[Research of suppression of the herpes simplex virus reproduction with drug resistance using a combination 15-Lys-bis-Nt with some antiherpetic drugs].

The antiviral effect of combinations of netropsin derivative 15-Lys-bis-Nt with the known antiherpetic compounds, whose activity does not depend on viral TK and which are able to inhibit replication of HSV in most cases, including strains resistant to acyclovir and pencyclovir, was studied. The combinations evoking additive, synergistic and significant synergistic effects of interaction of tested compounds were observed. The results obtained in this work indicated the possibility of significant reduction of concentrations of high toxic compounds in case of the combined use.

[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.

[Estimation of activity of bis-netropsin derivatives based on a model of an experimental cutaneous herpes simplex virus disease of guinea pigs].

Using the model of an experimental cutaneous infection of guinea pig males caused by herpes simple virus type 1, it is shown that application of dimerico derivatives of netropsin Lys-bis Nt and 15Lys-bis Nt in the form of polietilenglicol-based ointment suppresses viral infection more effectively than acyclovir.

[Antiherpetic activity of netropsin derivatives as tested in experiments in laboratory animals].

Two dimeric netropsin derivatives (Lys-bis-Nt 15Lys-bis-Nt) were comprehensively tested for antiviral and toxic activity in cell cultures and laboratory animals. The two compounds were found to provide effective and selective inhibition of reproduction of herpes simplex I both in cell culture Vero E6 and in brain of infected white mice, thereby increasing the survival rate and mean life expectation of treated animals as compared to control.

[Inhibition of herpes simplex virus helicase UL9 by netropsin derivatives and antiviral activities of bis-netropsins].

Data obtained show that antiviral activities of bis-linked netropsin derivatives are targeted by specific complexes formed by helicase UL9 of herpes simplex virus type 1 with viral DNA replication origins, represented by two OriS sites and one OriL site. According to the results of footprinting studies bis-netropsins get bound selectively to an A+T-cluster which separates interaction sites I and II for helicase UL9 in OriS. Upon binding to DNA bis-netropsins stabilize a structure of the A+T-cluster and inhibit thermal fluctuation-induced opening of AT- base pairs which is needed for local unwinding of DNA by helicase UL9. Kinetics of ATP-dependent DNA unwinding in the presence and absence of Pt-bis-netropsin are studied by measuring the efficiency of Forster resonance energy transfer (FRET) between the fluorescent probes attached covalently to 3?- and 5?-ends of the oligonucleotides in the minimal OriS duplex. Pt-bis-netropsin and related molecules inhibit unwinding of OriS duplex by helicase UL9. Pt-bis-netropsin is also able to reduce the rate of unwinding of the AT- rich hairpin formed by the upper strand in the minimal OriS duplex. The antiviral activities and toxicity of bis-linked netropsin derivatives are studied in cell cultured experiments and experiments with animals infected by herpes virus.

[Complex of the herpes simplex virus initiator protein UL9 with DNA as a platform for the design of a new type of antiviral drugs].

The protein binding to the origin of replication of the herpes simplex virus type 1 (HSV-1) is DNA helicase encoded by the UL9 gene of the herpes virus. The protein specifically binds to two binding sites in the viral DNA replication origins OriS or OriL. In order to determine the role of the UL9 protein in the initiation of replication and find efficient inhibitors of the UL9 activity, we have synthesized a recombinant UL9 protein expressed in E. coli cells. It was found that the recombinant UL9 protein binds to Boxes I and II in the OriS and possesses the DNA helicase and ATPase activities. In a complex with a fluorescent analog of ATP, two molecules of the ATP analog bind to one protein dimer molecule. It was also found that the UL9 protein in the dimer form can bind simultaneously to two DNA fragments, each containing specific binding sites for the protein. The interaction of the recombinant UL9 protein with the 63-mer double and single-stranded oligonucleotides OriS and OriS* has been investigated, which correspond to the origin of replication of herpes simplex virus. From the titrations of OriS and OriS* by ethidium bromide in the presence and absence of the UL9 protein, the equilibrium affinity constants of the protein binding to OriS and OriS* have been determined. A DNase I footprinting study showed that bis-linked netropsin derivatives exhibit preferences for binding to the AT-cluster in the origin of replication OriS and inhibit the fluctuation opening of AT-base pairs in the AT-cluster. The drugs also prevent the formation of an intermediate conformation of OriS* that involves a disordered tail at the 3'-end and stable Box I-Box III hairpin to which the UL9 helicase selectively binds. The stabilization by bis-netropsins of the AT-rich hairpin at its 3' end can inhibit the helicase activity. It was concluded that the antiviral activity of bis-netropsins may be associated with the inhibitory effects of bis-netropsins on these two stages of the reaction catalyzed by helicase UL9.

[Antiviral properties of the derivatives of netropsin and distamycin against herpes simplex viruses type 1 and variolovaccine].

