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1.
Molecules ; 25(3)2020 Feb 03.
Article in English | MEDLINE | ID: mdl-32028604

ABSTRACT

To minimize treatment toxicities, recent anti-cancer research efforts have switched from broad-based chemotherapy to targeted therapy, and emerging data show that altered cellular metabolism in cancerous cells can be exploited as new venues for targeted intervention. In this study, we focused on, among the altered metabolic processes in cancerous cells, altered glycosylation due to its documented roles in cancer tumorigenesis, metastasis and drug resistance. We hypothesize that the enzymes required for the biosynthesis of UDP-hexoses, glycosyl donors for glycan synthesis, could serve as therapeutic targets for cancers. Through structure-based virtual screening and kinetic assay, we identified a drug-like chemical fragment, GAL-012, that inhibit a small family of UDP-hexose pyrophosphorylases-galactose pyro-phosphorylase (GALT), UDP-glucose pyrophosphorylase (UGP2) and UDP-N-acetylglucosamine pyrophosphorylase (AGX1/UAP1) with an IC50 of 30 µM. The computational docking studies supported the interaction of GAL-012 to the binding sites of GALT at Trp190 and Ser192, UGP2 at Gly116 and Lys127, and AGX1/UAP1 at Asn327 and Lys407, respectively. One of GAL-012 derivatives GAL-012-2 also demonstrated the inhibitory activity against GALT and UGP2. Moreover, we showed that GAL-012 suppressed the growth of PC3 cells in a dose-dependent manner with an EC50 of 75 µM with no effects on normal skin fibroblasts at 200 µM. Western blot analysis revealed reduced expression of pAKT (Ser473), pAKT (Thr308) by 77% and 72%, respectively in the treated cells. siRNA experiments against the respective genes encoding the pyrophosphorylases were also performed and the results further validated the proposed roles in cancer growth inhibition. Finally, synergistic relationships between GAL-012 and tunicamycin, as well as bortezomib (BTZ) in killing cultured cancer cells were observed, respectively. With its unique scaffold and relatively small size, GAL-012 serves as a promising early chemotype for optimization to become a safe, effective, multi-target anti-cancer drug candidate which could be used alone or in combination with known therapeutics.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , UTP-Hexose-1-Phosphate Uridylyltransferase/antagonists & inhibitors , UTP-Hexose-1-Phosphate Uridylyltransferase/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Discovery/methods , Gene Knockout Techniques , Glycosylation , Humans , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Polysaccharides/metabolism , UTP-Hexose-1-Phosphate Uridylyltransferase/genetics
2.
Pediatr Res ; 31(5): 512-5, 1992 May.
Article in English | MEDLINE | ID: mdl-1603630

ABSTRACT

The sp act of galactose-1-phosphate uridyltransferase has been measured in individual regions of adult rat brain to see if site-specific differences in enzyme activity can aid in the understanding of brain abnormalities observed in well-treated galactosemic patients. The sp act in the cerebellum, brain stem, and midbrain were higher than in the cortex, hippocampus, and striatum. Activity in the cerebellum was 2-fold greater than that found in the cortex. Steady state levels of mRNA of the enzyme in the cerebellum were twice that of the cortex corresponding to the ratio of enzyme sp act in the two regions. Measurement of the kinetic parameters in tissue from the cerebellum and cortex revealed that the regional specificity in enzyme activity observed in the brain represents differences in the Vmax. Inhibition of the enzyme by uridine and uridine triphosphate was essentially the same for all regions and was not influenced by the 2-fold differences observed in the levels of enzyme. Inhibition by uridine was significantly greater than that for uridine triphosphate.


