[Modified nucleosides and nucleotides inhibiting HIV replication: analysis of the situation and potential prospects]. / Modifitsirovannye nukleozidy i nukleotidy, podavliaiushchie reproduktsiiu VICh: analiz situatsii i veroiatnaia perspektiva.

The results of an eight year search of blockaders of human immunodeficiency virus (HIV) among groups of modified nucleosides and nucleotides are reviewed. The molecular mechanism of action of these compounds is based on the inhibition of DNA polymerases activity. Attempts of systematic analysis of structure--anti-HIV activity relationship for modified substrates of DNA biosynthesis are made. In this analysis attention is focused on the evaluation of general properties of enzymes of the phosphorylation cascade and DNA polymerases. Such properties are analyzed on the basis of empiric rules which summarize electronic, steric and conformational properties of substrate analogs. The ability of phosphorylating nucleosides to block HIV reproduction are analyzed. The interdependence of structure of several most known inhibitors with their action on nucleic acids components metabolism as well as the structure--therapeutic properties relationship are discussed.

[Ways of finding inhibitors of HIV reproduction among nucleotides]. / Puti poiska ingibitorov reproduktsii HIV sredi nukleotidov.

In spite of continuous broad search for inhibitors of HIV reproduction among new nucleosides, there seems to be a certain crisis in the problem. One of the probable solutions could be based on the investigation of nucleotide and/or nucleoside 5'-triphosphates with modified carbohydrate and phosphate residues. The review analyzes the current state of the problem, examining of such types of compounds in cell-free systems with DNA polymerases and in HIV-infected cell cultures. The known data on the stability and metabolism of such compounds in cell cultures as well as in human serum are also analyzed.

[Non-canonical nucleotide exchange catalyzed by Escherichia coli DNA-polymerase I and the two-center model of the mechanism of action of this enzyme]. / Nekanonicheskii nukleotidnyi obmen, kataliziruemyi DNK-polimerazoiEscherichia coli, i dvukhtsentrovaia model' mekhanizma deistviia éto fermenta.

It is shown, that DNA hydrolysis catalyzed by E. coli DNA polymerase I is inhibited, when a reaction mixture contains one type of deoxynucleoside 5'-triphosphate (dNTP). When the reaction mixture contains [32P]dNTP, then [32P] is incorporated into DNA and v. v. (32P) from DNA is transferred into dNTP. The nucleotide exchange between DNA and dNTP in the assay mixture is observed only in the case, when the chemical nature of nucleotide residue of dNTP and that of the 3'-terminus of DNA is the same. Analysis of products of DNA hydrolysis in the presence of one type of dNTP using electrophoresis in polyacrylamide gel shows that most of the DNA molecules are terminated at the 3'-termini by the dNMP residue of the same chemical nature as the dNTP in the assay mixture. However, in some cases DNA molecules contain one additional nucleotide residue. This phenomenon can be explained by incorporation of one additional dNMP residue originating from dNTP only in those cases, when a non-typical base pairing of this nucleotide residue with a template residue readily takes place. The above-mentioned facts can be interpreted within the model for DNA hydrolysis with involvement of two intermediate covalent forms of dNMP residues with DNA polymerase I; one dNMP-intermediate should be placed at the elongation center and the other--at the hydrolysis center. The DNA hydrolysis by 3'----5' exonuclease activity of DNA polymerase I proceeds through these two covalent forms. DNA polymerases alpha from calf thymus and T4 phage do not catalyze the nucleotide exchange between DNA and dNTP from the reaction media.

[Peptidyltransferase center of ribosomes. Structure and relationship to other ribosomal functions]. / Peptidiltransferaznyi tsentr ribosom. Struktura i vzaimosviaz' s drugimi funktsiiami ribosom.

