Metabolic Heterogeneity, Plasticity, and Adaptation to "Glutamine Addiction" in Cancer Cells: The Role of Glutaminase and the GTωA [Glutamine Transaminase-ω-Amidase (Glutaminase II)] Pathway.

Many cancers utilize l-glutamine as a major energy source. Often cited in the literature as "l-glutamine addiction", this well-characterized pathway involves hydrolysis of l-glutamine by a glutaminase to l-glutamate, followed by oxidative deamination, or transamination, to α-ketoglutarate, which enters the tricarboxylic acid cycle. However, mammalian tissues/cancers possess a rarely mentioned, alternative pathway (the glutaminase II pathway): l-glutamine is transaminated to α-ketoglutaramate (KGM), followed by ω-amidase (ωA)-catalyzed hydrolysis of KGM to α-ketoglutarate. The name glutaminase II may be confused with the glutaminase 2 (GLS2) isozyme. Thus, we recently renamed the glutaminase II pathway the "glutamine transaminase-ω-amidase (GTωA)" pathway. Herein, we summarize the metabolic importance of the GTωA pathway, including its role in closing the methionine salvage pathway, and as a source of anaplerotic α-ketoglutarate. An advantage of the GTωA pathway is that there is no net change in redox status, permitting α-ketoglutarate production during hypoxia, diminishing cellular energy demands. We suggest that the ability to coordinate control of both pathways bestows a metabolic advantage to cancer cells. Finally, we discuss possible benefits of GTωA pathway inhibitors, not only as aids to studying the normal biological roles of the pathway but also as possible useful anticancer agents.

CTB-targeted protocells enhance ability of lanthionine ketenamine analogs to induce autophagy in motor neuron-like cells.

Impaired autophagy, a cellular digestion process that eliminates proteins and damaged organelles, has been implicated in neurodegenerative diseases, including motor neuron disorders. Motor neuron targeted upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue, activates autophagy in neuronal cell lines. We hypothesized that analogs of LK can be targeted to motor neurons using nanoparticles to improve autophagy flux. Using a mouse motor neuron-like hybrid cell line (NSC-34), we tested the effect of three different LK analogs on autophagy modulation, either alone or loaded in nanoparticles. For fluorescence visualization of autophagy flux, we used a mCherry-GFP-LC3 plasmid reporter. We also evaluated protein expression changes in LC3-II/LC3-I ratio obtained by western blot, as well as presence of autophagic vacuoles per cell obtained by electron microscopy. Delivering LK analogs with targeted nanoparticles significantly enhanced autophagy flux in differentiated motor neuron-like cells compared to LK analogs alone, suggesting the need of a delivery vehicle to enhance their efficacy. In conclusion, LK analogs loaded in nanoparticles targeting motor neurons constitute a promising treatment option to induce autophagy flux, which may serve to mitigate motor neuron degeneration/loss and preserve motor function in motor neuron disease.

Lanthionine Ketimine Ethyl Ester Accelerates Remyelination in a Mouse Model of Multiple Sclerosis.

Although over 20 disease modifying therapies are approved to treat Multiple Sclerosis (MS), these do not increase remyelination of demyelinated axons or mitigate axon damage. Previous studies showed that lanthionine ketenamine ethyl ester (LKE) reduces clinical signs in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and increased maturation of oligodendrocyte (OL) progenitor cells (OPCs) in vitro. In the current study, we used the cuprizone (CPZ) demyelination model of MS to test if LKE could increase remyelination. The corpus callosum (CC) and somatosensory cortex was examined by immunohistochemistry (IHC), electron microscopy and for mRNA expression changes in mice provided 5 weeks of CPZ diet followed by 2 weeks of normal diet in the presence of LKE or vehicle. A significant increase in the number of myelinated axons, and increased myelin thickness was observed in the CC of LKE-treated groups compared to vehicle-treated groups. LKE also increased myelin basic protein and proteolipid protein expression in the CC and cortex, and increased the number of mature OLs in the cortex. In contrast, LKE did not increase the percentage of proliferating OPCs suggesting effects on OPC survival and differentiation but not proliferation. The effects of LKE on OL maturation and remyelination were supported by similar changes in their relative mRNA levels. Interestingly, LKE did not have significant effects on GFAP or Iba1 immunostaining or mRNA levels. These findings suggest that remyelinating actions of LKE can potentially be formulated to induce remyelination in neurological diseases associated with demyelination including MS.

