DNA topoisomerase I has been shown to be an important therapeutic target in
cancer chemotherapy for the camptothecins as well as for indolocarbazole
antibiotics such as
BE-13793C and its synthetic derivatives
NB-506 and
ED-110 [Yoshinari et al. (1993)
Cancer Res. 53, 490-494]. To investigate the mechanism of
topoisomerase I inhibition by indolocarbazoles, we have studied the induction of DNA cleavage by purified mammalian
topoisomerase I mediated by the antitumor
antibiotic rebeccamycin 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 (R2), and (iii) different substituents on the maleimido function (R1 = H,
OH, NH2, NHCHO). Half of the
ligands have the same
carbohydrate moiety as
rebeccamycin whereas the other
ligands have no
sugar residue. The inhibitory potency of the test compounds was assessed in vitro by comparing the cleavage of [32P]-labeled restriction fragments by the
enzyme in the absence and presence of the
drug. In addition, the
DNA-binding properties of these compounds were investigated by means of complementary spectroscopic techniques including electric linear dichroism, and the DNA sequence selectivity was probed by
DNase I footprinting. The study shows that the
sugar residue attached to the indolocarbazole chromophore is critical for the
drug ability to interfere with
topoisomerase I as well as for the formation of intercalation complexes. Structure-activity relationships indicate that the presence of
chlorine atoms significantly reduces the effects on
topoisomerase I whereas 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 indolocarbazoles arises in part from their ability to interact with
DNA. Analysis of the base preferences around
topoisomerase I cleavage sites in various restriction fragments indicated that, in a manner similar to
camptothecin, the
rebeccamycin analogue R-3 stabilized
topoisomerase I preferentially at sites having a T and a G on the 5' and 3' sides of the cleaved bond, respectively. By analogy with models previously proposed for
camptothecin and numerous
topoisomerase II inhibitors which intercalate into
DNA, a stacking model for the interaction between
DNA, topoisomerase I and indolocarbazoles is proposed. These findings provide guidance for the development of new
topoisomerase I-targeted antitumor indolocarbazole derivatives.