Inhibitor Of Glutamine Synthetase Research Articles

Ammonium chloride reduces excitatory synaptic transmission onto CA1 pyramidal neurons of mouse organotypic slice cultures.

Acute liver dysfunction commonly leads to rapid increases in ammonia concentrations in both the serum and the cerebrospinal fluid. These elevations primarily affect brain astrocytes, causing modifications in their structure and function. However, its impact on neurons is not yet fully understood. In this study, we investigated the impact of elevated ammonium chloride levels (NH4Cl, 5 mM) on synaptic transmission onto CA1 pyramidal neurons in mouse organotypic entorhino-hippocampal tissue cultures. We found that acute exposure to NH4Cl reversibly reduced excitatory synaptic transmission and affected CA3-CA1 synapses. Notably, NH4Cl modified astrocytic, but not CA1 pyramidal neuron, passive intrinsic properties. To further explore the role of astrocytes in NH4Cl-induced attenuation of synaptic transmission, we used methionine sulfoximine to target glutamine synthetase, a key astrocytic enzyme for ammonia clearance in the central nervous system. Inhibition of glutamine synthetase effectively prevented the downregulation of excitatory synaptic activity, underscoring the significant role of astrocytes in adjusting excitatory synapses during acute ammonia elevation.

Abstract C042: Identification of a non-canonical pathway used by CAF to deliver gamma-aminobutyric acid in pancreatic cancer

Abstract Cancer-associated fibroblasts (CAFs) are cells responsible for deposition and remodeling of the extracellular matrix, and modification of the stromal microenvironment, which facilitates tumor growth. Moreover, CAFs are extremely immunosuppressive because they produce large amounts of inhibitory cytokines, growth factors and metabolites. Thus, understanding how CAFs impart severe immunosuppression in the tumor microenvironment is critical to make PDAC amenable to immunotherapies. The link between metabolism and immunosuppression in tumors has become clearer in recent years, and we postulate that one such metabolite, gamma-aminobutyric acid (GABA), a key inhibitory neurotransmitter, can act as a major immunosuppressive metabolite in PDAC. Our preliminary data shows that CAFs produce high levels of glutamate (Glu) and that metabolites derived from glutamate, such as GABA, are immunosuppressive to NK cells. Moreover, we found that CAFs could produce GABA, de novo, as determined by heavy isotope tracing. Thus, our goal is to uncover the mechanisms used by CAFs to produce GABA, and to evaluate the immunosuppressive mechanisms behind CAF- derived GABA. GABA production was measured in the conditioned media of CAFs grown in 3D culture, where it was observed that inhibition of glutaminase (GLS) and glutamine synthetase (GS) enzymes was not able to decrease the GABA levels. In addition, CAFs did not express glutamate decarboxylase (GAD), the canonical enzyme responsible for the conversion of Glu to GABA, which led us to explore the expression of a non-canonical GABA synthesis pathways in CAFs. Intriguingly, we found that CAFs derived from patient tumors expressed the enzymes of non-canonical pathway (ODC1, DAO, ALDH1A1 and ALDH9A1), and to a greater degree when compared to normal fibroblasts isolated from donor pancreata (Gift of Life Program). CRISPRi mediated knockdown (KD) of aldehyde dehydrogenase 1 (ALDH1a1) in CAFs decreased GABA production compared to control CAFs. On the other hand, CAFs knocked down for diamine oxidase (DAO), showed increased GABA production, suggesting a compensatory mechanism for GABA production upstream of ALDH1a1. Overall, our preliminary data demonstrate that CAFs produce GABA de novo, via a putative alternative GABA synthesis pathway. Our next steps are to identify the cytokine profile of KDs CAFs and perform functional assays utilizing our 3D co-culturing system, uncovering natural killer cell responses to tumor cells in the presence of CAFs with and without the enzymes necessary for GABA production. Translationally, we are analyzing the tumor interstitial fluid from PDAC patients to uncover metabolites/cytokines (secreted factors) that can be correlated with patient parameters (ex. overall survival) and immune cell activation and functionality. It is our hope that the insight provided by this study can lead to a novel set of prognostic or diagnostic factors to improve clinical outcomes in this devasting disease. Citation Format: Ariana Musa de Aquino, Myree Graves, Farrah Ali Ali, David A. Rangel, Langley Kyra, Julie Clark, David Kwon, Guillaume Cognet, Alex Muir, Débora Barbosa Vendramini Costa, Ralph Francescone. Identification of a non-canonical pathway used by CAF to deliver gamma-aminobutyric acid in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C042.

