ABSTRACT
Lactate transport is an important means of communication between astrocytes and neurons and is implicated in a variety of neurobiological processes. However, the connection between astrocyte-neuron lactate transport and nociceptive modulation has not been well established. Here, we found that Complete Freund's adjuvant (CFA)-induced inflammation pain leads to a significant increase in extracellular lactate levels in the anterior cingulate cortex (ACC). Inhibition of glycogenolysis and lactate release in the ACC disrupted the persistent, but not acute, inflammation pain induced by CFA, and this effect was reversed by exogenous L-lactate administration. Knocking down the expression of lactate transporters (MCT1, MCT4, or MCT2) also disrupted the long lasting inflammation pain induced by CFA. Moreover, glycogenolysis in the ACC is critical for the induction of molecular changes related to neuronal plasticity, including the induction of phospho- (p-) ERK, p-CREB, and Fos. Taken together, our findings indicate that astrocyte-neuron lactate transport in the ACC is critical for the occurrence of persistent inflammation pain, suggesting a novel mechanism underlying chronic pain.
Subject(s)
Arabinose/pharmacology , Cell Communication/immunology , Chronic Pain/immunology , Gyrus Cinguli/pathology , Imino Furanoses/pharmacology , Lactic Acid/metabolism , Sugar Alcohols/pharmacology , Animals , Arabinose/therapeutic use , Astrocytes/metabolism , Cell Communication/drug effects , Chronic Pain/drug therapy , Chronic Pain/pathology , Disease Models, Animal , Freund's Adjuvant/administration & dosage , Freund's Adjuvant/immunology , Glycogenolysis/drug effects , Glycogenolysis/immunology , Gyrus Cinguli/cytology , Gyrus Cinguli/drug effects , Gyrus Cinguli/immunology , Humans , Imino Furanoses/therapeutic use , Male , Mice , Neuronal Plasticity/drug effects , Neuronal Plasticity/immunology , Neurons/metabolism , Sugar Alcohols/therapeutic useABSTRACT
CONTEXT: The uric acid metabolism pathway is more similar in primates and humans than in rodents. However, there are no reports of using primates to establish animal models of hyperuricaemia (HUA). OBJECTIVES: To establish an animal model highly related to HUA in humans. MATERIALS AND METHODS: Inosine (75, 100 and 200 mg/kg) was intraperitoneally administered to adult male rhesus monkeys (n = 5/group). Blood samples were collected over 8 h, and serum uric acid (SUA) level was determined using commercial assay kits. XO and PNP expression in the liver and URAT1, OAT4 and ABCG2 expression in the kidneys were examined by qPCR and Western blotting to assess the effects of inosine on purine and uric acid metabolism. The validity of the acute HUA model was assessed using ulodesine, allopurinol and febuxostat. RESULTS: Inosine (200 mg/kg) effectively increased the SUA level in rhesus monkeys from 51.77 ± 14.48 at 0 h to 178.32 ± 14.47 µmol/L within 30 min and to peak levels (201.41 ± 42.73 µmol/L) at 1 h. PNP mRNA level was increased, whereas XO mRNA and protein levels in the liver were decreased by the inosine group compared with those in the control group. No changes in mRNA and protein levels of the renal uric acid transporter were observed. Ulodesine, allopurinol and febuxostat eliminated the inosine-induced elevation in SUA in tested monkeys. CONCLUSIONS: An acute HUA animal model with high reproducibility was induced; it can be applied to evaluate new anti-HUA drugs in vivo and explore the disease pathogenesis.
