Minor Groove Binder Distamycin Research Articles

Interactions of DNA binding ligands with PNA-DNA hybrids.

The interactions of two representative mixed-sequence (one with an AT-stretch) PNA-DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA-PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA-DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.

NMR characterization of a heterocomplex formed by distamycin (Dst) and its analog 2-imidazole-distamycin (2 ImD) with d(CGCAAGTTGGC):d(GCCAACTTGCG): preference for the 1:1:1 2-ImD:Dst:DNA complex over the 2:1 2-ImD:DNA and the 2:1 Dst:DNA complexes

The minor-groove binder distamycin (Dst), its pyrrole-imidazole-pyrrole analog, 2-imidazole-distamycin (2-ImD), and the oligonucleotide d(CGCAAGTTGGC):d(GCCAACTTGCG) were found to form a 1:1:1 2-ImD-Dst:DNA complex. As characterized by 2D NOE spectroscopy combined with molecular modeling, one 2-ImD and one Dst molecule bind simultaneously in a head-to-tail orientation contacting the minor groove of the central AAGTT:AACTT site. The 2-ImD ligand lies along the AAGTT strand with the imidazole nitrogen of the ligand specifically interacting with the guanine amino group. The distamycin ligand lies along the AACTT strand. The molecular structure of the 1:1:1 2-ImD:Dst:DNA complex is very similar to those of the complexes formed between two 2-ImD molecules (2:1 2-ImD:DNA complex) or two distamycin molecules (2:1 Dst:DNA complex) and the same oligonucleotide duplex. Competition titrations confirm that the 1:1:1 2-ImD:Dst:DNA complex forms preferentially over the 2:1 2-ImD:DNA complex, as well as over the newly discovered 2:1 Dst:DNA complex. These results indicate that (i) the hydrogen-bond-accepting imidazole nitrogen of the 2-ImD ligand in the 1:1:1 2-ImD:Dst:DNA complex is responsible for the strand-specific recognition of a GC base pair, (ii) the availability of a single hydrogen bond acceptor on the ligand molecule per guanine amino group enhanced both specificity and affinity of DNA binding, and (iii)more » different distamycin-like ligands can be combined in the 2:1 binding motif to expand the range of DNA sequences that can be specifically recognized through the minor groove. 18 refs., 7 figs., 4 tabs.« less

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.

Effects of minor groove binding drugs on camptothecin-induced DNA lesions in L1210 nuclei

Topoisomerase I inhibition detected in mammalian cells can be correlated with reduced tumor growth. Camptothecin specifically inhibits topoisomerase I by stabilization of a covalently linked DNA-enzyme complex and associated DNA single-strand breaks. Whether perturbations in nuclear DNA structure can alter camptothecin-induced DNA damage was examined using the non-intercalative DNA minor groove binders distamycin, Hoechst 33258 and DAPI (4', 6-diamidino-2-phenylindole). L1210 nuclei were treated with camptothecin alone or in the presence of single minor groove binders. DNA-protein crosslinks and single-strand breaks were determined using potassium-sodium dodecyl sulfate precipitation and alkaline elution respectively. Distamycin produced a dose-dependent decrease in DNA-protein crosslinks and strand breaks. This effect was reduced if nuclei were treated with camptothecin prior to distamycin addition. Distamycin was unable to reverse lesions once induced or to prevent repair of damage upon camptothecin removal. Hoechst 33258 and DAPI also decreased camptothecin-induced DNA damage. The order of inhibitory potency was: distamycin > Hoechst > DAPI. This order corresponded to the molecular weights as well as to the size of the nucleotide binding sites of the drugs. Identifying agents which alter such DNA lesions should provide better understanding of the chemotherapeutic activity of camptothecin as well as help elucidate new leads for drug combinations of improved therapeutic benefit.

Mammalian Topoisomerase II Activity Is Modulated by the DNA Minor Groove Binder Distamycin in Simian Virus 40 DNA

DNA topoisomerases II are nuclear enzymes that have been identified recently as targets for some of the most active anticancer drugs. Antitumor topoisomerase II inhibitors such as teniposide (VM-26) produce enzyme-induced DNA cleavage and inhibition of enzyme activity. By adding to such reactions distamycin, a compound whose effects on DNA have been extensively characterized, we investigated the effects of drug binding upon topoisomerase II-mediated DNA cleavage induced by VM-26. We have found a correspondence between distamycin binding (determined by footprinting analysis) and topoisomerase II-mediated cleavage of SV40 DNA (determined by sequencing gel analysis). Distamycin binding potentiated the cleavage of specific sites in the near proximity of distamycin-binding sites (within at least 25 base pairs), which indicates that DNA secondary structure is involved in topoisomerase II-DNA interactions. That distamycin potentiated cleavage only at sites that were recognized in the absence of distamycin and suppressed cleavage directly at distamycin-binding sites indicates that topoisomerase II recognizes DNA on the basis of primary sequence. In addition, distamycin stimulated topoisomerase II-mediated DNA relaxation and antagonized the inhibitory effect of VM-26. These results show that the DNA sequence-specific binding of distamycin produces local and propagated effects in the DNA which markedly affect topoisomerase II activity.