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BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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Ten-eleven translocation 1 (TET1) is a methylcytosine dioxygenase involved in active DNA demethylation. In our previous study, we demonstrated that TET1 reprogrammed the ovarian cancer epigenome, increased stem properties, and activated various regulatory networks, including metabolic networks. However, the role of TET1 in cancer metabolism remains poorly understood. Herein, we uncovered a demethylated metabolic gene network, especially oxidative phosphorylation (OXPHOS). Contrary to the concept of the Warburg effect in cancer cells, TET1 increased energy production mainly using OXPHOS rather than using glycolysis. Notably, TET1 increased the mitochondrial mass and DNA copy number. TET1 also activated mitochondrial biogenesis genes and adenosine triphosphate production. However, the reactive oxygen species levels were surprisingly decreased. In addition, TET1 increased the basal and maximal respiratory capacities. In an analysis of tricarboxylic acid cycle metabolites, TET1 increased the levels of α-ketoglutarate, which is a coenzyme of TET1 dioxygenase and may provide a positive feedback loop to modify the epigenomic landscape. TET1 also increased the mitochondrial complex I activity. Moreover, the mitochondrial complex I inhibitor, which had synergistic effects with the casein kinase 2 inhibitor, affected ovarian cancer growth. Altogether, TET1-reprogrammed ovarian cancer stem cells shifted the energy source to OXPHOS, which suggested that metabolic intervention might be a novel strategy for ovarian cancer treatment.

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Rotenone is a naturally occurring plant product used as an insecticide, pesticide and piscicide. It is lipophilic in nature and can cross the blood-brain barrier and induce the degeneration of neurons. It inhibits the mitochondrial respiratory chain complex I and stops the transfer of electrons. It induces ROS generation, which impairs mitochondrial activity. Rotenone is a toxic agent which causes the death of neurons. The present review describes the effect of rotenone on neurodegeneration with an emphasis on behavioral, pathological and neuropathological components carried out on various experimental models such as cell lines, Drosophila melanogaster, mice and rats.

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Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

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Mitochondrial complex I inhibitor (iC1) is a methylation-controlled J protein (MCJ) that decreases cellular respiration by inhibiting oxidative phosphorylation. Recent rodent studies showed that loss or inhibition of iC1 was associated with preventing lipid accumulation. A common metabolic disorder of dairy cattle is a fatty liver disease (FLD), which often occurs during the periparturient period. In humans and rodents, iC1 is expressed in the liver and acts as a mitochondrial "brake". However, iC1 expression in bovine liver and its possible role in FLD development have not yet been characterized. We hypothesized that iC1 is expressed in the bovine liver and that the expression of iC1 is correlated with FLD in periparturient dairy cattle. To test this hypothesis, we collected bovine liver tissue samples from an abattoir and isolated primary hepatic cells immediately following harvest. Utilizing an in vitro model of bovine FLD developed in our laboratory, we cultured primary hepatic cells in low-glucose DMEM supplemented with 10% FBS. The basal media was made to induce lipid accumulation and cytotoxicity in the primary liver cells with three treatments. To the basal media (control) we added 0.4 mM palmitate (treatment 1) or 20 ng/mL TNFα (treatment 2), or both 0.4 mM palmitate and 20 ng/mL TNFα (treatment 3). Consistent with our hypothesis, we present the novel characterization of iC1 expression in primary bovine liver cells cultured with or without the addition of lipotoxic factors made to emulate bovine FLD. We demonstrate both in situ and in vitro expression of iC1 in bovine liver and mRNA expression in hepatic cells and in the precipitates of conditioned media. The results of RT-qPCR, IHC, and western blot all demonstrated the expression of iC1 in bovine liver. In addition, we isolated precipitates of conditioned media further demonstrated iC1 expression by RT-qPCR. The transcript of iC1 tended to be more concentrated (4-fold; p > 0.05) in TNFα-treated conditioned media when compared with the control. Taken together, we present the novel finding that iC1 transcript and protein are expressed in liver tissue from dairy cattle, primary hepatic cells isolated from that liver tissue, and, finally, in the conditioned media derived from those cells. These novel findings and the prior findings on the role of iC1 in rodents and humans indicate that further investigation of the role of iC1 in the etiology and pathology of FLD in periparturient dairy cows is warranted.