The metabolic importance of the glutaminase II pathway in normal and cancerous cells.

In rapidly dividing cells, including many cancer cells, l-glutamine is a major energy source. Utilization of glutamine is usually depicted as: l-glutamine → l-glutamate (catalyzed by glutaminase isozymes; GLS1 and GLS2), followed by l-glutamate → α-ketoglutarate [catalyzed by glutamate-linked aminotransferases or by glutamate dehydrogenase (GDH)]. α-Ketoglutarate is a major anaplerotic component of the tricarboxylic acid (TCA) cycle. However, the glutaminase II pathway also converts l-glutamine to α-ketoglutarate. This pathway consists of a glutamine transaminase coupled to ω-amidase [Net reaction: l-Glutamine + α-keto acid + H2O → α-ketoglutarate + l-amino acid + NH4+]. This review focuses on the biological importance of the glutaminase II pathway, especially in relation to metabolism of cancer cells. Our studies suggest a component enzyme of the glutaminase II pathway, ω-amidase, is utilized by tumor cells to provide anaplerotic carbon. Inhibitors of GLS1 are currently in clinical trials as anti-cancer agents. However, this treatment will not prevent the glutaminase II pathway from providing anaplerotic carbon derived from glutamine. Specific inhibitors of ω-amidase, perhaps in combination with a GLS1 inhibitor, may provide greater therapeutic efficacy.

The metabolic importance of the overlooked asparaginase II pathway.

The asparaginase II pathway consists of an asparagine transaminase [l-asparagine + α-keto acid ⇆ α-ketosuccinamate + l-amino acid] coupled to ω-amidase [α-ketosuccinamate + H2O → oxaloacetate + NH4+]. The net reaction is: l-asparagine + α-keto acid + H2O → oxaloacetate + l-amino acid + NH4+. Thus, in the presence of a suitable α-keto acid substrate, the asparaginase II pathway generates anaplerotic oxaloacetate at the expense of readily dispensable asparagine. Several studies have shown that the asparaginase II pathway is important in photorespiration in plants. However, since its discovery in rat tissues in the 1950s, this pathway has been almost completely ignored as a conduit for asparagine metabolism in mammals. Several mammalian transaminases can catalyze transamination of asparagine, one of which - alanine-glyoxylate aminotransferase type 1 (AGT1) - is important in glyoxylate metabolism. Glyoxylate is a precursor of oxalate which, in the form of its calcium salt, is a major contributor to the formation of kidney stones. Thus, transamination of glyoxylate with asparagine may be physiologically important for the removal of potentially toxic glyoxylate. Asparaginase has been the mainstay treatment for certain childhood leukemias. We suggest that an inhibitor of ω-amidase may potentiate the therapeutic benefits of asparaginase treatment.

Liposomal Encapsulated FSC231, a PICK1 Inhibitor, Prevents the Ischemia/Reperfusion-Induced Degradation of GluA2-Containing AMPA Receptors.

Strokes remain one of the leading causes of disability within the United States. Despite an enormous amount of research effort within the scientific community, very few therapeutics are available for stroke patients. Cytotoxic accumulation of intracellular calcium is a well-studied phenomenon that occurs following ischemic stroke. This intracellular calcium overload results from excessive release of the excitatory neurotransmitter glutamate, a process known as excitotoxicity. Calcium-permeable AMPA receptors (AMPARs), lacking the GluA2 subunit, contribute to calcium cytotoxicity and subsequent neuronal death. The internalization and subsequent degradation of GluA2 AMPAR subunits following oxygen-glucose deprivation/reperfusion (OGD/R) is, at least in part, mediated by protein-interacting with C kinase-1 (PICK1). The purpose of the present study is to evaluate whether treatment with a PICK1 inhibitor, FSC231, prevents the OGD/R-induced degradation of the GluA2 AMPAR subunit. Utilizing an acute rodent hippocampal slice model system, we determined that pretreatment with FSC231 prevented the OGD/R-induced association of PICK1-GluA2. FSC231 treatment during OGD/R rescues total GluA2 AMPAR subunit protein levels. This suggests that the interaction between GluA2 and PICK1 serves as an important step in the ischemic/reperfusion-induced reduction in total GluA2 levels.

