AT 2433 B1

The antibiotic AT2433-B1 belongs to a therapeutically important class of antitumor agents. This natural product contains an indolocarbazole aglycone connected to a unique disaccharide consisting of a methoxyglucose and an amino sugar subunit, 2,4-dideoxy-4-methylamino-L-xylose. The configuration of the amino sugar distinguishes AT2433-B1 from its diastereoisomer iso-AT2433-B1. Here we have investigated the interaction of these two disaccharide indolocarbazole derivatives with different DNA sequences by means of DNase I footprinting and surface plasmon resonance (SPR). Accurate binding measurements performed at 4 and 25 degrees C using the BIAcore SPR method revealed that AT2433-B1 binds considerably more tightly to a hairpin oligomer containing a [CG](4) block than to an oligomer with a central [AT](4) tract. The kinetic analysis shows that the antibiotic dissociates much more slowly from the GC sequence compared to the AT one. Preferential binding of AT2433-B1 to GC-rich sequences in DNA was independently confirmed by DNase I footprinting experiments performed with a 117 bp DNA restriction fragment. The specific binding sequence 5'-AACGCCAG identified from the footprints was then converted into a biotin-labeled DNA hairpin duplex and compound interactions with this specific sequence were characterized by high resolution BIAcore SPR experiments. Such a combined approach provided a detailed understanding of the molecular basis of DNA recognition. The discovery that the glycosyl antibiotic AT2433-B1 preferentially recognizes defined sequences offers novel opportunities for the future design of sequence-specific DNA-reading small molecules.

Actinomadura melliaura ATCC 39691, a strain isolated from a soil sample collected in Bristol Cove, California, is a known producer of the disaccharide-substituted AT2433 indolocarbazoles (6-9). Reinvestigation of this strain using new media conditions led to >40-fold improvement in the production of previously reported AT2433 metabolites and the isolation and structure elucidation of the four new analogues, AT2433-A3, A4, A5, and B3 (1-4). The availability of this broader set of compounds enabled a subsequent small antibacterial/fungal/cancer SAR study that revealed disaccharyl substitution, N-6 methylation, and C-11 chlorination as key modulators of bioactivity. The slightly improved anticancer potency of the newly reported N-6-desmethyl 1 (compared to 6) contrasts extensive SAR of monoglycosylated rebeccamycin-type topoisomerase I inhibitors where N-6 alkylation has contributed to improved potency and ADME. Complete 2D NMR assignments for the known metabolite BMY-41219 (5) and (13)C NMR spectroscopic data for the known analogue AT2433-B1 (7) are also provided for the first time.

The antibiotics AT2433-A1 and AT2433-B1 are two indolocarbazole diglycosides related to the antitumor drug rebeccamycin known to stabilize topoisomerase I-DNA complexes. This structural analogy prompted us to explore the binding of four indolocarbazole diglycosides with DNA and their capacity to interfere with the DNA cleavage-reunion reaction catalyzed by topoisomerase I. The molecular basis of the drug interaction with double-stranded DNA and with purified chromatin, with particular emphasis on the role of the carbohydrate moiety, was investigated by means of complementary spectroscopic techniques, including surface plasmon resonance and electric linear dichroism. We compared the DNA binding properties, sequence recognition, and effects on topoisomerase I-mediated DNA relaxation and cleavage of AT2433-A1 bearing a 2,4-dideoxy-4-methylamino-L-xylose residue, its dechlorinated analog AT2433-B1, the diastereoisomer iso-AT2433-B1 with an inverted aminosugar residue, and compounds 5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, 12-beta-D-glucopyranosyl-12,13-dihydro-6-methyl (JDC-108) and 5H-indolo[2,3-a]pyrrolo[3, 4-c]carbazole-5,7(6H)-dione, 12-(6-O-alpha-D-galacto-pyranosyl-beta-D-glucopyranosyl)-12,13-dihydro-6-methyl (JDC-277) with an uncharged mono- and disaccharide, respectively. The two antibiotics AT2433-A1 and AT2433-B1 proved to be highly cytotoxic to leukemia cells and this may be a consequence of their tight intercalative binding to DNA, preferentially into GC-rich sequences as inferred from DNase I footprinting studies and surface plasmon resonance measurements. Like the diastereoisomer iso-AT2433-B1, they have no inhibitory effect on topoisomerase I, in contrast to the uncharged diglycoside JDC-277, which stimulates DNA cleavage by the enzyme mainly at TG sites, as observed with camptothecin. Cytotoxicity measurements with CEM and CEM/C2 human leukemia cell lines sensitive and resistant to camptothecin, respectively, also suggested that topoisomerase I contributes, at least partially, to the mechanism of action of the neutral diglycoside JDC-277 but not to that of the cationic AT2433 compounds. Together, the results indicate that sequence-selective DNA interaction and topoisomerase I inhibition is controlled to a large extent by the stereochemistry of the diglycoside moiety.