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The biological effects of the pesticide and mitochondrial complex I inhibitor tebufenpyrad (TEBU) on liver cells were investigated by combining proteomics and metabolomics. Both cell culture media and cellular lysates were analyzed in dose-response and kinetic experiments on the HepaRG cell line. Responses were compared with those obtained on primary human and rat hepatocytes. A multitude of phase I and II metabolites (>80) mainly common to HepaRG cells and primary hepatocytes and an increase in metabolization enzymes were observed. Synthesis of mitochondrion and oxidative phosphorylation complex constituents, fatty acid oxidation, and cellular uptake of lipids were induced to compensate for complex I inhibition and the decrease in ATP intracellular contents caused by TEBU. Secretion of the 20 S circulating proteasome and overall inhibition of acute inflammation followed by IL-6 secretion in later stages were observed in HepaRG cells. These effects were associated with a decrease in STAT1 and STAT3 transcription factor abundances, but with different kinetics. Based on identified TEBU targets, docking experiments, and nuclear receptor reporter assays, we concluded that liver cell response to TEBU is mediated by its interaction with the PPARγ transcription factor.

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Preclinical models suggest anticancer activity of IM156, a novel biguanide mitochondrial protein complex 1 inhibitor of oxidative phosphorylation (OXPHOS). This first-in-human dose-escalation study enrolled patients with refractory advanced solid tumors to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). Eligible patients received oral IM156 every other day (QOD) or daily (QD) and were assessed for safety, dose-limiting toxicities (DLTs), pharmacokinetics, and preliminary signals of efficacy. 22 patients with advanced cancers (gastric, n = 8; colorectal, n = 3; ovarian, n = 3; other, n = 8) received IM156 100 to 1,200 mg either QOD or QD. There were no DLTs. However, 1,200 mg QD was not well tolerated due to nausea; 800 mg QD was determined as the RP2D. The most frequent treatment-related AEs (TRAEs) were nausea (n = 15; 68%), diarrhea (n = 10; 46%), emesis (n = 9; 41%), fatigue (n = 4; 18%) and abdominal pain, constipation, and blood lactate increased (n = 2 each; 9%). Grade 3 nausea (n = 3; 14%) was the only grade ≥ 3 TRAE. Plasma exposures increased dose proportionally; mean Day 27 area under the curve (AUC0-24) values were higher following QD administration compared to the respective QOD regimen. Stable disease (SD), observed in 7 (32%) patients (confirmed in 2 [9%]), was the best response. To our knowledge, this is the first phase 1 study of an OXPHOS inhibitor that established a RP2D for further clinical development in cancer. Observed AEs of IM156 were manageable and SD was the best response.

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Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a high mortality rate and relapse risk. Although progress on the genetic and molecular understanding of this disease has been made, the standard of care has changed minimally for the past 40 years and the five-year survival rate remains poor, warranting new treatment strategies. Here, we applied a two-step screening platform consisting of a primary cell viability screening and a secondary metabolomics-based phenotypic screening to find synergistic drug combinations to treat AML. A novel synergy between the oxidative phosphorylation inhibitor IACS-010759 and the FMS-like tyrosine kinase 3 (FLT3) inhibitor AC220 (quizartinib) was discovered in AML and then validated by ATP bioluminescence and apoptosis assays. In-depth stable isotope tracer metabolic flux analysis revealed that IACS-010759 and AC220 synergistically reduced glucose and glutamine enrichment in glycolysis and the TCA cycle, leading to impaired energy production and de novo nucleotide biosynthesis. In summary, we identified a novel drug combination, AC220 and IACS-010759, which synergistically inhibits cell growth in AML cells due to a major disruption of cell metabolism, regardless of FLT3 mutation status.

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Background We assessed the safety, tolerability, and pharmacokinetics of mitochondrial complex 1 inhibitor ASP4132. Methods This phase I dose-escalation/dose-expansion study enrolled patients with treatment refractory advanced solid tumors to assess safety, dose-limiting toxicities (DLTs), efficacy and pharmacokinetic or oral ASP4132. Results Overall, 39 patients received ASP4132. Acceptable tolerability of ASP4132 5 mg in the first patient led to enrollment in the 10-mg dose cohort. After two DLTs at the 10-mg dose, additional patients were enrolled in the 5-mg cohort; a 7.5-mg cohort and two intermittent-dosing cohorts (ASP4132 10 mg for 3 days, then 4 days off; ASP4132 15 mg for 1 day, then 6 days off). ASP4132 5 mg was well tolerated; however, multiple DLTs such as fatigue, mental status changes, dizziness, lactic acidosis, enteritis, and posterior reversible encephalopathy syndrome were observed in higher dose cohorts (7.5-mg and intermittent 10-mg and 15-mg dose cohorts). Stable disease (+ 4 % to + 15 %) was observed in 8/39 (20.5 %) patients. ASP4132 plasma pharmacokinetics were characterized by high variability, with rapid absorption and accumulation from slow elimination. Conclusions ASP4132 showed limited clinical activity, and DLTs prohibited dose escalation. Further research is required to determine if DLTs will limit clinical activity of other mitochondrial complex I inhibitors. Clinical Trial ID (clinicaltrials.gov): NCT02383368, March 9, 2015.

