Structure and Dynamics of Ligand-Template Interactions of Topoisomerase Inhibitory Analogs of Hoechst 33258: High Field¹H-NMR and Restrained Molecular Mechanics Studies

Abstract

The binding characteristics of Hoechst 33258 (1), a synthetic bis-benzimidazole, and its structural analog 2, with one of the benzimidazoles replaced by a pyridoimidazole, to the self-complementary decadeoxyribonucleotide sequences d(CGCAATTGCG)2 (A) and d-(CATGGCCATG)2 (B) respectively, were examined using high field 1H-NMR techniques. Selective complexation induced chemical shift changes, the presence of exchange signals and intermolecular NOE contacts between the ligands and the minor groove protons of the oligonucleotides suggest the preferred binding sites as the centrally located AATT segment for complex A1, and the CCAT segment for complex B2. The B-type conformations of the two DNA duplexes are preserved upon complexation, as confirmed by the 2D-NOESY based sequential connectivities involving DNA base and sugar protons. Close intermolecular NOE based contacts between the ligands and their respective DNA sequences were further refined to model the ligand-DNA complexes starting from the computer generated B-type structures for the oligonucleotides. Force field calculations of ligand-DNA interaction energies indicate a more favorable contribution from the van der Waals energy component in the case of complex A1 consistent with its stronger net binding compared with the complex B2. Overall, the incorporation of a pyridinic nitrogen in Hoechst 33258 structure alters its selectivity for base pair recognition from A.T to G.C, resulting largely from the formation of a hydrogen bond between the new basic center and the 2-NH2 group of a guanosine moiety. The rates for the exchange of ligands between the two equivalent binding sites (AATT for 1, and CCAT for 2) of the self-complementary DNA sequences, are estimated from analyses of coalescence of NMR signals to be 189s-1 at 301 K for A1 and 79s-1 at 297 K for B2; which correspond to delta G++ of 13.8 and 18.6 kcal.mol-1 respectively.

Synthesis and sequence-specific DNA binding of a topoisomerase inhibitory analog of Hoechst 33258 designed for altered base and sequence recognition

“…Besides the identification of the cytosine aromatic protons in the complexed form of the oligonucleotide, an interesting feature pertains to a significant reduction in the cross peak intensities (compared with those for the ligand-free oligomer) of C6 and C7 residues. The reason for this is not entirely clear, but such characteristic changes upon ligand binding have been consistently observed in a number of previous investigations (16,17,30,31). Borah et al (48) have postulated that the intensity of cytidine H5-H6 cross peaks is quite sensitive to the local environment and mobility of cytosine residues in drug-DNA complexes and is reduced upon binding of ligands in close proximity to these residues.…”

“…The roman numerals identified for the oligonucleotide sequence designate the imino protons at each of the basepair steps and reflect the 2-fold symmetry of the selfcomplementary duplex sequence. The numbering scheme for the ligand is based on our previous NMR studies on Hoechst 33258 and its analogs (30,31).…”

“…In order to begin exploring the structure of the threecomponent systems of protein-template-ligand, we report the details of the high-field NMR study of interactions between ligand 1 and a synthetic decadeoxynucleotide sequence. The selection of the DNA sequence d(CATG-GCCATG>2 was based on our previous experience with its utility in probing the binding interactions of the modified ligands with high affinity for G-C sites (16,17,30,31).…”

Molecular mechanics calculations on the complexes between analogues of Hoechst 33258 and d(CGCGAAT-TCGCG)2: influence of bulky group substitution on base pair preference of DNA minor groove binders

“…According to computational (21 and footnote 2), spectroscopic (23), and thermodynamic (24) For personal use only. 'The values are relative to the reference AATT-a.…”

<title>Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation</title>

“…It is now known form X-ray crystallography [11] and NMR spectroscopy [12][13][14] that Hoechst 33342 binds in the minor groove of DNA, and specifically interacts with an AATT sequence. It is now known form X-ray crystallography [11] and NMR spectroscopy [12][13][14] that Hoechst 33342 binds in the minor groove of DNA, and specifically interacts with an AATT sequence.…”