ABSTRACT
Anti-HIV activity and cytotoxicity were tested for novel phosphonate derivatives of AZT, d4T and ddA. For d4T phosphonate derivatives the most active was 2',3'-Dideoxy-2',3'-didehydrothymidine 5'-isopropylphosphite and among the AZT phosphonate derivatives highest activity was shown by 2',3'-Dideoxy-3'-azidothymidine 5'-cyclohexylphosphite.
Subject(s)
Anti-HIV Agents/pharmacology , Dideoxynucleosides/pharmacology , Anti-HIV Agents/toxicity , Cell Line , Cell Survival/drug effects , Dideoxyadenosine/analogs & derivatives , Dideoxynucleosides/toxicity , HIV Core Protein p24/analysis , HIV-1/drug effects , HIV-1/physiology , Humans , Immunoenzyme Techniques , Organophosphonates/pharmacology , Organophosphonates/toxicity , Stavudine/analogs & derivatives , Virus Replication/drug effects , Zidovudine/analogs & derivativesABSTRACT
Bovine tryptophanyl-tRNA synthetase (E.C.6.1.1.2) lacking Zn2+ ions removed by chelation with phosphonate analog of P1,P4-bis-(5'-adenosyl)tetraphosphate (Ap4A) was obtained (E-Zn). E-Zn lost the ability to form tryptophanyl adenylate, however it hydrolyses ATP to ADP and further on to AMP and Pi. GTP serves as a substrate with Km approximately 0.6 mM. It is proposed that the hydrolysable nucleotides bind to a nucleotide binding site(s) distinguishable from the substrate (catalytic) ones. After incubation of E-Zn with Zn2+ and Mg2+ the initial catalytic activity (ATP-PPi exchange and amino-acylation reactions) is restored whereas the hydrolytic activity becomes fully suppressed.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Tryptophan-tRNA Ligase/metabolism , Zinc , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Catalysis , Cattle , Dinucleoside Phosphates , Hydrolysis , Kinetics , Pancreas/enzymology , Phosphates/metabolismABSTRACT
The modification of tyrosine residues of the human placenta DNA-polymerase alpha by N-acetylimidazole was investigated. The poly(dT)-template and the r(pA)10-primer a each added separately or simultaneously do not influence the rate of enzyme inactivation. In the presence of poly(dT)-r(pA)10 no effect of dCTP and dTTP (noncomplementary to template) and of dAMP and dADP (complementary to template) on the rate and the level of the enzyme inactivation was found. However dATP revealed practically complete protection. Orthophosphate, pyrophosphate each taken separately do not influence the rate of enzyme inactivation with this reagent. The presence of dADP with either ortho- or pyrophosphate, or dAMP with the one of these ligands leads to half protective action in comparison with dATP. Imidazolides of phosphonoacetic acid and 5'-adenylyl++ 1(phosphonoacetic acid) do not inactivate DNA-polymerase alpha from human placenta and the Klenov fragment of DNA-polymerase I from E. coli. All data obtained allow to suggest that the tyrosine residue in the dNTP binding site of DNA-polymerase reveals stacking with the nucleotide only if dNTP is complementary to the template.
Subject(s)
DNA Polymerase II/metabolism , Imidazoles , Sulfhydryl Reagents , Deoxyadenine Nucleotides , Diphosphates , Escherichia coli/enzymology , Female , Humans , Kinetics , Placenta/enzymology , Pregnancy , Tyrosine/metabolismABSTRACT
The reaction of pyrophosphorolysis catalyzed by Escherichia coli DNA polymerase I Klenov fragment, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and AMV reverse transcriptase was studied. Some pyrophosphate (PPi) analogs were taken as low molecular weight substrates. It was shown that only imidodiphosphonic acid acted as the PPi substrate analog for the reactions catalyzed by DNA polymerases I and alpha, both imidodiphosphonic acid and methylenediphosphonic acid were active in the case of DNA polymerase beta and reverse transcriptase. Other analogs tested were neither nucleotide residue acceptors, nor inhibitors of the pyrophosphorolysis reaction with PPi. The abilities of some PPi analogs to inhibit the DNA elongation catalyzed by reverse transcriptase were investigated. The principles of specificity of low molecular substrates recognition by DNA polymerases and some problems concerning the mechanisms of DNA synthesis inhibition by PPi analogues are discussed.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Diphosphates/metabolism , Animals , Catalysis , Cattle , Chromatography, Thin Layer , DNA/metabolism , Escherichia coli/enzymology , Hydrolysis , Liver/enzymology , Rats , Substrate Specificity , Thymus Gland/enzymologyABSTRACT
The interaction of rat liver Ac-CoA-carboxylase with reactive and stable analogs of carbon dioxide and phosphoric acid mixed anhydrides--hypothetic intermediate of the enzyme reaction--has been studied. Carbamoylphosphate showed substrate properties, whereas phosphonacetic acid and beta-oxopropyl-alpha, alpha-diphosphonate inhibited this enzyme (Ki 3.0 and 3.5 mM correspondingly). The analog of another possible intermediate in the reaction of ATP and carbon dioxide, Appp (CH2COOH) also inhibited Ac-CoA-carboxylase (Ki = 0.7 mM).
