Step Of Glycolysis Research Articles

Pyruvate Kinase M2: Regulatory Circuits and Potential for Therapeutic Intervention

Cancer cells are characterized by reprogramming of energy metabolism. Over the last decade, understanding of the metabolic changes that occur in cancer has increased dramatically, with great interest in targeting metabolism for cancer therapy. Pyruvate kinase isoenzyme type M2 (abbreviations: PKM2, M2-PK) plays a key role in modulating glucose metabolism to support cell proliferation. PKM2, like other PK isoforms, catalyzes the last energy-generating step in glycolysis, but is unique in its capacity to be regulated. PKM2 is regulated at several cellular levels, including gene expression, alternative splicing and post-translational modification. In addition, PKM2 is regulated by key metabolic intermediates and interacts with more than twenty different proteins. Hence, this isoenzyme is an important regulator of glycolysis, and additionally functions in other novel roles that have recently emerged. Recent evidence indicates that intervening with the complex regulatory network of PKM2 has severe consequences on tumor cell proliferation, indicating the potential of this enzyme as a target for tumor therapy.

Pyruvate kinase M2 and cancer: an updated assessment.

Cancer cells are characterized by high glycolytic rates to support energy regeneration and anabolic metabolism, along with the expression of pyruvate kinase isoenzyme M2 (PKM2). The latter catalyzes the last step of glycolysis and reprograms the glycolytic flux to feed the special metabolic demands of proliferating cells. Besides, PKM2 has moonlight functions, such as gene transcription, favoring cancer. Accumulating evidence suggests a critical role played by the low-activity-dimeric PKM2 in tumor progression, supported by the identification of mutations which result in the down-regulation of its activity and tumorigenesis in a nude mouse model. This review discusses PKM2 regulation and the benefits it confers to cancer cells. Further, conflicting views on PKM2's role in cancer, its therapeutic relevance and future directions in the field are also discussed.

A novel function of TIMP1/CD63 signaling in the regulation of tumor metabolism (LB68)

A distinctive feature of cancer cells that sets them apart from normal non‐transformed cells is a unique metabolic profile. In the 1920s, Otto Warburg recognized that carcinogenesis was accompanied by a striking shift to glycolysis as the major energy‐generating pathway. Nearly a century later, it is still unclear if this “glycolytic switch” is an epiphenomenon or a mechanistic determinant for malignancy. During the course of studying tissue inhibitor of metalloproteinase (TIMP)‐1 signaling, we found that TIMP‐1 upregulates the rate of the aerobic glycolysis as evidenced by increased glucose uptake and lactate production. TIMP‐1 upregulated hexokinaseII RNA level, known to be the rate‐limiting step of glycolysis. Interestingly, TIMP‐1‐induced glycolysis was accompanied with reduced mitochondrial respiration as shown by reduced cytochrome c oxidase activity and a low rate of oxygen consumption. ShRNA‐mediated knockdown of the tetraspanin CD63, a cell surface binding partner of TIMP‐1, reversed the TIMP‐1‐mediated glycolytic switch. Interestingly, TIMP‐1 activation of the CD63 signaling complex resulted in constitutive activation of HIF1α and protected breast epithelial cells from hypoxia‐induced cell death. In contrast, TIMP‐1‐stimulated cells rapidly underwent cell death upon glucose withdrawal. Taken together, the present study demonstrates a novel function of TIMP‐1 in the regulation of tumor metabolism in a CD63‐dependent manner.

