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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.

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.

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.

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*.

Cleavages of double-stranded DNA fragments of known base pair sequence upon ultrasound irradiation at 22 and 44 kHz were studied by gel electrophoresis. The cleavage rate is found to be strongly dependent on the DNA fragment length, pH, temperature and ionic strength of the solution under study. The cleavage of double-stranded DNA occurs predominantly at sites containing alternating 5'-CpG-3' sequences. The breakage of phosphatediester bond takes place between C and G in such a way that phosphate group at the 5'-end of the guanine residue remain intact. The cleavage rate at a given DNA site is found to depend on base pair sequences at adjacent sites. Distinctly different cleavage patterns are observed when free DNA and DNA complexes with cys-diammine-Pt-bridged bis-netropsin were irradiated by ultrasound. The observed effect can be attributed to local DNA conformation changes induced upon complex formation between ligand and DNA.

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.

The kinetics of the hydrolysis of the lactone ring of a hybrid molecule containing the molecules of the antitumor drug camptothecin and a derivative of the antibiotic netropsines, which is highly affine and specific to the DNA A-T sequences was investigated. It was shown that intramolecular interaction significantly slows down the rate of hydrolysis but does not change the equilibrium ratio of concentrations of the lactone and carboxylate forms of the camptothecin fragment of the hybrid molecule, which corresponds to the pH value. The use of intramolecular interaction for controlling the kinetics of the lactone/carboxylate transition makes it possible to create the drugs of the camptothecin family, which preserve the biologically active lactone form under the physiological conditions for a longer time and, therefore, are more effective as anticancer agents.

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A.T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5'-A4T(4)-3' sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5'-T4A(4)-3' sequences. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.

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'.

An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units linked covalently to a copper-chelating tripeptide Gly-Gly-L-His by means of two and three glycine residues. Binding to DNA and synthetic polynucleotides of netropsin analogue containing three glycine residues between Gly-Gly-L-His tripeptide and the N-end of netropsin analogue (His-Nt) has been studied. It is shown that this netropsin analogue chelates a copper ion with 1:1 stoichiometry, similar to a free Gly-Gly-L-His peptide. It is found that this netropsin analogue occupies 3 to 4 base pairs upon binding to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers, irrespective of whether it binds in Cu(2+)-ligated or unligated forms. Binding constants and binding site sizes have been calculated for netropsin analogue complexes with DNA, poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers at the [Cu2+]/[His-Nt] ratio equal to 0 and 1.0. In the three-component system including His-Nt and Cu(2+)-His-Nt, cooperative effects are recognized which can be explained by heterodimer generation on interaction of His-Nt and Cu(2+)-His-Nt at adjacent binding sites.

Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.

In search for compounds capable of forming covalent bonds with DNA AT-pair clusters, distamycin A and netropsin analogues containing DL-sarcolysin or platinum (II) atom at the N-terminus of the molecule were synthesized, as well as bis-netropsin and bis-distamycin in which two netropsin- or distamycin-like fragments are bound via a cis-diammineplatinum (II) residue. It is shown that these substances can be used for the DNA selective cleavage.

The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases inhibition data was performed on the same DNA end-labelled fragment. The inhibition induced by netropsin, a number of bis-netropsins and distamycin A was investigated. These experiments led us to the following conclusions. The restriction endonucleases inhibition by the ligands is caused by the ligand molecules binding in the close vicinity to the restriction endonuclease recognition sequence. The zone of +/- 4 bp from the center of the restriction endonuclease recognition sequence can be defined as the zone of the influence of the bounded ligand on the restriction endonuclease. But in this case the intersection of recognition sequence and the binding site occupied by a single ligand molecule is not sufficient for the inhibition to occur. Restriction endonuclease cutting sites protected by netropsin can be predicted basing upon known nucleotide sequence specificity of netropsin. Netropsin and bis-netropsins show different nucleotide sequence specificity. This fact can be used for selective inhibition of restriction endonucleases.

In the present communication, synthesis and DNA binding activities of three analogs of the antibiotic netropsin are reported. Each analog contains two N-propylpyrrolecarboxamide units linked covalently to either Dns-Gly-Val-Val-Val-Gly-Gly- (I), Val-Val-Val-Gly-Gly (II) or Gly-Gly (III). It is shown that analogs I and II can self-associate in aqueous solution and methanol as revealed from the fact that UV absorbance and circular dichroism spectra obtained for these analogs are concentration-dependent. By contrast, analogs III exists as a monomer, even at concentration levels of the order of 1.10(-3) M. Determination of the apparent sizes of intramolecular aggregates by gel-filtration shows that analog I in aqueous solution at concentration levels of the order of 1.10(-3) M forms a series of aggregates containing from 2 to 12 monomers. Analog II exhibits a lower tendency to form intermolecular aggregates as compared with that of analog I. Dimerization constants are determined for analogs I and II in aqueous solution and methanol. The binding of N-propylpyrrolecarboxamide units and peptide fragments of analog I to DNA can be independently monitored by circular dichroism and fluorescence methods. If self-associated species of analog I (or II) are present in solution, the ligand exhibits a markedly different order of base pair sequence preferencies as compared with that of analog III. The results obtained are consistent with the inference that analogs I and II in a beta-associated form recognizes base pair sequences containing two runs of 3 AT pairs separated by two GC pairs.

In the present communication design, synthesis and DNA binding activities of three bis-netropsins and two netropsin analogs containing two N-propylpyrrolecarboxamide fragments linked covalently to peptides Gly-Gly-(analog I) and Val-Val-Val-Gly-Gly-(analog II) are reported. Each bis-netropsin consists of two netropsin-like fragments attached to peptides -Gly-Cys-Gly-NH2 (compound IIIa), H-Gly-Cys-Gly-Gly-Gly-(compound IV) or Gly-Cys-Sar-NH2 (compound IIIb) which are linked symmetrically via S-S bonds. Physico-chemical studies show that each bis-netropsin carries 6 AT-specific reaction centers and covers approximately 10 base pairs upon binding to poly(dA).poly(dT). This indicates that two netropsin-like fragments of the bis-netropsin molecule are implicated in specific interaction with DNA base pairs. The peptide fragments of bis-netropsins IIIa and IV form small beta-sheets containing two-GC-specific reaction centers. The DNase I cleavage patterns of bis-netropsin-DNA complexes visualized by high resolution gel electrophoresis show that the preferred binding sites for bis-netropsins IIIa and IV are identical and contain two runs of three or more AT pairs separated by two GC pairs. Specificity determinants of netropsin analog II binding in the beta-associated dimeric form are identical to those of bis-netropsin IIIa thereby indicating that there is a similarity in the structure of complexes formed by these ligands with DNA. In the monomeric form analog II exhibits binding specificity identical to that of analog I. Replacement of C-terminal glycine residues by sarcosines in the peptide fragments of bis-netropsin IIIa leads to a decrease in the affinity of ligand for DNA.

It has been found that the effect of AT-specific ligand and Zn2+ on GC-alternating polymer brings about transfer of the latter into Z-conformation.

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.