RESUMO
A variety of spectroscopic and biochemical techniques have been employed to investigate the extent to which binding to DNA of an intercalator (amsacrine or its 4-carboxamide derivative SN16713) affects the binding of netropsin, a minor groove-targeted ligand, and vice versa. In general, rather little mutual interference has been found and the binding of one drug is compatible with binding of the other. The anilinoacridines exert little or no effect on the positioning of netropsin in the minor groove, judged by circular dichroism spectroscopy and electric linear dichroism, whereas netropsin has a perceptible effect on the intercalative binding of amsacrine, but not that of SN16713. Neither acridine drug prevents the netropsin-induced Z-->B structure reversion observed with poly(dG-dC).poly(dG-dC) in buffer containing 60% ethanol. The kinetics of dissociation of any one drug from its DNA complex are affected little, if at all, by the simultaneous presence of the other. Footprinting experiments with the several drugs singly or in combination reveal a certain amount of mutual interference, but the selective recognition of AT-rich sequences by netropsin tends to dominate the recognition pattern and is largely maintained in the presence of a considerable excess of amsacrine or its 4-carboxamide derivative.
Assuntos
Amsacrina/metabolismo , DNA/metabolismo , Desenho de Fármacos , Substâncias Intercalantes/metabolismo , Netropsina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Dicroísmo Circular , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Análise EspectralRESUMO
A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.