Biochemical characterisation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the liver fluke, Fasciola hepatica

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyses one of the two steps in glycolysis which generate the reduced coenzyme NADH. This reaction precedes the two ATP generating steps. Thus, inhibition of GAPDH will lead to substantially reduced energy generation. Consequently, there has been considerable interest in developing GAPDH inhibitors as anti-cancer and anti-parasitic agents. Here, we describe the biochemical characterisation of GAPDH from the common liver fluke Fasciola hepatica (FhGAPDH). The primary sequence of FhGAPDH is similar to that from other trematodes and the predicted structure shows high similarity to those from other animals including the mammalian hosts. FhGAPDH lacks a binding pocket which has been exploited in the design of novel antitrypanosomal compounds. The protein can be expressed in, and purified from Escherichia coli; the recombinant protein was active and showed no cooperativity towards glyceraldehyde 3-phosphate as a substrate. In the absence of ligands, FhGAPDH was a mixture of homodimers and tetramers, as judged by protein–protein crosslinking and analytical gel filtration. The addition of either NAD+ or glyceraldehyde 3-phosphate shifted this equilibrium towards a compact dimer. Thermal scanning fluorimetry demonstrated that this form was considerably more stable than the unliganded one. These responses to ligand binding differ from those seen in mammalian enzymes. These differences could be exploited in the discovery of reagents which selectively disrupt the function of FhGAPDH.

Metabolic control at the cytosol–mitochondria interface in different growth phases of CHO cells

Metabolism at the cytosol–mitochondria interface and its regulation is of major importance particularly for efficient production of biopharmaceuticals in Chinese hamster ovary (CHO) cells but also in many diseases. We used a novel systems-oriented approach combining dynamic metabolic flux analysis and determination of compartmental enzyme activities to obtain systems level information with functional, spatial and temporal resolution. Integrating these multiple levels of information, we were able to investigate the interaction of glycolysis and TCA cycle and its metabolic control. We characterized metabolic phases in CHO batch cultivation and assessed metabolic efficiency extending the concept of metabolic ratios. Comparing in situ enzyme activities including their compartmental localization with in vivo metabolic fluxes, we were able to identify limiting steps in glycolysis and TCA cycle. Our data point to a significant contribution of substrate channeling to glycolytic regulation. We show how glycolytic channeling heavily affects the availability of pyruvate for the mitochondria. Finally, we show that the activities of transaminases and anaplerotic enzymes are tailored to permit a balanced supply of pyruvate and oxaloacetate to the TCA cycle in the respective metabolic states. We demonstrate that knowledge about metabolic control can be gained by correlating in vivo metabolic flux dynamics with time and space resolved in situ enzyme activities.

Hexokinase-II Positively Regulates Glucose Starvation-Induced Autophagy through TORC1 Inhibition

Hexokinase-II (HK-II) catalyzes the first step of glycolysis and also functions as a protective molecule; however, its role in protective autophagy has not been determined. Results showed that inhibition of HK-II diminished, while overexpression of HK-II potentiated, autophagy induced by glucose deprivation in cardiomyocyte and noncardiomyocyte cells. Immunoprecipitation studies revealed that HK-II binds to and inhibits the autophagy suppressor, mTOR complex 1 (TORC1), and that this binding was increased by glucose deprivation. The TOS motif, a scaffold sequence responsible for binding TORC1 substrates, is present in HK-II, and mutating it blocked its ability to bind to TORC1 and regulate protective autophagy. The transition from glycolysis to autophagy appears to be regulated by a decrease in glucose-6 phosphate. We suggest that HK-II binds TORC1 as a decoy substrate and provides a previously unrecognized mechanism for switching cells from a metabolic economy, based on plentiful energy, to one of conservation, under starvation.

Chapter Six - Measurement of Enolase Activity in Cell Lysates

Enolase (EC 4.2.1.11) is a cytosolic metalloenzyme responsible for the conversion of 2-phosphoglycerate into phosphoenolpyruvate, the second to last step in glycolysis. In mammals, enolase is encoded by three homologous genes. These gene products not only possess distinct biochemical and immunological properties but also show different tissue distribution. Besides its glycolytic function, α-enolase plays a variety of roles in pathophysiological settings including oncogenesis, tumor progression, ischemia, and bacterial infection. The expression levels of α-enolase have been attributed diagnostic and prognostic value in a number of tumors. Furthermore, neuron-specific α-enolase is released into the cerebrospinal fluid as well as in the systemic circulation upon traumatic brain injury and ischemic episodes. Thus, the measurement of the enzymatic activity of enolase is relevant for diverse fields of investigation, including oncometabolism. Here, we described simple and rapid protocols to measure the activity of enolase in lysates from mammalian cells and tissues.

