Ihtshamul Haq

Thermodynamics of drug–DNA interactions

Many anticancer, antibiotic, and antiviral drugs exert their primary biological effects by reversibly interacting with nucleic acids. Therefore, these biomolecules represent a major target in drug development strategies designed to produce next generation therapeutics for diseases such as cancer. In order to improve the clinical efficacy of existing drugs and also to design new ones it is necessary to understand the molecular basis of drug-DNA interactions in structural, thermodynamic, and kinetic detail. The past decade has witnessed an increase in the number of rigorous biophysical studies of drug-DNA systems and considerable knowledge has been gained in the energetics of these binding reactions. This is, in part, due to the increased availability of high-sensitivity calorimetric techniques, which have allowed the thermodynamics of drug-DNA interactions to be probed directly and accurately. The focus of this article is to review thermodynamic approaches to examining drug-DNA recognition. Specifically, an overview of a recently developed method of analysis that dissects the binding free energy of these reactions into five component terms is presented. The results of applying this analysis to the DNA binding interactions of both minor groove drugs and intercalators are discussed. The solvent water plays a key role in nucleic acid structure and consequently in the binding of ligands to these biomolecules. Any rational approach to DNA-targeted drug design requires an understanding of how water participates in recognition and binding events. Recent studies examining hydration changes that accompany DNA binding by intercalators will be reviewed. Finally some aspects of cooperativity in drug-DNA interactions are described and the importance of considering cooperative effects when examining these reactions is highlighted.

H-NS Oligomerization Domain Structure Reveals the Mechanism for High Order Self-association of the Intact Protein

H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.

Drug-DNA recognition: Energetics and implications for design

In this article we review thermodynamic studies designed to examine the interaction of low molecular weight ligands or drugs with DNA. Over the past 10 years there has been an increase in the number of rigorous biophysical studies of DNA–drug interactions and considerable insight has been gained into the energetics of these binding reactions. The advent of high‐sensitivity calorimetric techniques has meant that the energetics of DNA–drug association reactions can be probed directly and enthalpic and entropic contributions to the binding free energy established. There are two principal consequences arising from this type of work, firstly three‐dimensional structures of DNA–drug complexes from X‐ray and NMR studies can be put into a thermodynamic context and the energetics responsible for stabilizing the observed structures can be more fully understood. Secondly, any rational approach to structure‐based drug design requires a fundamental base of knowledge where structural detail and thermodynamic data on complex formation are intimately linked. Therefore these types of studies allow a set of general guidelines to be established, which can then be used to develop drug design algorithms. In this review we describe recent breakthroughs in duplex DNA‐directed drug design and also discuss how similar principles are now being used to target higher‐order DNA molecules, for example, triplex (three‐stranded) and tetraplex (four‐stranded) structures. Copyright

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.

Calorimetric and Spectroscopic Studies of Hoechst 33258: Self-association and Binding to Non-cognate DNA

Sequence and structure-specific molecular recognition of DNA by small molecules is an important goal in biophysical chemistry and drug discovery. Many candidate ligands possess flat aromatic surfaces and other molecular features that allow them to self-associate. In addition, non-specific binding to the target is a complicating feature of these interactions. Therefore, multiple equilibria are present and need to be accounted for in data analysis in order to obtain meaningful thermodynamic parameters. In order to address these issues we have systematically examined the bis-benzimidazole dye Hoechst 33258 (H33258) in terms of self-aggregation and binding to DNA oligonucleotides lacking any cognate minor groove A.T sites. This model system has been interrogated using isothermal titration calorimetry (ITC), circular dichroism (CD), fluorescence spectroscopy and pulsed gradient spin echo NMR. Three distinct binding events and ligand self-aggregation have been identified and, where possible, quantified. H33258 self-aggregation involves a step-wise aggregation mechanism, driven by stacking interactions. The DNA binding process includes two specific binding modes and non-specific DNA-templated H33258 stacking. We have written novel ITC data-fitting software (IC-ITC; freely available to the biophysics community), which simultaneously fits ligand aggregation and ligand-DNA binding. Here, this numerical analysis, which uses simulated annealing of complex calorimetric data representing multiple coupled equilibria, is described.

Singular value decomposition of 3-D DNA melting curves reveals complexity in the melting process

Abstract The thermal denaturation of synthetic deoxypolynucleotides of defined sequence was studied by a three dimensional melting technique in which complete UV absorbance spectra were recorded as a function of temperature. The results of such an experiment defined a surface bounded by absorbance, wavelength, and temperature. A matrix of the experimental data was built, and analyzed by the method of singular value decomposition (SVD). SVD provides a rigorous, model-free analytical tool for evaluating the number of significant spectral species required to account for the changes in UV absorbance accompany-ing the duplex – to – single strand transition. For all of the polynucleotides studied (Poly dA – Poly dT; [Poly (dAdT)]2; Poly dG – Poly dC; [Poly(dGdC)]2), SVD indicated the existence of at least 4 – 5 significant spectral species. The DNA melting transition for even these simple repeating sequences cannot, therefore, be a simple two-state process. The basis spectra obtained by SVD analysis were found to be unique for each polynucleotide studied. Differential scanning calorimetry was used to obtain model free estimates for the enthalpy of melting for the polynucleotides studied, with results in good agreement with previously published values.

Roles of divalent metal ions in flap endonuclease–substrate interactions

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.

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.

A ruthenium dipyridophenazine complex that binds preferentially to GC sequences

Uniquely, a Ru11 complex of the dppz ligand shows a preference for GC sequences of DNA.

Water-soluble organic dppz analogues—tuning DNA binding affinities, luminescence, and photo-redox properties

Three new water-soluble dppz derivatives are reported, one of which binds to DNA with an affinity comparable to any mononuclear metal complex and also displays a high selectivity for GC sites.