Drug development and the process of transitioning to team-based learning in a qualitative way.

BACKGROUND AND PURPOSE: The North American Pharmacist Licensure Examination and the Pharmacy Curriculum Outcomes Assessment are two standardized tests that evaluate students' preparedness to progress into pharmacy practice. Pharmacy educators are responsible for ensuring individual learners are engaged in course material and take appropriate steps to succeed in meeting learning outcomes. Whenever a new pedagogy is adopted in a previously existing course, understanding the impact on learners is critical. EDUCATIONAL ACTIVITY AND SETTING: Team-based learning (TBL) was implemented within a novel Drug Discovery and Development course to measure the impact in active participation and student performance within the second year of pharmacy school. Survey data was collected to gather pharmacy students' perspectives regarding the pedagogy change. Examination competency scores and active participation were tracked to measure student engagement. FINDINGS: Survey results revealed students agreed or strongly agreed that Drug Discovery and Development was enjoyable when taught with a TBL model, created a conductive learning environment, and improved their perceived knowledge, communication skills, and confidence. Average attendance scores were > 90% in a TBL setting. No change in block exam competency scores were noted across the three academic cohorts. SUMMARY: Students agreed that Drug Discovery and Development was enjoyable when taught using TBL, created a conductive learning environment, and improved their perceived knowledge, communication skills, and confidence. Additional research should be conducted to quantify student engagement and active attendance within similar courses. Similar styles of TBL implementation could be incorporated at other colleges of pharmacy to measure its potential benefits.

Selective linkage of mitochondrial enzymes to intracellular calcium stores differs between human-induced pluripotent stem cells, neural stem cells, and neurons.

Mitochondria and releasable endoplasmic reticulum (ER) calcium modulate neuronal calcium signaling, and both change in Alzheimer's disease (AD). The releasable calcium stores in the ER are exaggerated in fibroblasts from AD patients and in multiple models of AD. The activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key mitochondrial enzyme complex, is diminished in brains from AD patients, and can be plausibly linked to plaques and tangles. Our previous studies in cell lines and mouse neurons demonstrate that reductions in KGDHC increase the ER releasable calcium stores. The goal of these studies was to test whether the relationship was true in human iPSC-derived neurons. Inhibition of KGDHC for one or 24 hr increased the ER releasable calcium store in human neurons by 69% and 144%, respectively. The effect was mitochondrial enzyme specific because inhibiting the pyruvate dehydrogenase complex, another key mitochondrial enzyme complex, diminished the ER releasable calcium stores. The link of KGDHC to ER releasable calcium stores was cell type specific as the interaction was not present in iPSC or neural stem cells. Thus, these studies in human neurons verify a link between KGDHC and releasable ER calcium stores, and support the use of human neurons to examine mechanisms and potential therapies for AD.

Synthesis of α-Ketoglutaramic acid.

α-Ketoglutaramic acid (KGM, α-ketoglutaramate), also known as 2-oxoglutaramic acid (OGM, 2-oxoglutaramate), is a substrate of ω-amidase, also known as Nitrilase 2 (NIT2), and is essential for studying the canonical role of ω-amidase, as well as its role in multiple diseases. Until now, KGM used for biological studies has been prepared most often by the enzymatic oxidation of l-glutamine using snake venom l-amino acid oxidase, which provides KGM as an aqueous solution, containing by-products including 5-oxoproline and α-ketoglutarate. The enzymatic method for KGM preparation, therefore, cannot provide pure product or an accurate percent yield evaluation. Here, we report a synthetic method for the preparation of this important substrate, KGM, in 3 steps, from l-2-hydroxyglutaramic acid, in pure form, in 53% overall yield.

