Efficient conjugation and characterization of distamycin-based peptides with selected oligonucleotide stretches

Selected sequences of oligodeoxyribonucleotides (ODNs) have been conjugated efficiently with distamycin-based peptides containing reactive cysteine and oxyamine functionalities at the C-terminus. The conjugation was performed easily within 30-60 min, using individual modified oligonucleotide stretches having sequences of 5'-d(GCTTTTTTCG)-3', 5'-d(GCTATATACG)-3', and 5'-AGCGCGCGCA-3'. Two types of linkages were used for making the covalent connection: (i) a five-membered thiazolidine ring and (ii) an oxime. These distamycin-like polyamide-ODN conjugates were then converted to the corresponding DNA duplexes using complementary oligonucleotide sequences. To elucidate the binding specificity of the distamycin-oligonucleotide conjugates, UV-melting temperature measurements were performed. These studies indicated that the distamycin-ODN conjugate favored binding with the duplex with sequence 5'-d(GCTTTTTTCG)-3' rather than 5'-d(GCTATATACG)-3'. On the other hand, no stabilization of the duplex with sequence 5'-d(AGCGCGCGCA)-3' was observed. UV results also suggest that the thiazolidine and oxime linkages do not significantly influence the process of distamycin binding to the minor groove surface of the DNA duplex. The results obtained from duplex UV-melting studies were further corroborated by a temperature-dependent study of the circular dichroism spectra of the conjugates and a fluorescence displacement titration assay using Hoechst 33258 fluorophore as a competitive binder for the minor groove. All these studies reinforce the fact that the specific stabilization of A/T rich DNA-DNA duplexes by distamycin was preserved upon conjugation with oligonucleotide stretches.     

DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footprinting.

The technique of DNAase I footprinting has been used to investigate preferred binding sites for actinomycin D and distamycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Only sites containing the dinucleotide step GpC are protected by binding of actinomycin, and all such sites are protected. Distamycin recognizes four major regions rich in A + T residues. Both antibiotics induce enhanced rates of cleavage at certain regions flanking their binding sites. These effects are not restricted to any particular base sequence since they are produced in runs of A and T by actinomycin and in GC-rich sequences by distamycin. The observed increases in susceptibility to nuclease attack are attributed to DNA structural variations induced in the vicinity of the ligand binding site, most probably involving changes in the width of the helical minor groove.     

Combinatorial identification of a novel consensus sequence for the covalent DNA-binding polyamide tallimustine

Many agents successfully used in cancer chemotherapy either directly or indirectly covalently modify DNA. Examples include cisplatin, which forms a covalent adduct with guanines, and doxorubicin, which traps a cleavage intermediate between topoisomerase II and torsionally strained DNA. In most cases, the efficacy of these drugs depends on the efficiency and specificity of their DNA binding, as well as the discrimination between normal and neoplastic cells in their handling of the drug-DNA adducts. While much is known about the chemistry of drug-DNA adducts, little is known regarding the overall specificity of their formation, especially in the context of a whole human genome, where potentially billions of binding sites are possible. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA-binding specificity of the semisynthetic covalent DNA-binding polyamide tallimustine, which contains a benzoic acid nitrogen mustard appended to the minor groove DNA-binding natural product distamycin A. After investigating over 134 million possible sequences, we found that the highest affinity tallimustine binding sites contained one of two consensus sequences, either the expected distamycin hexamer binding sites followed by a CG base pair (e.g., 5'-TTTTTTC-3' and 5'-AAATTTC-3') or the unexpected sequence 5'-TAGAAC-3'. Curiously, we found that tallimustine preferentially alkylated the N7 position of guanines located on the periphery of these consensus sequences. These findings suggested a cooperative binding model for tallimustine in which one molecule noncovalently resides in the DNA minor groove and locally perturbs the DNA structure, thereby facilitating alkylation by a second tallimustine of an exposed guanine on another side of the DNA.     

Nucleotide sequence specificity of DNA cleavage by iron-bleomycin. Alteration on ethidium bromide-, actinomycin-, and distamycin-intercalated DNA

The specific nucleotide recognition and sequence-specific cleavage of DNA by bleomycin (BLM) antibiotics are a typical example of macromolecular receptor-drug interaction in the field of chemotherapy. The present results demonstrate that ethidium bromide, distamycin A, and actinomycin D evidently altered the nucleotide sequence-specific mode of DNA breakage by the iron-BLM system, which cleaves isolated DNA preferentially at G-C (5' leads to 3') and G-T (5' leads to 3') sequences. In the presence of ethidium bromide, the most preferred cleavage site was the sequence G-T at position 52 to 53. Of special interest is marked alteration of the nucleotide sequence-specific mode by distamycin A. This intercalator masked the cleavages at G-T and G-A sequences, and produced higher specificity for G-C sequences than that of iron-BLM only. In the case of actinomycin D, the preferred sequence groups of DNA breakage were shifted from G-C sequences to G-A (43 to 44) and G-T (52 to 53) sequences. Certain intercalating agents are very available for the investigations of site-specific recognition and cleavage of DNA by DNA-cleaving drugs such as BLM.     

