[The suppression of a herpes simplex virus reproduction with drug resistance by combination 15lys-bis-nt and phosphate of acycloguanosine with some antiherpetic drugs].

Antiherpetic activity of the double and triple combinations, including original connections 15Lys-bis-Nt and phosphate of acycloguanosine (P-ACG), was studied in vitro. For the first time, it was demonstrated that in case of their combined use with known antiherpetic agents, whose activity does not depend on TK of HSV (PFA, AraA, CDV, Rib, GLN, αa-IFN), synergistic or additive effects of interaction was observed. The antiviral effect of the tested combinations was studied on the model of ACG-resistant viral strain. The tested combinations could be of interest for practical medicine.

[Investigation of active center of deoxynucleoside monophosphate kinase of bacteriophage T5 by site-directed mutagenesis].

Based on the computer model of active center of bacteriophage T5 deoxyribonucleoside monophosphate kinase amino acid residues essential for the enzyme activity were determined. As the result of site-directed mutagenesis, cloning and expression of the gene in E. coli series of proteins were obtained with single amino acid substitutions of conservative active center residues--S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, E176Q. Electrophoretically homogeneous preparations of mutant forms were purified using ion exchange and affinity chromatographic steps. Measuring of the specific enzyme activities of these enzymes for the natural acceptors of phosphoryl group (dAMP, dCMP, dGMP, dTMP) revealed that substitutions of charged residues of NMP-binding domain-namely, R130, R172, D170 and E176-lead to almost complete loss of enzyme activity. It was shown that presence of OH-group at position 17 is also important for catalytic activity. Based on the changes in specific activities we suppose that arginine residues at positions 130 and 172 participate in binding of γ-phosphoryl of donor and α-phosphoryl of acceptor. Also, aspartic acid at 16 position of ATP-binding site (P-loop) probably assists in the binding of acceptor, first of all dTMP. Unequal decrease in enzyme activities for different substrates of partially active mutants--G137A, T138A, T17N, Q134A, S13A, D16N--indicate that in the binding of various substrates different amino acid residues take part.

[Molecular-genetic analysis of DNA pol and TK of HSV-1 population using NGS technology].

It was determined the ratio of viral DNA and DNA from Vero cells using the polymerase chain reaction in real time in Vero cell lysate, infected with L2 strain of the herpes simplex virus type 1. Copy number of the virus reached a maximum after 24 hours of incubation of infection. Total DNA was isolated and sequenced using NGS technology by Ion Torrent device. Nucleotide sequences of the thymidine kinase gene (UL23) and DNA polymerase (UL30) were determined for a population of HSV-1 strain L2. Comparison of the primary structure of these genes with the corresponding nucleotide sequences of known strains of HSV-1 KOS and 17 was conducted. Differences in the structure of genes UL23 and UL30 between strain L2 and reference strains KOS and 17 are not important, because changes are found in non-conservative regions.

[Antiherpesviral activity of acycloguanosine H-phosphonate in experiments using laboratory animals].

A study of the antiherpesviral activity of acycloguanosine (ACG) H-phosphonate (ACG-P) on a model of fatal herpesvirus infection in inbred BALB/c albino mice has established that ACG-P reduces death rates in the animals, considerably increases their average lifespan, and significantly decreases brain virus titers with both 60% mortality in the control and 92% mortality in the control group. There was also a significant inhibition of herpes simplex virus type 1 (HSV-1) replication in the brain tissue of animals receiving ACG-P on a model of ACG-resistant HSV-1/L2/RACG(TK-).

[Cloning, expression, isolation and properties of thymidine kinase herpes simplex virus, strain L2].

A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.

[Enzymatic activity of thymidine kinase of herpes simlex virus strain resistant to H-phosphonates of Acv].