A standardized method for incorporation of drugs into food for use with Drosophila melanogaster.

With any in vivo model, diet plays an important role, even in an organism as simple as the fruit fly - Drosophila melanogaster. Flies serve as good surrogates to study human diseases as approximately 77% of human disease genes are orthologous in the fly. Though breeding and caring for fruit flies is simple, the use of this organism in drug discovery is wide-ranging, especially in the administration of drugs to flies, via their food. We present a standard method for preparing fly food containing drugs for administration to Drosophila melanogaster, from a chemist's perspective.

The Novel CYP2A6 Inhibitor, DLCI-1, Decreases Nicotine Self-Administration in Mice.

During tobacco and e-cigarette use, nicotine is mainly metabolized in the human liver by cytochrome P450 2A6 (CYP2A6). Given that a slower CYP2A6 metabolism has been associated with less vulnerability to develop nicotine dependence, the current studies sought to validate a novel CYP2A6 inhibitor, (5-(4-ethylpyridin-3-yl)thiophen-2-yl)methanamine (DLCI-1), for its effects on intravenous nicotine self-administration. Male and female mice were trained to self-administer nicotine across daily sessions. Once stable responding was achieved, DLCI-1 or vehicle control was administered prior to nicotine sessions. We found that the lower 25 mg/kg and moderate 50 mg/kg doses of DLCI-1 induced a significant decrease in nicotine intake for both males and females. DLCI-1 was further shown to be more effective than a moderate 1 mg/kg dose of bupropion on reducing nicotine intake and did not exert the adverse behavioral effects found with a high 75 mg/kg dose of bupropion. Although mice treated with DLCI-1 self-administered significantly less nicotine, similar nicotine-mediated behavioral effects on locomotion were observed. Together, along with the analysis of nicotine metabolites during self-administration, these findings support the contention that blocking hepatic nicotine metabolism would allow for similar activation of nicotinic acetylcholine receptors at lower nicotine doses. Moreover, these effects of DLCI-1 were specific to nicotine self-administration, as DLCI-1 did not result in any behavioral changes during food self-administration. Taken together, these studies validate DLCI-1 as a novel compound to decrease nicotine consumption, which may thereby promote tobacco and nicotine product cessation. SIGNIFICANCE STATEMENT: Current pharmacological approaches for nicotine and tobacco cessation have only been able to achieve limited efficaciousness in promoting long-term abstinence. In this work, we characterize the effects of a novel compound, (5-(4-ethylpyridin-3-yl)thiophen-2-yl)methanamine (DLCI-1), which inhibits the main enzyme that metabolizes nicotine, and we report a significant decrease in intravenous nicotine self-administration in male and female mice, supporting the potential of DLCI-1 as a novel tobacco cessation pharmacotherapeutic.

Identification of the 4-Position of 3-Alkynyl and 3-Heteroaromatic Substituted Pyridine Methanamines as a Key Modification Site Eliciting Increased Potency and Enhanced Selectivity for Cytochrome P-450 2A6 Inhibition.

Cigarette smoking causes nearly one in every five deaths in the United States. The development of a specific inhibitor of cytochrome P450 2A6 (CYP2A6), the major nicotine-metabolizing enzyme in humans, which could be prescribed for the cessation of cigarette smoking, has been undertaken. To further refine the structure activity relationship of CYP2A6, previously synthesized 3-alkynyl and 3-heteroaromatic substituted pyridine methanamines were used as lead compounds. Isosteric pyridine replacement and appendage of all available positions around the pyridine ring with a methyl group was performed to identify a modification that would increase CYP2A6 inhibition potency, which led to 4g (IC50 = 0.055 mM) as a primary analogue. Potent compounds were evaluated for CYP selectivity, human liver microsomal half-life, and compliance with the rules of five. Top compounds (i.e., 6i, IC50 = 0.017 mM, >120 min half-life) are poised for further development as treatments for smoking and tobacco use cessation.

