Molecular Basis for the DNA Sequence Selectivity of Ecteinascidin 736 and 743:  Evidence for the Dominant Role of Direct Readout via Hydrogen Bonding

Abstract

The marine natural product ecteinascidin 743 (Et 743) is currently in phase II clinical trials. We have undertaken parallel structural and modeling studies of an Et 743−(N2-guanine) 12-mer DNA adduct and an adduct involving the structurally related Et 736 of the same sequence in order to ascertain the structural basis for the ecteinascidin−DNA sequence selectivity. In contrast to the C-subunit differences found in Et 736 and Et 743, they have identical A−B-subunit scaffolds, which are the principal sites of interaction with DNA bases. These identical scaffolds generate parallel networks of drug−DNA hydrogen bonds that associate the drugs with the three base pairs at the recognition site. We propose that these parallel hydrogen bonding networks stabilize the Et 736 and Et 743 A- and B-subunit prealkylation binding complex with the three base pairs and are the major factors governing sequence recognition and reactivity. The possibility that a unique hydrogen-bonding network directs the course of sequence recognition was examined by first characterizing the hydrogen-bonding substituents using 1H NMR properties of the exchangeable protons attached to the hydrogen-bond donor and other protons near the proposed acceptor. Using these experimental findings as indicators of hydrogen bonding, Et 736−12-mer duplex adduct models (binding and covalent forms) containing the favored sequences 5‘-AGC and 5‘-CGG were examined by molecular dynamics (MD) in order to evaluate the stability of the hydrogen bonds in the resulting conformations. The MD-generated models of these favored sequences display optimal donor/acceptor positions for maximizing the number of drug−DNA hydrogen bonds prior to covalent reaction. The results of MD analysis of the carbinolamine (binding) forms of the sequences 5‘-GGG (moderately reactive) and 5‘-AGT (poorly reactive) suggested reasons for their diminished hydrogen-bonding capability. These experimental and modeling results provide the structural basis for the following sequence specificity rules: For the target sequence 5‘-XGY, the favored base to the 3‘-side, Y, is either G or C. When Y is G, then a pyrimidine base (T or C) is favored for X. When Y is C, a purine (A or G) is favored for X.

Unique Features of the Mode of Action of ET-743

“…Gel shift assays combined with high-field NMR have demonstrated that the alkylation of ET-743 occurs at guanines located either in the sequence 5′-PuGC-3′ or 5′-PyrGG-3′ [10][11][12][13]. Other adducts are formed, but are less stable and are reversible [12][13][14], suggesting that, after prolonged exposure, ET-743 would be released from these less stable adducts and a progressively higher percentage of ET-743 would be found bound to guanines located in the frame of bases conferring high thermodynamic stability. The reported structural changes induced by ET-743, such as widening of the minor groove and bending of DNA toward its major groove, could be important in affecting the recognition and binding of transcription factors or DNA-binding proteins (e.g., DNA repair proteins).…”

“…This observation prompted us to speculate that this part of the molecule is crucial for the mechanisms responsible for the antitumor activity. According to the model proposed by Hurley and coworkers [11,12,14], it seems that a C subunit not bound to DNA but projecting out from the minor groove might be able to interfere with DNA-binding factors. This hypothesis led us to investigate whether transcription factors that bind to DNA were inhibited by ET-743, which also binds to DNA [15].…”

Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove

“…Interactions of ecteinascidins with DNA have been proposed on the basis of structural similarities with other tetrahydroisoquinoline-containing antibiotics, biochemistry, x-ray crystallography, NMR spectroscopy, and molecular modeling data (2,5,(7)(8)(9). The pattern of potential hydrogen bond acceptors and donors indicates that the drug is likely to bind to the DNA minor groove (5,8,9).…”

“…The pattern of potential hydrogen bond acceptors and donors indicates that the drug is likely to bind to the DNA minor groove (5,8,9). Et743 contains a carbinolamine center at the N2 position, and elimination of the adjacent hydroxyl group (at position 21) results in a Schiff base vulnerable to nucleophilic attack, leading to DNA alkylation (Fig.…”

“…Remarkably, the alkylation reaction is DNA sequence specific (10). It requires noncovalent binding of Et743 in the DNA minor groove (5,8,9), and it is reversed on DNA denaturation (7). These characteristics set Et743 apart from the DNA alkylating agents presently used in cancer chemotherapy.…”