NMR studies of the interaction of the antibiotic nogalamycin with the hexadeoxyribonucleotide duplex d(5'-GCATGC)2

Abstract

1H resonance assignments in the NMR spectra of the self-complementary hexadeoxyribonucleoside pentaphosphate d(5'-GCATGC)2 and its complex with the antibiotic nogalamycin, together with interproton distance constraints obtained from two-dimensional nuclear Overhauser effect (NOE) spectra, have enabled us to characterize the three-dimensional structure of these species in solution. In the complex described, two drug molecules are bound per duplex, in each of two equivalent binding sites, with full retention of the dyad symmetry. Twenty-eight NOE distance constraints between antibiotic and nucleotide protons define the position and orientation of the bound drug molecule. Nogalamycin intercalates at the 5'-CA and 5'-TG steps with the major axis of the anthracycline chromophore aligned approximately at right angles to the major axes of the base pairs. The nogalose sugar occupies the minor groove of the helix and makes many contacts with the deoxyribose moieties of three nucleotides along one strand of the duplex in the 5'-TGC segment. The charged dimethylamino group and hydroxyl functions of the bicyclic sugar lie in the major groove juxtaposed to the guanine base, the bridging atoms of the bicyclic sugar making contacts with the methyl group of the thymine. Thus the antibiotic is not symmetrically disposed in the intercalation site but is in close contact in both grooves with atoms comprising the 5'-TGC strand. The intercalation cavity is wedge-shaped, the major axes of the base pairs forming the site being tilted with respect to one another. All base-pair hydrogen-bonding interactions are maintained in the complex, and there is no evidence for Hoogsteen pairing. The free duplex adopts a regular right-handed B-type conformation in which all glycosidic bond angles are anti and all sugar puckers lie in the C2'-endo range. In the complex the glycosidic bond angles and the sugar puckers deviate little from those observed for the duplex alone. The presence of two bound nogalamycin molecules substantially slows the "breathing" motions of the base pairs forming the intercalation cavity, and the observation of two downfield-shifted resonances in the 31P NMR spectrum of the complex suggests a pronounced local helix unwinding at the drug binding site. The footprinting data of Fox and Waring [Fox, K.R., & Waring, M.J. (1986) Biochemistry 25, 4349-4356] imply that the highest affinity binding sites of nogalamycin have the sequence 5'-GCA (or 5'-TGC).(ABSTRACT TRUNCATED AT 400 WORDS)

31P NMR investigation of the backbone conformation and dynamics of the hexamer duplex d(5′‐GCATGC)2 in its complex with the antibiotic nogalamycin

“…The interaction of the anthracyclin¢ antibiotic, nogalamycin, with the hexamer duplex d(GCATGC)~ has previously been shown to induce significant perturbations of base and sugar proton chemical shifts [3], alter the position of the conformational equilibrium of the deoxyribose rings [14] and cause large down-field shifts of two of the five phosphate resonances [3]. The data are consistent with a significant drug-induced alteration of the average solution conformation of the hexamer duplex.…”

Sequence‐Selective Intercalation of Antitumour Bis‐Naphthalimides into DNA

“…It does not follow that drugs will consistently adopt a common mode of interaction at all possible sites, as Fox and Waring (1986) were forced to conclude in respect of the orientation of nogalamycin bound to DNA and others have since clarified (Searle et al, 1988;Egli et al, 1991 ;van Houte et al, Definitive evidence for bis-intercalation via the major groove of the helix will have to await the determination of a high-resolution structure for an oligonucleotidehis-naphthalimide complex by NMR or X-ray crystallography, as has been achieved for ditercalinium (Williams and Gao, 1992). Efforts to obtain structural data by these means are under way.…”

Structure of nogalamycin bound to a DNA hexamer.

“…The alternative arrangement with the drug rotated by 1800 relative to the DNA was also proposed (17). However, with 1H NMR spectroscopy Wakelin and coworkers (14) have shown that nogalamycin intercalates at the TpG step of [d(GCATGC)]2 in the orientation proposed by Arora, with the nogalose in the minor groove and the fused bicyclo amino sugar in the major groove.…”

“…Nogalamycin has been shown by a variety of techniques to penetrate the DNA helix and form a stable, intercalative complex (8)(9)(10)(11)(12)(13)(14) with slow association and dissociation rates (17). The crystal structure and relative stereochemistry of nogalamycin were determined by Arora (18), who proposed a model DNA-nogalamycin-complex.…”