Search

The bisdioxopiperazines are catalytic inhibitors of eukaryotic type II DNA topoisomerases capable of trapping these enzymes as a salt-stable closed-clamp complex on circular DNA. The various bisdioxopiperazine analogs differ from each other because of structural differences in the linker connecting the two dioxopiperazine rings. Although the composition of this linker region has been found to be important for potency, the structural basis for this is largely unknown. To elucidate the role of the linker region in drug action, we have analyzed the effect of different linker substituents in otherwise identical analogs by studying their interaction with wild-type and mutant human topoisomerase II alpha. Two mutations, L169I and R162Q, displayed differential sensitivity toward closely related analogs, suggesting that the linker region in these compounds plays a highly specific role in protein drug interaction. The finding that the L169I mutation, which probably represents a subtle structural change, was sufficient to confer resistance further emphases the importance of this region of the protein for bisdioxopiperazine inhibition of topoisomerase II. Comparing the sensitivity profiles of different bisdioxopiperazines against wild-type and mutant proteins with that of mitindomide, we observed a spectrum of sensitivity closely resembling that of ICRF-154, a bisdioxopiperazine with no linker substituents. We discuss the implications of these observations for the understanding of the mechanism of bisdioxopiperazine action on topoisomerase II.

Maleimide, N-ethyl-maleimide (NEM), and N-methyl-maleimide (NMM) were identified as potent catalytic inhibitors of purified human topoisomerase IIalpha, whereas the ring-saturated analog succinimide was completely inactive. Catalytic inhibition was not abrogated by topoisomerase II mutations that totally abolish the effect of bisdioxopiperazine compounds on catalytic inhibition, suggesting a different mode of action by these maleimides. Furthermore, in DNA cleavage assay maleimide and NEM could antagonize etoposide-induced DNA double-strand breaks. Consistently, maleimide could antagonize the effect of topoisomerase II poisons in three different in vivo assays: 1) In an alkaline elution assay maleimide protected against etoposide-induced DNA damage. 2) In a band depletion assay maleimide reduced etoposide-induced trapping of topoisomerase IIalpha and beta on DNA. 3) In a clonogenic assay maleimide antagonized the cytotoxicity of etoposide and daunorubicin on four different cell lines of human and murine origin. at-MDR cell lines with reduced nuclear topoisomerase IIalpha content are fully sensitive to maleimide, indicating that it is not a topoisomerase II poison in vivo. Our finding that topoisomerase II is sensitive to maleimide, NMM, and NEM but insensitive to succinimide demonstrates a strict requirement for the unsaturated ring bond for activity. We suggest that the observed antagonism in vitro and in vivo is caused by covalent modification of topoisomerase II cysteine residues reducing the amount of catalytically active enzyme sensitive to the action of topoisomerase II poisons.