Yu. S. Skoblov

MODIFIED DINUCLEOSIDE TETRAPHOSPHONATES, NEW POTENTIAL INHIBITORS OF HIV REVERSE TRANSCRIPTASE

New γ-substituted analogues of dNTP were synthesized and their enzymatic stability and antiviral properties were evaluated.

Mutations in the DNA polymerase and thymidine kinase genes of herpes simplex virus clinical isolates resistant to antiherpetic drugs

Primary structures of DNA polymerase (ul30) and thymidine kinase (ul23) genes from several herpes simplex virus type 1 (HSV-1) clinical isolates di ffering in sensitivity to several antiherpetic drugs were determined and compared to those of two laboratory HSV-1 strains one of which (L2) was sensitive and the other (L2/R) was resistant to acyclovir. The phylogenetic sequence analysis showed that the ul30 and ul23 sequences of clinical isolates were close to those of L2, and that ul30 conserved regions differed between HSV-1 isolates and L2 only in point mutations and degenerated substitutions. Several new mutations in the HSV-1 DNA polymerase and thymidine kinase functional domains were identified as substitutions associated with strain resistance to ACV and other antiherpetic drugs.

The Study of Coupling Agents for Phosphorylation of 3′-Azido-3′-deoxythymidine with [32P]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 with high specific radioactivity (>100 Ci/mmol).

Radioligand method of assessment of human T-lymphocytes’ β-adrenoceptors activity

A new method of evaluation of beta-receptor’s activity on the surface of human T-lymphocytes has been proposed based on the radioligand method. Optimal conditions for evaluation of specific binding to β2-adrenoceptors of 0.5 fmol ligand per 1 million cells using [125I] -cyanopindolol were found. The possibility of using β2-adrenoceptor’s activity assessment in clinical settings was demonstrated on human T-lymphocytes.

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

Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d2CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, km and kcat 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 [α-32P]rNTP and [α-32P]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 β,γ-substituted analogues were synthesized. Their substrate properties toward a number of reverse transcriptases of the human immunodeficiency and avian myeloblastosis viruses, human DNA polymerases α and β, and the Klenow fragment of Escherichia coli DNA polymerase I were studied.

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 10µg 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.

The genome nucleotide sequence of herpes simplex virus 1 strain L2

The genome nucleotide sequence of the reference strain of herpes simplex virus type 1 was obtained using the technique of full size sequencing. For the virus genome structure determination, 402444 reads with an average length of 202 bp were performed, which corresponded to the 542-fold genome coverage. The data were collected to 52 contigs with N50-4518 and the total contig length of 120929 bp. The sequence obtained was deposited into the GenBank database.

Changes in the receptor activity of β2-adrenoreceptors of human T-lymphocytes under the effect of β2-agonists

Changes in the activity of β2-adrenergic receptors of human T-lymphocytes under the effect of salbutamol (a short-acting β2-agonist) have been evaluated with a new modified radioligand method utilizing [125I]cyanopindolol and a specific ligand ICI 118551. In healthy volunteers, the receptor activity decreased after 30 min upon the inhalation of salbutamol and restored to the initial level after 2 h. At the same time, there were changes in the transcription level of the ADRB2 gene, which encodes the protein component of the β2-adrenoreceptor. The dynamics of β2-adrenergic receptor activity of T-lymphocytes after salbutamol treatment in patients with cardiorespiratory pathology significantly differed from that in healthy volunteers.

The study of the bacteriophage T5 deoxynucleoside monophosphate kinase active site by site-directed mutagenesis

The amino acid residues essential for the enzymatic activity of bacteriophage T5 deoxyribonucleoside monophosphate kinase were determined using a computer model of the enzyme active site. By site-directed mutagenesis, cloning, and gene expression in E. coli, a series of proteins were obtained with single substitutions of the conserved active site amino acid residues—S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, and E176Q. After purification by ion exchange and affine chromatography electrophoretically homogeneous preparations were obtained. The study of the enzymatic activity with natural acceptors of the phosphoryl group (dAMP, dCMP, dGMP, and dTMP) demonstrated that the substitutions of charged amino acid residues of the NMP binding domain (R130, R172, D170, and E176) caused nearly complete loss of enzymatic properties. It was found that the presence of the OH-group at position 17 was also important for the catalytic activity. On the basis of the analysis of specific activity variations we assumed that arginine residues at positions 130 and 172 were involved in the binding to the donor γ-phosphoryl and acceptor α-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. Disproportional changes in enzymatic activities of partially active mutants, G137A, T138A, T17N, Q134A, S13A, and D16N, toward different substrates may indicate that different amino acid residues participate in the binding to various substrates.