Structural analysis of the ribosomal peptidyl transferase center using model substrates "minimal" peptide acceptors and donors is reported in the present work. Recent data on the ribosomal proteins and rRNA organizing the peptidyl transferase center and other functional centers of the large ribosomal subunits are given and the know facts about the catalytic mechanism of the peptide bond formation are considered. An analysis of the interaction of the peptidyl transferase center and the centers binding the cytoplasmic translocation factors and stringent-factor (for E. coli ribosome) is presented; a possible contribution of the peptidyl transferase center ot the translocation is discussed.

[Analogs of natural nucleosides and their 5'-triphosphates carrying the amino group in 3'-position--highly effective inhibitors of replication, repair and transcription]. / Analogi prirodnykh nukleozidov i ikh 5'-trifosfatov, nesushchie v 3'-polozhenii aminogruppu,--vysokoéffektivnye ingibitory replikatsii, reparatsii i transkriptsii.

The new type of DNA and RNA synthesis inhibitors is found. These compounds are active in synthesis, catalyzed by bacterial DNA and RNA polymerases and by eukaryotic DNA polymerases in vitro and in DNA replication in vivo.

[Reactions of 5'-H-phosphonates, 5'-F-phosphates, and 5'-phosphates of modified thymidines in human blood plasma]. / Reaktsii 5'-H-fosfonataov, 5'-F-fosfatov i 5'-fosfatov modifitsirovannykh timidinov v plazme krovi cheloveka.

Mechanisms and rates of hydrolytic dephosphorylation of 5'-hydrogen phosphonates, 5'-fluorophosphates, and 5'-phosphates of 3'-azido-3'-deoxythymidine, 3'-fluoro-3'-deoxythymidine, and thymidine in human blood serum were investigated. 5'-Hydrogen phosphonates of 3'-substituted thymidines are dephosphorylated 50-100 times slower than the corresponding 5'-phosphates. 5'-fluorophosphates of 3'-substituted thymidines are dephosphorylated 2 times slower than corresponding 5'-phosphates; first, substituted thymidine 5'-phosphates are formed, which are later dephosphorylated into substituted thymidines. These data illustrate probable molecular mechanisms of anti-HIV action of such nucleotides. 5'-hydrogen phosphonates of thymidines can serve as depot forms of corresponding thymidines, but other metabolic pathways are not excluded. Thymidine 5'-fluorophosphates can serve as depot-forms of both thymidines and their phosphates. Their fate in cells depends probably on their diffusion and on the activities of dephosphorylating and phosphorylating enzymes.

[New nucleotide inhibitors of human DNA polymerase alpha]. / Novye nukleotidnye ingibitory DNK-polimerazy alpha cheloveka.

2'-Deoxythymidine 5'-triphosphate and 2'-deoxyadenosine 5'-triphosphate analogs containing a methylene group between the alpha phosphorus and 5' oxygen were synthesized. The substrate properties of these compounds toward some mammalian DNA polymerases and retroviral reverse transcriptases were evaluated using a system containing phage M13mp10 DNA, a synthetic oligonucleotide, and the enzyme. The compounds containing a hydroxyl at the 3' position were incorporated into the DNA chain by DNA polymerase alpha and terminal deoxynucleotidyl transferase, but were not recognized by retroviral reverse transcriptases and mammalian DNA polymerases epsilon and beta. The selectivity of the compounds synthesized was capitalized on during simultaneous isolation of DNA polymerases alpha and epsilon from human placenta. A methylene group was also introduced into the acyclovir molecule. It was shown that this modification inactivates furanose-related nucleotide analogs, but has a minor effect on the substrate properties of acyclic nucleotide analogs.

[Substrate inhibitors of DNA biosynthesis]. / Substratnye ingibitory biosinteza DNK.

We present an account of the data on the inhibition analysis of DNA biosynthesis by substrate analogs with DNA polymerases of different origin. An attempt has been made to substantiate the factors that underline the specificity of inhibitors with respect to DNA synthesis that is catalyzed by various DNA polymerases.

[The effect of 3'-azido-2',3'-dideoxythymidine on experimental viral infections]. / Deistvie 3'-azido-2',3'-didezoksitimidina na éksperimental'nye virusnye infektsii.