Subject(s)
Disease Models, Animal , Hyperuricemia/chemically induced , Inosine/pharmacology , Uric Acid/blood , Acute Disease , Allopurinol/pharmacology , Animals , Dose-Response Relationship, Drug , Febuxostat/pharmacology , Hyperuricemia/drug therapy , Hyperuricemia/physiopathology , Imino Furanoses/pharmacology , Inosine/administration & dosage , Macaca mulatta , Male , Pyrimidinones/pharmacology , Reproducibility of ResultsABSTRACT
During cognitive efforts mediated by local neuronal networks, approximately 20% of additional energy is required; this is mediated by chemical messengers such as noradrenaline (NA). NA targets astroglial aerobic glycolysis, the hallmark of which is the end product l-lactate, a fuel for neurons. Biochemical studies have revealed that astrocytes exhibit a prominent glycogen shunt, in which a portion of d-glucose molecules entering the cytoplasm is transiently incorporated into glycogen, a buffer and source of d-glucose during increased energy demand. Here, we studied single astrocytes by measuring cytosolic L-lactate ([lac]i ) with the FRET nanosensor Laconic. We examined whether NA-induced increase in [lac]i is influenced by: (a) 2-deoxy-d-glucose (2-DG, 3 mM), a molecule that enters the cytosol and inhibits the glycolytic pathway; (b) 1,4-dideoxy-1,4-imino-d-arabinitol (DAB, 300 µM), a potent inhibitor of glycogen phosphorylase and glycogen degradation; and (c) 3-nitropropionic acid (3-NPA, 1 mM), an inhibitor of the Krebs cycle. The results of these pharmacological experiments revealed that d-glucose uptake is essential for the NA-induced increase in [lac]i , and that this exclusively arises from glycogen degradation, indicating that most, if not all, d-glucose molecules in NA-stimulated cells transit the glycogen shunt during glycolysis. Moreover, under the defined transmembrane d-glucose gradient, the glycolytic intermediates were not only used to produce l-lactate, but also to significantly support oxidative phosphorylation, as demonstrated by an elevation in [lac]i when Krebs cycle was inhibited. We conclude that l-lactate production via aerobic glycolysis is an essential energy pathway in NA-stimulated astrocytes; however, oxidative metabolism is important at rest.
Subject(s)
Astrocytes/metabolism , Glucose/metabolism , Glycogen/metabolism , Lactic Acid/biosynthesis , Norepinephrine/pharmacology , Animals , Animals, Newborn , Arabinose/pharmacology , Brain/metabolism , Citric Acid Cycle/drug effects , Deoxyglucose/pharmacology , Energy Metabolism , Fluorescence Resonance Energy Transfer , Imino Furanoses/pharmacology , Nitro Compounds/pharmacology , Oxidative Phosphorylation , Primary Cell Culture , Propionates/pharmacology , Rats , Rats, Wistar , Sugar Alcohols/pharmacology , TransfectionABSTRACT
α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.
Subject(s)
Glycoside Hydrolases/chemistry , Polyporales/enzymology , Arabinose/chemistry , Catalytic Domain , Imino Furanoses/chemistry , Ligands , Models, Molecular , Protein Binding , Sugar Alcohols/chemistryABSTRACT
A strategy towards the synthesis of three different target molecules, namely 1,4-dideoxy-1,4-imino-l-xylitol, deacetyl (+)-anisomycin and amino-substituted piperidine iminosugars, molecules of potential biological and medicinal significance, is reported from a common amino-vicinal diol intermediate derived from tri-O-benzyl-d-glucal. Construction of the key pyrrolidine ring present in 1,4-dideoxy-1,4-imino-l-xylitol and (+)-anisomycin was a consequence of thermodynamically driven concomitant intramolecular nucleophilic addition reaction of the amino group to the resultant aldehyde obtained by oxidative cleavage of the amino-vicinal diol. Alternatively, double nucleophilic substitution on an amino-diol, after mesylation, with various amines delivered amino-substituted piperidine iminosugars in good yields.