Minor groove to major groove, an unusual DNA sequence-dependent change in bend directionality by a distamycin dimer

DNA sequence-dependent conformational changes induced by the minor groove binder, distamycin, have been evaluated by polyacrylamide gel electrophoresis. The distamycin binding affinity, cooperativity, and stoichiometry with three target DNA sequences that have different sizes of alternating AT sites, ATAT, ATATA, and ATATAT, have been determined by mass spectrometry and surface plasmon resonance to help explain the conformational changes. The results show that distamycin binds strongly to and bends five or six AT base pair minor groove sites as a dimer with positive cooperativity, while it binds to ATAT as a weak, slightly anticooperative dimer. The bending direction was evaluated with an in phase A-tract reference sequence. Unlike other similar monomer minor groove binding compounds, such as netropsin, the distamycin dimer changes the directionality of the overall curvature away from the minor groove to the major groove. This distinct structural effect may allow designed distamycin derivatives to have selective therapeutic effects.     

DNA sequence dependent binding modes of 4',6-diamidino-2-phenylindole (DAPI)

The interactions of DAPI with natural DNA and synthetic polymers have been investigated by hydrodynamic, DNase I footprinting, spectroscopic, binding, and kinetic methods. Footprinting results at low ratios (compound to base pair) are similar for DAPI and distamycin. At high ratios, however, GC regions are blocked from enzyme cleavage by DAPI but not by distamycin. Both poly[d(G-C)]2 and poly[d(A-T)]2 induce hypochromism and shifts of the DAPI absorption band to longer wavelengths, but the effects are larger with the GC polymer. NMR shifts of DAPI protons in the presence of excess AT and GC polymers are significantly different, upfield for GC and mixed small shifts for AT. The dissociation rate constants and effects of salt concentration on the rate constants are also quite different for the AT and the GC polymer complexes. The DAPI dissociation rate constant is larger with the GC polymer but is less sensitive to changes in salt concentration than with the AT complex. Binding of DAPI to the GC polymer and to poly[d(A-C)].poly[d(G-T)] exhibits slight negative cooperativity, characteristic of a neighbor-exclusion binding mode. DAPI binding to the AT polymer is unusually strong and exhibits significant positive cooperativity. DAPI has very different effects on the bleomycin-catalyzed cleavage of the AT and GC polymers, a strong inhibition with the AT polymer but enhanced cleavage with the GC polymer. All of these results are consistent with two totally different DNA binding modes for DAPI in regions containing consecutive AT base pairs versus regions containing GC or mixed GC and AT base pair sequences. The binding mode at AT sites has characteristics which are similar to those of the distamycin-AT complex, and all results are consistent with a cooperative, very strong minor groove binding mode. In GC and mixed-sequence regions the results are very similar to those observed with classical intercalators such as ethidium and indicate that DAPI intercalates in DNA sequences which do not contain at least three consecutive AT base pairs.     

Distamycin-induced inhibition of homeodomain-DNA complexes.

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.     

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.     

Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2.

We have designed and synthesized a series of novel DNA photocleaving agents which break DNA with high sequence specificity. These compounds contain the non-diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analogues of the DNA binding portion of the antibiotic bleomycin A2. By using a variety of 5' or 3' 32P-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazole compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce DNA cleavage 3' of the site. Analysis of cleavage sites on the complementary DNA strand and inhibition of DNA breakage by distamycin A indicates these bithiazole derivatives bind and attack the minor groove of DNA. A photoactive unithiazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequence recognition of these derivatives appears to be determined by the thiazole moiety rather than the p-nitrobenzoyl group: use of a tetramethylene group in place of a dimethylene spacer shifted the position of DNA breakage by one base pair. Moreover, much less specific DNA photocleavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spacer. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives contrasts with that of bleomycin A2 which cleaves DNA most frequently at 5'-GPy-3' sequences. These results suggest that the cleavage specificity exhibited by bleomycin is not simply determined by its bithiazole/sulphonium terminus, and the contributions from other features, e.g. its metal-chelating domain, must be considered. The novel thiazole-based DNA cleavage agents described here should prove useful as reagents for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands.     