3'-Azido-2',3'-dideoxythymidine (az-T) inhibited effectively the reproduction of some retroviruses; among these viruses were the four serological subgroups of sarcoma Raus virus in chicken embryo, avian myeloblastosis virus and erythroblastosis virus in chicken. This inhibition was specific towards retroviruses and practically was not observed in the case of infections DNA- and RNA-genome model viruses of vaccinia and influenza, at whose reproduction reverse transcriptase is not involved. Three other 3'-modified nucleosides did not block the above-listed retroviruses. For chickens, az-T showed low toxicity. The molecular mechanisms of the action of az-T are discussed.

[Selective inhibition by 3'-azido-2',3-dideoxythymidine-5'- triphosphate of reverse transcription in retrovirus A particles from the rat liver]. / Selektivnoe tormozhenie 3'-azido-2',3-didezoksitimidin-5'- trifosfatom obratnoi transkriptsii v sostave retrovirusnykh chastits A-tipa iz pecheni krys.

The data presented demonstrate that 3'-azido-2',3'-dideoxythymidine 5'-triphosphate [dTTP (3N3)] specifically inhibits reverse transcription of viral RNA in the endogenous retroviral A-type particles isolated from the rat liver. The inhibitory action of dTTP (3N3) is concerned the termination of DNA synthesis obviously due to the incorporation of this dideoxynucleotide into the 3'-end of the growing DNA chain.

[Adducts of 3'-azido-2,3'-dideoxythymidine 5'-phosphate or 5'-phosphonate as inhibitors of cytopathic effect and transformation of cells under the influence of retroviruses in cell culture]. / Addukty 5'-fosfata ili 5'-fosfonata 3'-azido-2,3'-didezoksitimidina kak ingibitory tsitopaticheskogo éffekta i transformatsii kletok pod deistviem retrovirusov v kletochnykh kul'turakh.

Inhibition of HIV-1- or HIV-2-induced cytopathicity and (Moloney) murine sarcoma virus (MSV)-induced cell transformation by amino acid and amino alcohol adducts of either 3'-azido-2',3'-dideoxythymidine 5'-monophosphate (AZTMP) or 5'-hydrogenphosphonate (AZTHP) were investigated. Both types of nucleotide adducts inhibited replication of HIV-1 and HIV-2 in MT-4 cells at a 1.5- to 3-fold higher EC50 (50% effective concentration) than AZT; and, also, selectivity indexes of these adducts were approximately 1.5 to 3-fold lower than that of AZT. The activity of the AZTMP and AZTHP adducts against MSV-induced transformation of C3H/3T3 cells was equal to or only slightly inferior than that of AZT, but their toxicity was 10-fold lower, so that their selectivity indexes were 2- to 7-fold higher. The nature of the aminoacyl component of the adducts significantly influence the antiretroviral activity of the test compounds.

[Acyclic analogs of 2',3'-dideoxy-2',3'-didehydronucleoside-5'-triphosphates--terminators of DNA synthesis, catalyzed by a broad set of DNA polymerases]. / Atsiklicheskie analogi 2',3'-didezoksi-2',3'-didegidronukleozid-5'-trifosfatov--terminatory sinteza DNK, kataliziruemogo shirokim naborom DNK-polimeraz.

O-4'-nor-2', 3'-dideoxy-2', 3'-didehydronucleoside 5'-triphosphates are shown to be effective termination substrates of DNA biosynthesis catalyzed by human placental DNA polymerases alpha and epsilon, rat liver DNA polymerase beta, reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus, and calf thymus terminal deoxynucleotidyl transferase. These compounds do not interact only with the Escherichia coli DNA polymerase I (Klenow fragment). The probable reasons of interaction of acyclo-d4NTP with the DNA synthesizing complexes are discussed.

[Inhibition of replication of human hepatitis B virus]. / Ingibirovanie replikatsii virusa gepatita v cheloveka.