Rewiring of Glutamine Metabolism Is a Bioenergetic Adaptation of Human Cells with Mitochondrial DNA Mutations.

Using molecular, biochemical, and untargeted stable isotope tracing approaches, we identify a previously unappreciated glutamine-derived α-ketoglutarate (αKG) energy-generating anaplerotic flux to be critical in mitochondrial DNA (mtDNA) mutant cells that harbor human disease-associated oxidative phosphorylation defects. Stimulating this flux with αKG supplementation enables the survival of diverse mtDNA mutant cells under otherwise lethal obligatory oxidative conditions. Strikingly, we demonstrate that when residual mitochondrial respiration in mtDNA mutant cells exceeds 45% of control levels, αKG oxidative flux prevails over reductive carboxylation. Furthermore, in a mouse model of mitochondrial myopathy, we show that increased oxidative αKG flux in muscle arises from enhanced alanine synthesis and release into blood, concomitant with accelerated amino acid catabolism from protein breakdown. Importantly, in this mouse model of mitochondriopathy, muscle amino acid imbalance is normalized by αKG supplementation. Taken together, our findings provide a rationale for αKG supplementation as a therapeutic strategy for mitochondrial myopathies.

Multiple-step, one-pot synthesis of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates and their corresponding ethyl esters.

The multiple-step, one-pot procedure for a series of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates, analogues of the natural, sulfur amino acid metabolite lanthionine ketimine (LK), its 5-ethyl ester (LKE) and 2-substituted LKEs is described. Initiating the synthesis with the Michaelis-Arbuzov preparation of α-ketophosphonates allows for a wide range of functional variation at the 2-position of the products. Nine new compounds were synthesized with overall yields range from 40 to 62%. In addition, the newly prepared 2-isopropyl-LK-P, 2-n-hexyl-LKE-P and 2-ethyl-LKE were shown to stimulate autophagy in cultured cells better than that of the parent compound, LKE.

Mild metabolic perturbations alter succinylation of mitochondrial proteins.

Succinylation of proteins is widespread, modifies both the charge and size of the molecules, and can alter their function. For example, liver mitochondrial proteins have 1,190 unique succinylation sites representing multiple metabolic pathways. Succinylation is sensitive to both increases and decreases of the NAD+ -dependent desuccinylase, SIRT5. Although the succinyl group for succinylation is derived from metabolism, the effects of systematic variation of metabolism on mitochondrial succinylation are not known. Changes in succinylation of mitochondrial proteins following variations in metabolism were compared against the mitochondrial redox state as estimated by the mitochondrial NAD+ /NADH ratio using fluorescent probes. The ratio was decreased by reduced glycolysis and/or glutathione depletion (iodoacetic acid; 2-deoxyglucose), depressed tricarboxylic acid cycle activity (carboxyethyl ester of succinyl phosphonate), and impairment of electron transport (antimycin) or ATP synthase (oligomycin), while uncouplers of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine or tyrphostin) increased the NAD+ /NADH ratio. All of the conditions decreased succinylation. In contrast, reducing the oxygen from 20% to 2.4% increased succinylation. The results demonstrate that succinylation varies with metabolic states, is not correlated to the mitochondrial NAD+ /NADH ratio, and may help coordinate the response to metabolic challenge.

The Enzymology of 2-Hydroxyglutarate, 2-Hydroxyglutaramate and 2-Hydroxysuccinamate and Their Relationship to Oncometabolites.

