Substituted Purine Analogues Define a Novel Structural Class of Catalytic Topoisomerase II Inhibitors

Abstract

By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIA, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S 6 -substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIA, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O 6 -substituted guanine analogues, O 6 -benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells. (Cancer Res 2005; 65(16): 7470-7)

Probing Conformational Changes in Human DNA Topoisomerase IIα by Pulsed Alkylation Mass Spectrometry

“…For instance, thimerosal, a mercury-containing compound that rapidly reacts with thiol group, inhibited the topoisomerase II-decatenating activity (39). Thiopurines also inhibited topoisomerase II ATPase activity by alkylating thiol groups of cysteines (40). Both inhibitory effects can be abolished by adding reducing reagents, implicating cysteine modification in such reactions.…”

A Three-Dimensional Quantitative Structure-Activity Analysis of a New Class of Bisphenol Topoisomerase IIα Inhibitors

“…These results suggest that most of the bisphenols exerted their activity as catalytic inhibitors of topoisomerase II, but that they did not act as topoisomerase II poisons. Topoisomerase II catalytic inhibitors such as the bisdioxopiperazines dexrazoxane and ICRF-193 (Sehested et al, 1993;Hasinoff et al, 1996) and a newly identified "purine class" (NSC35866) of compounds (Jensen et al, 2005) can antagonize the growth inhibitory effects of topoisomerase II poisons. However, not all of the purines acted like NSC35866, because our follow-up study showed that most of the purines were not capable of antagonizing etoposide-induced cytotoxicity and DNA strand breaks in cells (Jensen et al, 2006).…”

“…As exemplified by dexrazoxane (Fattman et al, 1996;Hasinoff et al, 1997), catalytic inhibitors of topoisomerase II may inhibit cleavable complex formation by topoisomerase II poisons such as etoposide (Andoh and Ishida, 1998;Larsen et al, 2003;Jensen et al, 2005;Jensen et al, 2006). Experiments were thus carried out to see whether bisphenol pretreatment could antagonize etoposide-induced linear DNA formation by topoisomerase II␣.…”

“…Likewise, the purine NSC35866 (Jensen et al, 2005) can antagonize etoposideinduced growth inhibitory effects. For dexrazoxane, it may do this by trapping the enzyme in the form of a closed protein clamp, thereby preventing the formation or stabilization of the topoisomerase II-DNA intermediate Tanabe et al, 1991;Sehested et al, 1993).…”

“…To determine whether the bisphenols acted similarly to dexrazoxane and NSC35866 (Jensen et al, 2005) in antagonizing doxorubicin or etoposide growth inhibitory effects on K562 cells, experiments were carried out in which K562 cells were pretreated with either 5 M O3OH or S4OH for 30 min before treatment with doxorubicin (Fig. 5,A and B) and then continuously incubated with both drugs for 72 h before the MTS assay.…”