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Novel therapeutic strategies for Alzheimer's disease (AD) are of the greatest priority given the consistent failure of recent clinical trials focused on Aß or pTau. Earlier, we demonstrated that mild mitochondrial complex I inhibitor CP2 blocks neurodegeneration and cognitive decline in multiple mouse models of AD. To evaluate the safety of CP2 in humans, we performed a genome-wide association study (GWAS) using 196 lymphoblastoid cell lines and identified 11 SNP loci and 64 mRNA expression probe sets that potentially associate with CP2 susceptibility. Using primary mouse neurons and pharmacokinetic study, we show that CP2 is generally safe at a therapeutic dose.

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The successful treatment of Helicobacter pylori infections is becoming increasingly difficult due to the rise of resistance against current broad spectrum triple therapy regimens. In the search for narrow-spectrum agents against H. pylori, a high-throughput screen identified two structurally related thienopyrimidine compounds that selectively inhibited H. pylori over commensal members of the gut microbiota. To develop the structure-activity relationship (SAR) of the thienopyrimidines against H. pylori, this study employed four series of modifications in which systematic substitution to the thienopyrimidine core was explored and ultimately side-chain elements optimized from the two original hits were merged into lead compounds. During the development of this series, the mode of action studies identified H. pylori's respiratory complex I subunit NuoD as the target for lead thienopyrimidines. As this enzyme complex is uniquely essential for ATP synthesis in H. pylori, a homology model of the H. pylori NuoB-NuoD binding interface was generated to help rationalize the SAR and guide further development of the series. From these studies, lead compounds emerged with increased potency against H. pylori, improved safety indices, and a good overall pharmacokinetic profile with the exception of high protein binding and poor solubility. Although lead compounds in the series demonstrated efficacy in an ex vivo infection model, the compounds had no efficacy in a mouse model of H. pylori infection. Additional optimization of pharmacological properties of the series to increase solubility and free-drug levels at the sequestered sites of H. pylori infection would potentially result in a gain of in vivo efficacy. The thienopyrimidine series developed in this study demonstrates that NuoB-NuoD of the respiratory complex I can be targeted for development of novel narrow spectrum agents against H. pylori and that thienopyrimines can serve as the basis for future advancement of these studies.

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NADH provides electrons for aerobic ATP production. In cells deprived of oxygen or with impaired electron transport chain activity, NADH accumulation can be toxic. To minimize such toxicity, elevated NADH inhibits the classical NADH-producing pathways: glucose, glutamine, and fat oxidation. Here, through deuterium-tracing studies in cultured cells and mice, we show that folate-dependent serine catabolism also produces substantial NADH. Strikingly, when respiration is impaired, serine catabolism through methylene tetrahydrofolate dehydrogenase (MTHFD2) becomes a major NADH source. In cells whose respiration is slowed by hypoxia, metformin, or genetic lesions, mitochondrial serine catabolism inhibition partially normalizes NADH levels and facilitates cell growth. In mice with engineered mitochondrial complex I deficiency (NDUSF4-/-), serine's contribution to NADH is elevated, and progression of spasticity is modestly slowed by pharmacological blockade of serine degradation. Thus, when respiration is impaired, serine catabolism contributes to toxic NADH accumulation.

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Epidemiological studies have observed an association between pesticide exposure and the development of Parkinson's disease, but have not established causality. The concept of an adverse outcome pathway (AOP) has been developed as a framework for the organization of available information linking the modulation of a molecular target [molecular initiating event (MIE)], via a sequence of essential biological key events (KEs), with an adverse outcome (AO). Here, we present an AOP covering the toxicological pathways that link the binding of an inhibitor to mitochondrial complex I (i.e., the MIE) with the onset of parkinsonian motor deficits (i.e., the AO). This AOP was developed according to the Organisation for Economic Co-operation and Development guidelines and uploaded to the AOP database. The KEs linking complex I inhibition to parkinsonian motor deficits are mitochondrial dysfunction, impaired proteostasis, neuroinflammation, and the degeneration of dopaminergic neurons of the substantia nigra. These KEs, by convention, were linearly organized. However, there was also evidence of additional feed-forward connections and shortcuts between the KEs, possibly depending on the intensity of the insult and the model system applied. The present AOP demonstrates mechanistic plausibility for epidemiological observations on a relationship between pesticide exposure and an elevated risk for Parkinson's disease development.