Subject(s)
Anisomycin/chemical synthesis , Imino Sugars/chemical synthesis , Piperidines/chemical synthesis , Xylitol/analogs & derivatives , Anisomycin/chemistry , Imino Furanoses/chemical synthesis , Imino Furanoses/chemistry , Imino Sugars/chemistry , Molecular Conformation , Piperidines/chemistry , Stereoisomerism , Xylitol/chemical synthesis , Xylitol/chemistryABSTRACT
Venetoclax, an FDA-approved Bcl-2 selective inhibitor for the treatment of chronic lymphocytic leukemia and acute myeloid leukemia (AML), is tolerated well in elderly patients with AML and has good overall response rates; however, resistance remains a concern. In this study, we show that targeting CDK9 with voruciclib in combination with venetoclax results in synergistic antileukemic activity against AML cell lines and primary patient samples. CDK9 inhibition enhances venetoclax activity through downregulation of Mcl-1 and c-Myc. However, downregulation of Mcl-1 is transient, which necessitates an intermittent treatment schedule to allow for repeated downregulation of Mcl-1. Accordingly, an every other day schedule of the CDK9 inhibitor is effective in vitro and in vivo in enhancing the efficacy of venetoclax. Our preclinical data provide a rationale for an intermittent drug administration schedule for the clinical evaluation of the combination treatment for AML.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Cyclin-Dependent Kinase 9/genetics , Leukemia, Myeloid, Acute/drug therapy , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Sulfonamides/administration & dosage , Adolescent , Adult , Animals , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Benzopyrans/administration & dosage , Cell Death/drug effects , Cell Line, Tumor , Female , Heterografts , Humans , Imino Furanoses/administration & dosage , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Male , Mice , Middle Aged , Proto-Oncogene Proteins c-myc/genetics , Young AdultABSTRACT
N-Substituted derivatives of 1,4-dideoxy-1,4-imino-d-mannitol (DIM), the pyrrolidine core of swainsonine, have been synthesized efficiently and stereoselectively from d-mannose with 2,3:5,6-di-O-isopropylidene DIM (10) as a key intermediate. These N-substituted derivatives include N-alkylated, N-alkenylated, N-hydroxyalkylated and N-aralkylated DIMs with the carbon number of the alkyl chain ranging from one to nine. The obtained 33 N-substituted DIM derivatives were assayed against various glycosidases, which allowed a systematic evaluation of their glycosidase inhibition profiles. Though N-substitution of DIM decreased their α-mannosidase inhibitory activities, some of the derivatives showed significant inhibition of other glycosidases.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Glycoside Hydrolases/antagonists & inhibitors , Mannitol/analogs & derivatives , Animals , Enzyme Inhibitors/chemistry , Glycoside Hydrolases/metabolism , Humans , Imino Furanoses/chemical synthesis , Imino Furanoses/chemistry , Imino Furanoses/pharmacology , Inhibitory Concentration 50 , Mannitol/chemical synthesis , Mannitol/chemistry , Mannitol/pharmacology , Rats , Swainsonine/chemistryABSTRACT
The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.
Subject(s)
Arabinose/pharmacology , Endoplasmic Reticulum/enzymology , Glycoside Hydrolase Inhibitors/pharmacology , Imino Furanoses/pharmacology , Pyrrolizidine Alkaloids/pharmacology , Sugar Alcohols/pharmacology , alpha-Glucosidases/metabolism , Animals , Arabinose/chemistry , Dogs , Glycoside Hydrolase Inhibitors/chemistry , Humans , Imino Furanoses/chemistry , Mice , Microsomes/drug effects , Microsomes/metabolism , Pyrrolizidine Alkaloids/chemistry , Sugar Alcohols/chemistryABSTRACT
A P(V)-N activation method based on nucleoside phosphoropiperidate/DCI system has been developed for improved synthesis of diverse UDP-furanoses. The reaction conditions including temperature, amount of activator, and reaction time were optimized to alleviate the degradation of UDP-furanoses to cyclic phosphates. In addition, an efficient and facile phosphoramidite route was employed for the preparation of furanosyl-1-phosphates.
Subject(s)
Arabinose/analogs & derivatives , Imidazoles/chemistry , Imino Furanoses/chemical synthesis , Arabinose/chemical synthesis , Arabinose/chemistry , Imino Furanoses/chemistry , Nucleosides/chemistry , Phosphates/chemistry , Piperidines/chemistry , Uridine/chemistryABSTRACT
How glia affect neurite outgrowth during neural development has not been well elucidated. In the present study, we found that disruption of lactate production using 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and isofagomine significantly interfered with neurite outgrowth and that exogenous application of L-lactate rescued neurite growth failure. Monocarboxylate transporter-2-knockout, which blocked the lactate shuttle in neurons, showed a remarkable decrease in the length of axons and dendrites. We further demonstrated that Akt activity was decreased while glycogen synthase kinase 3ß (GSK3ß) activity was increased after astrocytic glycogen phosphorylase blockade. Additionally, GSK3ßSer9 mutation reversed neurite growth failure caused by DAB and isofagomine. Our results suggested that lactate transportation played a critical role in neural development and disruption of the lactate shuttle in quiescent condition also affected neurite outgrowth in the central nervous system.