Subject(s)
Acetate-CoA Ligase/metabolism , Adenosine Triphosphate/metabolism , Carbon Dioxide/metabolism , Coenzyme A Ligases/metabolism , Liver/enzymology , Adenosine Triphosphate/analogs & derivatives , Animals , Carbamyl Phosphate/metabolism , RatsABSTRACT
Effects of P1,P4-bis(5'-adenosyl)tetraphosphate and its phosphonate analogs on the ADP-ribosylation of H1 catalyzed by bovine testis ADP-ribose polymerase was investigated. Analogs App[CH(COCH3)]ppA and Ap[CH2]pppA as well as Ap4A inhibited poly(ADP)-ribosylation of histone H1 and at the same time accepted the ADP-ribosyl moiety of NAD. It was shown that inhibition of ADP-ribosylation of histone H1 is due to the competition of nucleotides with histone H1 for accepting ADP-ribosyl moiety of NAD on the one hand, and alteration of acceptor properties of the histone H1 on the other.
Subject(s)
Dinucleoside Phosphates/pharmacology , Histones/metabolism , Nucleoside Diphosphate Sugars/metabolism , Organophosphorus Compounds/pharmacology , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Animals , Cattle , In Vitro Techniques , KineticsABSTRACT
AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.
Subject(s)
DNA Polymerase II/metabolism , DNA Polymerase I/metabolism , DNA-Directed DNA Polymerase/metabolism , Diphosphates/metabolism , Escherichia coli/enzymology , Nucleotides/metabolism , Catalysis , Humans , Kinetics , Polymers , Sodium Fluoride , Templates, GeneticABSTRACT
Processive pyrophosphorolysis of RNA from ternary RNA polymerase-nascent RNA-delta D111 T7 DNA complex has been followed in the absence of nucleoside triphosphates. Series of inorganic pyrophosphate analogs were investigated for their ability to sustain the reaction and to compete with inorganic pyrophosphate for the reaction. Methylenediphosphonic, imidodiphosphonic, phosphonacetic acids, inorganic triphosphate, methylenediphosphonic and phosphate were found to be capable of substituting the inorganic pyrophosphate in RNA degradation reaction with tantamount efficiency. They give rise to nucleoside monophosphates for phosphonoacetic acid, nucleoside triphosphates for inorganic pyrophosphate and inorganic triphosphate, nucleoside triphosphates analogs for methylenediphosphonic, imidodiphosphonic acids and methylenediphosphonic acid phosphate as the low molecular weight product of the reaction. The problem of specific interaction of RNA polymerase with nucleoside triphosphates and inorganic pyrophosphate is discussed in the terms of structural requirements for the compounds to be a potent substrate for RNA polymerase.
