General features of the energetics of complex formation between ligand and nucleic acids

The analysis of the energy contributions of various physical factors to the complex formation between biologically active compounds and nucleic acids in aqueous solution was performed. A comparison of the energy parameters was made for ligand-ligand, intercalator-DNA, MGB-DNA and ligand-RNA groups. It was shown that the energetics of these reactions is of compensatory nature. Physical factors exerting the most pronounced influence on the energy parameters were identified. Correlation of the energy contributions to MGB-DNA complex formation and its biological effect was foun

Metallodrugs as inducers and inhibitors of chemical nuclease activity

Manipulation of DNA is both an intrinsic and essential component of molecular biology and biotechnology. Reagents capable of cutting DNA are applied within these fields as probes for DNA structure and function, with the ultimate aim being the design of target-specific—customised endonucleases—capable of modifying genomic DNA. Thus, DNA cleaving reagents are essential tools for both sequence analysis and genome engineering. Furthermore, the discovery of new molecular mechanisms by which small molecules modify DNA structure, reactivity, and biological repair contributes significantly to potential drug development. The chemical nuclease of [Cu(Phen)2]2+ (where Phen = 1,10-phenanthroline), is a well studied reagent which randomly cleaves nucleic acids in the presence of molecular oxygen (or hydrogen peroxide) upon reduction to Cu+. In addition, compounds based on this chemotype have found application in the biological field as antimicrobial and anticancer agents, DNA intercalators, and as nucleoside constituents for incorporation into the DNA backbone. [Cu(Phen)2]+ oxidises duplex DNA without specificity, predominately at the minor groove with C-H bonds at C1′, C4′, and C5′ being the main targets of hydrogen atom abstraction. The aim of this research was to extend structure-activity relationships of Cu2+-Phen complexes containing sterically functionalized pendant carboxylates and to investigate how synthetic extension of the ligated phenazine ligand within this complex model influences DNA recognition and oxidative degradation. These compounds have shown an enhanced DNA recognition relative to the well-studied chemical nuclease, [Cu(Phen)2]+. Furthermore, the effects of nuclearity on DNA oxidation were elucidated using the [Cu(-terephthalate)(Phen)4]2+ cation with results showing potent DNA oxidation in the absence of exogenous reductant. Many compounds developed in this work constitute a series of novel anticancer leads capable of intracellular DNA oxidation leading to genomic double strand breaks. In addition to the application of developmental metallodrugs as inducers of chemical nuclease activity, the effects of cytotoxic trinuclear platinum(II) complexes as high-affinity DNA binders that inhibit—or block—endonuclease enzyme recognition and excision are reported through a wide variety of biophysical and molecular biological methods

Sequence Specific Complexation of B DNA at Sites Containing G,C Base Pairs

A series of eight related analogs of distamycin A has been synthesized. Footprinting and affinity cleaving reveal that only two of the analogs, pyridine-2- car box amide-netropsin (2-Py N) and 1-methylimidazole-2-carboxamide-netrops in (2-ImN), bind to DNA with a specificity different from that of the parent compound. A new class of sites, represented by a TGACT sequence, is a strong site for 2-PyN binding, and the major recognition site for 2-ImN on DNA. Both compounds recognize the G•C bp specifically, although A's and T's in the site may be interchanged without penalty. Additional A•T bp outside the binding site increase the binding affinity. The compounds bind in the minor groove of the DNA sequence, but protect both grooves from dimethylsulfate. The binding evidence suggests that 2-PyN or 2-ImN binding induces a DNA conformational change. In order to understand this sequence specific complexation better, the Ackers quantitative footprinting method for measuring individual site affinity constants has been extended to small molecules. MPE•Fe(II) cleavage reactions over a 10⁵ range of free ligand concentrations are analyzed by gel electrophoresis. The decrease in cleavage is calculated by densitometry of a gel autoradiogram. The apparent fraction of DNA bound is then calculated from the amount of cleavage protection. The data is fitted to a theoretical curve using non-linear least squares techniques. Affinity constants at four individual sites are determined simultaneously. The distamycin A analog binds solely at A•T rich sites. Affinities range from 10⁶-10⁷ M⁻¹ The data for parent compound D fit closely to a monomeric binding curve. 2-PyN binds both A•T sites and the TGTCA site with an apparent affinity constant of 10⁵ M⁻¹. 2-ImN binds A•T sites with affinities less than 5 x 10⁴ M⁻¹. The affinity of 2-ImN for the TGTCA site does not change significantly from the 2-PyN value. At the TGTCA site, the experimental data fit a dimeric binding curve better than a monomeric curve. Both 2-PyN and 2-ImN have substantially lower DNA affinities than closely related compounds. In order to probe the requirements of this new binding site, fourteen other derivatives have been synthesized and tested. All compounds that recognize the TGTCA site have a heterocyclic aromatic nitrogen ortho to the N or C-terminal amide of the netropsin subunit. Specificity is strongly affected by the overall length of the small molecule. Only compounds that consist of at least three aromatic rings linked by amides exhibit TGTCA site binding. Specificity is only weakly altered by substitution on the pyridine ring, which correlates best with steric factors. A model is proposed for TGTCA site binding that has as its key feature hydrogen bonding to both G's by the small molecule. The specificity is determined by the sequence dependence of the distance between G's. One derivative of 2-PyN exhibits pH dependent sequence specificity. At low pH, 4-dimethylaminopyridine-2-carboxamide-netropsin binds tightly to A•T sites. At high pH, 4-Me₂NPyN binds most tightly to the TGTCA site. In aqueous solution, this compound protonates at the pyridine nitrogen at pH 6. Thus presence of the protonated form correlates with A•T specificity. The binding site of a class of eukaryotic transcriptional activators typified by yeast protein GCN4 and the mammalian oncogene jun contains a strong 2-ImN binding site. Specificity requirements for the protein and small molecule are similar. GCN4 and 2-lmN bind simultaneously to the same binding site. GCN4 alters the cleavage pattern of 2-ImN-EDTA derivative at only one of its binding sites. The details of the interaction suggest that GCN4 alters the conformation of an AAAAAAA sequence adjacent to its binding site. The presence of a yeast counterpart to jun partially blocks 2-lmN binding. The differences do not appear to be caused by direct interactions between 2-lmN and the proteins, but by induced conformational changes in the DNA protein complex. It is likely that the observed differences in complexation are involved in the varying sequence specificity of these proteins.</p