Subject(s)
Glycogen Synthase Kinase 3 beta/genetics , Lactic Acid/metabolism , Monocarboxylic Acid Transporters/genetics , Neurogenesis/genetics , Animals , Arabinose/pharmacology , Astrocytes/drug effects , Axons/enzymology , Axons/metabolism , Biological Transport/genetics , Imino Furanoses/pharmacology , Imino Pyranoses/pharmacology , Neurites/metabolism , Neurogenesis/drug effects , Neuroglia/drug effects , Neuroglia/metabolism , Neuronal Outgrowth/genetics , Neurons/drug effects , Neurons/metabolism , Phosphatidylinositol 3-Kinases , Primary Cell Culture , Proto-Oncogene Proteins c-akt/genetics , Rats , Signal Transduction/drug effects , Sugar Alcohols/pharmacologyABSTRACT
BACKGROUND/AIMS: The overexpression of ATP-Binding Cassette (ABC) transporters has known to be one of the major obstacles impeding the success of chemotherapy in drug resistant cancers. In this study, we evaluated voruciclib, a CDK 4/6 inhibitor, for its chemo-sensitizing activity in ABCB1- and ABCG2- overexpressing cells. METHODS: Cytotoxicity and reversal effect of voruciclib was determined by MTT assay. The intracellular accumulation and efflux of ABCB1 and ABCG2 substrates were measured by scintillation counter. The effects on expression and intracellular localization of ABCB1 and ABCG2 proteins were determined by Western blotting and immunofluorescence, respectively. Vanadate-sensitive ATPase assay was done to determine the effect of voruciclib on the ATPase activity of ABCB1 and ABCG2. Flow cytometric analysis was done to determine the effect of voruciclib on apoptosis of ABCB1 and ABCG2-overexpressing cells and docking analysis was done to determine the interaction of voruciclib with ABCB1 and ACBG2 protein. RESULTS: Voruciclib significantly potentiated the effect of paclitaxel and doxorubicin in ABCB1-overexpressing cells, as well as mitoxantrone and SN-38 in ABCG2-overexpressing cells. Voruciclib moderately sensitized ABCC10- overexpressing cells to paclitaxel, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, voruciclib increased the intracellular accumulation and decreased the efflux of substrate anti-cancer drugs from ABCB1- or ABCG2-overexpressing cells. However, voruciclib did not alter the expression or the sub-cellular localization of ABCB1 or ABCG2. Voruciclib stimulated the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner. Lastly, voruciclib exhibited a drug-induced apoptotic effect in ABCB1- or ABCG2- overexpressing cells. CONCLUSION: Voruciclib is currently a phase I clinical trial drug. Our findings strongly support its potential use in combination with conventional anti-cancer drugs for cancer chemotherapy.
Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Antineoplastic Agents/pharmacology , Benzopyrans/pharmacology , Drug Resistance, Neoplasm/drug effects , Imino Furanoses/pharmacology , Neoplasm Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Benzopyrans/chemistry , Binding Sites , Cell Line, Tumor , Cell Survival/drug effects , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Doxorubicin/pharmacology , HEK293 Cells , Humans , Imino Furanoses/chemistry , Mitoxantrone/pharmacology , Molecular Docking Simulation , Multidrug Resistance-Associated Proteins/metabolism , Mutation , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Paclitaxel/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Structure, TertiaryABSTRACT
OBJECTIVE: Glycogen in astrocyte processes contributes to maintenance of low extracellular glutamate and K+ concentrations around excitatory synapses. Sleep deprivation (SD), a common migraine trigger, induces transcriptional changes in astrocytes, reducing glycogen breakdown. We hypothesize that when glycogen utilization cannot match synaptic energy demand, extracellular K+ can rise to levels that activate neuronal pannexin-1 channels and downstream inflammatory pathway, which might be one of the mechanisms initiating migraine headaches. METHODS: We suppressed glycogen breakdown by inhibiting glycogen phosphorylation with 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and by SD. RESULTS: DAB caused neuronal pannexin-1 large pore opening and activation of the downstream inflammatory pathway as shown by procaspase-1 cleavage and HMGB1 release from neurons. Six-hour SD induced pannexin-1 mRNA. DAB and SD also lowered the cortical spreading depression (CSD) induction threshold, which was reversed by glucose or lactate supplement, suggesting that glycogen-derived energy substrates are needed to prevent CSD generation. Supporting this, knocking down the neuronal lactate transporter MCT2 with an antisense oligonucleotide or inhibiting glucose transport from vessels to astrocytes with intracerebroventricularly delivered phloretin reduced the CSD threshold. In vivo recordings with a K+ -sensitive/selective fluoroprobe, Asante Potassium Green-4, revealed that DAB treatment or SD caused a significant rise in extracellular K+ during whisker stimulation, illustrating the critical role of glycogen in extracellular K+ clearance. INTERPRETATION: Synaptic metabolic stress caused by insufficient glycogen-derived energy substrate supply can activate neuronal pannexin-1 channels as well as lower the CSD threshold. Therefore, conditions that limit energy supply to synapses (eg, SD) may predispose to migraine attacks, as suggested by genetic studies associating glucose or lactate transporter deficiency with migraine. Ann Neurol 2018;83:61-73.
