Current Medicinal Chemistry - Volume 7, Issue 1, 2000

DNA secondary structures containing regions of single-stranded DNA have now been identified in the genomic DNA of a number of prokaryotic and eukaryotic species, including humans. Many of these secondary structures are associated with regions of DNA involved in regulation of transcription: promoters or upstream elements. The secondary structures involved appear likely to be hairpin or cruciform structures that may be recognition sites for binding of transcription factors. In the case of the coliphage N4 virion RNA polymerase, a defined hairpin in the polymerase promoter necessary for binding of the polymerase and regulation of transcription has been shown to be extruded under physiological conditions in plasmid DNA. The presence of single-stranded DNA in the promoters of several species suggests that regulatory hairpins may be involved in transcription of a number of genes. In support of this, hairpin- or cruciform-binding proteins have been identified from several species. These results imply that secondary structures in regulatory regions may be targets for drugs that bind and either block or enhance binding of proteins involved in transcription. In this review, we discuss the evidence for DNA secondary structures, particularly hairpins and cruciforms, in genomic DNA and review the studies to date of development of small molecules that can selectively bind these structures.

The formation of intermolecular DNA triple helices offers the possibility of designing compounds with extensive sequence recognition properties which may be useful as antigene agents or tools in molecular biology. In these structures a third strand oligonucleotide binds in the DNA major groove, making specific contacts with substituents on the exposed faces of the base pairs. Although triplexes form with exquisite specificity their use suffers from several drawbacks. Two limitations of this approach, which are considered in this review are, firstly that conditions of low pH are necessary for formation of the C +l GC triplet, and secondly that these structures are often less stable than their duplex counterparts. This review outlines the strategies that have been employed to overcome these drawbacks. The pH problem is addressed by considering the various DNA base analogues that have been used to recognise GC base pairs in a pH independent fashion, and discusses the benefits and limitations of each analogue. Triplex stability can be increased by using novel base analogues, backbone modifications and the use of triplex-specific binding ligands.

Inhibitors of topoisomerase I constitute a novel family of antitumor agents. The camptothecin derivatives topotecan and irinotecan represent new weapons in our arsenal for battling human cancer. These two drugs act specifically at the level of the topoisomerase I-DNA complex and stimulate DNA cleavage. This mechanism of action is not restricted to the camptothecins. Numerous topoisomerase I poisons including DNA minor groove binders such as Hoechst 33258 and DNA intercalators such as ben-zophenanthridine alkaloids and indolocarbazole derivatives have been discovered and developed. Another important group of topoisomerase I inhibitors contains drugs which prevent or reverse topoisomerase I-DNA complex formation. Many of these topoisomerase I suppressors are natural products (b-lapachone, diospyrin, topostatin, topostin, favonoids) which are believed to interact directly with the enzyme. This review is concerned with the different families of topoisomerase I poisons and suppressors. Their origin, chemical nature and mechanism of action are presented. The relationships between drug binding to DNA and topoisomerase I inhibition are discussed.

We characterize intercalative complexes as either high charge and low charge. In low charge complexes, stacking interactions appear to dominate stability and structure. The dominance of stacking is evident in structures of daunomycin, nogalamycin, ethidium, and triostin A/echinomycin. By contrast in a DNA complex with the tetracationic metalloporphyrin CuTMPyP4 [copper (II) meso-tetra(N-methyl-4-pyridyl)porphyrin], electrostatic interactions appear to draw the porphyrin into the duplex interior, extending the DNA along its axis, and unstacking the DNA. Similarly, DNA complexes of tetracationic ditercalinium and tetracationic flexi-di show significant unstacking. Here we report x-ray structures of complexes of the tetracationic bis-intercalator D232 bound to DNA fragments d(CGTACG) and d(Br CGTA Br CG). D232 is analogous to ditercalinium but with three methylene groups inserted between the piperidinium groups. The extension of the D232 linker allows it to sandwich four base pairs rather than two. In comparison to CuTMPyP4, flexi-di and ditercalinium, stacking interactions of D232 are significantly improved. We conclude that it is not sufficient to characterize intercalators simply by net charge. One anticipates strong electrostatic forces when cationic charge is focused to a small volume or region near DNA and so must consider the extent to which cationic charge is focused or distributed. In sum, ditercalinium, with a relatively short linker, focuses cationic charge more narrowly than does D232. So even though the net charges are equivalent, electrostatic charges are expected to be of greater structural significance in the ditercalinium complex than in the D232 complex.

Therapeutic targeting of RNA is not as well-developed as with DNA and proteins, and the many structures and functions of RNA suggest that it is an underutilized target. As with DNA, RNA has heterocyclic bases and base pairs with a highly anionic backbone, but as with proteins, RNA can fold into complex tertiary structures that create unique binding pockets for small molecules. Aminoglycoside targeting of ribosomal RNA is a well-known success story, and mRNAs and tRNAs have also served as therapeutic targets as well as model systems for understanding RNA-ligand interactions. The unique, species-specific structures and chemistry involved in splicing and ribozyme activity makes this RNA function an attractive target, and inhibitors of ribozyme activity have been discovered. The numerous serious human diseases caused by RNA viruses highlight the importance of developing new compounds that can target RNA structures in viral genomes. Considerable effort has been directed at finding compounds that target HIV-1 RNAs that control viral replication and frameshifting. As part of these efforts very useful new assays have been developed for small molecule-RNA interactions. The assays have led to the discovery of new inhibitors for different steps in viral replication. The next phase of research in RNA targeting will not only focus on the discovery of new compounds, but also on how to develop small molecules with high affinity and selectively for RNA that can penetrate effectively into a wide array of cell types.

The design of agents targeted toward a structure-specific molecular recognition of DNA triplexes or tetraplexes (quadruplexes) is discussed, where such structures are relevant to antigene-based chemotherapies and the in situ cellular inhibition of telomerase function, respectively. Using principles that stem from the development of earlier synthetic duplex-binding ligands, together with recent findings that probe structure thermodynamic linkages and kinetic features of stability, a rational approach is developed to exploit the distinct molecular templates offered by these high-order nucleic acid biotarget systems. Such analytical techniques can usefully augment conventional drug design methods, particularly where detailed structural information is unavailable or the mode of binding to form a persistent DNA biotarget ligand complex is not established. Examples from the authors laboratory are used to illustrate structure-specific (or structure-preferential) recognition and subsequent stabilization of DNA triplexes using intercalative or groove-mediated binding mechanisms, and the successful targeting of DNA tetraplexes using planar extended-aromatic ligands. In each case, chemical manipulation of the molecule by exploiting either (i) geometric isomers, (ii) redistribution of charged groups and/or H-bond donors/acceptors, or (iii) optimization of intermolecular p-overlap can be used to improve the affinity or specificity of the underlying DNA drug binding events.