A 2D gel electrophoresis-based snapshot of the phosphoproteome in the cyanobacterium Synechocystis sp. strain PCC 6803

Cyanobacteria are photoautotrophic prokaryotes that occur in highly variable environments. Protein phosphorylation is one of the most widespread means to adjust cell metabolism and gene expression to the demands of changing growth conditions. Using a 2D gel electrophoresis-based approach and a phosphoprotein-specific dye, we investigated the protein phosphorylation pattern in cells of the model cyanobacterium Synechocystis sp. strain PCC 6803. The comparison of gels stained for total and phosphorylated proteins revealed that approximately 5 % of the protein spots seemed to be phosphoproteins, from which 32 were identified using MALDI-TOF MS. For eight of them the phosphorylated amino acid residues were mapped by subsequent mass spectrometric investigations of isolated phosphopeptides. Among the phosphoproteins, we found regulatory proteins, mostly putative anti-sigma factor antagonists, and proteins involved in translation. Moreover, a number of enzymes catalysing steps in glycolysis or the Calvin-Benson cycle were found to be phosphorylated, implying that protein phosphorylation might represent an important mechanism for the regulation of the primary carbon metabolism in cyanobacterial cells.

Glucose Phosphate Isomerase Deficiency In 2 Patients With Novel Mutations Presenting As Severe Neurologic Abnormalities and Transfusion Dependent Hemolytic Anemia

▪ IntroductionGlucose phosphate isomerase (GPI) deficiency is the third most common red cell enzymopathy. GPI is an enzyme that reversibly catalyzes the conversion of glucose-6-phosphate into fructose 6-phosphate in the second step of glycolysis. Patients afflicted by GPI deficiency have chronic hemolysis and may also suffer from acute hemolytic crises. There are 184 known mutations of the GPI gene and to date, a neurological deficit is found in only five patients and only two of these have been characterized at a molecular level. We report 2 patients with previously unknown mutations of the GPI gene associated with, severe neurologic abnormalities and hemolytic anemia. Case 1He was born at 38 weeks gestation; marked pallor and hepatosplenomegaly were noted at birth. The bilirubin was elevated at birth (indirect 7.5 mg/dl and direct 2.2mg/dl) requiring phototherapy. He has transfusion dependent anemia since birth. Enzymes studied were performed which showed GPI levels of 2.02 EU/gm hb ( normal range 16.3-24.7 ) and elevated glucose 6 phosphate dehydrogenase , pyruvate kinase and hexokinase. The pyrimidine 5'-nucleotidase screen was normal. In his subsequent course, he started to have seizures at 6 months of age, refractory to anticonvulsant therapy. He has severe hypotonia and global developmental delay. Magnetic resonance imaging of the brain showed generalized cerebral atrophy with no evidence of kernicterus. Case 2He was noted to have anemia and marked hepatosplenomegaly at birth. He required exchange transfusions and phototherapy in the neonatal period. He has subsequently suffered from lifelong transfusion-dependent hemolytic anemia. He also suffers from developmental delay, ataxia, spasticity, and seizures. Of note, MRI did not exhibit evidence of kernicterus.Table. Mutations of the GPI gene in both the patientsMutation : Change in amino acid and locationLocation of amino acid in GPI crystal structureCarrier parentCASE 1Histidine to arginine at codon 191 (H191R)located in alpha helix tenFatherGlycine to tryptophan at codon 381 (G381W)located in beta sheet fourMotherCASE 2Arginine to glutamine at codon 104 (R104Q)boundary of alpha helix sevenMotherLeucine to phenylalanine at codon 297 (L297F).located in alpha helix fifteenFatherThe mutations were predicted to be pathogenic (probably damaging) by PolyPhen. Both these mutations were in a highly conserved residue. Genetic probe for preimplantation diagnosis is being used for selection of an embryo which is does not have GPI deficiency and is also a potential HLA match with the hope of undergoing hematopoietic stem cell transplantation to avoid the complications of chronic transfusions and iron overload. DiscussionGPI has many functions. In dimeric form, it exhibits its catalytic function. In monomeric form, it acts as a neurotrophic growth factor, neuroleukin, which in vitro promotes survival of neurons. Abnormalities in neuroleukin have been found in motor neuron disease and in patients with central nervous system abnormalities in patients with acquired immunodeficiency disease. These effects of GPI/neuroleukin and relative deficiency in brain and neurons of this protein may explain the neurologic presentation. Decreased phosphatide phosphatase 1 activity, a lipogeneic enzyme due to mTOR activation by accumulated glucose-6-phosphate has been suggested to contribute to the neurologic symptoms. Why some of these mutation are associated with neurologic deficits while most others are not is not known. It has been speculated that the mutations which affect the folding may cause altered structure and function causing neurologic symptoms as well as hemolytic anemia while mutations affecting the catalytic site presents only as hemolytic anemia without neurologic symptoms. Disclosures:No relevant conflicts of interest to declare.