Several nucleoside 5'-triphosphate analogs were investigated as inhibitors of human hepatitis B virus replication. Different analogs inhibited DNA synthesis differently, 3'-azido-2',3'-dideoxythymidine 5'triphosphate being the most active compound. This inhibitor blocked DNA synthesis by 50% at inhibitor: substrate molar ratio 1:8, and by 80% - at 1:1. The hypothesis is formulated that 3'-azido-2',3'-dideoxythymidine 5'-triphosphate inhibits RNA directed viral DNA replication due to incorporation of this compound into 3'-termini of newly synthesized DNA chains. The phenomenon observed opens new possibilities for chemotherapy of acute and chronic human hepatitis B.

[The effect of 3'-azido-3'-deoxynucleosides on the reproduction of AIDS virus in cell culture]. / Deistvie 3'-azido-3'-dezoksinukleozidov na reproduktsiiu virusa SPID v kul'ture kletok.

Action of three nucleoside analogues with azido-group on AIDS virus (human immunodeficiency virus--HIV) reproduction was studied in two cell species. Among these compounds there were 3'-azido-3'-deoxythymidine (Az-T), 3'-azido-3'-deoxyarabinothymidine (Az-AT) and 3'-azido-2',3'-dideoxy-5-methylcytidine (Az-dCMe). All compounds prevent cells against HIV infection, but Az-T action was expressed more strongly. The reason for the lower activity of the Az-AT and Az-dCMe bases is probably stipulated by their poor conversion to the corresponding 5'-triphosphates. Az-T 5'-triphosphate blocks HIV reproduction due to the inhibition of the viral reverse transcriptase.

[DNA polymerase I, Klenow fragment of Escherichia coli: hydrolysis and pyrophosphorolysis of DNA containing phosphothioate groups]. / DNK-polimeraza I Escherichia coli, fragment Klenova: gidroliz i pirofosforoliz DNK, soderzhashchei fosfotioatnye gruppy.

Reaction of DNA synthesis catalyzed by DNA polymerase I KF in the presence of 2'-deoxynucleoside 5'-alpha-thiotriphosphates (dNTP alpha S) was investigated. DNA with thiophosphate groups (DNA[P=S]) obtained by such a way was studied in reactions of hydrolysis and pyrophosphorolysis catalyzed by DNA polymerase I KF. It is shown that the rate of DNA elongation is decreased both on the step of incorporation of dNMP alpha S residues and on the step of incorporation of the next dNMP residue. The rate of pyrophosphorolysis of 3'-terminal dNMP alpha S was demonstrated to be one order of magnitude less in comparison with the corresponding reaction with the natural dNMP residue. Contrary, the rate of 3'----5'-exonuclease hydrolysis of both DNA[P=S] and DNA of the same structure revealed no distinguishable differences.

[Modified nucleoside-5'-triphosphates with an additional conjugated through the 2'-3'-fused ring as DNA polymerase substrates]. / Modifitsirovannye nukleozid-5'-trifosfaty s doponitel'nym sopriazhennym po 2'-3'-sviazi tsiklom kak substraty DNK-polimeraz.

5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D-lyxofuranosyl) thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl) thymine and 2',3'-lyxoanhydrothymidine have been shown to be terminator substrates of human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases as well as DNA polymerase I from E. coli. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. The KM values of ltTTP, rtTTP and laTTP incorporation into DNA chain agree closely with each other, being 1.5-2.5 times higher than KM for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times less than Vmax for dTTP. The evidence favours the hypothesis of a great affinity of modified nucleosides with flattened ribose ring of glycone for DNA polymerases active sites.

[Peptidyltransferase center of ribosomes. II. Effect of temperature on the activity of model peptide donors and a study of oligopeptide formation in a matrix-free system with ribosomes]. / Peptidiltransferaznyi tsentr ribosom. II. Vliianie temperatury na aktivnost' model'nykh donorov peptida i issledovanie obrazovaniia oligopeptidov v bezmatrichnoi sisteme s ribosomami.

