CML mice with a HIF-1 inhibitor, Echinomycin, in combination with TKI treatment, resulted in significant depletion of CML stem cells compared to TKI alone. The combination of HIF-1 inhibitor with TKI treatment also depleted human CML stem cells in xenografted mice. Conclusions: Our results demonstrate metabolic adaptation in CML and progenitor stem cells following TKI treatment, and identify HIF-1 activation as a targetable mechanism to enhance elimination of CML stem cell that persist after TKI treatment.

Discovery of 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as new cytochrome bd oxidase inhibitors for tuberculosis therapy

The cytochrome bcc-aa3 oxidase (Cyt-bcc) of Mycobacterium tuberculosis (Mtb) is a promising anti-tuberculosis target. However, when Cyt-bcc is inhibited, cytochrome bd terminal oxidase (Cyt-bd) can still maintain the activity of the respiratory chain and drive ATP synthesis. Through virtual screening and biological validation, we discovered two FDA-approved drugs, ivacaftor and roquinimex, exhibited moderate binding affinity to Cyt-bd. Structural modifications of them led to 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as potent new Cyt-bd inhibitors. Compound 8d binds to Cyt-bd with a Kd value of 4.17 μM and inhibits the growth of the Cyt-bcc knock-out strain (ΔqcrCAB, Cyt-bd+) with a MIC value of 6.25 μM. The combination of 8d with the Cyt-bcc inhibitor Q203 completely inhibited oxygen consumption of the wild-type strain and the inverted-membrane vesicles expressing M. tuberculosis Cyt-bd (ΔcydAB::MtbCydAB+). Our study provides a promising starting point for the development of novel dual chemotherapies for tuberculosis.

THE CLUE IS IN THE BLOOD GAS: A RARE MANIFESTATION OF LACTIC ACIDOSIS

THE CLUE IS IN THE BLOOD GAS: A RARE MANIFESTATION OF LACTIC ACIDOSIS

Design, synthesis and biological evaluation of (Quinazoline 4-yloxy)acetamide and (4-oxoquinazoline-3(4H)-yl)acetamide derivatives as inhibitors of Mycobacterium tuberculosis bd oxidase

New chemical scaffolds with novel mechanism of action are urgently needed for the treatment of drug resistant tuberculosis. The oxidative phosphorylation pathway of Mycobacterium tuberculosis consists of multiple clinically validated drug targets. This pathway can function through any one of the two terminal oxidases-the proton pumping cytochrome bc1-aa3 supercomplex, or the less energy efficient but high affinity cytochrome bd oxidase. Inhibiting the bc1 complex alone has been found bacteriostatic and not bactericidal. On the other hand, inhibition of both these oxidases turns lethal to the pathogen. In the present study, we used a bc1 complex mutant of M. tuberculosis to screen (Quinazoline 4-yloxy)acetamide and (4-oxoquinazoline-3(4H)-yl)acetamide derivatives against the alternate oxidase, i.e., cytochrome bd oxidase. Two molecules, S-021-0601 and S-021-0607 were found to inhibit the mutant with MICs 8 and 16 μM respectively, compared to MICs of 128 and 256 μM against the wild type M. tuberculosis. In the wild type, one of the compounds showed synergism with Q203, an inhibitor of bc1 complex, in inhibiting growth under aerobic conditions. Both compounds showed synergism with Q203 in depleting bacterial ATP and inhibiting oxygen consumption. Both the compounds at 32 μM (one-fourth or one-eighth of their MICs for wild type) were bactericidal to wild type bacteria under hypoxic condition, causing ∼1.9 log10 reduction in viable counts which increased to ∼4-log10 when combined with Q203.

Tumoral oxygenation and biodistribution of Lonidamine oxygen microbubbles following localized ultrasound-triggered delivery

Prior work has shown that microbubble-assisted delivery of oxygen improves tumor oxygenation and radiosensitivity, albeit over a limited duration. Lonidamine (LND) has been investigated because of its ability to stimulate glycolysis, lactate production, inhibit mitochondrial respiration, and inhibit oxygen consumption rates in tumors but suffers from poor bioavailability. The goal of this work was to characterize LND-loaded oxygen microbubbles and assess their ability to oxygenate a human head and neck squamous cell carcinoma (HNSCC) tumor model, while also assessing LND biodistribution. In tumors treated with surfactant-shelled microbubbles with oxygen core (SE61O2) and ultrasound, pO2 levels increased to a peak 19.5 ± 9.7 mmHg, 50 s after injection and returning to baseline after 120 s. In comparison, in tumors treated with SE61O2/LND and ultrasound, pO2 levels showed a peak increase of 29.0 ± 8.3 mmHg, which was achieved 70 s after injection returning to baseline after 300 s (p < 0.001). The co-delivery of O2andLNDvia SE61 also showed an improvement of LND biodistribution in both plasma and tumor tissues (p < 0.001). In summary, ultrasound-sensitive microbubbles loaded with O2 and LND provided prolonged oxygenation relative to oxygenated microbubbles alone, as well as provided an ability to locally deliver LND, making them more appropriate for clinical translation.

