Biomedical subjects
Publications and source records attributed to A A Kraevskiĭ.
Chemical reactions catalyzed by various DNA polymerases are discussed, including DNA chain extension, the 3'-->5'-exonuclease proofreading activity, and some other pathways of replicative repair. The contribution of DNA polymerases to the fidelity of the template-dependent synthesis is analyzed by the examples of some most typical DNA polymerases.
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[3H]5'-O-Phosphonylmethylthymidine with a specific activity of 71 Ci/mmol was obtained by isotope exchange. Its incubation with a HeLa cell culture resulted in the formation of [3H]-labeled 5'-O-(beta-phosphoryl-alpha-phosphonylmethyl)thymidine, 5'-O-(beta,gamma-diphosphoryl-alpha-phosphonylmethyl)thymidine, and [3H]DNA. This proved the ability of 5'-O-phosphonylmethylthymidine to undergo phosphorylation and incorporation into the DNA of human cells.
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The long-term action of azidothymidine, reverse transcriptase inhibitor, on cultivated U-937 (human promyelocyte leukemia) and MeWo (human melanoma) cells led to the concentration-dependent decrease in the length of telomeric chromosomal repeats. Telomere shortening was accompanied by temporary retardation of cell proliferation. Combined with the data obtained previously, these results suggest that azidothymidine inhibits telomerase functioning in cultivated cells.
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In the course of our investigations on DNA polymerases a synthesis of new triphosphate analogues, 5'-mercapto-5'-deoxythymidine 5'-S-triphosphate and 5'-mercapto-3',5'-dideoxythymidine 5'-S-triphosphate, was brought about starting from 3'-O-acetyl-thymidine and 3'-deoxythymidine, respectively. The former compound proved to be a weak substrate for E. coli DNA polymerase I and AMV reverse transcriptase and inactive towards DNA polymerases alpha from placenta and beta from rat liver. The second synthesized triphosphate was incorporated into DNA chain by none of the tested enzymes. Probable reasons for so strong a decrease in the substrate properties of the prepared compounds are discussed.
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Azidothymidine (retrovir) and didesoxyinosine, which represent nucleoside agents, are major remedies in the treatment of HIV infection and acquired immunodeficiency syndrome (AIDS). The paper describes the molecular mechanism of their action. It implicates that triphosphates of these nucleosides selectively suppress the activity of reverse transcriptase (RNA-dependent DNA polymerase) of HIV by a termination mechanism. This results in effective inhibition of HIV reproduction and recovery of lymphocyte count and yields marked therapeutical benefits. The new generation anti-HIV agents are nucleoside-based phosphonates which were discovered by Russian investigators in 1987. The agents having a significant anti-HIV activity are low toxic. Emphasis is made on combined therapy of HIV infection, which holds much promise.
Several 5'-fluoroalkylphosphonates of 3'-azido-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine were synthesized as potential antiviral compounds. Fluoromethyl, difluoromethyl, fluorochloromethyl and vinyl phosphonates were used as phosphonylating components, with dicyclohexylcarbodiimide or triisopropylbenzenesulphonyl chloride as condensing reagents.
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3'-Mercapto-3'-deoxy-TTP was synthesized and tested as DNA chain terminator nucleotide for calf thymus alpha DNA polymerase, E. coli DNA polymerase I (Klenow fragment), terminal deoxyribonucleotidyltransferase (Bollum enzyme) and reverse transcriptase from AMV- and HIV-I-viruses. It was shown that the compound terminates DNA chain elongation by reverse transcriptases selectively and irreversibly. Other tested DNA polymerases do not use this nucleotide analogue as a substrate. 3'-Mercapto-3'-deoxythymidine was tested on lymphoblastoid T-cell line MT-4 with HIV-viruses and shown to suppress viruses as efficiently as 3'-azido-3'-deoxythymidine.
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.
An action of azidothymidine (AZT) on the acquisition of passive avoidance behaviour was studied in mice. Intraperitoneal administration of AZT (5-80 mg/kg) inhibited markedly the formation of passive avoidance behaviour. AZT had amnestic effect when administered 1 hour before learning, immediately after learning, 1 to 3 hours, but not 5 hours after learning. Administration of AZT 1 hour before learning had no influence on passive avoidance behaviour tested 24 hours later. The first signs of amnesia appeared 48 hours after learning and the effect was maximum during testing one week after learning. It is suggested that this effect of AZT may indicate the necessity of reverse transcription in the nervous cells for the consolidation of long-term memory.
Synthesis of 3'-chloro- and 3'-cyanothio-2',3'-dideoxythymidine by the reaction of 2,3'-anhydro-2'-deoxythymidine with ammonium chloride and lithium thiocyanate, respectively, has been developed. In addition, 3'-methanesulphonylamido- and 3'-sulphonylamido-2',3'-dideoxythymidines were synthesized starting from 3'-amino-2',3'-dideoxythymidine. All these compounds along with 2',3'-anhydroriboadenosine,2',3'-anhydrolyxoadenosine, 2',3'-O-isopropylidenecytidine, and 2,3'-anhydro-2'-deoxythymidine were transformed into 5'-triphosphates by treatment with phosphoryl tris-1,2,4-triazolide and then with bis(tri-n-butylammonium)pyrophosphate. All 5'-triphosphates of nucleoside analogues were tested as termination substrates in cell-free systems with various DNA polymerases.
Ability of some new substrates containing the 5'-alpha-thiotriphosphate residue to terminate the DNA synthesis catalyzed by several DNA polymerases has been investigated. The cell-free test system contained the M13mp10 phage single-stranded DNA and a synthetic oligonucleotide primer. Reverse transcriptase from avian myeloblastosis virus catalyzed termination of DNA synthesis by 3'-azido-3'-fluoro- and 3'-amino-2',3'-dideoxythymidine-5'-(alpha-thio)triphosphates, whereas rat liver DNA polymerase beta and E. coli DNA polymerase I (Klenow's fragment) utilized only the second and the third compounds, and calf thymus DNA polymerase alpha failed to utilize any of the substrates. Low specificity of reverse transcriptase to different moieties of the substrate molecules is discussed.
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2',3'-Dideoxy-2',3'-dehydrothymidine 5'-triphosphate (dddTTP) reveals the termination substrate properties in the DNA synthesis catalyzed by E. coli polymerase I (Klenow fragment), rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase, and reverse transcriptase of avian myeloblastosis virus but does not affect calf thymus DNA polymerase alpha. For DNA polymerase I, dddTTP by an order of magnitude is more effective than any known termination substrate. It is supposed that dddTTP models the conformational state of the substrate's carbohydrate moiety in the complex DNA polymerase + template-primer.