Cloned laboratory mutants of herpes simplex virus type I resistant to acycloguanosine H-phosphonate have been investigated. For all clones were shown that mutations resulted to increasing of sensitivity to acting of sidofovir. Thymidine kinase of mutant viruses partially preserves the ability to phosphorilate thymidine, but loses the ability to phosphorilate BVDU.

[Analysis of mutations in DNA polymerase and thymidine kinase genes of herpes simplex virus clinical isolates resistant to antiherpetic drugs].

The primary structures of DNA-polymerase (ul30) and thymidine kinase (ul23) genes from several herpes simplex virus type 1 (HSV-1) clinical isolates which differed in sensitivity for a number of antiherpetic drugs were determined and compared with those for two laboratory HSV-1 strains one of which was ACV-sensitive (L2), while the another was resistant (L2) to ACV. The phylogenetic analysis of the sequences showed that conserved regions of ul30 gene of HSV-1 clinical isolates and L2 strain were homologous with the exception of point mutations and degenerated substitutions. Several new mutations in the HSV-1 DNA-polymerase and thymidine kinase functional domains were established and identified as the substitutions associated with the strain-resistance to ACV and other drugs.

[DNA assay for genetically engineered pharmaceutical substances using the real-time PCR technique].

A real-time PCR procedure is proposed for assaying E. coli residual DNA in the pharmaceutical substance of human recombinant insulin. For the quantitative analysis of the DNA content, an amplification of fragments of the bla gene plasmid DNA and E. coli genomic DNA of the 16S RNA gene were used. The contents of plasmid and genomic DNA were detected both in intermediates at various stages of the insulin purification process and in the finished product.

[Comparative study of resistance to acycloguanosine and acycloguanosine H-phosphonate in herpes simplex virus].

The ability of acycloguanosine H-phosphate to inhibit the reproduction of herpes simplex virus type 1 (HSV-1) variants, including its acycloguanosine (acyclovir)-resistant ones, was studied. Acycloguanosine H-phosphate-resistant HSV-1 variants were obtained. It was found that these variants were cross-resistant to thymidine kinase-dependent HSV reproduction inhibitors, but preserved sensitivity to Apa-A and phosphonoacetic acid.

[Substrate specificity of T5 bacteriophage deoxyribonucleoside monophosphate kinase and its application for the synthesis of [alpha-(32)P]d/rNTP].

Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d(2)CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, K(m) and k(cat) were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [alpha-(32)P]rNTP and [alpha-(32)P]dNTP.

[Isosteric triphosphonate analogues of dNTP: synthesis and substrate properties toward various DNA polymerases].

Isosteric triphosphonate derivatives of 2',3'-dideoxy-2',3'-didehydroadenosine and 3'-deoxy-2',3'-didehydrothymidine and their beta,gamma-substituted analogues were synthesized. Their substrate properties toward a number of reverse transcriptases of the human immunodeficiency and bird myeloblastosis viruses, human DNA polymerases alpha and beta, and the Klenow fragment of Escherichia coli DNA polymerase I were studied.

[Enzymatic synthesis of bis(5'-nucleosidyl) tetra- or triphosphates].

The total fraction of aminoacyl-tRNA synthases from Escherichia coli has been shown to catalyze the synthesis of the bis(5'-nucleosidyl) oligophosphates Ap4AZT, Ap4d4T, Ap43TC, and Ap4ACV, as well as Ap3AZT and Ap3d4T, from [alpha-32P]ATP and the corresponding nucleoside-5'-tri(or di)phosphate. The resulting compounds, characterized by HPLC, are resistant to alkaline phosphatase. Ap4AZT, Ap4d4T, and Ap43TC are formed with approximately equal efficiency, whereas the efficiencies of the synthesis of Ap4ACV, Ap3AZT, and Ap3d4T are three- to fivefold lower. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

[The study of coupling agents for the phosphorylation of 3'-azido-3'-deoxythymidine with (P32)orthophosphoric acid].