Effects of Glutamine Synthetase on Neovascularization in Glioma: In Vivo MR Vessel Size Imaging and Histology.

Glutamine Synthetase (GS) could induce vascular sprouting through the improvement of endothelial cell migration in inflammatory diseases. MR vessel-size imaging has been proposed as a valuable approach for visualizing the underlying angiogenic processes in the brain. This study aims to investigate the role of GS in the neovascularization of gliomas through the utilization of MR vessel-size imaging and histopathological techniques. In this exploratory animal study, we randomly divided the C6 glioma rat models into a control group and an L-methionine sulfoximine (MSO) treatment group. Daily intraperitoneal injections were administered for three consecutive days, starting from day 10 following the implantation of C6 glioma cells in rats. Subsequently, MR vessel size imaging was conducted using a BRUKER 7 T/200 mm MRI scanner, and the MRI results were validated through histopathological examination. A significant decrease in microvessel density was observed in both the tumor periphery and center areas in the MSO treatment group compared to that in the control group. The mean vessel diameter (mVD) and vessel size index (VSI) did not exhibit significant changes compared to the control group. Moreover, the staining intensity of platelet endothelial cell adhesion molecule-1 (CD31) and GS in the tumor periphery was significantly decreased in the MSO treatment group. Additionally, the MSO treatment demonstrated a substantial inhibition of tumor growth. GS inhibitors significantly reduced angiogenesis in the periphery area of C6 glioma, exerting an inhibitory effect on tumor progression. Thus, GS inhibitors could be potential therapeutic agents for treating glioma. Additionally, in vivo MR vessel size imaging detects changes in vascularrelated parameters after tumor treatment, making it a promising method for detecting neovascularization in glioma.</p>.

Ammonia scavenger and glutamine synthetase inhibitors cocktail in targeting mTOR/β-catenin and MMP-14 for nitrogen homeostasis and liver cancer.

The glutamine synthetase (GS) facilitates cancer cell growth by catalyzing de novo glutamine synthesis. This enzyme removes ammonia waste from the liver following the urea cycle. Since cancer development is associated with dysregulated urea cycles, there has been no investigation of GS's role in ammonia clearance. Here, we demonstrate that, although GS expression is increased in the setting of β-catenin oncogenic activation, it is insufficient to clear the ammonia waste burden due to the dysregulated urea cycle and may thus be unable to prevent cancer formation. In vivo study, a total of 165 male Swiss albino mice allocated in 11 groups were used, and liver cancer was induced by p-DAB. The activity of GS was evaluated along with the relative expression of mTOR, β-catenin, MMP-14, and GS genes in liver samples and HepG2 cells using qRT-PCR. Moreover, the cytotoxicity of the NH3 scavenger phenyl acetate (PA) and/or GS-inhibitor L-methionine sulfoximine (MSO) and the migratory potential of cells was assessed by MTT and wound healing assays, respectively. The Swiss target prediction algorithm was used to screen the mentioned compounds for probable targets. The treatment of the HepG2 cell line with PA plus MSO demonstrated strong cytotoxicity. The post-scratch remaining wound area (%) in the untreated HepG2 cells was 2.0%. In contrast, the remaining wound area (%) in the cells treated with PA, MSO, and PA + MSO for 48 h was 61.1, 55.8, and 78.5%, respectively. The combination of the two drugs had the greatest effect, resulting in the greatest decrease in the GS activity, β-catenin, and mTOR expression. MSO and PA are both capable of suppressing mTOR, a key player in the development of HCC, and MMP-14, a key player in the development of HCC. PA inhibited the MMP-14 enzyme more effectively than MSO, implying that PA might be a better way to target HCC as it inhibited MMP-14 more effectively than MSO. A large number of abnormal hepatocytes (5%) were found to be present in the HCC mice compared to mice in the control group as determined by the histopathological lesions scores. In contrast, PA, MSO, and PA + MSO showed a significant reduction in the hepatic lesions score either when protecting the liver or when treating the liver. The molecular docking study indicated that PA and MSO form a three-dimensional structure with NF-κB and COX-II, blocking their ability to promote cancer and cause gene mutations. PA and MSO could be used to manipulate GS activities to modulate ammonia levels, thus providing a potential treatment for ammonia homeostasis.

