Drug-DNA interactions: spectroscopic and footprinting studies of site and sequence specificity of elliptinium.
The binding of the antitumoral ellipticine derivative 2-methyl-9-hydroxyellipticinium acetate (elliptinium; NMHE) to DNA was analyzed by the combined use of DNase I footprinting and spectroscopic methods. Using two fragments of pBR322 DNA, five discrete NMHE binding sites of 5-7 protected base pairs (bp) were detected by footprinting at 4 degrees C on the analyzed regions. These corresponded to alternating pyrimidines and purines. The inactive derivative 2-methyl ellipticinium acetate L(NME) lacking a hydroxy group failed to demonstrate DNA protection even at low temperature. Ultraviolet-absorption and 1H-nmr analysis was performed using two autocomplementary octanucleotides d(TGACGTCA) (I) and d(ACTGCAGT) (II). The uv-absorption titrations resulted in an intercalative binding mode for NMHE in the oligomers. Analysis of the derived biphasic Scatchard plots yielded two binding sites corresponding to approximately 6-bp and 2-bp sizes and characterized by apparent association constants K1 approximately 10(8) M-1 and K2 approximately 10(6) M-1, respectively. The 1H-nmr analysis of exchangeable (imino) protons and nonexchangeable protons performed in the one- and two-dimensional modes confirmed the intercalation of NMHE, and further revealed the existence of multiple sites on DNA. Assuming that imino resonance line width concerned the sole kinetic effects, 10-ms order lifetimes were estimated for the drug-oligonucleotide complexes at 7 degrees C, pH 7, and 0.1 ionic strength. Finally, examination of every drug-DNA spectra in the light of the footprinting results indicated that there was a preference for binding of NMHE to the CpG (octamer I) and TpG (octamers I and II) steps.
Mauffret
O
O
Laboratoire de Biochimie-Enzymologie, INSERM U 140, Institut Gustave Roussy, Villejuif, France.
Rene
B
B
Convert
O
O
Monnot
M
M
Lescot
E
E
Fermandjian
S
S
D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004611:Ellipticines / Q000737:chemistry / Q000378:metabolism*; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000970:Antineoplastic Agents; D004611:Ellipticines; D007364:Intercalating Agents; D009838:Oligodeoxyribonucleotides; C027767:elliptinium; D004247:DNA; D003850:Deoxyribonuclease I