A. L. Zhuze

Binding of Symmetrical Cyanine Dyes into the DNA Minor Groove

Abstract Optical methods, such as fluorescence, circular dichroism and linear flow dichroism, were used to study the binding to DNA of four symmetrical cyanine dyes, each consisting of two identical quinoline, benzthiazole, indole, or benzoxazole fragments connected by a trime-thine bridge. The ligands were shown to form a monomer type complex into the DNA minor groove. The complex of quinoline-containing ligand with calf thymus DNA appeared to be the most resistant to ionic strength, and it did not dissociate completely even in 1 M NaCl. Binding of cyanine dyes to DNA could also be characterized by possibility to form ligand dimers into the DNA minor groove, by slight preference of binding to AT pairs, as well as by possible intercalation between base pairs of poly(dG)·poly(dC). The correlation found between the binding constants to DNA and the extent of cyanine dyes hydrophobicity estimated as the n-octanol/water partition coefficient is indicative of a significant role of hydrophobic interactions for the ligand binding into the DNA minor groove.

Interaction of Topotecan, DNA Topoisomerase I Inhibitor, with Double-Stranded Polydeoxyribonucleotides. 1. Topotecan Dimerization in Solution

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5–11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa ≈ 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10–5M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10–6M) to 7.1 ([TPT] = 10–4M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 ± 0.7) · 103M–1.

The hoechst 33258 covalent dimer covers a total turn of the double-stranded DNA

Abstract With the goal to design ligands recognizing extended regions on dsDNA, a covalent dimer of the fluorescent dye Hoechst 33258 [bis-HT(NMe)] composed of two dye molecules linked via the phenol oxygen atoms with a (CH2)3-N+H(CH3)-(CH2)3 fragment was constructed using computer modeling and then synthesized. Its interactions with the double-stranded DNA (dsDNA) were studied by fluorescent and UV-Vis spectroscopy and circular (CD) and linear dichroism (LD). Based on variations in the affinity to the dsDNA, it was shown that complexes of three types are formed. The first type complexes result from binding of a bis- HT(NMe) monomer in the open conformation; in this case the ligand covers the total dsDNA turn and is located in the minor groove according to the positive value of CD at 370 nm. In addition, the ability to form bis-HT(NMe)-bridges between two dsDNA molecules, i.e., each of the two bis-HT(NMe) ends binds to two different dsDNA molecules, was demonstrated for the first type complexes. Spectral characteristics (maximal absorption at 362 nm, positive sign, and maximal value of CD at 370 nm) of the first type complexes conform to those of the specific Hoechst 33258 complex with poly[d(A-T)] · poly[d(A-T]. The second type complexes correspond to the bis-HT(NMe) sandwich (as an inter- or intramolecular) binding to dsDNA with stoichiometry ≥ 5 bp. Thereby, a negative LD at 360 nm and the location of bis- HT(NMe) sandwiches in the minor groove of B form dsDNA seems contradictory. Spectral characteristics (maximal positive CD at 345 nm, a dramatic decrease in fluorescence intensity and the shift of its maximum to 490 nm) of these complexes favor a suggestion that this binding correlates to the formation of nonspecific dimeric Hoechst 33258 complex with dsDNA. The third type complexes are characterized by stoichiometry of one bis-HT(NMe) molecule per ≈ 2 bp and the tendency to zero of LD values at 270 and 360 nm. We assume that in these complexes bis-HT(NMe) sandwich dimers are formed on dsDNA. The complexes of this type conform to the aggregation type complex of Hoechst 33258 with dsDNA. The ability of bis-HT(NMe) to cover the whole dsDNA turn or form bridges with two dsDNA upon the formation of the first type complexes essentially distinguishes it from Hoechst 33258, which can only occupy 5 bp and does not form such bridges. This specific property of bis-HT(NMe) may support new biological activities.

Dimeric bisbenzimidazoles inhibit the DNA methylation catalyzed by the murine Dnmt3a catalytic domain

When located in the DNA minor groove, dimeric bisbenzimidazoles DB(n) effectively inhibited in vitro the Dnmt3a catalytic domain (IC50 5–77 μM). The lowest IC50 value was observed for compound DB(11) with an 11-unit methylene linker joining the bisbenzimidazole fragments. Increased time of incubation of DNA with DB(n) as well as the presence of AT-clusters in DNA enhances the inhibitory effect.

Inhibition of the helicase activity of the HCV NS3 protein by symmetrical dimeric bis-benzimidazoles

Dimeric bis-benzimidazoles (DBn) are the compounds specifically binding to A-T enriched sequences in the DNA minor groove. Due to this property they can inhibit DNA-dependent enzymes. We show that inhibition of the helicase activity of HCV NS3 protein by DBn was due to a novel mechanism, which involved direct binding of the ligands to the enzyme. The binding potency and inhibition efficacy depended on the length of the linker between the benzimidazole fragments. The most effective inhibitor DB11 partially prevented activation of NTPase activity of NS3 by poly(U) and increased affinity of the enzyme to the helicase substrate DNA.

