Differential Rates of Reversibility of Ecteinascidin 743−DNA Covalent Adducts from Different Sequences Lead to Migration to Favored Bonding Sites

Abstract

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.

Devising a Structural Basis for the Potent Cytotoxic Effects of Ecteinascidin 743

“…The substrates were constructed by ligating a duplex oligonucleotide modified with Et 743 at a single guanine to flanking duplex oligonucleotides on both the 5 0 -and 3 0 -sides. Although we succeeded at creating a site-directed adduct at a 5 0 -AGC sequence, attempts at generating a site-directed adduct at the 5 0 -AGT sequence failed [30]. After several ligation and purification steps, it was observed that the ligated 60-mer no longer contained Et 743 covalently bound at the 5 0 -AGT sequence.…”

“…A band shift assay was used to monitor the amount of drug-modified duplex in order to determine if the covalent adduct is stable over time [30]. A 22-mer oligonucleotide containing a single modification site, either 5 0 -AGT or 5 0 -AGC, surrounded by a common sequence (Fig.…”

“…To determine if Et 743 can rebond at other recognition sequences, Et 743-modified oligonucleotides (22-mer) containing the sitedirected adducts at either the 5 0 -AGC or 5 0 -AGT target sequence were incubated with a different size oligonucleotide (30-mer) (Fig. 23.9a) containing either the favored or the non-favored Et 743 target sequences, respectively [30]. Figure 23.9b shows that as the level of Et 743-modified 5 0 -AGT adduct decreases over time, the other duplex (30-mer) containing the favored drug target sites becomes modified with Et 743.…”

DNA Structural Similarity in the 2:1 Complexes of the Antitumor Drugs Trabectedin (Yondelis) and Chromomycin A3 with an Oligonucleotide Sequence Containing Two Adjacent TGG Binding Sites on Opposing Strands

“…Given the amount of nonpolar surface area that is buried upon complex formation (ϳ525 Å 2 ), we would expect an important hydrophobic contribution not only to the free energy of trabectedin-DNA association but also to the ensuing stabilization of the double helix in the aqueous medium. For a complex of the type reported here it seems safe to assume that the increase in the temperature of DNA thermal denaturation will be larger than that obtained for a DNA oligonucleotide containing a single trabectedin adduct, which has been shown to be of 19°for a 5Ј-AGC site (Zewail-Foote and Hurley, 2001a). Therefore, it is not unreasonable to think that the known ability of trabectedin to block the activity of exonuclease III (ExoIII) (Dziegielewska et al, 2004) or the helicase activities of both the simian virus 40 (SV40) large tumor antigen (T-antigen) (Zewail-Foote and Hurley, 2001a) and the UvrA 2 B complex (Zewail-Foote and Hurley, 2001b) derives from this substantial stabilization, which is likely to be amplified if two or more suitable triplets are tandemly or separately arranged in the oligonucleotide sequence.…”

Transcriptional Inhibition of Type I Collagen Gene Expression in Scleroderma Fibroblasts by the Antineoplastic Drug Ecteinascidin 743

“…The stability of these complexes is governed by DNA target sequences, since under some conditions the drug is not capable of forming optimally stable bonds with its corresponding recognition sites in the DNA leading to the formation of unstable products. The same authors in other work (25) demonstrated that ET-743 can migrate from a nonfavored bonding sequence (5Ј-AGT) to the favored sequence site (5Ј-AGC) and that the ET-743-AGT adduct is less stable, displays a higher degree of molecular motion, and produces dif- ferent conformational changes in DNA than the more stable alkylation product ET-743-AGC.…”