Inhibition of IMP Dehydrogenase by Mycophenolic Acid in Molt F4 Human Malignant Lymphoblasts

1994 ◽  
Vol 31 (2) ◽  
pp. 174-180 ◽  
Author(s):  
Elisabet H Stet ◽  
Ronney A De Abreu ◽  
Jos P M Bökkerink ◽  
Lambert H J Lambooy ◽  
Trade M Vogels-Mentink ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Growth ◽  
Mycophenolic Acid ◽  
De Novo ◽  
Specific Inhibitor ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Inosine Monophosphate Dehydrogenase ◽  
De Novo Synthesis ◽  
Rate Limiting

The effects of inhibition of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide de novo synthesis, on cell growth, cell viability, endogenous nucleotide concentrations and concentrations of extracellular nucleosides and bases were studied in Molt F4 human malignant lymphoblasts. Mycophenolic acid (MPA) was used as a specific inhibitor of the enzyme activity. IMPDH activity was maximally inhibited with 0–5 μM MPA. After a 2 h exposure of the cells to 0–5 μM MPA, guanine nucleotides were depleted to approximately 50% of control values, whereas 5-phosphoribosyl-1-pyrophosphate levels increased to approximately 200%. Under these conditions, cytotoxicity became obvious after 24 h. Depletion of guanine nucleotides and cytotoxicity were prevented by addition of guanosine to MPA treatment. Daily supplements of guanosine were required to prevent MPA cytotoxicity during the entire incubation period of 72 h. We conclude that depletion of guanine nucleotides, induced by treatment with MPA, induces a severe and rapid cytotoxicity in Molt F4 cells.

scholarly journals GTP metabolic reprogramming by IMPDH2: unlocking cancer cells’ fuelling mechanism

2020 ◽  
Vol 168 (4) ◽  
pp. 319-328 ◽  
Author(s):  
Satoshi Kofuji ◽  
Atsuo T Sasaki
Keyword(s):  
De Novo ◽  
Current Knowledge ◽  
Metabolic Reprogramming ◽  
Inosine Monophosphate Dehydrogenase ◽  
Metabolic Demand ◽  
Adaptive Mechanisms ◽  
Key Aspects ◽  
Polymerase I ◽  
Rate Limiting

Abstract Growing cells increase multiple biosynthetic processes in response to the high metabolic demands needed to sustain proliferation. The even higher metabolic requirements in the setting of cancer provoke proportionately greater biosynthesis. Underappreciated key aspects of this increased metabolic demand are guanine nucleotides and adaptive mechanisms to regulate their concentration. Using the malignant brain tumour, glioblastoma, as a model, we have demonstrated that one of the rate-limiting enzymes for guanosine triphosphate (GTP) synthesis, inosine monophosphate dehydrogenase-2 (IMPDH2), is increased and IMPDH2 expression is necessary for the activation of de novo GTP biosynthesis. Moreover, increased IMPDH2 enhances RNA polymerase I and III transcription directly linking GTP metabolism to both anabolic capacity as well as nucleolar enlargement historically observed as associated with cancer. In this review, we will review in detail the basis of these new discoveries and, more generally, summarize the current knowledge on the role of GTP metabolism in cancer.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide

2021 ◽  
Author(s):  
Yao Li ◽  
Li Yi ◽  
Sipeng Cheng ◽  
Yongshan Wang ◽  
Jiongjiong Wang ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Active Metabolite ◽  
Canine Distemper Virus ◽  
De Novo ◽  
Specific Inhibitor ◽  
Vero Cells ◽  
Canine Distemper ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide ◽  
Rate Limiting

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

scholarly journals Host cell amplification of nutritional stress contributes to persistence in Chlamydia trachomatis

2021 ◽  
Author(s):  
Nick D. Pokorzynski ◽  
REY CARABEO
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
De Novo ◽  
Host Cells ◽  
Guanine Nucleotide ◽  
Nucleotide Synthesis ◽  
Rate Limiting Step ◽  
Metabolic Consequence ◽  
Rate Limiting ◽  
Insight Into