1H NMR and optical spectroscopic investigation of the sequence-dependent dimerization of a symmetrical cyanine dye in the DNA minor groove

A symmetrical cyanine dye was previously shown to bind as a cofacial dimer to alternating A-T sequences of duplex DNA. Indirect evidence suggested that dimerization of the dye occurred in the minor groove. 1H NMR experiments reported here verify this model based on broadening and shifting of signals due to protons on carbon 2 of adenine and imino protons at the central five A-T pairs of the 11 base pair duplex: 5'-GCGTATATGCG-3'/3'-CGCATATACGC-5'. This binding mode is similar to that of distamycin A, even though the dye lacks the hydrogen-bonding groups used by distamycin for sequence-specific recognition. Surprisingly, the third base pair (G-C) was also implicated in the binding site. UV-vis experiments were used to compare the extent of dimerization of the dye for 11 different sequence variants. These experiments verified the importance of a G-C pair at the third position: replacing this pair with A-T suppressed dimerization. These results indicate that the dye binding site spans six base pairs: 5'-GTATAT-3'. The initial G-C pair seems to be important for widening the minor groove rather than for making important contacts with the dye molecules since inverting its orientation to C-G or replacing it with I-C still led to favorable dimerization of the dye.     

Distamycin A affects the stability of NF-kappaB p50-DNA complexes in a sequence-dependent manner

The effect of two different DNA minor groove binding molecules, Hoechst 33258 and distamycin A, on the binding kinetics of NF-kappaB p50 to three different specific DNA sequences was studied at various salt concentrations. Distamycin A was shown to significantly increase the dissociation rate constant of p50 from the sequences PRDII (5'-GGGAAATTCC-3') and Ig-kappa B (5'-GGGACTTTCC-3') but had a negligible effect on the dissociation from the palindromic target-kappaB binding site (5'-GGGAATTCCC-3'). By comparison, the effect of Hoechst 33258 on binding of p50 to each sequence was found to be minimal. The dissociation rates for the protein--DNA complexes increased at higher potassium chloride concentrations for the PRDII and Ig-kappaB binding motifs and this effect was magnified by distamycin A. In contrast, p50 bound to the palindromic target-kappaB site with a much higher intrinsic affinity and exhibited a significantly reduced salt dependence of binding over the ionic strength range studied, retaining a K(D) of less than 10 pM at 150 mM KCl. Our results demonstrate that the DNA binding kinetics of p50 and their salt dependence is strongly sequence-dependent and, in addition, that the binding of p50 to DNA can be influenced by the addition of minor groove-binding drugs in a sequence-dependent manner.     

Probing the interaction of distamycin A with S100β: the “unexpected” ability of S100β to bind to DNA‐binding ligands

DNA‐minor‐groove‐binding ligands are potent antineoplastic molecules. The antibiotic distamycin A is the prototype of one class of these DNA‐interfering molecules that have been largely used in vitro. The affinity of distamycin A for DNA is well known, and the structural details of the complexes with some B‐DNA and G‐quadruplex‐forming DNA sequences have been already elucidated. Here, we show that distamycin A binds S100β, a protein involved in the regulation of several cellular processes. The reported affinity of distamycin A for the calcium(II)‐loaded S100β reinforces the idea that some biological activities of the DNA‐minor‐groove‐binding ligands arise from the binding to cellular proteins. Copyright © 2015 John Wiley & Sons, Ltd.     

DNA minor groove binding agents interfere with topoisomerase II mediated lesions induced by epipodophyllotoxin derivative VM-26 and acridine derivative m-AMSA in nuclei from L1210 cells

This study demonstrated that agents capable of interacting with the minor groove in nuclear DNA interfere with topoisomerase II mediated effects of antitumor drugs such as VM-26 and m-AMSA. Distamycin, Hoechst 33258, and DAPI were used as agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range distortion of DNA structure. In intact nuclei from L1210 cells, these minor groove binders inhibited the induction of topoisomerase II mediated DNA damage (DNA-protein cross-links and DNA double-strand breaks) by VM-26 and m-AMSA. The inhibitory effects of distamycin reflected prevention of formation of new lesions but not reversal of preexisting damage. The minor groove binders did not differentiate between lesions induced by an intercalator, m-AMSA, or by a DNA-nonbinding drug, VM-26. All three groove binders inhibited DNA breaks more strongly than DNA-protein cross-links. The inhibitory potency correlated with the size of minor groove binders and the size of their DNA-binding sites: distamycin (5 bp) greater than Hoechst 33258 (4 bp) greater than DAPI (3 bp). The results showed that DNA minor groove binders are a new type of modulators of the action of topoisomerase II targeted drugs.     

DNA conformational analysis in solution by uranyl mediated photocleavage.

Uranyl mediated photocleavage of double stranded DNA is proposed as a general probing for DNA helix conformation in terms of minor groove width/electronegative potential. Specifically, it is found that A/T-tracts known to constitute strong distamycin binding sites are preferentially photocleaved by uranyl in a way indicating strongest uranyl binding at the center of the minor groove of the AT-region. The A-tracts of kinetoplast DNA show the highest reactivity at the 3'-end of the tract--as opposed to cleavage by EDTA/Fell--in accordance with the minor groove being more narrow at this end. Finally, uranyl photocleavage of the internal control region (ICR) of the 5S-RNA gene yields a cleavage modulation pattern fully compatible with that obtained by DNase I which also--in a more complex way--senses DNA minor groove width.     

