Among its many properties, amiloride is a DNA intercalator and topoisomerase II 
inhibitor. Previous work has indicated that the most stable conformation for 
amiloride is a planar, hydrogen-bonded, tricyclic structure. To determine 
whether the ability of amiloride to intercalate into DNA and to inhibit DNA 
topoisomerase II was dependent on the ability to assume a cyclized conformation, 
we studied the structure-activity relationship for 12 amiloride analogs. These 
analogs contained structural modifications which could be expected to allow or 
impede formation of a cyclized conformation. Empirical assays consisting of 
biophysical, biochemical, and cell biological approaches, as well as 
computational molecular modeling approaches, were used to determine 
conformational properties for these molecules, and to determine whether they 
intercalated into DNA and inhibited topoisomerase II. Specifically, we measured 
the ability of these compounds to 1) alter the thermal denaturation profile of 
DNA, 2) modify the hydrodynamic behavior of DNA, 3) inhibit the catalytic 
activity of purified DNA topoisomerase II in vitro, 4) promote the topoisomerase 
II-dependent cleavage of DNA, and 5) inhibit functions associated with DNA 
topoisomerase II in intact cells. Results indicated that only those analogs 
capable of cyclization could intercalate into DNA and inhibit topoisomerase II. 
Thus, the ability of amiloride and the 12 analogs studied to intercalate into 
DNA and to inhibit topoisomerase II appears dependent on the ability to exist in 
a planar, hydrogen-bonded, tricyclic conformation.