Amsacrine as a Topoisomerase II Poison: Importance of Drug–DNA Interactions

Amsacrine (<i>m</i>-AMSA) is an anticancer agent that displays activity against refractory acute leukemias as well as Hodgkin’s and non-Hodgkin’s lymphomas. The drug is comprised of an intercalative acridine moiety coupled to a 4′-amino-methanesulfon-<i>m</i>-anisidide headgroup. <i>m</i>-AMSA is historically significant in that it was the first drug demonstrated to function as a topoisomerase II poison. Although <i>m</i>-AMSA was designed as a DNA binding agent, the ability to intercalate does not appear to be the sole determinant of drug activity. Therefore, to more fully analyze structure–function relationships and the role of DNA binding in the action of <i>m</i>-AMSA, we analyzed a series of derivatives for the ability to enhance DNA cleavage mediated by human topoisomerase IIα and topoisomerase IIβ and to intercalate DNA. Results indicate that the 3′-methoxy (<i>m</i>-AMSA) positively affects drug function, potentially by restricting the rotation of the headgroup in a favorable orientation. Shifting the methoxy to the 2′-position (<i>o</i>-AMSA), which abrogates drug function, appears to increase the degree of rotational freedom of the headgroup and may impair interactions of the 1′-substituent or other portions of the headgroup within the ternary complex. Finally, the nonintercalative <i>m</i>-AMSA headgroup enhanced enzyme-mediated DNA cleavage when it was detached from the acridine moiety, albeit with 100-fold lower affinity. Taken together, our results suggest that much of the activity and specificity of <i>m</i>-AMSA as a topoisomerase II poison is embodied in the headgroup, while DNA intercalation is used primarily to increase the affinity of <i>m</i>-AMSA for the topoisomerase II–DNA cleavage complex