The antiherpesvirus activity of newly synthesized DNA-binding compounds for cultured Vero E6 cells was examined. The compounds were found to have selective antiherpesviral activity. Their antiviral activity was shown against the virus strains isolated from clinical specimens. The test compounds were ascertained to have also a high activity against herpes simplex virus type 1 (HSV-1/L2 TC-) that was very resistant to acyclovir. The authors' data demonstrated an obvious dose-dependent antiviral effect, which was representatively seen when Pt-bis-Dst and Lys-bis-Nt were used. The findings have offered the challenge to test some of these compounds in experiments on laboratory animals.

[Statistical fluctuations of the filling level of operator by repressor determine the level of noise of reporter gene expression].

The regulation of the reporter gene activity in a single bacterial cell by means of lambda-phage C1 repressor has been described by the methods of statistical thermodynamics. The equations for the calculation of the mean production rate of the reporter protein and its standard deviation as a function of C1 repressor concentration in the cell have been obtained. The stochastic nature of C1 repressor binding with OR1 and OR2 operator sites becomes apparent when both repressor molecules and operators are present in the bacterial cell in a small number of copies. In this case, the number of repressor molecules that bind to OR1 and OR2 sites fluctuates considerably. The in vitro binding of C1 repressor to OR1 and OR2 sites, their mutant forms, and nonspecific DNA regions has been well studied. Using the binding constants of in vitro binding of C1 repressor to OR1, OR2 and nonspecific DNA regions and also the value of the cooperativity parameter for C1 repressor binding to OR1 and OR2 sites, we calculated the mean rate of synthesis of the reporter protein and its standard deviation as a function of repressor concentration in cell. The theoretical relations fit well the experimental results. The results of calculations confirm the assumption that gene expression noise in a single cell at a repressor concentration exceeding 100 nM is related to the stochastic nature of binding of repressor dimers to OR1 and OR2 sites. Other mechanisms of the generation of gene expression noise (for example, monomer-dimer balance) make a significant contribution at concentrations less than 100 nM.

[DNA-binding activity of bis-netropsins containing a cis-diaminoplatinum group between two netropsin fragments].

The binding to DNA of Pt-bis-Nt and its modified analogue (Pt*-bis-Nt), which differs from Pt-bis-Nt by the fact that the connecting chain between two netropsin fragments contains two additional glycine residues, has been studied. Elongating the chain in the bis-netropsin molecule increases the cytotoxicity and leads to a complete disappearance of the antiherpetic activity of bis-netropsin. A study of the binding of two bis-netropsins with the oligonucleotide duplex containing an AT cluster, which is present at the replication initiation site of herpes virus (OriS), revealed significant structural differences between complexes of bis-netropsins with this DNA oligomer. It was shown by CD spectroscopy that the binding of Pt-bis-Nt in the elongated conformation and in the form of a hair-pin with the parallel orientation of two bis-netropsin fragments makes a greater contribution than it is the case in the complex formation with Pt*-bis-Nt. At high binding rates, Pt*-bis-Nt binds to the AT cluster in OriS predominantly in the form of associates based on the antiparallel double-stranded pyrrolcarboxyamide motif. The interaction of Pt-bis-Nt and Pt*-bis-Nt with the single-stranded oligonucleotide (64 nt), which corresponds to the upper strand at the replication initiation site of herpes virus (OriS*), was also studied. Substantial differences in the binding of bis-netropsins with OriS* and thermostability of the resulting complexes were found by CD spectroscopy and by studying the melting of complexes of bis-netropsins with OriS*.

[The interaction of lamda-Cro repressor and its mutant covalent S-S-dimer lamda-CroV55C with symmetric and asymmetric DNA].

Binding of lamda-Cro protein and mutant CroV55C disulfide bonded dimer to synthetic olygonucleotide duplexes were studied using a competition with distamycin A. The equilibrium binding constants for lamda operator OR3 and duplexes contained symmetry left or right halves of OR3 with one base pair deletion or insertion in center of duplex were calculated. The higher binding constant for Cro was detected with 17 bp symmetry duplex consist two left halves of OR3, for the mutant CroV55C higher binding constant was detected with 16 bp derivate of this duplex with the central GC base pair deletion.

[DNA-binding and antiviral activities of bis-netropsins].

The DNA-binding and antiviral activitus of bis-netropsins in which two monomers are attached covalently via three glycin residue were studied. These compounds have the same C-end groups but contain clusters with different numbers of lysine residues at the N-end of the molecule. In the homologous series of these compounds, bis-neropsins containing 15 and 31 branched lysine residues at the N-end of the molecule appear to be the most effective inhibitors of reproduction of the simplex herpes virus of type I in the Vero cell culture, including the virus versions resistant to aciclovir, ganciclovir, and other medicinal preparations. It was shown that the cytotoxicity of all the compounds studied is much lower than that of netropsin. The antiviral activity of the compounds correlates with their ability to selectively interact with the expanded clusters of the AT-pairs of DNA bases in the form of a monomer or a dimer, stabilized by interaction between the C-end halves of two bis-netropsin molecules bound at the neighboring overlapping binding sites on the DNA. The possible sites of their binding are the expanded clusters of AT-pairs at the origin of replication of OriS and OriL of the herpes virus.