Subject(s)
Diphosphates/pharmacology , Escherichia coli/enzymology , Phosphates/pharmacology , Binding Sites , DNA-Directed RNA Polymerases/metabolism , Kinetics , Structure-Activity RelationshipABSTRACT
3'(2')-O-acyl derivatives of the uridine triphosphate were synthesized. Acyl residues contained fluorescent dye; fluoresceine or rodamine C. Optical properties and stability of UTP analogues were studied. Their ability to serve as the substrates for calf thymus terminal deoxyribonucleotidyl transferase and E. coli RNA polymerase was also examined. It was shown that both enzymes were able to use tested analogues as substrates. Incorporation of the analogues into nascent RNA and DNA chains inhibited the synthetic reaction because of primer inactivation. The rate of the incorporation of the analogues showed an exponential time dependence
Subject(s)
DNA/biosynthesis , Fluoresceins , RNA/biosynthesis , Rhodamines , Uracil Nucleotides/analogs & derivatives , Uridine Triphosphate/analogs & derivatives , Xanthenes , Animals , Cattle , DNA Nucleotidyltransferases/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/enzymology , KineticsABSTRACT
The antiviral activity of 3'-azido-2',3'-dideoxynucleoside 5'-phosphate analogues: 5'-phosphonomethylene-3'-azido-2',3'-dideoxythymidine, 5'-methylphosphonate and 5'-phosphite of 3'-azido-2',3'-dideoxythymidine, 5'-phosphites of 3'-azido-2',3'-dideoxyadenosine and guanosine was investigated in HIV-infected cell cultures (human lymphoblastoid cells). The effectivity of inhibition of HIV-reproduction in cells by these substances was close or even higher than that for the corresponding 3'-azido-2',3'-dideoxynucleosides, whereas their toxicity was lower than that of nucleosides. These substances are supposed to be transported into the cells and to be transformed into the corresponding 5'-triphosphate analogues under the action of cell kinases. It is possible that such agents are terminator substrates of virus reverse transcriptases and thus inhibit the biosynthesis of DNA chains.
Subject(s)
Antiviral Agents , Azides/pharmacology , Dideoxynucleosides/pharmacology , HIV-1/drug effects , Virus Replication/drug effects , Antiviral Agents/chemical synthesis , Azides/chemical synthesis , Cells, Cultured , Chemical Phenomena , Chemistry , Dideoxyadenosine/analogs & derivatives , Dideoxyadenosine/chemical synthesis , Dideoxyadenosine/pharmacology , Dideoxynucleosides/chemical synthesis , HIV-1/physiology , Humans , Organophosphonates/chemical synthesis , Organophosphonates/pharmacology , Zidovudine/analogs & derivatives , Zidovudine/chemical synthesis , Zidovudine/pharmacologyABSTRACT
The hydrolysis of 5'-phosphonates of 2'-deoxythymidine and its 3'-modified analogs, inhibiting the HIV reproduction, by the E. coli alkaline, calf intestine and human placenta phosphatases as well as by the Crotalus atrox venom 5'-nucleotidase were studied. Transformations of 5'-phosphonates of adenosine and its analogs during incubation with human and fetal calf blood sera were investigated. The nucleotide derivatives modified at the phosphate residue were not hydrolyzed by any of the phosphatases studied except for the cobra venom 5'-nucleotidase, the effectiveness of the latter depended on the substitutes at both phosphate and sugar residues. 2'-Deoxyadenosine incubation with blood sera resulted in its transformation to 2'-deoxyinosine and then to hypoxanthine. 2'-Deoxyadenosine 5'-phosphonates were stable during incubation with blood sera under the same conditions.
Subject(s)
Antiviral Agents/metabolism , Blood , Nucleotides/metabolism , Phosphoric Monoester Hydrolases/metabolism , 5'-Nucleotidase/metabolism , Animals , Cattle , Crotalid Venoms/enzymology , Escherichia coli/enzymology , Humans , Hydrolysis , Intestines/enzymology , Magnetic Resonance Spectroscopy , Placenta/enzymologyABSTRACT
5'-Phosphites (5'-hydrogenphosphonates) of 3'-azido-2'-, 3'-dideoxynucleosides are shown to be effective inhibitors of the human immunodeficiency virus (HIV-1) in MT4 cell culture. 5'-Phosphite of 3'-azido-2', 3'-dideoxythymidine was the most active among these compounds and even a little more active as compared to the well-known anti-AIDS drug 3'-azido-2',3'-dideoxythymidine; at the same time 5'-phosphites of 3'-azido-2',3' -dideoxynucleosides with adenine, guanine and cytosine bases were more active than the corresponding nucleosides. The toxicity of all four phosphites was comparatively low and the equimolar mixture of all four phosphites was 2-3 fold less toxic than each of them separately. Data on the decreased toxicity of the phosphite mixture are explained from the viewpoint of a decreased pool disbalance of natural 2'-deoxynucleoside 5'-triphosphates in cells; a significant pool disbalance is developed in the case of 3'-azido-2',3'-dideoxythymidine action.