Subject(s)
Brain Chemistry , Cortical Spreading Depression/genetics , Glycogen/metabolism , Sleep Deprivation/physiopathology , Animals , Arabinose/pharmacology , Astrocytes/drug effects , Astrocytes/metabolism , Connexins/drug effects , Connexins/metabolism , Energy Metabolism , Gene Knockdown Techniques , HMGB1 Protein/metabolism , Imino Furanoses/pharmacology , Injections, Intraventricular , Mice , Monocarboxylic Acid Transporters/antagonists & inhibitors , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/metabolism , Oligonucleotides, Antisense/pharmacology , Phloretin/pharmacology , Potassium/physiology , Sugar Alcohols/pharmacology , Vibrissae/innervationABSTRACT
The glucose polymer glycogen is a vital fuel reserve in the brain. The mediobasal hypothalamic energy sensor AMP-activated protein kinase (AMPK) maintains glucostasis via neurotransmitter mechanisms that suppress [γ-aminobutyric acid; GABA] or stimulate [nitric oxide; steroidogenic factor-1 (SF1)] counter-regulatory outflow. This study investigated whether glycogen-derived fuel supply is a critical screened variable in ventromedial hypothalamic nucleus (VMN) monitoring of neuro-metabolic stability during glucostasis and/or insulin (I)-induced hypoglycemia. Adult male rats were pretreated by intra-VMN infusion of the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) before sc vehicle or I injection. Western blot analyses of micropunch-dissected VMN tissue from euglycemic animals showed DAB augmentation of phosphoAMPK (pAMPK), neuronal nitric oxide synthase (nNOS), and SF-1, but not glutamate decarboxylase65/67 (GAD) protein. Combinatory DAB/I treatment did not further enhance AMPK activity but significantly amplified nNOS expression relative to DAB alone. Hypoglycemic stimulation of corticosterone, but not glucagon release was prevented by DAB Results imply that glycogen-derived substrate fuel provision represses VMN AMPK activity and neurotransmitter signals of metabolic deficiency. Progressive augmentation of nNOS protein by DAB/I versus DAB/V intimates that "fuel-inhibited" nitrergic neurons may exhibit increasing sensitivity to disrupted glycogen breakdown during glucoprivation versus glucostasis. nNOS and GAD reactivity to DAB/I, but not I implies that acute glycogen utilization during hypoglycemia may be sufficiently robust to avert effects on local metabolic sensory signaling. DAB/I upregulation of GAD alongside prevention of hypercorticosteronemia suggests that indicators of metabolic sufficiency may occur secondary to local compensatory adaptations to severe restriction of glucose-derived energy.
Subject(s)
Glycogen Phosphorylase/metabolism , Hypoglycemia/metabolism , Nitric Oxide Synthase Type I/metabolism , Ventromedial Hypothalamic Nucleus/metabolism , AMP-Activated Protein Kinase Kinases , Animals , Arabinose/pharmacology , Enzyme Inhibitors/pharmacology , Glutamate Decarboxylase/metabolism , Glycogen/metabolism , Glycogen Phosphorylase/antagonists & inhibitors , Imino Furanoses/pharmacology , Male , Nitric Oxide Synthase Type I/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , Rats , Rats, Sprague-Dawley , Sugar Alcohols/pharmacology , Ventromedial Hypothalamic Nucleus/drug effectsABSTRACT
Aberrant regulation of BCL-2 family members enables evasion of apoptosis and tumor resistance to chemotherapy. BCL-2 and functionally redundant counterpart, MCL-1, are frequently over-expressed in high-risk diffuse large B-cell lymphoma (DLBCL). While clinical inhibition of BCL-2 has been achieved with the BH3 mimetic venetoclax, anti-tumor efficacy is limited by compensatory induction of MCL-1. Voruciclib, an orally bioavailable clinical stage CDK-selective inhibitor, potently blocks CDK9, the transcriptional regulator of MCL-1. Here, we demonstrate that voruciclib represses MCL-1 protein expression in preclinical models of DLBCL. When combined with venetoclax in vivo, voruciclib leads to model-dependent tumor cell apoptosis and tumor growth inhibition. Strongest responses were observed in two models representing high-risk activated B-cell (ABC) DLBCL, while no response was observed in a third ABC model, and intermediate responses were observed in two models of germinal center B-cell like (GCB) DLBCL. Given the range of responses, we show that CIVO, a multiplexed tumor micro-dosing technology, represents a viable functional precision medicine approach for differentiating responders from non-responders to BCL-2/MCL-1 targeted therapy. These findings suggest that the combination of voruciclib and venetoclax holds promise as a novel, exclusively oral combination therapy for a subset of high-risk DLBCL patients.