Inhibition of respiratory complex I by 6-ketocholestanol: Relevance to recoupling action in mitochondria

6-Ketocholestanol (kCh) is known as a mitochondrial recoupler, i.e. it abolishes uncoupling of mitochondria by such potent agents as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) [Starkov et al., 1997]. Here, we report data on the kCh-induced inhibition of both NADH-oxidase and NADH-ubiquinone oxidoreductase activities of the respiratory complex I in bovine heart submitochondrial particles (SMP). Based on the absence of such inhibition with hexaammineruthenium (III) (HAR) as the complex I electron acceptor, the kCh effect could be associated with the ubiquinone-binding centre of this respiratory enzyme. In isolated rat liver mitochondria (RLM), kCh inhibited oxygen consumption with the glutamate/malate, substrates of NAD-linked dehydrogenases, while no inhibition of RLM respiration was observed with succinate, in agreement with the absence of the kCh effect on the succinate oxidase activity in SMP. Three kCh analogs (cholesterol, 6α-hydroxycholesterol, and 5α,6α-epoxycholesterol) exhibited no effect on the NADH oxidase activities in both SMP and RLM. Importantly, the kCh analogs were ineffective in the recoupling of RLM treated with CCCP or SF6847. Therefore, interaction of kCh with the complex I may be involved in the kCh-mediated mitochondrial recoupling.

Temperature increases induce metabolic adjustments in the early developmental stages of bigfin reef squid (Sepioteuthis lessoniana)

Climate changes, such as extreme temperature shifts, can have a direct and significant impact on animals living in the ocean system. Ectothermic animals may undergo concerted metabolic shifts in response to ambient temperature changes. The physiological and molecular adaptations in cephalopods during their early life stages are largely unknown due to the challenge of rearing them outside of a natural marine environment. To overcome this obstacle, we established a pelagic bigfin reef squid (Sepioteuthis lessoniana) culture facility, which allowed us to monitor the effects of ambient thermal elevation and fluctuation on cephalopod embryos/larvae. By carefully observing embryonic development in the breeding facility, we defined 23 stages of bigfin reef squid embryonic development, beginning at stage 12 (blastocyst; 72 h post-egg laying) and continuing through hatching (~1 month post-egg laying). Since temperature recordings from the bigfin reef squid natural habitats have shown a steady rise over the past decade, we examined energy substrate utilization and cellular/metabolic responses in developing animals under different temperature conditions. As the ambient temperature increased by 7 °C, hatching larvae favored aerobic metabolism by about 2.3-fold. Short-term environmental warming stress inhibited oxygen consumption but did not affect ammonium excretion in stage (St.) 25 larvae. Meanwhile, an aerobic metabolism-related marker (CoxI) and a cellular stress-responsive marker (HSP70) were rapidly up-regulated upon acute warming treatments. In addition, our simulations of temperature oscillations mimicking natural daily rhythms did not result in significant changes in metabolic processes in St. 25 animals. As the ambient temperature increased by 7 °C, referred to as heatwave conditions, CoxI, HSP70, and antioxidant molecule (SOD) were stimulated, indicating the importance of cellular and metabolic adjustments. As with other aquatic species with high metabolic rates, squid larvae in the tropical/sub-tropical climate zone undergo adaptive metabolic shifts to maintain physiological functions and prevent excessive oxidative stress under environmental warming.

Abstract 2386: Investigating cyclopamine tartrate as a potential suppressor of lung adenocarcinoma and squamous cell carcinoma

Abstract Lung cancer is the leading cause of death by cancer, and non-small cell lung cancer comprises of 85-90% of lung cancer cases. Previous studies have demonstrated the importance of heme in mitochondrial metabolism and OXPHOS. Heme is an essential molecule in the oxidative phosphorylation pathway especially since Complex II-IV all contain heme. During lung tumorigenesis there is a marked increase in heme synthesis and uptake; other protein and enzymes relating to heme were also upregulated. Heme-sequestration proteins (HeSPs) have shown high effectiveness in suppressing the development and progression of lung adenocarcinoma and squamous cell carcinoma, therefore targeting heme using CycT should yield similar results. Previous studies have indicated that cyclopamine tartrate (CycT) can effectively arrest lung tumor growth and progression. CycT does this by inhibiting the levels of proteins and enzymes involved in heme uptake, synthesis, as well as reducing heme synthesis and degradation in adenocarcinoma. We therefore aim to examine the effectiveness of CycT in both, adenocarcinoma and squamous cell carcinoma.In vitro experiments using NSCLC adenocarcinoma cell line A549 and squamous cell carcinoma HCC15 are in progression with initial results supporting the hypothesis. Cell proliferation assays have shown a marked decrease in CycT-treated groups in both cell lines. Based on colony formation assays, results indicate that CycT inhibits colony formation. Heme synthesis and degradation have also shown the effectiveness of CycT in suppressing tumorigenesis in both cell types. Oxygen consumption rates of both cell types were examined with CycT inhibiting oxygen consumption. Overall, in vitro experiments support the idea that CycT can suppress or arrest lung adenocarcinoma and squamous cell carcinoma. Citation Format: Eranda Berisha, Adnin Ashrafi, Maria Del Carmen Chacon Castro, Li Zhang. Investigating cyclopamine tartrate as a potential suppressor of lung adenocarcinoma and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2386.