DNA CLEAVAGE BY TOPOISOMERASE-I IN THE PRESENCE OF INDOLOCARBAZOLE DERIVATIVES OF REBECCAMYCIN

DNA topoisomerase I has been shown to be an important therapeutic targ et in cancer chemotherapy for the camptothecins as well as for indoloc arbazole antibiotics such as BE-13793C and its synthetic derivatives N B-506 and ED-110 [Yoshinari et al. (1993) Cancer Res. 53, 490-494]. To investigate the mechanism of topoisomerase I inhibition by indolocarb azoles, we have studied the induction of DNA cleavage by purified mamm alian topoisomerase I mediated by the antitumor antibiotic rebeccamyci n and a series of 20 indolocarbazole derivatives. The compounds tested bear (i) various functional groups on the non-indolic moiety (X = CO, CH2, CHOH), (ii) a hydrogen or a chlorine atom at positions 1 and 11 (R(2)), and (iii) different substituents on the maleimido function (R( 1) = H, OH, NH2, NHCHO). Half of the ligands have the same carbohydrat e moiety as rebeccamycin whereas the other ligands have no sugar resid ue. The inhibitory potency of the test compounds was assessed in vitro by comparing the cleavage of [P-32]-labeled restriction fragments by the enzyme in the absence and presence of the drug. In addition, the D NA-binding properties of these compounds were investigated by means of complementary spectroscopic techniques including electric linear dich roism, and the DNA sequence selectivity was probed by DNase I footprin ting, The study shows that the sugar residue attached to the indolocar bazole chromophore is critical for the drug ability to interfere with topoisomerase I as well as for the formation of intercalation complexe s. Structure-activity relationships indicate that the presence of chlo rine atoms significantly reduces the effects on topoisomerase I wherea s the substituents on the maleimido function and the functional group on the non-indolic moiety can be varied without reduction of activity. The results suggest that the inhibition of topoisomerase I by indoloc arbazoles arises in part from their ability to interact with DNA. Anal ysis of the base preferences around topoisomerase I cleavage sites in various restriction fragments indicated that, in a manner similar to c amptothecin, the rebeccamycin analogue R-3 stabilized topoisomerase I preferentially at sites having a T and a G on the 5' and 3' sides of t he cleaved bond, respectively. By analogy with models previously propo sed for camptothecin and numerous topoisomerase II inhibitors which in tercalate into DNA, a stacking model for the interaction between DNA, topoisomerase I and indolocarbazoles is proposed. These findings provi de guidance for the development of new topoisomerase I-targeted antitu mor indolocarbazole derivatives.