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INTRODUCTION: Rotenone, a mitochondrial complex I inhibitor is used as a neurotoxin agent to reproduce the neuropathological, and behavioural feature of Parkinson's Disease (PD) in rat. Due to acute and chronic exposure of rotenone with various doses through different routes of administration, mortality is being reported. Low dose takes a longer duration to produce PD symptoms in animals. This present study was designed to create hemiparkinsonian 'partial' lesion model by rotenone at a single moderate dose in two sites of striatum in albino rats and also to assess its toxicity by behavioural parameters and by microscopic study. AIM: To assess all the motor deficits in lesioned animals that are due to the depletion of dopaminergic neurons or its terminals, the lesioned animals were administered with anti-parkinsonian drug, Levodopa which should counteract motor deficits in rats. MATERIALS AND METHODS: The unilateral partially lesioned PD model was induced by rotenone stereotaxically into two sites of striatum of male Wistar albino rats at a dosage of 25 µg of rotenone/site. Rats were tested for its neurobehavioural activity on 7th day, 14th day, 21st day and on 30th day after rotenone infusion and compared with the sham group and sacrificed on 21st and 30th day for microscopic studies. L-DOPA was administered from 21st day to 30th day after lesion and compared with the lesioned group for the motor performance and sacrificed on 30th day for histology. Statistical analysis using One-way Analysis of variance followed by Tukey's test was applied for behavioural studies. RESULTS: Statistical analysis showed that the signs and symptoms like motor in-coordination and postural disturbances are highly significant (p<0.05) on 14th and 21st day after administration of rotenone when compared to sham group. In L-DOPA treated rats, all the motor deficits were reversed. The neuronal cell death was minimal and sprouting of nerve terminals was detected. In lesioned group, the degeneration of nerve terminals and striatal neurons were in progressive manner. CONCLUSION: These findings suggest that intrastriatal infusion of rotenone at a moderate dose could be used for producing hemiparkinsonian partially lesioned animal model without any mortality. Hence, this model is suitable for evaluating behavioural studies and in drug screening programs even for a long term study.

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Deguelin is one of four major naturally occurring rotenoids isolated from root extracts and is best recognized as a NADH: ubiquinone oxidoreductase (complex I) inhibitor, resulting in significant alterations in mitochondrial function. Deguelin has also been implicated as a regulator of apoptosis through signaling pathways, such as the (PI3K)/Akt pathway, as well as an initiator of cell cycle arrest. Consequently, this compound has accrued great interest as a potential chemopreventive and chemotherapeutic. Additionally, deguelin exposure has been linked to Parkinson's disease (PD). PD is a neurodegenerative disorder, characterized by a substantial loss of dopaminergic neurons in the substantia nigra, as well the manifestation of symptoms such as bradykinesia, rigidity, and rest tremor. While exploring the genetic impact of PD is imperative, environmental factors, such as exposure to pesticides, herbicides, and insecticides, have also been connected to the development of PD. The etiology and pathogenesis of PD are yet to be fully understood and elucidated, but mitochondrial dysfunction is gaining recognition as a molecular hallmark of PD. In fact, deguelin has been reported to elicit PD-like symptoms (degeneration of the dopaminergic pathway) in rats administered with deguelin (6 mg/kg/day for 6 days), possibly through the inhibition of mitochondrial complex I. Further research investigating the mechanisms by which deguelin inhibits central cellular processes is essential in order to advance any prospective research addressing potential applications and risks of deguelin.

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Parkinson's disease (PD) is the second most common neurodegenerative disease, which is characterized by loss of dopaminergic (DA) neurons in the substantia nigra pars compacta and the formation of Lewy bodies and Lewy neurites in surviving DA neurons in most cases. Although the cause of PD is still unclear, the remarkable advances have been made in understanding the possible causative mechanisms of PD pathogenesis. Numerous studies showed that dysfunction of mitochondria may play key roles in DA neuronal loss. Both genetic and environmental factors that are associated with PD contribute to mitochondrial dysfunction and PD pathogenesis. The induction of PD by neurotoxins that inhibit mitochondrial complex I provides direct evidence linking mitochondrial dysfunction to PD. Decrease of mitochondrial complex I activity is present in PD brain and in neurotoxin- or genetic factor-induced PD cellular and animal models. Moreover, PINK1 and parkin, two autosomal recessive PD gene products, have important roles in mitophagy, a cellular process to clear damaged mitochondria. PINK1 activates parkin to ubiquitinate outer mitochondrial membrane proteins to induce a selective degradation of damaged mitochondria by autophagy. In this review, we summarize the factors associated with PD and recent advances in understanding mitochondrial dysfunction in PD.

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Nafuredin-γ (2), converted from nafuredin (1) under mild basic conditions, demonstrates potent and selective inhibitory activity against helminth complex I. However, 2 is unstable in air because the conjugated dienes are oxygen-labile. To address this, we designed and synthesized air-stable nafuredin-γ analogs. Although the complex I inhibitory activities of all the new nafuredin-γ analogs were lower than that of 2, all were in the high nM range (IC50: 300-820nM).

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