Abstract C156: PKM2 activation sensitizes cancer cells to growth inhibition by 2-deoxyglucose.

Abstract Introduction: This study proposes the use 2-deoxyglucose (2DG) with the PKM2 activator, TEPP46 as a novel drug combination for cancer chemotherapy. Pyruvate kinase converts phosphoenolpyruvate to pyruvate in the last step of glycolysis. The M2 isoform of pyruvate kinase, PKM2, is expressed in all cancer cell lines tested so far. PKM2 exists either as a dimer or a more active, tetrameric form. The less active dimer is thought to favor rapid cell division by the diversion of glycolytic intermediates into biosynthetic pathways. Recently, small molecule activators of PKM2 have been shown to inhibit xenograft tumor growth. However, these activators do not inhibit cell growth in culture at physiological conditions and have only been shown to prevent tumor formation in mice. As PKM2 activation accelerates the last step of glycolysis, we hypothesize that activator treatment will also increase overall glucose consumption. 2DG is a glucose analog that acts as a competitive inhibitor of glucose metabolism and is currently in Phase I/II clinical trial for several solid tumors. However, 2DG monotherapy has shown limited efficacy and at high doses, 2DG causes hypoglycemia in patients. The use of 2DG with TEPP46 has the potential of lowering the dose of 2DG needed for therapeutic effects as well as a possible synergistic effect on cancer growth inhibition. Methods: MDA-MB-231, 468, and MCF7 breast cancer cells were grown in DMEM with 25 mM glucose, 4 mM glutamine, and 10% fetal bovine serum. Viability assays were performed using the CellTiter 96 Aqueous One Solution (Promega, WI) in 96-well plates seeded with 2,000 cells per well. 2DG was used at 1 mM in all experiments while TEPP46 was diluted from a 1000x stock in DMSO to a final concentration of 30 μM in cell culture medium. Results: 72 hr post-treatment, cells dosed with TEPP46 alone and 2DG alone demonstrated no appreciable difference in viability compared to DMSO (vehicle) treated cells. However, all three breast cancer cell lines showed a reduced viability, ranging from 56 to 80% of control when treated with a combination of TEPP46 and 2DG. This was most pronounced in the MCF7 cell line that displayed a 55% reduction in viability compared to vehicle treatment. Discussion: PKM2 has recently received much attention as a tumor-specific drug target and small molecule activators such as TEPP46 have been demonstrated to prevent tumor formation in mice. TEPP46 limited efficacy in physiological cell culture conditions and is unlikely to have a therapeutic effect in established tumors. This study is the first to combine a PKM2 activator with another chemotherapeutic, 2DG that is currently in clinical trials. Cell culture studies in three different lines have been encouraging; combination treatment resulted in a reduction of viability that is not evident when either drug is used in isolation. Encouragingly, all studies were performed in cell culture media with a high concentration of glucose and glutamine. The efficacy of this combination is currently be investigated in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C156. Citation Format: Sui-Seng Tee, Jae Mo Park, Daniel Spielman. PKM2 activation sensitizes cancer cells to growth inhibition by 2-deoxyglucose. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C156.

