STRUCTURAL STUDIES OF ANTICANCER DRUG MITOXANTRONE AND ITS COMPLEX WITH DNA

Abstract

Nature has evolved a diverse set ofantibiotics that bind to DNA in a variety of ways, but with the common ability to act as potent inhibitors ofDNA 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 ofaction are generally unavailable. Asubstantial 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 ofthe microscopic structural homogeneity of DNA and the molecular basis for drug-DNA interactions. The purpose of present study understands the molecular basis ofaction of these drugs enabled by solution studies using nuclear magnetic resonance spectroscopy. Here, we have studied mechanism ofanticancer drug mitoxantrone with DNA which has been developed as an alternative to existing drugs namely daunomycin, adriamycin etc. owing to their lethal effects such as cardiotoxicity. Mitoxantrone has shown comparable activity and less cardiotoxic effect. Various analogues of the drugs have also been studied through computer modeling techniques. Chapter 1contains brief introduction of the subject as well as an overview the work carried out in literature. Chapter 2deals with the materials and methods being used. Chapters 3and 4deals with the study ofthe mitoxantrone by Nuclear Magnetic Resonance techniques in three different solvents namely D2O, H2O and DMSO. Various homonuclear 2D experiments DQF COSY, 2D ROESY and 'H - 13C (proton - carbon) heteronuclear experiments namely HSQC and HMBC have been carried for complete, unambiguous assignment and determination of structure of drug. Structural refinement of has been carried out using NOE distance constraints by restrained Molecular Dynamics (rMD) with different starting structures, potential functions and rMD protocols for all three solvents. Chapters 5 and 6 deal with Phosphorus-31 and proton NMR investigations on binding of mitoxantrone with DNA hexamer sequence d-(CGATCG)2. Titrations of drug with DNA have been carried out by adding increasing amounts of drug to a fixed concentration of DNAand recording one dimensional NMR spectra at 278 K, 2D 31 P exchange NOESY spectra and 2D protonNOESYspectra have been recorded for 1:1, 1.5: 1 and 1.75:1 and 2:1 drug to DNA stoichiometric ratios at 278 K. New resonance peaks corresponding to bound drug / DNA in the complex are found be in slow exchange with the corresponding resonances in free drug / DNA. The intramolecular and intermolecular distances have been used to analyze the conformational aspects of drug/ DNA in complex. Chapter 7 describes the structure of complex obtained by restrained energy minimized and molecular dynamics simulations. The helicoidal parameters and backbone torsional angles have been obtained using curves software. Chapter 8 summarizes results obtained and their implications in understandingmolecular basis of drug action.