STUDY OF DRUG-DNA INTERACTIONS BY NMR SPECTROSCOPY

Abstract

Nature has evolved a diverse set of antibiotics that bind to DNA in a variety of ways, but with the common ability to act as potent inhibitors of DNA transcription and replication. As a consequence, these natural products have been of considerable interest as potential anti cancer agents. Many synthetic compounds have been added to this list in the search for more potent drugs for use in chemotherapy. While it is appreciated that DNA is a primary target for many potent antitumor agents, data that pinpoint the exact mechanism of action are generally unavailable. A substantial body of research has been directed towards understanding the molecular basis for DNA sequence specificity for binding, by identifying the preferred binding sequences of many key drugs with DNA. Structural tools such as X-ray crystallography and NMR spectroscopy, coupled with molecular modeling techniques have had considerable impact in advancing our understanding of the microscopic structural homogeneity of DNA and the molecular basis for drug-DNA interactions. The present study is a significant step towards understanding the molecular basis of action of these drugs enabled by solution studies using nuclear magnetic resonance spectroscopy. Chapter 1 contains introduction of the subject as well as highlights the work carried out in literature. Chapter 2 deals with the materials and methods being used. In chapter 3 structural refinement of d-(CGATCG)2 has been carried out using a total of 10 spin-spin coupling constants and 112 NOE intensities by restrained Molecular Dynamics (rMD) with different starting structures, potential functions and rMD protocols. Refinement using different methods resulted in essentially the same structure, indicating that the structure obtained is defined by experimental restraints. The structural details have been analyzed with respect to torsional angles, base pair geometries and helicoidal parameters. The final structure, representing a timeaveraged structure, shows base-sequence dependent variations and hence strong local structural heterogeneity. In chapter 4, the structural features of duplex d-TGATCA has been investigated by restrained molecular dynamics up to 100 ps using atotal of 12 torsional angles and 121 distance constraints. The structure is characterized by a large positive roll at TpG/CpA base pair step and large negative propeller twist for AT and TA base pairs. There is evidence of significant flexibility of the sugar-phosphate backbone with rapid inter conversion between two different conformers at TpG/CpA base pair step. The base sequence dependent variations and local structural heterogeneity have important implications in specific recognition of DNA by ligands. Chapter^, 6and 7deals with the restrained molecular dynamics simulations of three complexes, that is, adriamycin-d-(CGATCG)2, daunomycin-d-(TGATCA)2 and daunomycin-d-(CGATCG)2, respectively. The restraint data set consists of several intramolecular and intermodular nuclear Overhauser enhancement cross peaks obtained from two-dimensional nuclear magnetic resonance spectroscopy data. The drug is found to intercalate between CG and GC base pairs at two d-CpG sites in adriamycin-d-(CGATCG)2 and daunomycin-d-(CGATCG)2 complexes. In daunomycin-d-(TGATCA)2 complex the drug intercalates at TlpG2 and C5pA6 sites. The drug-DNA complex is stabilized via specific hydrogen bonding and van der waal's interactions involving 40CH3, 05, 013, 60H, NH3+ moiety of daunosamine sugar and ring Aprotons. The glycosidic bond C7-07-Cl'-C2' lies in the range 138°- 160° during the course of simulations. The role of various functional groups leading to molecular basis of drug action is discussed. Acomparison of structural features of the complexes shows that some of the related interactions are similar and may be related to a common mode of binding. Several other interactions are either DNA sequence specific or drug specific. These may be attributed to difference in biological action of adriamycin and daunomycin.