Subject(s)
Brain/enzymology , UTP-Hexose-1-Phosphate Uridylyltransferase/metabolism , Animals , Brain/metabolism , Cerebellum/enzymology , Cerebral Cortex/enzymology , Galactosemias/enzymology , Kinetics , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains , Tissue Distribution , UTP-Hexose-1-Phosphate Uridylyltransferase/antagonists & inhibitors
3.
Pediatr Res ; 30(3): 222-6, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1945559

ABSTRACT

Uridine-containing sugar nucleotides, uridine diphosphate (UDP)-glucose and UDPgalactose are important intermediates in galactose metabolism, and tissue UDPgalactose may be a salient factor in the etiology of the long-term clinical manifestations of patients with galactose-1-phosphate uridyltransferase deficient galactosemia. Because uridine and uridine nucleotides such as uridine triphosphate (UTP) are known inhibitors of rat hepatic transferase, we have examined the effects of these compounds on the activity of the enzyme in homogenates of rat brain and ovary which are target organs of galactose toxicity in classical galactosemia. In addition, the concerted effect of uridine and UTP together on hepatic transferase has been assessed. These investigations have been prompted by considerations that uridine administration may have a therapeutic role in the long-term treatment of classical galactosemia. Both uridine and UTP have been found to be potent inhibitors of brain and ovarian transferase activity. Brain enzyme activity is more sensitive to these compounds than is that of the ovary. They are competitive inhibitors of UDPglucose in both newborn and adult brain enzyme preparations with a ki of 0.15 to 0.20 mM. Uridine and UTP at low concentrations were found to have an additive effect on rat hepatic transferase activity, which is especially significant in that uridine administration is known to increase hepatic UTP concentration. These findings suggest judicious caution should be used in giving uridine to patients with genetically limited transferase activity because the possibility exists of inhibiting small amounts of residual enzyme in the tissues of affected subjects.


Subject(s)
UTP-Hexose-1-Phosphate Uridylyltransferase/antagonists & inhibitors , Uridine Triphosphate/pharmacology , Uridine/pharmacology , Animals , Animals, Newborn , Brain/drug effects , Brain/enzymology , Female , Galactose/toxicity , Galactosemias/drug therapy , Galactosemias/enzymology , Ovary/drug effects , Ovary/enzymology , Pregnancy , Rats
4.
Anal Biochem ; 187(1): 104-8, 1990 May 15.
Article in English | MEDLINE | ID: mdl-2142585

ABSTRACT

UDP-GlcN was synthesized from GlcN and UTP by a two step hollow fiber enzyme reactor method. In step 1, GlcN was converted to GlcN 6-P and then to GlcN 1-P by hexokinase and phosphoglucomutase, respectively, and UTP was used as the phosphate donor. In step 2, GlcN 1-P was converted to UDP-GlcN by UDP glucose pyrophosphorylase. All the enzymes required for the synthesis of UDP-GlcN were enclosed in hollow fiber bundles which allow for the free diffusion of substrates and products across the membranes to and from the enzymes, allow for the reutilization of the enzymes, and simplify the isolation of the product, UDP-GlcN. We show that both UTP and GlcN 6-P are inhibitors of the yeast UDPG pyrophosphorylase and therefore their concentrations must be regulated to obtain maximum yields of UDP-GlcN. The UDP-GlcN produced can be N-acetylated with [14C]acetic anhydride to produce UDP-[14C]GlcNAc. This method can also be used to synthesize [32P]UDP-GlcN and [32P]UDP-GlcNAc from [alpha-32P]UTP and GlcN 1-P.


Subject(s)
Uridine Diphosphate Glucose/chemical synthesis , Uridine Diphosphate Sugars/chemical synthesis , Acetylglucosamine/metabolism , Chromatography, High Pressure Liquid , Glucosamine/analogs & derivatives , Hexokinase/metabolism , Phosphoglucomutase/metabolism , Substrate Specificity , UTP-Hexose-1-Phosphate Uridylyltransferase/antagonists & inhibitors , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Triphosphate/metabolism
5.
Enzyme ; 42(1): 53-60, 1989.
Article in English | MEDLINE | ID: mdl-2550217

ABSTRACT

Uridine sugar nucleotides are important intermediates in galactose metabolism and may play a role in the long-term galactose toxicity in human galactose-1-phosphate uridyltransferase deficiency galactosemia. Since administration of uridine, a precursor of uridine nucleotides, has been considered as a therapeutic measure, we have investigated the effects of this compound on the activity of rat hepatic transferase. Uridine has been found to be an inhibitor of the enzyme in in vitro studies and to cause an increase in galactose-1-phosphate in liver perfused with galactose which is consistent with physiologic inhibition of the enzyme. Uridine is a partial linear competitive inhibitor of UDPglucose and an uncompetitive inhibitor of galactose-1-phosphate. These findings suggest caution should be applied in giving the compound to subjects with genetically limited transferase activity because of the possibility of inhibiting the small amount of residual enzyme.