Hormonal Status of Transgenic Birch with a Pine Glutamine Synthetase Gene during Rooting In Vitro and Budburst Outdoors.

Improving nitrogen use efficiency (NUE) is one of the main ways of increasing plant productivity through genetic engineering. The modification of nitrogen (N) metabolism can affect the hormonal content, but in transgenic plants, this aspect has not been sufficiently studied. Transgenic birch (Betula pubescens) plants with the pine glutamine synthetase gene GS1 were evaluated for hormone levels during rooting in vitro and budburst under outdoor conditions. In the shoots of the transgenic lines, the content of indoleacetic acid (IAA) was 1.5-3 times higher than in the wild type. The addition of phosphinothricin (PPT), a glutamine synthetase (GS) inhibitor, to the medium reduced the IAA content in transgenic plants, but it did not change in the control. In the roots of birch plants, PPT had the opposite effect. PPT decreased the content of free amino acids in the leaves of nontransgenic birch, but their content increased in GS-overexpressing plants. A three-year pot experiment with different N availability showed that the productivity of the transgenic birch line was significantly higher than in the control under N deficiency, but not excess, conditions. Nitrogen availability did not affect budburst in the spring of the fourth year; however, bud breaking in transgenic plants was delayed compared to the control. The IAA and abscisic acid (ABA) contents in the buds of birch plants at dormancy and budburst depended both on N availability and the transgenic status. These results enable a better understanding of the interaction between phytohormones and nutrients in woody plants.

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

Cyanidin-3-O-glucoside alleviates trimethyltin chloride-induced neurodegeneration by maintaining glutamate homeostasis through modulation of the gut microbiota

Trimethyltin chloride (TMT) is a potent neurotoxin to cause neurodegeneration, especially in hippocampus. This study aimed to identify dietary components that can effectively attenuate TMT-induced neurodegeneration in humans. The predominant anthocyanin in human diets, cyanidin-3-O-glucoside (C3G, 5 or 50 mg/kg), was given to mice for 16 days, and TMT (2.7 mg/kg) was injected intraperitoneally once on the eighth day. C3G (50 mg/kg) significantly alleviated TMT-induced seizures and subsequent cognitive impairment by ameliorating hippocampal neurodegeneration and synaptic dysfunction. Furthermore, C3G treatment restored glutamate homeostasis in brain and reversed glutamine synthetase (GS) inhibition in reactive astrogliosis and neuroinflammation, which are critical for C3G’s neuroprotective effects. Notably, C3G decreased the lipopolysaccharide, tumor necrosis factor-α, interleukin-6, and interleukin-1β levels in the mice, which potentially by modulating the relative abundance of Atopobiaceae and Lachnospiraceae in the gut. C3G may be a promising and practical dietary component for reducing TMT-induced neurodegeneration.

Effect of MTA3 Inhibition of Glutamine Synthetase-Mediated Glutaminolysis on Radiosensitivity of Patients with Esophageal Cancer

