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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 ( ) or aliphatic pentamethylene chain ( ), 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 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 and binding as a hairpin has been found to be DNA duplex 5'-CGTATACG-3'.

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 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: TATATTTATTTTAATTTTTTAATTT, 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 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.

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.

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.

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.

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.