Topoisomerase II Site-directed Alkylation of DNA by Psorospermin and Its Effect on Topoisomerase II-mediated DNA Cleavage

Abstract

Psorospermin, a plant-derived antitumor agent, has been shown to selectively alkylate a guanine at the topoisomerase II cleavage site to trap the topoisomerase II-DNA cleaved complex. The results of this study provide further important insight into the mechanism of the topoisomerase II site-directed alkylation of DNA by psorospermin and its subsequent effects on the topoisomerase II-induced DNA cleavage. First, we demonstrate that the topoisomerase II-induced alkylation of DNA by psorospermin occurs at a time preceding the topoisomerase II-mediated strand cleavage event, because it occurs in the absence of Mg 2؉. We confirm that the alkylation of DNA by psorospermin takes place at N-7 of guanine in the presence of topoisomerase II, because substitution of the target guanine by 7-deazaguanine prevents alkylation. Because the stimulation of the topoisomerase II-induced DNA cleavage by psorospermin can be slowly reversed by the addition of excess salt, this indicates that alkylation of DNA by psorospermin traps a reversible topoisomerase II-DNA complex. Both the DNA alkylation by psorospermin in the presence of topoisomerase II and the enzyme-mediated DNA cleavage elevated by psorospermin are more enhanced at acidic pH values, in accordance with the increased stability of the topoisomerase II-DNA complex at acidic pH values. Finally, our results suggest that it is the psorospermin-DNA adducts, not the abasic sites resulting from depurination, that are responsible for the stimulation of the topoisomerase II-mediated cleavage. Because the precise location of the psorospermin within the topoisomerase II cleavage site is known, together with the covalent DNA linkage chemistry and the conformation of the psorospermin-DNA adduct, this structural insight provides an excellent opportunity for the design and synthesis of new, more effective topoisomerase II poisons.Psorospermin (Fig. 1), a natural product isolated from the roots and stem bark of the African plant Psorospermum febrifugum (1, 2), has been shown to intercalate into the DNA helix and to covalently modify guanine at the N-7 position in the major groove through an epoxide-mediated electrophilic attack (3). It is active against drug-resistant human leukemia lines and AIDS-related lymphoma. 1 In the initial mechanistic studies using recovered SV40 DNA from psorospermin-treated cells, it was found that numerous abasic sites were generated on the DNA. The extensive loss of DNA bases was proposed to lead to DNA strand breaks and protein-DNA cross-links, which might be responsible for the cytotoxicity and antitumor activity of this compound (5). Furthermore, although topoisomerase I and II can form cross-links to DNA in the presence of psorospermin, it was suggested that these cross-links are not specific (5). However, we have recently demonstrated that psorospermin alkylation at specific sites on DNA was greatly enhanced in the presence of topoisomerase II, 2 indicating that the antitumor activity of psorospermin might be related to its specific interaction with the t...

Determination of the importance of the stereochemistry of psorospermin in topoisomerase II–induced alkylation of DNA and in vitro and in vivo biological activity

“…This suggests that topoisomerase II -mediated psorospermin alkylation occurs in the initial noncovalent binding step in the topoisomerase II catalytic cycle. We also showed that N7 of guanine is still the alkylation site for psorospermin in the presence of topoisomerase II (10). Furthermore, the topoisomerase II-DNA complex trapped by psorospermin is slowly reversible on the addition of excess salt (10).…”

“…We have shown that the enhanced DNA alkylation by psorospermin in the presence of topoisomerase II is dependent on pH but is independent of Mg 2+ or ATP (10). This suggests that topoisomerase II -mediated psorospermin alkylation occurs in the initial noncovalent binding step in the topoisomerase II catalytic cycle.…”

“…-methyl psorospermin and its three chiral isomers (2VS,3VS), (2VR,3VS), and (2VS,3VR) was determined by a strand breakage assay (4). A 60-bp duplex DNA, which contained a single topoisomerase II cleavage site, was incubated with increasing concentrations of all compounds in the presence and absence of human topoisomerase II (10). Piperidine heat treatment of the samples was then used to generate strand breakage products, which results from the depurination of the N7-alkylated guanine (Fig.…”

Topoisomerase II-mediated site-directed alkylation of DNA by psorospermin and its use in mapping other topoisomerase II poison binding sites

“…The intercalation and enhanced covalent alkylation site for psorospermin in the presence of topoisomerase II are within the gate site, with alkylation occurring at the ϩ4Ј position. Finally, psorospermin is a topoisomerase II poison that traps the enzyme-DNA complex, suggesting that psorospermin also may interact with topoisomerase II or, alternatively, distort the DNA on covalent reaction to slow down the religation step (35).…”

Mechanistic Basis of Sensitivity/Resistance Towards Anti-cancer Drugs Targeting Topoisomerase II

“…Studies have shown that topo II can be poisoned by various mechanisms. (Liu, Rowe et al 1983;Zechiedrich, Christiansen et al 1989;Frydman, Marton et al 1997;Kwok and Hurley 1998). A mechanism for topo II poisoning was recently discovered in which thiol alkylation of topo II stimulates topo ll-dependent DNA cleavage (Frydman, Marton et al 1997;Wang, Mao et al 2001).…”