Exploiting mechanistic differences between drug classes to define functional drug interaction domains on topoisomerase II. Evidence that several diverse DNA cleavage-enhancing agents share a common site of action on the enzyme.

Abstract

To fully understand the mechanism of action of topoisomerase II-targeted agents, the effects of these drugs on the catalytic cycle of the enzyme must be well characterized. The present study utilized a nonturnover DNA catenation assay to determine the effects of several drugs (etoposide, genistein, CP-115,953, amsacrine, and novobiocin) on the DNA strand passage event mediated by topoisomerase II. With the exception of etoposide, all of the drugs inhibited the DNA strand passage step of the topoisomerase II catalytic cycle. A series of drug competition experiments that exploited this mechanistic difference was used to determine relationships between drug interaction domains on the enzyme. While the inclusion of etoposide in nonturnover DNA catenation assays reversed the inhibition of strand passage induced by genistein, CP-115,953, and amsacrine, it had no effect on the inhibition induced by novobiocin. These results strongly suggest that etoposide can displace other DNA cleavage-enhancing agents from the enzyme.DNA complex. Therefore, it is concluded that the interaction domain of etoposide overlaps those of several DNA cleavage-enhancing drugs but, consistent with previous observations (Robinson, M. J., Corbett, A. H., and Osheroff, N. (1993) Biochemistry 32, 3638-3643), is distinct from that of novobiocin.