NMR-STUDIES ON THE BINDING OF ANTITUMOR DRUG NOGALAMYCIN TO DNA HEXAMER D(CGTACG)

The interactions between a novel antitumor drug nogalamycin with the self-complementary DNA hexamer d(CGTACG) have been studied by 500 MHz two dimensional proton nuclear magnetic resonance spectroscopy. When two nogalamycins are mixed with the DNA hexamer duplex in a 2: 1 ratio, a symmetrical complex is formed. All non-exchangeable proton resonances (except H5′ & H5″) of this complex have been assigned using 2D-COSY and 2D-NOESY methods at pH 7.0. The observed NOE cross peaks are fully consistent with the 1.3 A resolution x-ray crystal structure (Liaw et al., Biochemistry 28, 9913-9918, 1989) in which the elongated aglycone chromophore is intercalated between the CpG steps at both ends of the helix. The aglycone chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. The binding conformation suggests that specific hydrogen bonds exist in the complex between the drug and guanine-cytosine bases in both grooves of the helix. When only one drug per DNA duplex is present in solution, there are three molecular species (free DNA, 1: 1 complex and 2: 1 complex) in slow exchange on the NMR time scale. This equilibrium is temperature dependent. At high temperature the free DNA hexamer duplex and the 1: 1 complex are completely destabilized such that at 65°C only free single-stranded DNA and the 2: 1 complex co-exist. At 35°C the equilibrium between free DNA and the 1: 1 complex is relatively fast, while that between the 1: 1 complex and the 2: 1 complex is slow. This may be rationalized by the fact that the binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. A separate study of the 2: 1 complex at low pH showed that the terminal GC base pair is destabilized. © 1990 Oxford University Press.