The reaction of 3'(2')-O-(N-formylmethionyl)-adenosine 5'-phosphate with Phe-tRNA or CACCA-Phe catalysed with E. coli MRE-600 ribosomes and stimulated with cytidine 5'-phosphate was investigated. It was shown that the reaction with Phe-tRNA was stimulated within 2--3 times when the temperature has been raised from 0 to 40 degrees. On the contrary when the peptide acceptor was CACCA-Phe the yield of peptides synthesis dropped 5 times and more under the same conditions. Similar temperature influence was observed in the reaction with 50S subunits. The inhibition of peptide bond formation with pA and CpA at 0 degrees was achieved up to 80--90% but it was very low at 40 degrees. The synthesis of tri- and tetrapeptides was observed when the reaction of 3'(2')-O-(N-formylmethionyl)-adenosine 5'-phosphate was carried out either with Phe-tRNA or with CACCA-Phe.

[Oxytetracycline binding to E. coli ribosomes]. / Sviazyvanie oksitetratsiklina s ribosomami E. coli

Binding of oxytetracycline to E. coli ribosomes was studied by equilibrium dialysis. The results are consistent with the existence of two classes of binding sites for the antibiotic on ribosomes having different reactivities. There is one strong binding site as well as about 500 weak ones. The association constant for strong complexes is about 10(3) times greater than the value for weak ones. Oxytetracycline and tetracycline bind to ribosomes as magnesium chelates. Increase of the concentration of Mg2+ leads to the formation of two types of magnesium chelates of the antibiotic: chelate 1 which is formed at a relatively low concentration of Mg2+ and has a stiochiometry 1:1, and chelate 2 which probably corresponds to the attachment of second ion to the antibiotic molecule. The strong binding of oxytetracycline to ribosomes prevents the template dependent association of aminoacyl-tRNA with ribosomes. However, no changes in the extent of the antibiotic binding were found upon addition of aminoacyl-tRNA, poly(U) and chloramphenicol to oxytetracycline-ribosome complexes. It has been suggested that inhibiting effect of oxytetracycline on the protein synthesis involves an allosteric mechanism.

[Comparative conformational analysis of dinucleoside phosphate CpA and its analog C(3'NH)pA]. / Sravnitel'nyi konformatsionnyi analiz dinukleozidfosfata CpA i ego analoga C(3'NH)pA.

By the method of theoretical conformational analysis, on the examples of CpA and C(3'NH)pA comparison is made of conformational flexibility of dinucleoside phosphates with the natural O-P-O and abnormal N-P-O internucleotide bonds. The conformational flexibility of sugar cycles is accounted for. It is shown that substitution of the phosphodiester bond into amide ester leads to a noticeable limitation of favorable areas in conformational space of molecules and, as consequence, to the increase of the equilibrium ratio of conformers with the Pba and Mbb type of base stacking. The obtained results are used for discussion of the binding experiments of acylamino acid derivatives of CpA and C(3'NH)pA to the donor site of the peptidyl transferase center of ribosomes.

[Donor site of E. coli ribosomal peptidyltransferase]. / Issledovanie donornogo uchastka peptidiltransferazy ribosom E. coli.

The mechanism of 5'-cytidilic acid stimulation of the reaction between 2'(3')-O-formylmethionine ester of 5'-adenylic acid and phenylalanyl-tRNA catalyzed by E. coli ribosomes has been studied. It has been shown that cytidilic acid binds to the donor site of the peptidyltransferase in the area which is usually occupied by the second nucleotide residue of the peptidyl-tRNA 3'-end. After the binding cytidilic acid stimulates effectively the donor activity of formylmethionine ester of adenylic acid. A number of compounds have been tested as possible stimulants. Both the chemical nature of stimulant and its conformation are important for the stimulating action. A hypothetic scheme is suggested explaining possible causative factors of peptidyl-tRNA translocation from the acceptor site to the donor site after peptide bond formation.