Metastasis-associated protein 3 (MTA3) can serve as a tumor suppressor in many cancer types. However, the role of MTA3 in radiosensitivity of patients with esophageal squamous cell cancer (ESCC) remains unclear. We thus investigated the function of MTA3 in radiosensitivity for ESCC, one of the most common digestive cancers. The colony formation assay and nude mice xenograft tumor assay were performed to investigate the effect of MTA3 on radiosensitivity in ESCC. Glutamine consumption assay kit and glutamate production assay kit were used to assess the glutaminolysis. Glutaminase (GLS) Activity Assay Kit and Glutamine Synthetase (GS) Activity Assay Kit were used to analyze the activity of specific metabolic enzymes dominate glutaminolysis. The regulatory mechanism of glutaminolysis by MTA3 was confirmed using Chromatin immunoprecipitation assay and Gaussia luciferase assay. The expression levels of MTA3 and GS in ESCC primary tissues were evaluated using immunohistochemistry. Survival curves were plotted with the Kaplan-Meier method and compared by log-rank test. The colony formation assay showed that MTA3 depletion and overexpression caused significantly higher and lower clonogenic survival after different doses of irradiation (IR), respectively. When these cells were subcutaneously injected into nude mice, the tumors derived from the cells with MTA3 overexpression and MTA3 knockdown were significantly smaller and bigger after IR, respectively. These findings suggest that MTA3 can enhance radiosensitivity in vitro and in vivo. Meanwhile, overexpressed and knockdown MTA3 can repress and expedite glutamine consumption and glutamate production uniformly, respectively. To determine how MTA3 acts on glutaminolysis, the activity of two specific metabolic enzymes dominate this metabolism, GS and GLS, were evaluated. It found that overexpressed and knockdown MTA3 can restrain and enhance the activity of GS, respectively, but have less effect on GLS. Moreover, the decreased radiosensitivity mediated by MTA3 knockdown is significantly increased when treated with GS inhibitor, suggesting that GS plays a crucial role in MTA3-mediated radiosensitivity enhancement. Mechanistically, Chromatin immunoprecipitation assay and Gaussia luciferase assay showed that MTA3 was recruited to the promoter of GS and suppressed GS transcription. However, knockdown of GATA3 abolished MTA3's repressive effect on GS and inhibited the MTA3's occupation on the promoter region of GS. These results collectively demonstrated that, in ESCC cells, MTA3 is recruited by GATA3 to inhibit GS expression, then ultimately represses glutaminolysis and enhances radiosensitivity. Finally, we showed that the ESCC patients in the MTA3low/GShigh group is significantly associated with shorter overall survival. MTA3 is capable of enhancing radiosensitivity through downregulating GS and MTA3low/GShigh might be a potential prognostic factor for ESCC patients.

Bladder microenvironment actuated proteomotors with ammonia amplification for enhanced cancer treatment.

Enzyme-driven micro/nanomotors consuming in situ chemical fuels have attracted lots of attention for biomedical applications. However, motor systems composed by organism-derived organics that maximize the therapeutic efficacy of enzymatic products remain challenging. Herein, swimming proteomotors based on biocompatible urease and human serum albumin are constructed for enhanced antitumor therapy via active motion and ammonia amplification. By decomposing urea into carbon dioxide and ammonia, the designed proteomotors are endowed with self-propulsive capability, which leads to improved internalization and enhanced penetration invitro. As a glutamine synthetase inhibitor, the loaded l-methionine sulfoximine further prevents the conversion of toxic ammonia into non-toxic glutamine in both tumor and stromal cells, resulting in local ammonia amplification. After intravesical instillation, the proteomotors achieve longer bladder retention and thus significantly inhibit the growth of orthotopic bladder tumor invivo without adverse effects. We envision that the as-developed swimming proteomotors with amplification of the product toxicity may be a potential platform for active cancer treatment.

Modulation of host glutamine anabolism enhances the sensitivity of small cell lung cancer to chemotherapy

Small cell lung cancer (SCLC) is one of the deadliest human cancers, with a 5-year survival rate of ∼7%. Here, we performed a targeted proteomics analysis of human SCLC samples and thereby identified hypoxanthine phosphoribosyltransferase 1 (HPRT1) in the salvage purine synthesis pathway as a factor that contributes to SCLC malignancy by promoting cell survival in a glutamine-starved environment. Inhibition of HPRT1 by 6-mercaptopurine (6-MP) in combination with methotrexate (MTX), which blocks the de novo purine synthesis pathway, attenuated the growth of SCLC in mouse xenograft models. Moreover, modulation of host glutamine anabolism with the glutamine synthetase inhibitor methionine sulfoximine (MSO) in combination with 6-MP and MTX treatment resulted in marked tumor suppression and prolongation of host survival. Our results thus suggest that modulation of host glutamine anabolism combined with simultaneous inhibition of the de novo and salvage purine synthesis pathways may be of therapeutic benefit for SCLC.