Persistence, a viable, but non-replicating state has been implicated in diseases caused by Chlamydia trachomatis. Multiple nutritional stressors produce a superficially similar "persistent" state, yet no systematic comparison has been made to determine their likeness. We employed host-pathogen dual RNA-sequencing under both iron- and tryptophan-starved conditions to gain insight into chlamydial persistence and identify contributions by the host cell. Analysis of the transcriptome of iron- or tryptophan-starved Chlamydia revealed a common "core" component and a stress-specific "accessory" subset. Despite the overall transcriptomic differences of host cells starved for either iron or tryptophan, both stressors induced persistence. A common metabolic consequence of the stressors was a reduction in intracellular GTP levels. Mizoribine inhibition of IMDPH1, which catalyzes the rate-limiting step in de novo guanine nucleotide synthesis reproduced to a similar extent GTP depletion, and inhibited chlamydial growth as expected for a pathogen that is auxotrophic for GTP. Thus, the reduction of guanine nucleotide synthesis manifests amplification of either iron or tryptophan starvation contributing to persistence. These findings illustrate that a nutritionally stressed host cell remains effective in arresting growth of Chlamydia by targeting metabolic pathways required by the pathogen.

Thymidylate kinase from Acetabularia. II. Regulation during the life cycle

1983 ◽  
Vol 64 (1) ◽  
pp. 27-36
Author(s):  
E.J. de Groot ◽  
H.G. Schweiger
Keyword(s):  
Enzyme Activity ◽  
Life Cycle ◽  
Kinase Activity ◽  
De Novo ◽  
Nuclear Genome ◽  
De Novo Synthesis ◽  
Cell Extracts ◽  
Phase Regulation ◽  
Generative Phase ◽  
Low Activity

The activity of dTMP kinase (EC 2.7.4.9) was estimated during the development of Acetabularia. Enzyme activity was increased at the beginning of the generative phase. Regulation of the dTMP kinase activity was observed even in the absence of the nucleus. More than 30 days after enucleation the enzyme activity increased two- to threefold. The increase in activity was inhibited by puromycin and cycloheximide but not by rifampicin and chloramphenicol. These results indicate that the enzyme is coded by the nuclear genome and translated on 80 S ribosomes. From mixing experiments with low-activity and high-activity cell extracts it is concluded that the regulation is due to de novo synthesis of the enzyme.

scholarly journals IMMU-43. POLYAMINE METABOLISM REGULATES MYELOID IMMUNE SUPPRESSION IN GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi128-vi128
Author(s):  
Jason Miska ◽  
Catalina Lee Chang ◽  
Aida Rashidi ◽  
Yu Han ◽  
Aurora Lopez-Rosas ◽  
...  
Keyword(s):  
Immune Suppression ◽  
De Novo ◽  
Specific Inhibitor ◽  
Suppressor Cells ◽  
Polyamine Metabolism ◽  
Arginase 1 ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Abstract Tumor-associated myeloid cells, which consist of tumor associated macrophages and myeloid-derived suppressor cells (MDSCs), make up a majority of cellular infiltrates in glioma. Glioma infiltrating MDSCs highly express arginase-1 (Arg-1), a catabolic enzyme thought to deplete arginine from the tumor microenvironment. Despite being a well-known marker of immunosuppressive cells, the metabolic reasons for this choice are not clear. Examination of MDSC phenotype in murine glioma models using: RNA-seq, bulk metabolomics, and Carbon-13 arginine flux revealed that two separate pathways of arginine catabolism converge on the generation of ornithine. Ornithine is the prerequisite substrate for the de-novo generation of polyamines, a group of nitrogen-rich metabolites with foundational importance to all mammalian, bacterial, and plant biology. Importantly, we found that the rate-limiting step of polyamine generation, ornithine decarboxylase 1 (ODC1), is dramatically upregulated by glioma infiltrating MDSCs, suggesting de-novo polyamine generation is important for MDSC function. Treatment with a specific inhibitor of de-novo polyamine synthesis, difluoromethylornithine (DMFO), inhibited the immunosuppressive function of in-vitro generated glioma associated MDSCs. However, DFMO only exerted effects before differentiation, as DFMO treatment post-generation did not change their suppressive functions. This suggests that the generation of the polyamine pool is critical to immune suppression by MDSCs. Interestingly the expression of the rate limiting step of polyamine degradation (SAT1) is inversely correlated with (ODC1) during MDSC differentiation, suggesting that utilization of this polyamine pool may be required for the suppressive functions of these cells. Inhibition of SAT1 after MDSC generation blunted MDSC mediated T-cell suppression. The results of this study show that the role of arginine metabolism in tumor infiltrating MDSCs is to generate pools of polyamines which maintain MDSC function in glioma. Therapeutic targeting of this pathway may be a novel and powerful tool to combat immunosuppression in glioma.