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzopyrans/pharmacology , Gene Expression Regulation/drug effects , Imino Furanoses/pharmacology , Lymphoma, Large B-Cell, Diffuse/drug therapy , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Antineoplastic Agents/therapeutic use , Benzopyrans/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cell Line, Tumor , Drug Synergism , Humans , Imino Furanoses/therapeutic use , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Sulfonamides/pharmacology , Sulfonamides/therapeutic useABSTRACT
Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC-rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU-positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate-mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α-cyano-4-hydroxy-cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. J. Cell. Physiol. 232: 986-995, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
Subject(s)
Cell Cycle , Cell Differentiation , Lactic Acid/metabolism , Oligodendroglia/cytology , Stem Cells/cytology , Animals , Arabinose/pharmacology , Cell Cycle/drug effects , Cell Death/drug effects , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Corpus Callosum/pathology , Cuprizone , Demyelinating Diseases/metabolism , Demyelinating Diseases/pathology , Female , Glucose/pharmacology , Imino Furanoses/pharmacology , Lactic Acid/pharmacology , Male , Mice, Inbred C57BL , Models, Biological , Monocarboxylic Acid Transporters/antagonists & inhibitors , Monocarboxylic Acid Transporters/metabolism , Myelin Sheath/drug effects , Myelin Sheath/metabolism , Stem Cells/drug effects , Stem Cells/metabolism , Sugar Alcohols/pharmacologyABSTRACT
Glycogen synthase (GS) and glycogen phosphorylase (GP) are the key enzymes that control, respectively, the synthesis and degradation of glycogen, a multi-branched glucose polymer that serves as a form of energy storage in bacteria, fungi and animals. An abnormal glycogen metabolism is associated with several human diseases. Thus, GS and GP constitute adequate pharmacological targets to modulate cellular glycogen levels by means of their selective inhibition. The compound 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) is a known potent inhibitor of GP. We studied the inhibitory effect of DAB, its enantiomer LAB, and 29 DAB derivatives on the activity of rat muscle glycogen phosphorylase (RMGP) and E. coli glycogen synthase (EcGS). The isoform 4 of sucrose synthase (SuSy4) from Solanum tuberosum L. was also included in the study for comparative purposes. Although these three enzymes possess highly conserved catalytic site architectures, the DAB derivatives analysed showed extremely diverse inhibitory potential. Subtle changes in the positions of crucial residues in their active sites are sufficient to discriminate among the structural differences of the tested inhibitors. For the two Leloir-type enzymes, EcGS and SuSy4, which use sugar nucleotides as donors, the inhibitory potency of the compounds analysed was synergistically enhanced by more than three orders of magnitude in the presence of ADP and UDP, respectively. Our results are consistent with a model in which these compounds bind to the subsite in the active centre of the enzymes that is normally occupied by the glucosyl residue which is transferred between donor and acceptor substrates. The ability to selectively inhibit the catalytic activity of the key enzymes of the glycogen metabolism may represent a new approach for the treatment of disorders of the glycogen metabolism.
Subject(s)
Arabinose/chemistry , Arabinose/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glycogen/metabolism , Imino Furanoses/chemistry , Imino Furanoses/pharmacology , Sugar Alcohols/chemistry , Sugar Alcohols/pharmacology , Animals , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/metabolism , Glucosyltransferases/antagonists & inhibitors , Glucosyltransferases/metabolism , Glycogen Phosphorylase/antagonists & inhibitors , Glycogen Phosphorylase/metabolism , Glycogen Synthase/antagonists & inhibitors , Glycogen Synthase/metabolism , Molecular Docking Simulation , Rats , Solanum tuberosum/drug effects , Solanum tuberosum/enzymology , Solanum tuberosum/metabolismABSTRACT
Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and ß-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth.
