This project will explore the biophysical properties associated with the interactions of selected antitumor agents (and their structurally relevant analogs) with nucleic acids. During the course of this research, efforts will be focused towards correlating the physical- chemical nature of these drug-DNA interactions with their biological effectiveness as topoisomerase II inhibitors. Experiments will be designed to address questions concerning the physical and/or chemical properties which dictate topoisomerase II inhibition, whether these properties are related to the DNA binding of these compounds, and finally, the mechanism(s) by which they elicit topoisomerase II inhibition. The proposed mechanism by which several of the leading antitumor agents, including m-AMSA, VP-16, VM-26, adriamycin, daunorubicin, and mitoxantrone mediate antitumor activity is through the inhibition of topoisomerase II activity. Information concerning the actual mechanism by which the drugs exert this type of inhibition effect is limited. However, the ability for these compounds to interact with DNA does appear to be an essential requirement for inhibition of the topoisomerase II to occur. We have designed, synthesized, and characterized a series of anilinoacridine compounds with modifications to both the N-phenyl side chain and to the acridine ring to use as probes for examining the structural requirements necessary for drug- induced topoisomerase II inhibition to occur. This series of acridine analogs provide a unique opportunity for examining and characterizing both the physical-chemical properties associated with ternary complex formation and evaluating the influence of chemical substituent type and position on mediating topoisomerase II activity. The activities of these compounds is presumed to reside in formation of a ternary complex between the topoisomerase II-DNA and drug. We will probe the structural and functional properties of this ternary complex, using a variety of methods including photoaffinity crosslinking of the antitumor agent to the DNA (and/or topoisomerase II). The 3-azido-m-AMSA will provide an ideal probe for examining the topoisomerase-DNA-drug ternary complex and provide insight into the mechanism(s) by which m-AMSA exerts its biological effects. Prior to photolysis, this compound has been demonstrated to bind DNA in a manner identical to the parent m-AMSA. Upon photolysis, the azido is converted to the reactive nitrene which forms a covalent attachment in situ. Our laboratory has recently observed that the noncovalent addition of 3-azido-m-AMSA was just as effective in eliciting topoisomerase II inhibition as the parent m- AMSA; indicative of its effectiveness as a probe for examining the mechanistic properties of m-AMSA. With this probe, we will attempt to obtain insight into the overall geometry of the ternary complex (i.e., the location of drug with respect to topoisomerase II and DNA). In addition, our studies will examine the binding properties and topological specificities associated with topoisomerase II-DNA interactions in the absence and presence of topoisomerase II inhibiting antibiotics. These studies on several selected antitumor agents (analogs) will provide insight into our overall understanding of how these compounds exert their potent biological effects and the nature of the drug-DNA complexes, thus leading to a more rational design of novel antitumor agents.
