The ecteinascidins (Ets), which are natural products derived from marine tunicates, exhibit potent antitumor activity. Of the numerous Ets isolated, Et 743 is presently being evaluated in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine. Although structurally similar to saframycin, which exhibits poor activity in cellular assays, Et 743 has shown good efficacy as an antitumor agent. In this study, DNA structural distortions induced by Et 743 were examined to provide insight into the molecular basis for the antitumor activity of Et 743. Electrophoretic mobility shifts of ligated oligomers containing site-directed adducts were used to examine the extent and direction of the Et 743-induced bend. Surprisingly, we find that Et 743 bends DNA toward the major groove, which is a unique feature among DNA-interactive agents that occupy the minor groove.

Ecteinascidin 743 (Et 743), one of a series of structurally related antitumor antibiotics isolated from a marine tunicate, is currently in phase II clinical trials. Et 743 alkylates guanine N2 through the minor groove of DNA. Hydrogen-bonding networks that associate the drug with a three base pair DNA recognition site have been proposed to contribute to both the reactivity and the stability of the Et 743-DNA adduct. Here, we report that the reaction of Et 743 with DNA is reversible under nondenaturing conditions and that the rate of this reverse reaction depends critically upon the DNA-modified sequence. Quite unexpectedly, it was found that although the rates of alkylation are similar for the 5'-AGT and 5'-AGC sequences, reversal from the 5'-AGT sequence occurs faster than from the 5'-AGC sequence. Consequently, it is the differences in the rate of the reverse reaction that dictate the sequence selectivity of Et 743 toward its favored target sequence. As a direct consequence of the reversible nature of Et 743 with DNA, Et 743 can migrate from the nonfavored bonding sequence (e.g., 5'-AGT) to the favored DNA target site (e.g., 5'-AGC). The data suggest that the observed differences in the rate of reversibility arise from differences in the stability of the Et 743-DNA adduct at the 5'-AGT and 5'-AGC target sequences. On the basis of gel electrophoresis and (1)H NMR experiments, the Et 743-AGT adduct is less stable, has more dynamic motion, and produces different conformational changes in the DNA than the more stable Et 743-AGC adduct. The shuffling of Et 743-DNA adducts to the more stable alkylation sites has important implications for understanding the underlying relationship between the structural modification of DNA by Et 743 and its biological potency and efficacy in tumor cells.

A long-lived and sequence specific ligand-DNA complex would make possible the modulation of biological processes for extended periods. We have been investigating the threading polyintercalation approach to DNA recognition in which chains of aromatic units thread back and forth repeatedly through the double helix. Here we report the preliminary sequence specificity and detailed kinetic analysis of a structurally characterized threading tetraintercalator. Specific binding on a relatively long DNA strand was observed, strongly favoring a predicted 14-base pair sequence. Kinetic studies revealed a multi-step association process and specificity was found to derive primarily from large differences in dissociation rates. Importantly, the rate-limiting dissociation rate constant of the tetraintercalator complex dissociating from its preferred binding site was extremely slow, corresponding to a 16 day half-life, making it one of the longer non-covalent complex half-lives yet measured, and, to the best of our knowledge, the longest for a DNA binding molecule.

The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.