1581-P: Systemic Alteration of Amino Acids Metabolism in Glutamate Dehydrogenase Gain-of-Function Mutation Knockin Mice

Patients with gain-of-function (GoF) mutations of glutamate dehydrogenase (GDH) have protein-sensitive hypoglycemia, hyperammonemia, epilepsy, and intellectual disability (HI/HA syndrome). Studies of mouse models with a beta-cell specific expression of H454Y hGDH suggest that the mechanisms of HI are caused by increased glutaminolysis induced by GDH GoF in islets. However, the mechanism of hyperammonemia is still unknown. In order to study the systemic effects of GDH GoF, H454Y GDH knockin mice (H454Y-GDH-KI) were created. Enzyme kinetic of GDH in the brain, kidney and liver confirmed that H454Y was successfully expressed with impaired GTP inhibition. H454Y-GDH-KI mice have a ~2.5-fold elevation of plasma ammonia and fasting hypoglycemia, similar to HI/HA patients. Tissues (brain, liver and kidney) and plasma amino acids profiles were determined by LC-MS. Compared to wildtype, the amino acids profile in the kidney was unaltered in KI mice. In contrast, KI liver had 60% lower glutamine, unchanged glutamate and aspartate, increased branch chain amino acids, etc. The glutamine of KI brain was 40% lower than the wildtype, glutamate and GABA was unchanged. Interestingly, plasma glutamine in KI mice was 30% higher compared to controls. A similar phenomenon has also been observed in HI/HA patients. We hypothesize that hyperammonemia is mainly caused by the increased glutaminolysis in KI liver. Higher plasma glutamine can be explained by the increased activity of tissue glutamine synthetase (GS). GS and glutamine serve as an ammonia sink, which is the main mechanism for the removal of increased ammonia in KI mice. We therefore applied the GS inhibitor, methionine sulfoximine (MSO) to KI mice. MSO significantly increased ammonia levels in KI mice after a single dose of injection. These studies in H454Y-GDH-KI mice suggest that GDH GoF in the liver is the main cause of hyperammonemia. GS plays an important role in ammonia removal. Disclosure C.Su: None. D.Han: Employee; Nanjing AscendRare Pharmaceutical Technology. S.Meng: Employee; Nanjing AscendRare Pharmaceutical Technology. Q.Zhu: Employee; Nanjing AscendRare Pharmaceutical Technology. X.Zhou: Employee; Nanjing AscendRare Pharmaceutical Technology. H.Jiang: None. J.Li: None. C.Gong: None. C.Li: Employee; Hua Medicine, Nanjing AscendRare Pharmaceutical Technology. Funding Beijing Natural Science Foundation (L212037)

Improving anti-tumor CD8+ T cell function through manipulating glutamine metabolism

Abstract Immunotherapies that bolster the anti-tumor effects of cytotoxic CD8 T lymphocytes (CTLs) have improved outcomes for many, yet most patients fail to achieve durable responses. Investigations into strategies to further enhance the cancer killing capacity of these cells are crucial in the effort to improve immunotherapies. T cells radically alter their metabolism upon activation, and prior studies have shown pan-inhibition of glutamine metabolism (glutaminolysis) using the inhibitor 6-Diazo-5-oxo-norleucine (DON) enhances the function of CTLs in the tumor microenvironment. However, DON’s toxicity combined with this drug’s lack of specificity demonstrate a need for a more directed glutaminolysis targeting approach. I utilized a targeted glutaminolysis CRISPR library to investigate the role of individual enzymes targeted by DON. To interrogate the importance of these enzymes specifically in anti-tumor CTLs, I developed a model in which CRISPR-library edited antigen-specific CTLs were adoptively transferred into tumor-bearing mice. After 7 days of in vivo selection, next generation sequencing was performed on isolated CTLs to evaluate the survival advantage or disadvantage of these gene knockouts. While multiple DON targets resulted in decreased CTL fitness, deletion of the gene encoding for glutamine synthetase (GS) – which converts glutamate to glutamine – conferred an advantage to CTLs. Upon inhibition of GS, cells may increase cellular levels of glutamate, and preliminary data suggest this results in a decrease in glycolysis and increase in mitochondrial respiration capacity. Lastly, in vitro data demonstrate increased inflammatory cytokine production of CTLs upon genetic knockout or pharmacologic inhibition of GS. Supported by grants from NIH (T32 GM007347, R01 CA217987)

The Arabidopsis Target of Rapamycin kinase regulates ammonium assimilation and glutamine metabolism.