Abstract A138: Identification of PKM2 as a potential biomarker of high-grade ovarian serous tumor.

Abstract The M2 splice isoform of pyruvate kinase (PKM2), that catalyzes the later step of glycolysis, is a key regulator of aerobic glycolysis (Warburg effect) in cancer cells. Expression and low enzymatic activity of PKM2 confer on cancer cells the glycolytic phenotype, which promotes rapid energy production and flow of glycolytic intermediates into collateral pathways to synthesize nucleic acids, amino acids, and lipids without the accumulation of reactive oxygen species [1]. Furthermore, studies indicate that the PKM2 expression might be an early event in carcinogenesis [2]. Thus, PKM2 can be a useful biomarker for the early detection of tumors. Although the presence of PKM2 is described for different types of cancer, there is no information specific PKM2 in ovarian cancer. Thus, to investigate the presence of PKM2 in ovarian cancer, we performed proteomic analysis in a pool of ovarian serous adenocarcinoma (malignant) fluids (n=10) and in a pool of ovarian serous cystadenoma (benign) fluids (n=10) using immunodepletion of albumin and IgG, isotopic labeling with acrylamide, separation by SDS-PAGE, in situ digestion with trypsin and liquid chromatography ion exchange followed by reverse phase coupled to a mass spectrometer LTQ-ORBITRAP (LC-MS/MS). Moreover, evaluated the expression of PKM2 in plasma and tumor fluid of ovarian serous adenocarcinoma (n=14) and of ovarian serous cystadenoma (n=13) using the kit ELISA EDTA-Plasma Test (ScheBo® Biotech AG), and evaluated the expression of PKM2 in tissue microarrays of 71 ovarian serous adenocarcinoma samples (different stages and grades) using Tumor M2-PK Antibodies (monoclonal, ScheBo®, Biotech AG). The PKM2 protein was detected in proteomic analysis of ovarian serous adenocarcinoma. In ELISA test, PKM2 can be confirmed in both plasma and tumor fluid of ovarian serous adenocarcinoma and ovarian serous cystadenoma, with significant difference between fluids (p<0.0001). Concentrations of PKM2 in plasma and tumor fluid are higher in ovarian serous adenocarcinoma. The tissue microarrays analysis confirmed that PKM2 is highly expressed in adenocarcinoma of all stages and grades. The presence of PKM2 in different biological samples of ovarian cancer and in all stages and grades suggests that your inhibition is a viable strategy in the treatment of this malignancy and conducting research in tumor metabolism will benefit cancer treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A138. Citation Format: Aline Poersch, Lucas Oliveira Souza, Vitor Marcel Faça, Lewis Joel Greene, Francisco Jose Candido dos Reis. Identification of PKM2 as a potential biomarker of high-grade ovarian serous tumor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A138.

Hexokinase II in breast carcinoma: A potent prognostic factor associated with hypoxia‐inducible factor‐1α and Ki‐67

Hypoxia-inducible factor-1α (HIF-1α) mediates adaptive responses to changes under tissue hypoxia in carcinoma cells by controlling the expression of various target genes. Previous studies have demonstrated that HIF-1α is associated with adverse clinical outcome in breast carcinoma patients, but details of HIF-1α's role have remained largely unknown. Therefore, in this study, we examined the expression profiles of HIF-1α-induced genes in 10 breast carcinoma cases using microarray data. As a result, we demonstrated that the status of hexokinase II (HKII) was associated with carcinoma recurrence in patients with these genes. The enzyme HKII is involved in the first, and rate-limiting, step of glycolysis, but its clinical significance has not yet been examined in breast carcinoma. Therefore, we immunolocalized HKII in 118 breast carcinomas, and HKII immunoreactivity was detected in 44% of the cases. It is significantly associated with histological grade, Ki-67 labeling index and HIF-1α immunoreactivity. Also, HKII status is significantly associated with increased risk of recurrence and adverse clinical outcome in breast cancer patients. Subsequent multivariate analysis demonstrated that HKII status was an independent prognostic factor for disease-free survival of patients. These results all suggest that HKII is induced by HIF-1α and plays important roles in the proliferation and/or progression of breast carcinoma possibly through increased glycolytic activity. The status of HKII is therefore considered a potent prognostic factor in human breast cancer patients.

