Minor Groove-Directed and Intercalative Ligand−DNA Interactions in the Poisoning of Human DNA Topoisomerase I by Protoberberine Analogs

Abstract

Spectroscopic, calorimetric, DNA cleavage, electrophoretic, and computer modeling techniques have been employed to characterize the DNA binding and topoisomerase poisoning properties of three protoberberine analogs, 8-desmethylcoralyne (DMC), 5,6-dihydro-8-desmethylcoralyne (DHDMC), and palmatine, which differ in the chemical structures of their B- and/or D-rings. DNA topoisomerase-mediated cleavage assays revealed that these compounds were unable to poison mammalian type II topoisomerase. By contrast, the three protoberberine analogs poisoned human topoisomerase I according to the following hierarchy: DHDMC > DMC > palmatine. DNA binding by all three protoberberine analogs induced negative flow linear dichroism signals as well as unwinding of the host duplex. These two observations are consistent with an intercalative mode of protoberberine binding to duplex DNA. However, a comparison of the DNA binding properties for DMC and DHDMC, which differ only by the state of saturation at the 5,6 positions of the B-ring, revealed that the protoberberine analogs do not "behave" like classic DNA intercalators. Specifically, saturation of the 5-6 double bond in the B-ring of DMC, thereby converting it to the DHDMC molecule, was associated with enhanced DNA unwinding as well as a reversal of DNA binding preference from a DNA duplex with an inaccessible or occluded minor groove {poly[d(G-C)]2} to DNA duplexes with accessible or unobstructed minor grooves {poly[d(A-T)]2 and poly[d(I-C)]2}. In addition, a comparison of the DNA binding properties for DHDMC and palmatine revealed that transferring the 11-methoxy moiety on the D-ring of DHDMC to the 9 position, thereby converting it to palmatine, was associated with a reduction in binding affinity for both duplexes with unobstructed minor grooves as well as for duplexes with occluded minor grooves. These DNA binding properties are consistent with a "mixed-mode" DNA binding model for protoberberines in which a portion of the ligand molecule intercalates into the double helix, while the nonintercalated portion of the ligand molecule protrudes into the minor groove of the host duplex, where it is thereby available for interactions with atoms lining the floor and/or walls of the minor groove. Furthermore, saturation at the 5,6 positions of the B-ring, which causes the A-ring to be tilted relative to the plane formed by rings C and D, appears to stabilize the interaction between the host duplex and the minor groove-directed portion of the protoberberine ligand. Computer modeling studies on the DHDMC-poly[d(A-T)]2 complex suggest that this interaction may involve van der Waals contacts between the ligand A-ring and backbone sugar atoms lining the minor groove of the host duplex. The hierarchy of topoisomerase I poisoning noted above suggests that this minor groove-directed interaction may play an important role in topoisomerase I poisoning by protoberberine analogs. In the aggregate, our results presented here, coupled with the recent demonstration of topoisomerase I poisoning by minor ...

Apparent circular dichroism signature of stirring-oriented DNA and drug–DNA complexes

“…Addition of counterions renders the double helices more flexible so the flow gradient becomes insufficient to orient the DNA molecules and thus the anisotropic CD pattern vanishes. It is to be noted that in special devices suitable to generate much higher hydrodynamic flow gradient (e.g., Couette flow cell), the DNA helices can be aligned even in the presence of salts and display very similar spectral pattern to that reported here [3,8,9].…”

Down-Regulation of DNA Topoisomerase IIα in Human Colorectal Carcinoma Cells Resistant to a Protoberberine Alkaloid, Berberrubine

“…Coralyne and its derivatives were shown to be inducers of topoisomerase I-DNA cleavable complexes, whereas the structurally similar benzophenanthridine alkaloid nitidine showed a dual poison activity for topoisomerases I and II (Pilch et al, 1997;Sanders et al, 1998;Makhey et al, 2000). In previous work from our laboratory, we have demonstrated that berberrubine has a potent activity as DNA topoisomerase II poison by stabilizing topoisomerase II-mediated cleavable complex in vitro .…”

Molecular Characterization of Recombinant Pneumocystis carinii Topoisomerase I: Differential Interactions with Human Topoisomerase I Poisons and Pentamidine

“…Unfortunately, depletion of the genomic DNA sample prevented us from obtaining the intervening sequences at the 5Ј end of the gene. We were, however, able to identify seven introns with lengths of 61,45,50,46,48,43, and 44 bp, whose locations are shown in Fig. 1A.…”

“…In contrast to the strong P. carinii topoisomerase I-poisoning activity obtained with the benzimidazoles and nogalamycin, no poisoning was seen with coralyne or nitidine (Fig. 6), both of which are potent human topoisomerase I poisons (26,46). Coralyne (and other protoberberines) are also inactive against both the A. nidulans and S. cerevisiae topoisomerase I enzymes (26,29).…”

“…With the exception of CPT derivatives and the several antiCandida drugs characterized by Fostel and coworkers, the topoisomerase I poisons discussed above are cationic drugs that bind in the minor groove of DNA (12,46,69). The correlation between minor-groove binding and topoisomerase I poisoning activity has been noted with terbenzimidazoles (69) and other drug classes (11,12,59), although it is clear that strong minorgroove binding behavior is not the sole determinant for topoisomerase I poisoning activity, as several well-known minorgroove binding agents, including berenil and netropsin, show little or no activity as topoisomerase I poisons (reviewed in reference 46).…”