Indolocarbazole glycosides in inactive conformations.

Abstract

Indolocarbazole glycosides related to rebeccamycin represent a promising category of antitumor agents targeting DNA and topoisomerase I. These drugs prefer to adopt a closed conformation with an intramolecular hydrogen bond between the indole NH group and the pyranose oxygen atom. Three pairs of indolocarbazole monoglycosides bearing an NH or an N-methyl indole moiety were synthesized and their biological properties investigated at the molecular and cellular level. Replacing the indole NH proton with a methyl group reduces DNA interaction and abolishes activity against DNA topoisomerase I. Surface plasmon resonance studies performed with a pair of water-soluble indolocarbazole glycosides and two hairpin oligonucleotides containing an [AT]4 or a [CG]4 sequence indicate that both the NH and the N-methyl derivative maintain a relatively high affinity for DNA (Keq = 2 - 6 x 10(5) M(-1)) but the incorporation of the methyl group restricts access to the DNA. The number of ligand binding sites (n) on the oligonucleotides is about twice as high for the NH compound compared to its N-methyl analogue. Modeling and 1H NMR studies demonstrate that addition of the N-methyl group drives a radical change in conformation in which the orientation of the aglycone relative to the beta-glucoside is reversed. The loss of the closed conformation by the N-methyl derivatives perturbs thir ability to access DNA binding sites and prevents the drug from inhibiting topoisomerase I. As a consequence, the NH compounds exhibit potent cytotoxicity against CEM leukemia cells with an IC50 value in the 1 microM range, whereas the N-methyl analogues are 10 to 100 times less cytotoxic. These studies offer circumstantial evidence supporting the importance of the closed conformation in the interaction of indolocarbazole glycosides with their molecular targets, DNA and topoisomerase I.