The kinetics of 3'-azido-3'-deoxythymidine phosphorylation with [32P]orthophosphoric acid was studied in the presence of various coupling agents. The most effective method with the use of BrCN provided the isolation of the target 3'-azido-3'-deoxythymidine 5'-[32P]monophosphate in 46% yield and a high specific radioactivity (>100 Ci/mmol).

[A PCR-based semiquantitative assay of DNA impurities in recombinant protein preparations].

A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10 microg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.

[Photoaffinity modification of bacteriophage T7 DNA-dependent RNA polymerase by the reaction product containing the azido derivative of UTP]. / Fotoaffinnaia modifikatsiia DNK-zavisimoi RNK-polimerazy bakteriofaga T7 produktom polimeraznoi reaktsii, soderzhashchim azidoproizvodnoe UTP.

The possibility of using the 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)-propenyl-1]-UTP (N3-TFBP-UTP) as the affinity modificator of bacteriophage T7 DNA-dependent RNA polymerase was demonstrated. The UTP derivative used was rather efficient substrate substituting UTP in the transcription reaction performed by the enzyme. The UV treatment of "stopped" reaction complex formed using three of four substrate ribonucleotides, allow to obtain the covalent binding between the enzyme and the reaction product of 9 nucleotides length. The isolation and the analysis of the obtained "nucleotide-peptide" showed that the sequence of modified peptide corresponded to the fragment Tyr802-Lys826, which belonged to the conservative motif C in the enzyme structure. His811 or Asp812 residues belonging to this sequence are most probable targets of the modification.

[The synthesis and antiherpetic activity of acyclovir phosphonate esters]. / Sintez i antigerpeticheskaia aktivnost' fosfonatnykh éfirov atsiklovira.

Alkyl esters of acyclovir phosphite, alkoxycarbonylphosphonate, ethoxycarbonylmethylphosphonate, and aminocarbonylphosphonate were synthesized. Most of them were shown to inhibit the replication of type 1 herpes simplex virus in Vero cell culture. The stability in phosphate buffer and human blood serum was studied for several of the derivatives. A correlation between the stability and antiviral activity of the synthesized compounds is discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[New phosphonoformic acid derivatives of 3'-azido-3'-deoxythymidine]. / Novye proizvodnye 3'-azido-3'-dezoksitimidina i fosfonomurav'inoi kisloty.

New 5'-alkyl ethoxy- and aminocarbonylphosphonates of 3'-azido-3'-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5'-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

[Efficacy of 4'-thio-5-ethyl-2'-deoxyuridine (TEDU) and its derivatives in experimental infection in mice caused by herpes simplex virus]. / Izuchenie éffektivnosti 4'-tio-5-étil-2'-deoksiuridina (TEDU) i ego proizvodnykh, vyzvannoi virusom gerpesa prostogo.

It was demonstrated that several 5'-phosphonates of 4'-thio-5-ethyl-2'-deoxyuridine possessed antiviral activity in vitro and in the murine model of herpes simplex virus type 1 infection. It was shown that the derivatives after intraabdominal administration penetrated effectively into the brain tissue. The agents provided statistically significant increase of the average life span, lower virus titre in the brain and lower lethality when compared to the control group of the animals. It is emphasized that the derivatives were less toxic than the original compound.

[4'-Thio-5-ethyl-2'-deoxyuridine 5'-phosphonates: synthesis and antiviral activity]. / 5'-Fosfonaty 4'-tio-5-étil-2'-dezoksiuridina: sintez i antivirusnye svoistva.

A number of new 5'-phosphonate derivatives of 4'-thio-5-ethyl-2'-deoxyuridine (TEDU) were synthesized. These compounds displayed a low cytotoxicity and, except for TEDU 5'-fluorophosphate, antiherpes activity similar to that of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) and 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (pencyclovir). 5'-Ethoxycarbonylphosphonate and 5'-aminocarbonylphosphonate of TEDU were also found to suppress the reproduction of herpes simplex type 1 virus, which is resistant to acyclovir.