DNA Sequence- and Structure-Selective Alkylation of Guanine N2 in the DNA Minor Groove by Ecteinascidin 743, a Potent Antitumor Compound from the Caribbean Tunicate Ecteinascidia turbinata

Abstract

Ecteinascidin 743 is one of several related marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata. It is remarkably active and potent in a variety of in vitro and in vivo systems and has been selected for development as an anticancer agent. The present study investigates the interactions of ecteinascidin 743 with DNA. Ecteinascidin 743 retarded the electrophoretic migration of both supercoiled and relaxed simian virus 40 DNA even in the presence of sodium dodecyl sulfate and after ethanol precipitation, consistent with covalent DNA modifications. Similar results were obtained in a DNA oligonucleotide derived from ribosomal DNA. However, DNA denaturation reversed the DNA modifications. The homopolymeric oligonucleotide dG/dC was modified while neither the dI/dC nor the dA/dT oligonucleotides were, consistent with covalent attachment of ecteinascidin 743 to the exocyclic amino group at position 2 of guanine. Ecteinascidin 743 was then compared to another known DNA minor groove alkylating agent, anthramycin, which has also been shown to alkylate guanine N2. Footprinting analyses with DNase I and 1,10-phenanthroline-copper and exonuclease III digestions showed that ecteinascidin 743 covers three to five bases of DNA and exhibits a different sequence specificity than anthramycin in the Escherichia coli tyrosine tRNA promoter (tyrT DNA). The binding of ecteinascidin to DNA was abolished when guanines were substituted with inosines in this promoter. A band shift assay was designed to evaluate the influence of the bases flanking a centrally located guanine in an oligonucleotide containing inosines in place of guanines elsewhere. Ecteinascidin 743 and anthramycin showed similarities as well as differences in sequence selectivity. Ecteinascidin 743-guanine adducts appeared to require at least one flanking guanine and were strongest when the flanking guanine was 3' to the targeted guanine. These data indicate that ecteinascidin 743 is a novel DNA minor groove, guanine-specific alkylating agent.