In eukaryotes, a target of rapamycin (TOR) is a well-conserved kinase that controls cell metabolism and growth in response to nutrients and environmental factors. Nitrogen (N) is an essential element for plants, and TOR functions as a crucial N and amino acid sensor in animals and yeast. However, knowledge of the connections between TOR and the overall N metabolism and assimilation in plants is still limited. In this study, we investigated the regulation of TOR in Arabidopsis (Arabidopsis thaliana) by the N source as well as the impact of TOR deficiency on N metabolism. Inhibition of TOR globally decreased ammonium uptake while triggering a massive accumulation of amino acids, such as Gln, but also of polyamines. Consistently, TOR complex mutants were hypersensitive to Gln. We also showed that the glutamine synthetase inhibitor glufosinate abolishes Gln accumulation resulting from TOR inhibition and improves the growth of TOR complex mutants. These results suggest that a high level of Gln contributes to the reduction in plant growth resulting from TOR inhibition. Glutamine synthetase activity was reduced by TOR inhibition while the enzyme amount increased. In conclusion, our findings show that the TOR pathway is intimately connected to N metabolism and that a decrease in TOR activity results in glutamine synthetase-dependent Gln and amino acid accumulation.

L-asparaginase anti-tumor activity in pancreatic cancer is dependent on its glutaminase activity and resistance is mediated by glutamine synthetase

l-Asparaginase is a cornerstone of acute lymphoblastic leukemia (ALL) therapy since lymphoblasts lack asparagine synthetase (ASNS) and rely on extracellular asparagine availability for survival. Resistance mechanisms are associated with increased ASNS expression in ALL. However, the association between ASNS and l-Asparaginase efficacy in solid tumors remains unclear, thus limiting clinical development. Interestingly, l-Asparaginase also has a glutaminase co-activity that is crucial in pancreatic cancer where KRAS mutations activate glutamine metabolism. By developing l-Asparaginase-resistant pancreatic cancer cells and using OMICS approaches, we identified glutamine synthetase (GS) as a marker of resistance to l-Asparaginase. GS is the only enzyme able to synthesize glutamine, and its expression also correlates with l-Asparaginase efficacy in 27 human cell lines from 11 cancer indications. Finally, we further demonstrated that GS inhibition prevents cancer cell adaptation to l-Asparaginase-induced glutamine starvation. These findings could pave the way to the development of promising drug combinations to overcome l-Asparaginase resistance.

Attenuation of initial pilocarpine-induced electrographic seizures by methionine sulfoximine pretreatment tightly correlates with the reduction of extracellular taurine in the hippocampus.

Initiation and development of early seizures by chemical stimuli is associated with brain cell swelling resulting in edema of seizure-vulnerable brain regions. We previously reported that pretreatment with a nonconvulsive dose of glutamine (Gln) synthetase inhibitor methionine sulfoximine (MSO) mitigates the intensity of initial pilocarpine (Pilo)-induced seizures in juvenile rats. We hypothesized that MSO exerts its protective effect by preventing the seizure-initiating and seizure-propagating increase of cell volume. Taurine (Tau) is an osmosensitive amino acid, whose release reflects increased cell volume. Therefore, we tested whether the poststimulus rise of amplitude of Pilo-induced electrographic seizures and their attenuation by MSO are correlated with the release of Tau from seizure-affected hippocampus. Lithium-pretreated animals were administered MSO (75 mg/kg ip) 2.5h before the induction of convulsions by Pilo (40 mg/kg ip). Electroencephalographic (EEG) power was analyzed during 60 min post-Pilo, at 5-min intervals. Extracellular accumulation of Tau (eTau) served as a marker of cell swelling. eTau, extracellular Gln (eGln), and extracellular glutamate (eGlu) were assayed in the microdialysates of the ventral hippocampal CA1 region collected at 15-min intervals during the whole 3.5-h observation period. The first EEG signal became apparent at ~10min post-Pilo. The EEG amplitude across most frequency bands peaked at ~40 min post-Pilo, and showed strong (r~ .72-.96) temporal correlation with eTau, but no correlation with eGln or eGlu. MSO pretreatment delayed the first EEG signal in Pilo-treated rats by ~10min, and depressed the EEG amplitude across most frequency bands, to values that remained strongly correlated with eTau (r> .92) and moderately correlated (r~ -.59) with eGln, but not with eGlu. Strong correlation between attenuation of Pilo-induced seizures and Tau release indicates that the beneficial effect of MSO is due to the prevention of cell volume increase concurrent with the onset of seizures.