Persistent Overexpression of Phosphoglycerate Mutase, a Glycolytic Enzyme, Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice

BackgroundHeart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function.Methods and ResultsTransgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/βATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload.ConclusionsConstitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice.

Akt Phosphorylates HK-II at Thr-473 and Increases Mitochondrial HK-II Association to Protect Cardiomyocytes

Hexokinase II (HK-II) is an enzyme that catalyzes the first step in glycolysis and localizes not only in the cytosol but also at mitochondria. Akt, activated by insulin-like growth factor 1 (IGF-1) treatment in neonatal rat ventricular myocytes, translocates to mitochondria and increases mitochondrial HK-II binding. Expression of an HK-II-dissociating peptide diminished IGF-1-induced increases in mitochondrial HK-II as well as protection against hydrogen peroxide treatment, suggesting an important role of mitochondrial HK-II in IGF-1/Akt-mediated protection. We hypothesized, on the basis of an Akt phosphorylation consensus sequence present in HK-II, that Thr-473 is the target of Akt kinase activity. Indeed, recombinant kinase-active Akt robustly phosphorylates WT HK-II, but not Thr-473 mutants. Phosphomimetic (T473D)HK-II, but not non-phosphorylatable (T473A)HK-II, constitutively increased mitochondrial binding compared with WT HK-II and concomitantly confers greater protection against hydrogen peroxide. Glucose 6-phosphate (G-6P), a product of the catalytic activity of HK-II, is well known to dissociate HK-II from mitochondria. Addition of G-6P to isolated mitochondria dose-dependently dissociates WT HK-II, and this response is inhibited significantly in mitochondria isolated from cardiomyocytes expressing T473D HK-II. Pretreatment with IGF-1 also inhibits G-6P-induced overexpressed or endogenous HK-II dissociation, and this response was blocked by Akt inhibition. These results show that Akt phosphorylation of HK-II at Thr-473 is responsible for the Akt-mediated increase in HK-II binding to mitochondria. This increase is, at least in part, due to the decreased sensitivity to G-6P-induced dissociation. Thus, phosphorylation-mediated regulation of mitochondrial HK-II would be a critical component of the protective effect of Akt.

Synthetic Topsentin Analogues as Potent, Selective Inhibitors of Methicillin Resistant Staphylococcus Aureus Pyruvate Kinase

MRSA pyruvate kinase is an evolutionary conserved enzyme, responsible for catalysing the rate limiting final step in glycolysis. Sequence alignment and X-ray co-crystallography revealed amino acid sequence divergence from common human PK orthologs at the small interface, which is also the site of inhibitor binding. As part of an ongoing investigation of the MRSA inhibition activity and selectivity of marine bis-indole imidazole alkaloids we present the synthesis of dihalogenated analogues of the naturally occurring sponge metabolite topsentin, which display potent nanomolar activity against MRSA pyruvate kinase and 1000 – 10000 fold selectivity for the bacterial over human PK orthologs.

Tumor cell death induced by the inhibition of mitochondrial electron transport: The effect of 3-hydroxybakuchiol

Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells.

Interrogating a Hexokinase-Selected Small-Molecule Library for Inhibitors of Plasmodium falciparum Hexokinase

Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (~26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC50s) of <1 μM. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC50] of ≥ 0.6 and <6.8 μM). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.

Examination of the mode of action of the almiramide family of natural products against the kinetoplastid parasite Trypanosoma brucei.