[Thermodynamic models of binding ligands to nucleic acids]. / Termodinamicheskie modeli sviazyvaniia ligandov s nukleinovymi kislotami.

Models of adsorption were considered, which describe the binding of biologically active ligands on DNA templates. The binding is described most comprehensively and in greatest detail by the distribution function, which determines the probability of detecting the preset number of adsorbed ligands on the template. In the case of noncooperative binding, this function corresponds to the Gaussian distribution and is characterized by two quantities: the mean value of the occupation of the template by ligands and the dispersion of occupation. The accuracy of the occupation of the template by ligands is inversely proportional to dispersion. As the length of the template and the number of reaction sites covered by one ligand upon binding increase, the accuracy of the occupation of the template by ligands increases. An important characteristic of binding is the degree of coverage of the template by ligands. This characteristic represents the portion of template reaction sites covered by all ligands adsorbed on the template. If polycations are bound to nucleic acid molecules, the coverage of the template determines the transition of nucleic acids to a compact state. The degree of template coverage for extended ligands depends only slightly on the binding constant in a wide range of concentrations of a free ligand in solution. Different adsorption models are considered from the unified point of view. The classification of cooperative interactions for a wide class of systems is given, from situations when several ligands are bound on nucleic acid templates to a situation when templates change by the action of ligands and begin to interact with each other.

[Statistical fluctuations in processes of gene expression regulation: consideration of the problem from point of view of statistical mechanics]. / Statisticheskie fluktuatsii v protsessakh reguliatsii ékspressii genov: rassmotrenie s tochki zreniia statisticheskoi mekhaniki.

The regulation of gene expression is a basic problem of biology. In some cases, the gene activity is regulated by specific binding of regulatory proteins to DNA. In terms of statistical mechanics, this binding is described as the process of adsorption of ligands on the one-dimensional lattice and has a probability nature. As a random physical process, the adsorption of regulatory proteins on DNA introduces a noise to the regulation of gene activity. We derived equations, which make it possible to estimate this noise in the case of the binding of the lac repressor to the operator and showed that these estimates correspond to experimental data. Many ligands are able to bind nonspecifically to DNA. Nonspecific binding is characterized by a lesser equilibrium constant but a greater number of binding sites on the DNA, as compared with specific binding. Relations are presented, which enable one to estimate the probability of the binding of a ligand on a specific site and on nonspecific sites on DNA. The competition between specific and nonspecific binding of regulatory proteins plays a great role in the regulation of gene activity. Similar to the one-dimensional "lattice gas" of particles, ligands adsorbed on DNA produce "one-dimensional" pressure on proteins located at the termini of free regions of DNA. This pressure, an analog of osmotic pressure, may be of importance in processes leading to changes in chromatin structure and activation of gene expression.

[Effect of DNA local conformation on the affinity and binding specificity of bis-netropsins to DNA]. / Vliianie lokal'noi konformatsii DNK na srodstvo i spetsifichnost' sviazyvaniia bis-netropsinov s DNK.

The interaction of short nucleotide duplexes with bis-netropsins, in which netropsin fragments are linked in the tail-to-tail orientation via cis-diammineplatinum group (<--Nt-Pt(NH3)-Nt-->) or aliphatic pentamethylene chain (<--Nt-(CH2)5-Nt-->), has been studied. Both the bis-netropsins have been shown to bind to DNA oligomer 5'-CCTATATCC-3' (I) as a hairpin with parallel orientation of netropsin fragments in 1:1 stoichiometry. Monodentate binding has been detected upon binding of bis-netropsins to other duplexes of sequences 5'-CCXCC-3'--where X = TTATT (II), TTAAT (III), TTTTT (IV), and AATTT (V)--along with the binding of bis-netropsins as a hairpin. The formation of dimeric antiparallel motif between the halves of two bound bis-netropsin molecules has been observed in the complexes of <--Nt-(CH2)5-Nt--> with DNA oligomers IV and V. The ratio of binding constant of bis-netropsin as a hairpin (K2) to monodentate binding constant (K1) has been shown to correlate with the width and/or conformational lability of DNA in the binding site. The share of bis-netropsin bound as a hairpin decreases in the order: TATAT > TTATT > TTAAT > TTTTT > AATTT, whereas the contribution of monodentate binding rises. The minimal strong binding site for <--Nt-Pt(NH3)-Nt--> and <--Nt-(CH2)5-Nt--> binding as a hairpin has been found to be DNA duplex 5'-CGTATACG-3'.