Glutamine synthetase expression rescues human dendritic cell survival in a glutamine-deprived environment.

Glutamine deficiency is a well-known feature of the tumor environment. Here we analyzed the impact of glutamine deprivation on human myeloid cell survival and function. Different types of myeloid cells were cultured in the absence or presence of glutamine and/or with L-methionine-S-sulfoximine (MSO), an irreversible glutamine synthetase (GS) inhibitor. GS expression was analyzed on mRNA and protein level. GS activity and the conversion of glutamate to glutamine by myeloid cells was followed by 13C tracing analyses. The absence of extracellular glutamine only slightly affected postmitotic human monocyte to dendritic cell (DC) differentiation, function and survival. Similar results were obtained for monocyte-derived macrophages. In contrast, proliferation of the monocytic leukemia cell line THP-1 was significantly suppressed. While macrophages exhibited high constitutive GS expression, glutamine deprivation induced GS in DC and THP-1. Accordingly, proliferation of THP-1 was rescued by addition of the GS substrate glutamate and 13C tracing analyses revealed conversion of glutamate to glutamine. Supplementation with the GS inhibitor MSO reduced the survival of DC and macrophages and counteracted the proliferation rescue of THP-1 by glutamate. Our results show that GS supports myeloid cell survival in a glutamine poor environment. Notably, in addition to suppressing proliferation and survival of tumor cells, the blockade of GS also targets immune cells such as DCs and macrophages.

Molecular targets of insecticides and herbicides – Are there useful overlaps?

New insecticide modes of action are needed for insecticide resistance management strategies. The number of molecular targets of commercial herbicides and insecticides are fewer than 35 for both. Few commercial insecticide targets are found in plants, but ten targets of commercial herbicides are found in insects. For several of these commonly held targets, some compounds kill both plants and insects. For example, herbicidal inhibitors of p-hydroxyphenylpyruvate dioxygenase are effective insecticides on blood-fed insects. The glutamine synthetase-inhibiting herbicide glufosinate is insecticidal by the same mechanism of action, inhibition of glutamine synthetase. These and other examples of shared activities of commercial herbicides with insecticides through the same target site are discussed. Compounds with novel herbicide targets shared by insects that are not commercialized as pesticides (such as statins) are also discussed. Compounds that are both herbicidal and insecticidal can be used for insect pests not associated with crops or with crops made resistant to the compounds.

ADSCs-derived exosomes ameliorate hepatic fibrosis by suppressing stellate cell activation and remodeling hepatocellular glutamine synthetase-mediated glutamine and ammonia homeostasis

BackgroundHepatic fibrosis is a common pathologic stage in chronic liver disease development, which might ultimately lead to liver cirrhosis. Accumulating evidence suggests that adipose-derived stromal cells (ADSCs)-based therapies show excellent therapeutic potential in liver injury disease owing to its superior properties, including tissue repair ability and immunomodulation effect. However, cell-based therapy still limits to several problems, such as engraftment efficiency and immunoreaction, which impede the ADSCs-based therapeutics development. So, ADSCs-derived extracellular vesicles (EVs), especially for exosomes (ADSC-EXO), emerge as a promise cell-free therapeutics to ameliorate liver fibrosis. The effect and underlying mechanisms of ADSC-EXO in liver fibrosis remains blurred.MethodsHepatic fibrosis murine model was established by intraperitoneal sequential injecting the diethylnitrosamine (DEN) for two weeks and then carbon tetrachloride (CCl4) for six weeks. Subsequently, hepatic fibrosis mice were administrated with ADSC-EXO (10 μg/g) or PBS through tail vein infusion for three times in two weeks. To evaluate the anti-fibrotic capacity of ADSC-EXO, we detected liver morphology by histopathological examination, ECM deposition by serology test and Sirius Red staining, profibrogenic markers by qRT-PCR assay. LX-2 cells treated with TGF-β (10 ng/ml) for 12 h were conducted for evaluating ADSC-EXO effect on activated hepatic stellate cells (HSCs). RNA-seq was performed for further analysis of the underlying regulatory mechanisms of ADSC-EXO in liver fibrosis.ResultsIn this study, we obtained isolated ADSCs, collected and separated ADSCs-derived exosomes. We found that ADSC-EXO treatment could efficiently ameliorate DEN/CCl4-induced hepatic fibrosis by improving mice liver function and lessening hepatic ECM deposition. Moreover, ADSC-EXO intervention could reverse profibrogenic phenotypes both in vivo and in vitro, including HSCs activation depressed and profibrogenic markers inhibition. Additionally, RNA-seq analysis further determined that decreased glutamine synthetase (Glul) of perivenous hepatocytes in hepatic fibrosis mice could be dramatically up-regulated by ADSC-EXO treatment; meanwhile, glutamine and ammonia metabolism-associated key enzyme OAT was up-regulated and GLS2 was down-regulated by ADSC-EXO treatment in mice liver. In addition, glutamine synthetase inhibitor would erase ADSC-EXO therapeutic effect on hepatic fibrosis.ConclusionsThese findings demonstrated that ADSC-derived exosomes could efficiently alleviate hepatic fibrosis by suppressing HSCs activation and remodeling glutamine and ammonia metabolism mediated by hepatocellular glutamine synthetase, which might be a novel and promising anti-fibrotic therapeutics for hepatic fibrosis disease.