Lesion selectivity in blockage of lambda exonuclease by DNA damage.

Various kinds of DNA damage block the 3' to 5' exonuclease action of both E. coli exonuclease III and T4 DNA polymerase. This study shows that a variety of DNA damage likewise inhibits DNA digestion by lambda exonuclease, a 5' to 3' exonuclease. The processive degradation of DNA by the enzyme is blocked if the substrate DNA is treated with ultraviolet irradiation, anthramycin, distamycin, or benzo[a]-pyrene diol epoxide. Furthermore, as with the 3' to 5' exonucleases, the enzyme stops at discrete sites which are different for different DNA damaging agents. On the other hand, digestion of treated DNA by lambda exonuclease is only transiently inhibited at guanine residues alkylated with the acridine mustard ICR-170. The enzyme does not bypass benzo[a]-pyrene diol epoxide or anthramycin lesions even after extensive incubation. While both benzo[a]-pyrene diol epoxide and ICR-170 alkylate the guanine N-7 position, only benzo[a]-pyrene diol epoxide also reacts with the guanine N-2 position in the minor groove of DNA. Anthramycin and distamycin bind exclusively to sites in the minor groove of DNA. Thus lambda exonuclease may be particularly sensitive to obstructions in the minor groove of DNA; alternatively, the enzyme may be blocked by some local helix distortion caused by these adducts, but not by alkylation at guanine N-7 sites.     

Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I)

The bis(1,10-phenanthroline)copper(I) complex is a relatively simple molecule previously shown to cause DNA cleavage with a strong preference for gene control regions such as the Pribnow box. Sequence level mapping of sites of [(Phen)2CuI]+ cleavage in greater than 2000 bases in histone genes and the plasmid pUC9 showed that the specificity for control regions is related to a predominant preference for minor groove binding at TAT triplets, which were cleaved most strongly at the adenosine sugar ring. The related sequences TGT, TAAT, TAGPy, and CAGT (Py = pyrimidine) were moderately preferred, while CAT and TAC triplets, PyPuPuPu quartets, PuPuPuPy quartets, and CG-rich PyPuPuPy quartets were cleaved with low to average frequency. Polypurine and polypyrimidine sequences were cleaved with low frequency. The sequence preferences of [(Phen)2CuI]+ can be ascribed predominantly to (i) a requirement for binding in the minor groove at a pyrimidine 3'----5' step and (ii) stereoelectronic effects of the 2-amino group of guanine in the minor groove, which inhibit binding. Although the reagent appears primarily to recognize sequence features at the triplet or quartet level, lower than expected cleavage was observed for two TAT sequences adjacent to several other preferred sequences and higher than expected cleavage was observed at CAAGC sequences, suggesting that longer range sequence-dependent DNA conformational effects influence specificity in certain cases.     

Molecular dynamics investigation of the interaction between DNA and distamycin

The complex of the minor groove binding drug distamycin and the B-DNA oligomer d-(CGCAAATTTGCG) was investigated by molecular dynamics simulations. For this purpose, accurate atomic partial charges of distamycin were determined by extended quantum chemical calculations. The complex was simulated without water but with hydrated counterions. The oligomer without the drug was simulated in the same fashion and also with 1713 water molecules and sodium counterions. The simulations revealed that the binding of distamycin in the minor groove induces a stiffening of the DNA helix. The drug also prevents a transition from B-DNA to A-DNA that was found to occur rapidly (30 ps) in the segment without bound distamycin in a water-free environment but not in simulations including water. In other simulations, we investigated the relaxation processes after distamycin was moved from its preferred binding site, either radially or along the minor groove. Binding in the major groove was simulated as well and resulted in a bound configuration with the guanidinium end of distamycin close to two phosphate groups. We suggest that, in an aqueous environment, tight hydration shells covering the DNA backbone prevent such an arrangement and thus lead to distamycin's propensity for minor groove binding.     

Accessibility of the minor groove of DNA in chromatin to the binding of antibiotics netropsin and distamycin A

The interaction of the antibiotics distamycin A, distamycin analogue and netropsin with chromatin of calf thymus has been studied by circular dichroism measurements and by gel filtration. The minor groove of DNA in chromatin is accessible by 83–89% to the binding of these antibiotics as compared with that of free DNA. The present results combined with our data on the methylation of chromatin with dimethylsulphate [3] strongly suggest that the minor groove of DNA in chromatin is not occupied by chromatin proteins.Abbreviations DM distamycin A - DM₂ analogue of distamycin - Nt netropsin - CD spectra circular dichroism spectra