Subject(s)
Cell Wall/metabolism , Endosperm/metabolism , Hordeum/metabolism , Starch/metabolism , Arabinose/pharmacology , Cell Wall/drug effects , Endosperm/drug effects , Glycoside Hydrolases/antagonists & inhibitors , Glycoside Hydrolases/metabolism , Hordeum/growth & development , Imino Furanoses/pharmacology , Immunoblotting , Plant Proteins/antagonists & inhibitors , Plant Proteins/metabolism , Seedlings/genetics , Seedlings/growth & development , Seedlings/metabolism , Sugar Alcohols/pharmacology , Xylans/metabolismABSTRACT
The fungus Thelonectria discophora SANK 18292 produces the iminosugar nectrisine, which has a nitrogen-containing heterocyclic 5-membered ring and acts as a glycosidase inhibitor. In our previous study, an oxidase (designated NecC) that converts 4-amino-4-deoxyarabinitol to nectrisine was purified from T. discophora cultures. However, the genes required for nectrisine biosynthesis remained unclear. In this study, the nectrisine biosynthetic gene cluster in T. discophora was identified from the contiguous genome sequence around the necC gene. Gene disruption and complementation studies and heterologous expression of the gene showed that necA, necB, and necC could be involved in nectrisine biosynthesis, during which amination, dephosphorylation, and oxidation occur. It was also demonstrated that nectrisine could be produced by recombinant Escherichia coli coexpressing the necA, necB, and necC genes. These findings provide the foundation to develop a bacterial production system for nectrisine or its intermediates through genetic engineering. IMPORTANCE: Iminosugars might have great therapeutic potential for treatment of many diseases. However, information on the genes for their biosynthesis is limited. In this study, we report the identification of genes required for biosynthesis of the iminosugar nectrisine in Thelonectria discophora SANK 18292, which was verified by disruption, complementation, and heterologous expression of the genes involved. We also demonstrate heterologous production of nectrisine by recombinant E. coli, toward developing an efficient production system for nectrisine or its intermediates through genetic engineering.
Subject(s)
Genes, Fungal , Hypocreales/genetics , Imino Furanoses/isolation & purification , Imino Furanoses/metabolism , Amination , Escherichia coli/genetics , Genetic Complementation Test , Genetic Engineering , Genome, Fungal , Hypocreales/metabolism , Imino Furanoses/chemistry , Multigene Family , Oxidation-ReductionABSTRACT
A formal enantioselective synthesis of nectrisine, a potent α-glucosidase inhibitor, was carried out starting from butadiene monoepoxide through a synthetic sequence involving enantioselective allylic substitution, cross-metathesis, dihydroxylation, and cyclization.
Subject(s)
Glycoside Hydrolase Inhibitors/chemical synthesis , Imino Furanoses/chemical synthesis , Palladium/chemistry , Amination , Amines/chemical synthesis , Butadienes , Catalysis , Cyclization , Hydroxylation , Indicators and Reagents , Magnetic Resonance Spectroscopy , StereoisomerismABSTRACT
5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme that catalyzes the hydrolysis of the N-ribosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). MTAN activity has been linked to quorum sensing pathways, polyamine biosynthesis, and adenine salvage. Previously, the coding sequence of Rv0091 was annotated as a putative MTAN in Mycobacterium tuberculosis. Rv0091 was expressed in Escherichia coli, purified to homogeneity, and shown to be a homodimer, consistent with MTANs from other microorganisms. Substrate specificity for Rv0091 gave a preference for 5'-deoxyadenosine relative to MTA or SAH. Intrinsic kinetic isotope effects (KIEs) for the hydrolysis of [1'-(3)H], [1'-(14)C], [5'-(3)H2], [9-(15)N], and [7-(15)N]MTA were determined to be 1.207, 1.038, 0.998, 1.021, and 0.998, respectively. A model for the transition state structure of Rv0091 was determined by matching KIE values predicted via quantum chemical calculations to the intrinsic KIEs. The transition state shows a substantial loss of C1'-N9 bond order, well-developed oxocarbenium character of the ribosyl ring, and weak participation of the water nucleophile. Electrostatic potential surface maps for the Rv0091 transition state structure show similarity to DADMe-immucillin transition state analogues. DADMe-immucillin transition state analogues showed strong inhibition of Rv0091, with the most potent inhibitor (5'-hexylthio-DADMe-immucillinA) displaying a Ki value of 87 pM.