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

Abstract

Psorospermin is a plant natural product that shows significant in vivo activity against P388 mouse leukemia. The molecular basis for this selectivity is unknown, although psorospermin has been demonstrated to intercalate into DNA and alkylate N7 of guanine. Significantly, the alkylation reactivity of psorospermin at specific sites on DNA increased 25-fold in the presence of topoisomerase II. In addition, psorospermin trapped the topoisomerase II-cleaved complex formation at the same site. These results imply that the efficacy of psorospermin is related to its interaction with the topoisomerase II-DNA complex. Because thermal treatment of (N7 guanine)-DNA adducts leads to DNA strand breakage, we were able to determine the site of alkylation of psorospermin within the topoisomerase II gate site and infer that intercalation takes place at the gate site between base pairs at the ؉1 and ؉2 positions. These results provide not only additional mechanistic information on the mode of action of the anticancer agent psorospermin but also structural insights into the design of an additional class of topoisomerase II poisons. Because the alkylation site for psorospermin in the presence of topoisomerase II can be assigned unambiguously and the intercalation site inferred, this drug is a useful probe for other topoisomerase poisons where the sites for interaction are less well defined.Psorospermin, a natural product isolated from roots and stem bark of the African plant Psorospermum febrifugum, is mechanistically related to the pluramycin family of antitumor antibiotics ( Fig. 1) and has been shown to exhibit significant activity in vitro against various tumor cell lines and in vivo against P388 mouse leukemia (1, 2). Several studies have revealed that the pluramycins intercalate into the DNA helix and covalently modify guanine at the N7 position in the major groove through an epoxide-mediated electrophilic addition (3-7). Using high-field NMR and gel electrophoresis experiments, Hansen et al. (8) were able to show that these two compounds react with DNA in a similar fashion. However, despite the similarity of their mechanism of covalent modification of DNA, distinct differences exist between psorospermin and the pluramycin class of compounds in terms of sequence selectivity, relative alkylation reactivity, and orientation of the chromophore at the intercalation site (8).Protein-DNA complexes are the molecular targets of a number of antitumor agents (9). It has been demonstrated that pluramycin reactivity is enhanced at a specific site downstream of the TATA box, immobilizing the TATA box-binding protein on DNA (10). Studies using an alkylating analogue of camptothecin have suggested that the camptothecins inhibit topoisomerase I by binding at the enzyme-DNA interface (11). A radioisotopelabeled quinolone was found to form a complex with DNA and gyrase together, but not with either component alone (12). In a study carried out by Permana et al. (13), simian virus 40 DNA isolated from psorospermin-treated cells was cros...

The topoisomerase II poison clerocidin alkylates non-paired guanines of DNA: implications for irreversible stimulation of DNA cleavage

“…It is therefore conceivable that at least one of these electrophilic moieties is capable of reacting with appropriate nucleophiles in DNA and/or the enzyme component (5,8). Several dicarbonyl compounds and aldehydes can in fact react with DNA at specific sites (9,10) and epoxides can induce DNA damage with a mechanism similar to nitrogen mustards (11)(12)(13)(14).…”

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

“…The reaction was filtered and the precipitate was washed with H 2 O (5 mL) and acetone (2 mL) and collected to yield a diastereomeric mixture of (F)-diol (0.24 g, 96%). (F)-(2VR ,3VR )-4V-t -Butylsilyloxy-1,5-Dimethoxy-3V-Hydroxy Dihydrofuranoxanthone (5). A solution of diol (0.23 g, 0.62 mmol), t-butyldimethylsilylchloride (0.14 g, 0.93 mmol), imidazole (0.13 g, 1.9 mmol), and 4-dimethylaminopyridine (38 mg, 0.31 mmol) in dimethylformamide was heated with a heat gun to f85jC and stirred overnight.…”

“…It is active against drug-resistant human leukemia lines and AIDS-related lymphoma. 6 We have shown that psorospermin alkylation at specific sites on DNA is greatly enhanced in the presence of topoisomerase II (5), indicating that the abasic sites on DNA and antitumor activity of psorospermin might be related to its specific interaction with the topoisomerase II-DNA complex. It has also been proposed that psorospermin induces DNA strand breaks, abasic sites, and protein-DNA cross-links (6).…”

Psorospermin structural requirements for P-glycoprotein resistance reversal

“…This cytotoxicity has been attributed to its interaction with topoisomerase II and DNA (19). However, currently, treatment of doxorubicin-resistant 8226 cells with PS-RR has resulted in a significantly enhanced intracellular retention of doxorubicin.…”

“…We previously have shown that the alkylation activity of psorospermin is greatly increased in the presence of topoisomerase II, an enzyme that assists in DNA breakage and re-ligation. This increase is due to psorospermin's ability to trap the topoisomerase II–DNA complex in the initial noncovalent binding step of the topoisomerase II catalytic cycle, resulting in topoisomerase II–mediated DNA cleavage (19). Due to its mechanism of topoisomerase II–directed alkylation, psorospermin has been characterized as a novel topoisomerase II poison (18, 19).…”