Binding of etoposide to topoisomerase II in the absence of DNA: Decreased affinity as a mechanism of drug resistance

Despite the prevalence of topoisomerase II-targeted drugs in cancer chemoth erapy and the impact of drug resistance on the efficacy of treatment, inter actions between these agents and topoisomerase II are not well understood. Therefore, to further define interactions between anticancer drugs and the type II enzyme, a nitrocellulose filter assay was used to characterize the binding of etoposide to yeast topoisomerase II. Results indicate that etopo side binds to the enzyme in the absence of DNA. The apparent K-d value for the interaction was similar to 5 mu M drug. Etoposide also bound to ytop2H1 012Y, a mutant yeast type II enzyme that is similar to 3-4-fold resistant t o etoposide. However, the apparent K-d value for the drug (similar to 16 mu M) was similar to 3 times higher than that determined for wild-type topois omerase II. Although it has been widely speculated that resistance to topoi somerase II-targeted anticancer agents results from a decreased drug-enzyme binding affinity, these data provide the first direct evidence in support of this hypothesis. Finally, the ability of yeast topoisomerase II to bind etoposide was dependent on the presence of the hydroxyl moiety of Tyr783, s uggesting specific interactions between etoposide and the active site resid ue that is involved in DNA scission.