Subject(s)
Antiviral Agents/pharmacology , Dideoxynucleosides/pharmacology , HIV-1/drug effects , Cells, Cultured , Dideoxynucleosides/toxicity , Immunoenzyme Techniques , RNA-Directed DNA Polymerase/metabolism , Reverse Transcriptase Inhibitors , Zidovudine/analogs & derivatives , Zidovudine/pharmacology , Zidovudine/toxicityABSTRACT
We have investigated the substrate properties of deoxyribonucleoside 5'-triphosphate analogues, modified in the carbohydrate and triphosphate moieties, in DNA synthesis catalyzed by different DNA polymerases and reverse transcriptases. It was shown that (3'-azido-2',3'-dideoxythymidine-5'-O-methylenephosphonate) diphosphate, (3'-azido-2',3'-dideoxythymidine 5'-phosphate) dibromomethylenediphosphonate, (3'-azido-2',3'-dideoxythymidine 5'-phosphate) phosphonoacetate terminate DNA synthesis catalyzed by reverse transcriptases. (2'-Deoxythymidine 5'-phosphate) phosphonoacetate displays substrate properties for DNA polymerase beta, different reverse transcriptases, terminal deoxynucleotidyl transferase, but not for DNA polymerase alpha, Klenow's fragment DNA polymerase I. The Km value for this substance in DNA synthesis reactions catalyzed by reverse transcriptases was two of orders magnitude higher than that for native 2'-deoxythymidine 5'-triphosphate.
Subject(s)
Carbohydrates/chemistry , DNA/chemical synthesis , Deoxyribonucleotides/chemistry , Organophosphorus Compounds/chemistry , Base Sequence , DNA Polymerase I , DNA-Directed DNA Polymerase/chemistry , Molecular Sequence DataABSTRACT
5'-Phosphites (5'-hydrogenphosphonates) of 2',3'-dideoxynucleosides (T, A, G, C) were synthesized and studied as inhibitors of human immunodeficiency virus type 1 (HIV-1) in MT4 and CEM13 cell cultures. It was shown that all 5'-phosphites effectively inhibit the production of viral antigens and protect cells from the cytotoxic effect of HIV infection. 5'-Phosphites were more active antiviral compounds than the corresponding nucleosides.
Subject(s)
Antiviral Agents/pharmacology , Dideoxynucleosides/pharmacology , HIV-1/physiology , Thymine Nucleotides/pharmacology , Virus Replication/drug effects , Zidovudine/analogs & derivatives , Cells, Cultured , Dideoxynucleotides , Enzyme-Linked Immunosorbent Assay , HIV Antigens/analysis , Zidovudine/metabolism , Zidovudine/pharmacologyABSTRACT
We have investigated the ability of some nucleoside 5'-triphosphate analogues to terminate the DNA synthesis catalyzed by calf thymus DNA polymerase alpha and terminal deoxynucleotidyl transferase, rat liver DNA polymerase beta, E. coli DNA polymerase I (Klenow's fragment) and AMV reverse transcriptase. It has been shown that lyxoanhydronucleoside 5'-triphosphates terminate DNA synthesis catalyzed by reverse transcriptase and terminal deoxynucleotydil transferase. 2',3'-O-Isopropylidenecytidine 5'-triphosphate inhibits the DNA synthesis catalyzed by reverse transcriptase and DNA polymerase beta and its moiety was incorporated in the place of dTMP residue. Riboanhydroadenosine 5'-triphosphate reveals the properties of an effective termination substrate for all the DNA polymerases studied. This is the first attempt to investigate nucleotide analogues with the restricted conformation of the carbohydrate moiety as termination substrates for several prokaryotic and eukaryotic DNA polymerases.
Subject(s)
DNA Polymerase I/metabolism , DNA/biosynthesis , Nucleic Acid Conformation , Nucleotides/metabolism , Animals , Base Sequence , DNA Nucleotidylexotransferase/metabolism , DNA-Directed DNA Polymerase/metabolism , Molecular Sequence Data , RNA-Directed DNA Polymerase/metabolism , Substrate SpecificityABSTRACT
Halophosphonate ATP analogues pp[CHBr]pA and p[CHBr]ppA synthesised from bromomethylenebisphosphonate and adenosine derivatives, proved to be effective competitive inhibitors of Ac-CoA-carboxylase (CE 6.4.1.2) from rat liver (Ki = 0,2 mM). The inhibitory effects of both analogues were reversible and higher than those of some other ATP analogues. Another enzyme, Ac-CoA-synthetase (CE 6.2.1.1), with a different mode of ATP-cleavage showed wider specificity to ATP-analogues than Ac-CoA-carboxylase.