Subject(s)
Liver/enzymology , Nucleotidyltransferases/metabolism , UTP-Hexose-1-Phosphate Uridylyltransferase/metabolism , Uridine/pharmacology , Aging/metabolism , Animals , Galactose/metabolism , Galactosephosphates/metabolism , Glucosephosphates/metabolism , Liver/metabolism , Male , Rats , Rats, Inbred Strains , UTP-Hexose-1-Phosphate Uridylyltransferase/antagonists & inhibitors , Uridine Diphosphate Glucose/pharmacology
6.
Vopr Med Khim ; 31(4): 65-70, 1985.
Article in Russian | MEDLINE | ID: mdl-4049789

ABSTRACT

Administration of galactose into young rats within an early postnatal period led to alteration in activity of some enzymes involved in utilization of galactose (galactose-1-phosphaturidyl transferase, galactose-6-phosphate dehydrogenase etc) for a long period of the animals life. This stable alteration in activity of adaptive enzymes was characterized as the enzymatic imprinting. After administration of galactose into neonatal animals synthesis of RNA, matrix activity of chromatin, activities of DNA-dependent RNA polymerase and RNA-dependent DNA polymerase were shown to increase in liver tissue of these animals. These alterations are considered as a possible basis for the stable alterations in the genes expression. The elevated activities of DNA-dependent RNA-polymerase and reverse transcriptase were maintained within a long period of the animals life.


Subject(s)
Animals, Newborn , Galactose/pharmacology , Galactosemias/enzymology , Animals , DNA/biosynthesis , DNA-Directed RNA Polymerases/biosynthesis , DNA-Directed RNA Polymerases/genetics , Enzyme Induction/drug effects , Galactosemias/genetics , Gene Expression Regulation , Glucosephosphate Dehydrogenase/biosynthesis , Glucosephosphate Dehydrogenase/genetics , Liver/enzymology , Rats , UTP-Hexose-1-Phosphate Uridylyltransferase/antagonists & inhibitors , UTP-Hexose-1-Phosphate Uridylyltransferase/genetics
8.
Pediatr Res ; 14(5): 729-34, 1980 May.
Article in English | MEDLINE | ID: mdl-6247691

ABSTRACT

In the erythrocytes of a patient with the clinical symptoms of galactosemia, a galactose-1-phosphate uridylyltransferase with abnormal kinetics was observed. Under standard assay conditions, the uridylyltransferase activity was almost normal initially and became completely inactivated within 30 min. The abnormal kinetics could be ascribed to a product inhibition by glucose 1-phosphate. The inhibition was produced by a variety of sugar phosphates, the most potent of which proved to be glucose 1-phosphate, mannose 1-phosphate, and fructose 6-phosphate. The variant galactose-1-phosphate uridylyltransferase was further characterized by a lowered affinity towards galactose 1-phosphate, non-Michaelis-Menten kinetics towards UDP-glucose, an increased thermal stability, and complete inactivity upon Cellogel electrophoresis.


Subject(s)
Erythrocytes/enzymology , Galactosemias/enzymology , Nucleotidyltransferases/metabolism , UTP-Hexose-1-Phosphate Uridylyltransferase/metabolism , Child, Preschool , Galactosemias/blood , Galactosemias/genetics , Glucosephosphates/pharmacology , Humans , Kinetics , Male , Pedigree , UTP-Hexose-1-Phosphate Uridylyltransferase/antagonists & inhibitors
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