Identification of Concomitant Inhibitors against Glutamine Synthetase and Isocitrate Lyase in Mycobacterium tuberculosis from Natural Sources.

Tuberculosis (T.B.) is a disease that occurs due to infection by the bacterium, Mycobacterium tuberculosis (Mtb), which is responsible for millions of deaths every year. Due to the emergence of multidrug and extensive drug-resistant Mtb strains, there is an urgent need to develop more powerful drugs for inclusion in the current tuberculosis treatment regime. In this study, 1778 molecules from four medicinal plants, Azadirachta indica, Camellia sinensis, Adhatoda vasica, and Ginkgo biloba, were selected and docked against two chosen drug targets, namely, Glutamine Synthetase (G.S.) and Isocitrate Lyase (I.C.L.). Molecular Docking was performed using the Glide module of the Schrӧdinger suite to identify the best-performing ligands; the complexes formed by the best-performing ligands were further investigated for their binding stability via Molecular Dynamics Simulation of 100 ns. The present study suggests that Azadiradione from Azadirachta indica possesses the potential to inhibit Glutamine Synthetase and Isocitrate Lyase of M. tuberculosis concomitantly. The excellent docking score of the ligand and the stability of receptor-ligand complexes, coupled with the complete pharmacokinetic profile of Azadiradione, support the proposal of the small molecule, Azadiradione as a novel antitubercular agent. Further, wet lab analysis of Azadiradione may lead to the possible discovery of a novel antitubercular drug.

Involvement of glutamine synthetase 2 (GS2) amplification and overexpression in Amaranthus palmeri resistance to glufosinate

Main conclusionAmplification and overexpression of the target site glutamine synthetase, specifically the plastid-located isoform, confers resistance to glufosinate in Amaranthus palmeri. This mechanism is novel among glufosinate-resistant weeds.Amaranthus palmeri has recently evolved resistance to glufosinate herbicide. Several A. palmeri populations from Missouri and Mississippi, U.S.A. had survivors when sprayed with glufosinate-ammonium (GFA, 657 g ha−1). One population, MO#2 (fourfold resistant) and its progeny (sixfold resistant), were used to study the resistance mechanism, focusing on the herbicide target glutamine synthetase (GS). We identified four GS genes in A. palmeri; three were transcribed: one coding for the plastidic protein (GS2) and two coding for cytoplasmic isoforms (GS1.1 and GS1.2). These isoforms did not contain mutations associated with resistance. The 17 glufosinate survivors studied showed up to 21-fold increase in GS2 copies. GS2 was expressed up to 190-fold among glufosinate survivors. GS1.1 was overexpressed > twofold in only 3 of 17, and GS1.2 in 2 of 17 survivors. GS inhibition by GFA causes ammonia accumulation in susceptible plants. Ammonia level was analyzed in 12 F1 plants. GS2 expression was negatively correlated with ammonia level (r = – 0.712); therefore, plants with higher GS2 expression are less sensitive to GFA. The operating efficiency of photosystem II (ϕPSII) of Nicotiana benthamiana overexpressing GS2 was four times less inhibited by GFA compared to control plants. Therefore, increased copy and overexpression of GS2 confer resistance to GFA in A. palmeri (or other plants). We present novel understanding of the role of GS2 in resistance evolution to glufosinate.