Terence C. Jenkins

Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding.

The three‐dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti‐trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5′‐AAT‐3′ region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen‐bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen‐bonded contact with an amidinium nitrogen atom, N3 of the 5′ end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.

Parsing free energies of drug-DNA interactions

Publisher Summary This chapter discusses that a number of clinically important small molecules appear to act by binding directly to DNA, and subsequently inhibiting gene expression or replication by interfering with the enzymes that catalyze these functions. Thermodynamics provides quantitative information that can helps elucidate the principal driving forces for the interaction, which can provide insight to guide possible chemical modifications that might enhance both DNA-drug binding affinity and base sequence specificity. It reviews the power of thermodynamics is that it provides quantitative information that is independent of the details for the underlying molecular processes. Advances in instrumentation have enabled the acquisition of reliable thermodynamic data for reactions of biological interest. In parallel, significant advances have been made in the interpretation of thermodynamic data, making it possible to begin to parse free energy values into the contributions from a variety of forces and interactions. Considerable insight into the forces that drive association reactions can be gleaned from such interpretations. The chapter describes a framework and specific details for the application of these methods to DNA-drug binding reactions. While only relatively few compounds have thus far been examined by these approaches, it is clear from these initial successes that valuable new molecular insights will emerge as more DNA-binding drugs are also discussed.

Thermodynamics of nucleic acids and their interactions with ligands.

1. Introduction 255 1.1 General thermodynamics 256 2. Nucleic acid thermodynamics 260 2.1 DNA duplexes 261 2.2 RNA duplexes 263 2.3 Hybrid DNA–RNA duplexes 264 2.4 Hydration 267 2.5 Conformational flexibility 269 2.6 Thermodynamics 272 3. Nucleic acid–ligand interactions 277 3.1 Minor groove binders 278 3.2 DNA intercalators 284 3.3 Triple-helical systems 288 3.3.1 Structures 288 3.3.2 Hydration 291 3.3.3 Thermodynamics 291 4. Conclusions 295 5. Acknowledgements 298 6. References 298 In recent years the availability of large quantities of pure synthetic DNA and RNA has revolutionised the study of nucleic acids, such that it is now possible to study their conformations, dynamics and large-scale properties, and their interactions with small ligands, proteins and other nucleic acids in unprecedented detail. This has led to the (re)discovery of higher order structures such as triple helices and quartets, and also the catalytic activity of RNA contingent on three-dimensional folding, and the extraordinary specificity possible with DNA and RNA aptamers. Nucleic acids are quite different from proteins, even though they are both linear polymers formed from a small number of monomeric units. The major difference reflects the nature of the linkage between the monomers. The 5′–3′ phosphodiester linkage in nucleic acids carries a permanent negative charge, and affords a relatively large number of degrees of freedom, whereas the essentially rigid planar peptide linkage in proteins is neutral and provides only two degrees of torsional freedom per backbone residue. These two properties conspire to make nucleic acids relatively flexible and less likely to form extensive folded structures. Even when true 3D folded structures are formed from nucleic acids, the topology remains simple, with the anionic phosphates forming the surface of the molecule. Nevertheless, nucleic acids do occur in a variety of structures that includes single strands and high-order duplex, triplex or tetraplex (‘quadruplex’) forms. The principles of biological recognition and the related problem of understanding the forces that stabilise such folded structures are in some respects more straightforward than for proteins, making them attractive model systems for understanding general biophysical problems. This view is aided by the relatively facile chemical synthesis of pure nucleic acids of any desired size and defined sequence, and the ease of incorporation of a wide spectrum of chemically modified bases, sugars and backbone linkers. Such modifications are considerably more difficult to achieve with oligopeptides or proteins.

Targeting Multi-Stranded DNA Structures

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 author s 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 pi-overlap can be used to improve the affinity or specificity of the underlying DNA drug binding events.

Synthesis of a novel C2/C2′-exo unsaturated pyrrolobenzodiazepine cross-linking agent with remarkable DNA binding affinity and cytotoxicity

A C2/C2′-exo unsaturated pyrrolobenzodiazepine dimer 1 has been synthesised which is cytotoxic at the picomolar level and has remarkable covalent DNA binding affinity, raising the melting temperature of duplex-form calf thymus DNA by 34 °C after 18 h incubation.