Human DNA‐topoisomerase I activity is affected by bis‐netropsin's binding to DNA minor groove

Bis‐netropsins (bis‐Nts) are known to be efficient inhibitors of human DNA topoisomerase (topo) I with a higher antitumor activity than netropsin. New sequence‐specific derivatives of bis‐Nts were used for modulation of topo I‐mediated DNA cleavage with and without camptothecin (CPT). Relation between the bis‐Nts binding sites and topo I cleavage sites has been analyzed with the plasmid DNA constructs generated by insertion of synthetic oligonucleotides containing various topo I‐cleavage and bis‐Nt‐binding sites. These constructs offer an opportunity to study minor groove binders effects on the topo I reaction on DNA. Three major effects: (i) bis‐Nt ‐ mediated disappearance of some of the topo I cleavage sites; (ii) enhancement of some other sites, and, (iii) generation of new cleavage sites, have been fovnd and analyzed. These effects demonstrate that bis‐Nts drastically change the topo I‐mediated DNA cleavage.

Raman and surface-enhanced Raman scattering spectroscopy of bis-netropsins and their DNA complexes.

The interactions of three bis-netropsins (bis-Nts), which are potent catalytic inhibitors of DNA-binding enzymes, with three double-stranded oligonucleotides (OLIGs), which contain sites of different specific affinities for each bis-Nt, were analyzed. Raman spectroscopy was performed for selective monitoring of modifications of the bis-Nt or the OLIG structure upon bis-Nt-DNA binding, and surface-enhanced Raman scattering spectroscopy (SERS) was an additional tool for topology studies of ligand-DNA complexes. The spectral data showed conformational changes of both partners (bis-Nt and OLIG) upon complexation. Structural variations of bis-Nts appeared to be dependent on a bis-Nt-OLIG binding constant and were found to be small in the specific DNA binding and highest for nonspecific binding of bis-Nt with the corresponding OLIG. The conformational changes of the OLIGs were varied with a bis-Nt-OLIG binding constant in the same manner. The bis-Nts seemed to induce a perturbation in the OLIGs structure, as well as in the positions of their direct binding. These DNA structural modification effects may explain the inhibition of DNA-binding enzymes in the variety of very distinct DNA-enzyme binding sites by bis-Nts reported previously.

Hoechst 33258—poly(dG-dC)·poly(dG-dC) Complexes Of Three Types

Abstract It was found recently that Hoechst 33258, a dsDNA fluorescent dye used in cytological studies, is an efficient inhibitor of the interaction of TATA-box-binding protein with DNA, DNA topoisomerase I, and DNA helicases. In addition it proved to be a radioprotector. Biological activity of Hoechst 33258 may be associated with dsDNA complexes of not only monomeric, but also dimeric type. In this work, the Hoechst 33258 interaction with poly(dG-dC)·poly(dG- dC) was studied using UV-vis and fluorescent spectroscopy, circular and flow-type linear dichroism. It was found that Hoechst 33258 formed with poly(dG-dC)·poly(dG-dC) complexes of three types, namely, monomeric, dimeric, and, apparently, tetrameric, and their spectral properties were studied. Complexes of monomeric and dimeric types competed with distamycin A, a minor groove ligand, for binding to poly(dG-dC)·poly(dG-dC). We proposed that Hoechst 33258 both monomers and dimers form complexes of the external type with poly(dG-dC)·poly(dG-dC) from the side of the minor groove.

DNA sequence-specific ligands: XIII. New dimeric Hoechst 33258 molecules, inhibitors of HIV-1 integrase in vitro

Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta-or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N,N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of ligand chromophores in the CD spectrum of the DNA CLD indicates the formation of a DBBI-DNA complex in which ligand chromophores are arranged at an angle close to 54° relative to the helix axis of DNA, which suggests the localization of the DBBI in the narrow groove of DNA. All the DBBIs were found to be in vitro inhibitors of HIV-1 DNA integrase in the 3′-processing reaction, and, of the three DBBIs, two dimers inhibit HIV-1 integrase even in submicromolar concentrations.

Interaction of Topotecan, DNA Topoisomerase I Inhibitor, with Double-stranded Polydeoxyribonucleotides. 3. Binding at the Minor Groove

Interaction of topotecan (TPT) with calf thymus DNA, coliphage T4 DNA, and poly(dGdC) · poly(dG-dC) was studied by optical (linear flow dichroism, UV-vis spectroscopy) and quantum chemical methods. The linear dichroism signal of TPT bound to DNA was shown to have positive sign in the range 260–295 nm. This means that the plane of quinoline fragment (rings A and B) of TPT forms an angle less than 54° with the long axis of DNA, and hence the TPT molecule cannot intercalate between DNA base pairs. TPT was established to bind to calf thymus DNA as readily as to coliphage T4 DNA whose cytosines in the major groove were all glycosylated at the 5th position. Consequently, the DNA major groove does not participate in TPT binding. TPT molecule was shown to compete with distamycin for binding sites in the minor groove of DNA and poly(dG-dC) · poly(dG-dC). Thus, it was demonstrated for the first time that TPT binds to DNA at its minor groove.