Regulation of PTH receptor-adenylate cyclase system of canine kidney: influence of Mn2+ on effects of Ca2+, PTH, and GTP

1982 ◽  
Vol 242 (5) ◽  
pp. F457-F462
Author(s):  
E. Bellorin-Font ◽  
J. Tamayo ◽  
K. J. Martin
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Metal Ions ◽  
Guanine Nucleotides ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Adenylate Cyclase System ◽  
Guanine Nucleotide ◽  
Hormone Binding ◽  
Stimulation Of

Metal ions play important roles in the regulation of the activation of adenylate cyclase. Previous studies have suggested that an important site of action of metal ions is at or closely related to the nucleotide regulatory protein. The present studies examine the nature of the regulation of enzyme activity by divalent cations and the influence of Mn2+ on hormone binding and stimulation of adenylate cyclase. Studies were performed in canine renal cortical membranes. Substitution of Mg2+ by Mn2+ was associated with a progressive decline in the ability of GTP or PTH to stimulate adenylate cyclase activity. Mn2+ did not alter specific binding of an iodinated PTH analogue. However, in spite of the loss of guanine nucleotide stimulation of enzyme activity, the effects of guanine nucleotide on PTH binding were not altered in the presence of Mn2+. Substitution of Mg2+ by Mn2+ abolished the inhibitory effect of Ca2+ on basal adenylate cyclase activity. Similarly, the effects of GTP or PTH to enhance the inhibitory effects of Ca2+ on enzyme activity were abolished in the presence of Mn2+. Since Mg2+ and Ca2+ compete for a common allosteric site and Mn2+ abolished the effects of these cations, it would appear that Mn2+ also competes for the binding site of Mg2+ and Ca2+. The present studies demonstrating that Mn2+ does not affect hormone binding or the actions of guanine nucleotides on hormone binding yet totally eliminates the effect of GTP on enzyme activity indicate that the effect of Mn2+ occurs at the level of the interactions of the nucleotide regulatory component with the catalytic unit. In addition, these data suggest that there are two functionally distinct sites of guanine nucleotides with different ionic requirements.

scholarly journals Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations

2017 ◽  
Vol 28 (20) ◽  
pp. 2600-2608 ◽  
Author(s):  
Sajitha A. Anthony ◽  
Anika L. Burrell ◽  
Matthew C. Johnson ◽  
Krisna C. Duong-Ly ◽  
Yin-Ming Kuo ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Catalytic Activity ◽  
Mammalian Cells ◽  
Substrate Affinity ◽  
Guanine Nucleotide ◽  
Inosine Monophosphate Dehydrogenase ◽  
Biosynthetic Enzyme ◽  
Purine Nucleotides ◽  
Macromolecular Assemblies ◽  
Catalytically Active

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear, but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas guanosine triphosphate (GTP) promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and nonassembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.

scholarly journals Tight links between adenine and guanine nucleotide pools in mouse pancreatic islets: a study with mycophenolic acid

1997 ◽  
Vol 324 (2) ◽  
pp. 467-471 ◽  
Author(s):  
Philippe DETIMARY ◽  
Changqing XIAO ◽  
Jean-Claude HENQUIN
Keyword(s):  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Mycophenolic Acid ◽  
Adenine Nucleotide ◽  
Guanine Nucleotides ◽  
Specific Role ◽  
Guanine Nucleotide ◽  
Isolated Pancreatic Islets ◽  
Nucleotide Pools