Structure-activity relationships of monomeric C2-aryl pyrrolo[2,1-c][1,4]benzodiazepine (PBD) antitumor agents

A comprehensive SAR investigation of the C2-position of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) monomer antitumor agents is reported, establishing the molecular requirements for optimal in vitro cytotoxicity and DNA-binding affinity. Both carbocyclic and heterocyclic C2-aryl substituents have been studied ranging from single aryl rings to fused ring systems, and also styryl substituents, establishing across a library of 80 analogues that C2-aryl and styryl substituents significantly enhance both DNA-binding affinity and in vitro cytotoxicity, with a correlation between the two. The optimal C2-grouping for both DNA-binding affinity and cytotoxicity was found to be the C2-quinolinyl moiety which, according to molecular modeling, is due to the overall fit of the molecule in the DNA minor groove, and potential specific contacts with functional groups in the floor and walls of the groove. This analogue (14l) was shown to delay tumor growth in a HCT-116 (bowel) human tumor xenograft model.

Molecular Modelling and Cytotoxicity of Substituted Anthraquinones as Inhibitors of Human Telomerase

Molecular modelling has been carried out for a number of amine-functionalised anthraquinone derivatives to determine their extent of binding to G-tetraplex DNA and their ability to inhibit the enzymes telomerase and Taq polymerase. The results are compared to data obtained from a modified TRAP assay and show good correlation between the two methods. The findings suggest that anthraquinone derivatives of this type inhibit telomerase by stabilisation of four-stranded tetraplex structures associated with guanine-rich telomeric DNA regions.

Calorimetric techniques in the study of high-order DNA-drug interactions.

Publisher Summary Biophysical studies of DNA–drug (or DNA–ligand) interactions have an established and pivotal role in the rational development of novel ligands directed toward high-order nucleic acid system. To illustrate the importance and power of calorimetry as a biophysical tool this chapter describes how isothermal titration calorimetry (ITC) has been used to determine the energetics of ligand binding to triplex and tetraplex DNA, and how such studies can help to clarify the nature of the interactions and thereby provide feasible binding models. Complementary studies involving DSC can be used to examine nucleic acid stability under given conditions; thus, for example, protocols for using DSC to examine the differential stabilization of tetraplex DNA with Na + and K + ions are also discussed. The genuine strength of calorimetry becomes apparent when it is used in conjunction with parallel techniques such as UV-visible/CD/fluorescence spectrophotometry, NMR spectroscopy, X-ray crystallography, and stopped-flow kinetic methods. Such a multitechnique approach to studies of DNA–drug interactions (or indeed any binding interaction) can provide a comprehensive and cohesive picture for the underlying biomolecular events.

Induction of DNA strand breaks by RSU-1069, a nitroimidazole-aziridine radiosensitizer: Role of binding of both unreduced and radiation-reduced forms to DNA, in vitro

[2-14C]-RSU-1069 [1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol], either as a parent (unreduced) or following radiation reduction, binds to calf thymus DNA in vitro. Radiation-reduced RSU-1069 binds to a greater extent and more rapidly than the parent compound. RSU-1137, a nonaziridino analogue of RSU-1069, binds following radiation reduction. Radiation-reduced misonidazole (1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol) exhibits binding ratios a thousand-fold less than those of reduced RSU-1069. There is no evidence for binding of parent misonidazole. Both parent and reduced RSU-1069 cause single strand breaks (ssbs) in pSV2 gpt plasmid DNA with the reduced compound causing a greater number of breaks. Parent and reduced RSU-1137 and misonidazole do not cause ssbs. It is inferred that the aziridine moiety present in both parent and reduced RSU-1069 is required for ssb production. RSU-1069 reacts with inorganic phosphate probably via nucleophilic ring-opening of the aziridine fragment. Incubation of plasmid DNA with reduced RSU-1069 in the presence of either phosphate or deoxyribose-5-phosphate at concentrations greater than 0.35 mol dm-3 prevents strand breakage, whereas 1.2 mol dm-3 deoxyribose does not protect against strand breakage formation. From these findings it is proposed that the observed binding to DNA occurs via the aziridine and the reduced nitro group of RSU-1069 and that these two have different target sites. Binding to DNA via the reduced nitro group may serve to increase aziridine attack due to localization at or near its target.

Telomerase inhibitors for the treatment of cancer: the current perspective

Telomerase is a holoenzyme responsible for the maintenance of telomeres, the protein-nucleic acid complexes at the ends of eukaryotic chromosomes that serve to maintain chromosomal stability and integrity. Telomerase activity is essential for the sustained proliferation of most immortal cells, including cancer cells. Since the discovery that telomerase activity is detected in 85 - 90% of all human tumours and tumour-derived cell lines but not in most normal somatic cells, telomerase has become the focus of much attention as a novel and potentially highly-specific target for the development of new anticancer chemotherapeutics. Herein we review the current perspective for the development of telomerase inhibitors as cancer chemotherapeutics. These include antisense strategies, reverse transcriptase inhibitors and compounds capable of interacting with high-order telomeric DNA tetraplex (‘G-quadruplex’) structures, so as to prevent enzyme access to the necessary linear telomere substrate. Critical appraisal of each individual approach is provided together with highlighted areas of likely future development.