Many enzymes make "mistakes". Consequently, repair enzymes have evolved to correct these mistakes. For example, lactate dehydrogenase (LDH) and mitochondrial malate dehydrogenase (mMDH) slowly catalyze the reduction of 2-oxoglutarate (2-OG) to the oncometabolite l-2-hydroxyglutarate (l-2-HG). l-2-HG dehydrogenase corrects this error by converting l-2-HG to 2-OG. LDH also catalyzes the reduction of the oxo group of 2-oxoglutaramate (2-OGM; transamination product of l-glutamine). We show here that human glutamine synthetase (GS) catalyzes the amidation of the terminal carboxyl of both the l- and d- isomers of 2-HG. The reaction of 2-OGM with LDH and the reaction of l-2-HG with GS generate l-2-hydroxyglutaramate (l-2-HGM). We also show that l-2-HGM is a substrate of human ω-amidase. The product (l-2-HG) can then be converted to 2-OG by l-2-HG dehydrogenase. Previous work showed that 2-oxosuccinamate (2-OSM; transamination product of l-asparagine) is an excellent substrate of LDH. Finally, we also show that human ω-amidase converts the product of this reaction (i.e., l-2-hydroxysuccinamate; l-2-HSM) to l-malate. Thus, ω-amidase may act together with hydroxyglutarate dehydrogenases to repair certain "mistakes" of GS and LDH. The present findings suggest that non-productive pathways for nitrogen metabolism occur in mammalian tissues in vivo. Perturbations of these pathways may contribute to symptoms associated with hydroxyglutaric acidurias and to tumor progression. Finally, methods for the synthesis of l-2-HGM and l-2-HSM are described that should be useful in determining the roles of ω-amidase/4- and 5-C compounds in photorespiration in plants.

Reductions in the mitochondrial enzyme α-ketoglutarate dehydrogenase complex in neurodegenerative disease - beneficial or detrimental?

Reductions in metabolism and excess oxidative stress are prevalent in multiple neurodegenerative diseases. The activity of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) appears central to these abnormalities. KGDHC is diminished in multiple neurodegenerative diseases. KGDHC can not only be rate limiting for NADH production and for substrate level phosphorylation, but is also a source of reactive oxygen species (ROS). The goal of these studies was to determine how changes in KGDHC modify baseline ROS, the ability to buffer ROS, baseline glutathionylation, calcium modulation and cell death in response to external oxidants. In vivo, reducing KGDHC with adeno virus diminished neurogenesis and increased oxidative stress. In vitro, treatments of short duration increased ROS and glutathionylation and enhanced the ability of the cells to diminish the ROS from added oxidants. However, long-term reductions lessened the ability to diminish ROS, diminished glutathionylation and exaggerated oxidant-induced changes in calcium and cell death. Increasing KGDHC enhanced the ability of the cells to diminish externally added ROS and protected against oxidant-induced changes in calcium and cell death. The results suggest that brief periods of diminished KGDHC are protective, while prolonged reductions are harmful. Furthermore, elevated KGDHC activities are protective. Thus, mitogenic therapies that increase KGDHC may be beneficial in neurodegenerative diseases. Read the Editorial Highlight for this article on Page 689.

Mild mitochondrial metabolic deficits by α-ketoglutarate dehydrogenase inhibition cause prominent changes in intracellular autophagic signaling: Potential role in the pathobiology of Alzheimer's disease.

Brain activities of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) are reduced in Alzheimer's disease and other age-related neurodegenerative disorders. The goal of the present study was to test the consequences of mild impairment of KGDHC on the structure, protein signaling and dynamics (mitophagy, fusion, fission, biogenesis) of the mitochondria. Inhibition of KGDHC reduced its in situ activity by 23-53% in human neuroblastoma SH-SY5Y cells, but neither altered the mitochondrial membrane potential nor the ATP levels at any tested time-points. The attenuated KGDHC activity increased translocation of dynamin-related protein-1 (Drp1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) from the cytosol to the mitochondria, and promoted mitochondrial cytochrome c release. Inhibition of KGDHC also increased the negative surface charges (anionic phospholipids as assessed by Annexin V binding) on the mitochondria. Morphological assessments of the mitochondria revealed increased fission and mitophagy. Taken together, our results suggest the existence of the regulation of the mitochondrial dynamism including fission and fusion by the mitochondrial KGDHC activity via the involvement of the cytosolic and mitochondrial protein signaling molecules. A better understanding of the link among mild impairment of metabolism, induction of mitophagy/autophagy and altered protein signaling will help to identify new mechanisms of neurodegeneration and reveal potential new therapeutic approaches.