Glucose metabolism in pancreatic B-cells leads to an increase in the ATP/ADP ratio that might participate in the regulation of insulin secretion. Good correlations have also been observed between guanine nucleotide levels in isolated pancreatic islets and insulin secretion. To assess whether guanine nucleotides have a specific role in stimulus–secretion coupling, their concentration should be modified selectively. This was attempted by culturing mouse islets overnight in the presence of mycophenolic acid (MPA), an inhibitor of GMP synthesis at the level of IMP dehydrogenase. The drug (25–50 μg/ml) did not affect the insulin content but decreased the GTP content of the islets and inhibited insulin secretion during subsequent incubation in the presence of 15 mM glucose. However, MPA also decreased the ATP/ADP ratio in the islets. The addition of guanine to the culture medium (to stimulate the salvage pathway of GTP synthesis) restored normal GTP levels, corrected the ATP/ADP ratio and partly prevented the inhibition of insulin release. In contrast, attempts to stimulate ATP synthesis specifically (by provision of adenine or adenosine) failed to reverse any of the effects of MPA. It is concluded that guanine and adenine nucleotide pools are tightly linked and cannot be specifically affected by MPA in pancreatic islet cells, probably because of the activity of nucleoside diphosphate kinase and because of the role of GTP in several reactions leading to adenine nucleotide generation. Contrary to previous claims, MPA is not an adequate tool for evaluating a specific role of guanine nucleotides in the control of insulin secretion.

scholarly journals Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione

2000 ◽  
Vol 346 (2) ◽  
pp. 545-552 ◽  
Author(s):  
Kai LÜERSEN ◽  
Rolf D. WALTER ◽  
Sylke MÜLLER
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
De Novo ◽  
Specific Inhibitor ◽  
De Novo Synthesis ◽  
Glutamylcysteine Synthetase ◽  
Infected Rbcs ◽  
Erythrocytic Cycle ◽  
Gsh Metabolism

During the erythrocytic cycle, Plasmodium falciparum is highly dependent on an adequate thiol status for its survival. Glutathione reductase as well as de novo synthesis of GSH are responsible for the maintenance of the intracellular GSH level. The first and rate-limiting step of the synthetic pathway is catalysed by γ-glutamylcysteine synthetase (γ-GCS). Using L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of the γ-GCS, we show that the infection with P. falciparum causes drastic changes in the GSH metabolism of red blood cells (RBCs). Infected RBCs lose GSH at a rate 40-fold higher than non-infected RBCs. The de novo synthesis of the tripeptide was found to be essential for parasite survival. GSH depletion by BSO inhibits the development of P. falciparum with an IC50 of 73 μM. The effect of the drug is abolished by supplementation with GSH or GSH monoethyl ester. Our studies demonstrate that the plasmodicidal effect of the inhibitor BSO does not depend on its specificity towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite GSH in the P. falciparum-infected RBCs. Therefore the depletion of GSH is proposed as a chemotherapeutic strategy for malaria, and γ-GCS is proposed as a potential drug target.

scholarly journals CTP synthase forms cytoophidia in archaea

2019 ◽  
Author(s):  
Shuang Zhou ◽  
Hua Xiang ◽  
Ji-Long Liu
Keyword(s):  
De Novo ◽  
Stimulated Emission ◽  
Metabolic Enzyme ◽  
De Novo Synthesis ◽  
Low Salt ◽  
Ctp Synthase ◽  
Intracellular Compartments ◽  
Domains Of Life ◽  
Haloarcula Hispanica ◽  
Rate Limiting

AbstractCTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of de novo synthesis of the nucleotide CTP. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures termed cytoophidia in bacteria and eukaryotes. However, it remains unknown whether cytoophidia exist in archaea, the third domain of life. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaeal cells. Under stimulated emission depletion (STED) microscopy, we find that some HhCTPS compartments have elongated filamentous structures, resembling cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. Moreover, overexpression of CTPS or glutamine analog treatment promotes cytoophidium assembly in H. hispanica. Our study reveals that CTPS forms cytoophidia in all three domains of life, suggesting that this is an ancient property of CTPS.

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