Subject(s)
Acetate-CoA Ligase/antagonists & inhibitors , Acetyl-CoA Carboxylase/antagonists & inhibitors , Coenzyme A Ligases/antagonists & inhibitors , Ligases/antagonists & inhibitors , Adenosine , Adenosine Triphosphate , Animals , Chemical Phenomena , Chemistry , Cytosol/enzymology , Diphosphonates , In Vitro Techniques , Liver/enzymology , Myocardium/ultrastructure , Rabbits , RatsABSTRACT
Comparative study of DNA biosynthesis inhibition, catalyzed by avian myeloblastose virus (AMV) reverse transcriptase (RT), human immunodeficiency virus (HIV) recombinant and native RT, has been performed. 3'-Azido-2',3'-dideoxythymidine 5'-triphosphate (AzTTP); 3'-azido-2',3'-dideoxythymidine 5'-methylenephosphonate-diphosphate: 3'-azido-2',3'-dideoxythymidine 5'-phosphate-phosponoacetate; 3'-azido-2',3'-dideoxythymidine 5'-phosphate-dibromomethylenephosphonate; 2',3'-O-isopropylidenecytidine 5'-methylenephosphonate-diphosphate (rC-IP-MPDP) were used as inhibitors. AzTTP proved to by the most active inhibitor (its activity against HIV RT is higher than against AMV RT), although not selective as the phosphonates; only rC-iP-MPDP has low selectivity.
Subject(s)
DNA/biosynthesis , Retroviridae/enzymology , Reverse Transcriptase Inhibitors , Avian Myeloblastosis Virus/enzymology , Avian Sarcoma Viruses/enzymology , Catalysis , Chemical Phenomena , Chemistry , HIV/enzymology , Oligodeoxyribonucleotides/metabolism , Poly A , Templates, Genetic , Zidovudine/analogs & derivatives , Zidovudine/pharmacologyABSTRACT
Diphosphonic analogues of inorganic pyrophosphate (PPi): methylene-, oxyethylidene-, aminomethylenediphosphonic acids as well as phosphonacetic, imidodiphosphoric bis- (phosphonomethyl)-phosphonic acids and methylenediphosphonic and phosphonic acid monoanhydrides were studied for their effect on the RNA-synthesizing activity of thymocytes. DNA-dependent RNA-polymerases I and II from the calf thymus nuclei were used for these studies. The analogues and PPi under study are shown to be inhibitors of both RNA-polymerases in nuclei from calf thymus and of purified RNA-polymerase II, which is more sensitive to the effect of diphosphonates. Methylenediphosphonic acid is the strongest inhibitor among the studied analogues, and imidodiphosphoric and phosphonacetic acids are the weakest inhibitors. Inhibition of purified RNA-polymerase II by diphosphonates has a complex character and includes both interaction of the PPi analogues with enzymes and chelating by them of Mn ions which are cofactors for RNA polymerase.
Subject(s)
DNA-Directed RNA Polymerases/antagonists & inhibitors , Diphosphates/pharmacology , Organophosphonates/pharmacology , Thymus Gland/enzymology , Animals , Cattle , Chelating Agents/pharmacology , Manganese/metabolism , RNA Polymerase I/antagonists & inhibitors , RNA Polymerase II/antagonists & inhibitorsABSTRACT
The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect.
Subject(s)
Antibody-Producing Cells/enzymology , Diphosphonates/pharmacology , Pyrophosphatases/metabolism , Spleen/enzymology , Animals , Erythrocytes/immunology , Immunization , Inorganic Pyrophosphatase , Male , Mice , Mice, Inbred BALB C , Pyrophosphatases/antagonists & inhibitors , Spleen/immunology , Structure-Activity Relationship , Tissue DistributionABSTRACT
Effect of bis(adenosyl-5')oligophosphates on the ATP receptors in the somatic membranes of rat sensory neurons has been studied. Bis(adenosyl-5')tetraphosphate and bis(adenosyl-5')pentaphosphate are partial agonists of these receptors. They can activate currents of about an order lower than maximum ATP-activated currents.