MOLECULAR-INTERACTIONS OF DNA TOPOISOMERASE-I AND TOPOISOMERASE-II INHIBITOR WITH DNA AND TOPOISOMERASES AND IN TERNARY COMPLEXES - BINDING MODES AND BIOLOGICAL EFFECTS FOR INTOPLICINE DERIVATIVES

Molecular interactions of intoplicine, dual DNA-topoisomerases (Topo) I and II inhibitor, with topoisomerases, plasmid DNA, in ternary cleavable complexes with enzymes and plasmid DNA, and in the reversed cleavable complexes were examined by means of surface-enhanced Raman scattering (SERS) and CD spectroscopy and by biochemical techniques. Detailed spectral analysis of intoplicine derivatives allowed us to assign SERS vibrational modes of chromophores and to propose the models for these complexes. Intoplicine was found to be able to interact specifically with Topo II alone, but with Topo I only when in the presence of DNA. It shows at least two modes of binding to the DNA: the first was found to be dominant for its derivative 1c (most potent Topo I inhibitor), and the second was dominant for derivative 2a (most potent Topo II inhibitor). The possibility of forming these two types of complexes simultaneously is suggested to be one of the main factors enabling the drug to be a dual Topo I and Topo II inhibitor. The ''deep intercalation mode'' of the drug from the DNA minor groove with the long axis of the chromophore oriented roughly parallel to the dyad axis has been suggested to be responsible for induction of distortions of the DNA structure by the intercalating drug. Being involved in the formation of Topo I-mediated cleavable ternary complex, the molecules participating in the deep intercalation mode within the DNA do not change their molecular interactions as compared with their complex with the DNA alone. The stabilization of the Topo-I-mediated cleavable complex was shown to be followed by the local denaturation of DNA in the AT-rich regions of the helix. When the ternary cleavable complex was reversed, the drug was shown to be in the complex with the plasmid. The ''outside binding mode'' from the DNA major groove via the hydroxyl group of the A-ring of the chromophore has been suggested to be responsible for Topo II inhibition. These molecules did not induce significant distortions of the DNA structure. Being involved in the formation of Topo II-mediated cleavable ternary complex, the drug changed its molecular interactions as compared with the complex with DNA alone. Drug-Topo II interactions within the ternary complex involve the hydroxyl group of the A-ring of the chromophore, which was found to be easily accessible to the functional groups of enzyme when the outside binding mode of the drug to DNA is realized. Direct interactions between the drug and Topo II can play a critical role in the process of DNA recognition by the enzyme and Topo II inhibition. Intoplicine shows higher affinity to the enzyme than to the DNA: reversion of ternary cleavable complex led to elimination of drug-DNA interactions, whereas interactions between drug and Topo II seem to be unchanged. These results suggest that dual Topo I and II inhibition, being critical for the antitumor activity of intoplicine, should be explained not only by effects induced at the DNA level but also by molecular interactions displayed by the drug with DNA and enzymes, in cleavable ternary complexes and in reversed ternary complexes.