Almiramide C is a marine natural product with low micromolar activity against Leishmania donovani, the causative agent of leishmaniasis. We have now shown that almiramide C is also active against the related parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. A series of activity-based probes have been synthesized to explore both the molecular target of this compound series in T. brucei lysates and site localization through epifluorescence microscopy. These target identification studies indicate that the almiramides likely perturb glycosomal function through disruption of membrane assembly machinery. Glycosomes, which are organelles specific to kinetoplastid parasites, house the first seven steps of glycolysis and have been shown to be essential for parasite survival in the bloodstream stage. There are currently no reported small-molecule disruptors of glycosome function, making the almiramides unique molecular probes for this understudied parasite-specific organelle. Additionally, examination of toxicity in an in vivo zebrafish model has shown that these compounds have little effect on organism development, even at high concentrations, and has uncovered a potential side effect through localization of fluorescent derivatives to zebrafish neuromast cells. Combined, these results further our understanding of the potential value of this lead series as development candidates against T. brucei.

Characterization of phosphofructokinase activity in Mycobacterium tuberculosis reveals that a functional glycolytic carbon flow is necessary to limit the accumulation of toxic metabolic intermediates under hypoxia.

Metabolic versatility has been increasingly recognized as a major virulence mechanism that enables Mycobacterium tuberculosis to persist in many microenvironments encountered in its host. Glucose is one of the most abundant carbon sources that is exploited by many pathogenic bacteria in the human host. M. tuberculosis has an intact glycolytic pathway that is highly conserved in all clinical isolates sequenced to date suggesting that glucose may represent a non-negligible source of carbon and energy for this pathogen in vivo. Fructose-6-phosphate phosphorylation represents the key-committing step in glycolysis and is catalyzed by a phosphofructokinase (PFK) activity. Two genes, pfkA and pfkB have been annotated to encode putative PFK in M. tuberculosis. Here, we show that PFKA is the sole PFK enzyme in M. tuberculosis with no functional redundancy with PFKB. PFKA is required for growth on glucose as sole carbon source. In co-metabolism experiments, we report that disruption of the glycolytic pathway at the PFK step results in intracellular accumulation of sugar-phosphates that correlated with significant impairment of the cell viability. Concomitantly, we found that the presence of glucose is highly toxic for the long-term survival of hypoxic non-replicating mycobacteria, suggesting that accumulation of glucose-derived toxic metabolites does occur in the absence of sustained aerobic respiration. The culture medium traditionally used to study the physiology of hypoxic mycobacteria is supplemented with glucose. In this medium, M. tuberculosis can survive for only 7–10 days in a true non-replicating state before death is observed. By omitting glucose in the medium this period could be extended for up to at least 40 days without significant viability loss. Therefore, our study suggests that glycolysis leads to accumulation of glucose-derived toxic metabolites that limits long-term survival of hypoxic mycobacteria. Such toxic effect is exacerbated when the glycolytic pathway is disrupted at the PKF step.

Fructose: A Key Factor in the Development of Metabolic Syndrome and Hypertension

Diabetes mellitus and the metabolic syndrome are becoming leading causes of death in the world. Identifying the etiology of diabetes is key to prevention. Despite the similarity in their structures, fructose and glucose are metabolized in different ways. Uric acid, a byproduct of uncontrolled fructose metabolism is known risk factor for hypertension. In the liver, fructose bypasses the two highly regulated steps in glycolysis, glucokinase and phosphofructokinase, both of which are inhibited by increasing concentrations of their byproducts. Fructose is metabolized by fructokinase (KHK). KHK has no negative feedback system, and ATP is used for phosphorylation. This results in intracellular phosphate depletion and the rapid generation of uric acid due to activation of AMP deaminase. Uric acid, a byproduct of this reaction, has been linked to endothelial dysfunction, insulin resistance, and hypertension. We present possible mechanisms by which fructose causes insulin resistance and suggest actions based on this association that have therapeutic implications.