DRUG-DNA INTERACTIONS BY SPECTROSCOPIC AND MOLECULAR MODELING TECHNIQUES

Abstract

The natural products have been of considerable interest as potential anti cancer agents that bind to DNA, but with the common ability to act as potent inhibitors of DNA transcription and replication. Many synthetic compounds have been added to this list in the search for more potent drugs for use in chemotherapy in view of pronounced cytotoxicity of these drugs, resistance towards tumour cell lines and difference in their neoplastic potency. Structural tools such as X-ray crystallography and NMR spectroscopy, coupled with molecular modeling techniques have considerable impact in advancing our understanding of the microscopic structural homogeneity of DNA and the molecular basis for drug-DNA interactions. Besides above-mentioned techniques, theoretical studies using Density Functional method (DFT) by Gaussian 98 are done supported by Diffusion Ordered Spectroscopy (DOSY), Electron Spray Ionization Mass Spectrometry (ESIMS) and Fluorescence Life-Time Measurements Techniques. These studies help to understand the molecular basis for binding and identifying the preferred sequence specificity of many key drugs with DNA. The Ph.D. thesis work has been reported in the form of eight chapters. Chapter 1 contains introduction to the subject, a comprehensive review of the literature and scope of thesis. Chapter 2 deals with the materials and methods used. The detailed Nuclear Magnetic Resonance Spectroscopy- IDNMR, DQF COSY, TOCSY, lH - ]H NOESY for the proton assignment; HSQC ('H-13C) and HMBC ('H-13C) for the carbon assignment; !H - 31P HMBC, 31P - 31P NOESY for the phosphorus assignment; UV-Visible and Fluorescence studies is for calculating the dimerization constant (Kgq); Diffusion Ordered Spectroscopy (DOSY) and Electron Spray Ionization Mass Spectrometry (ESIMS) studies are to see the self-aggregation; Fluorescence Life-Time Measurements methods are explained to know the intercalated complex formation. The strategies used for restrained energy minimization, molecular dynamics simulations and quantum mechanical calculations involving GIAO method (for chemical shift calculation) and DFT method (for optimization) are also discussed. Chapter 3 deals with the NMR spectral assignment of 4'-epiadriamycin and restrained Molecular Dynamics simulation and quantum mechanical calculations of 4'- epiadriamycin, adriamycin and daunomycin. In this chapter, the structural and electronic properties of 4'-epiadriamycin, adriamycin and daunomycin have been studied using Density Functional Theory (DFT) employing B3LYP exchange correlation. The chemical shift of'H and 13C resonances in Nuclear Magnetic Resonance spectra have been calculated using Gauge-Invariant Atomic Orbital (GIAO) method as implemented in Gaussian 98 and compared with experimental NMR spectra recorded at 500 MHz. A restrained Molecular Dynamics approach was used to get the optimized solution structure of drugs using interproton distance constraints obtained from 2D NOESY spectra. The glycosidic angle C7-07- Cl'-C2' is found to show considerable flexibility by adopting 156-161 ° (I), 142-143 ° (II) and 38-78 ° (III) conformations, of which the biological relevant structure appears to be the conformer (II). The observed different conformations of the three drugs are correlated to the differential anticancer activity exhibited by these drugs. Chapter 4 deals with the studies on self-aggregation of 4'-epiadriamycin, adriamycin and daunomycin by restrained Molecular Dynamics approach using Nuclear Magnetic Resonance Spectroscopy supported by Absorption, Fluorescence, Diffusion Ordered Spectroscopy and Mass Spectrometry. Self association is a process which competes with binding to DNA and formation of heterocomplexes. The change in chemical shift shows the presence of stacked dimer in all the three drugs. The two-dimensional NOESY studies show several intra-molecular and intermolecular inter-proton connectivities suggesting specific stacking patterns of aromatic chromophores in parallel and anti-parallel orientation. Absorption, emission and diffusion ordered spectroscopy demonstrate formation of self aggregates. Electron Spray Ionization Mass Spectrometry study also proves the presence of dimer and absence of higher aggregates. Also there is cleavage of glycosidic bond in daunomycin and adriamycin but not in 4'- epiadriamycin which is a clear evidence of reduced cardiotoxicity by 4'-epiadriamycin, as compared to daunomycin and adriamycin. The restrained Molecular Dynamics simulations show structural differences between drugs which have been correlated to biological action. Chapter 5 deals with Phosphorus-31 NMR studies on binding of adriamycin with DNA hexamer sequence d-(TGATCA)2. Titration studies are performed by adding increasing amounts of drug to a fixed concentration of DNA at 275 K. Besides, the 5 phosphate group resonances observed in d-(TGATCA)2, additional peaks are seen, which are in slow exchange with those in the uncomplexed DNA. 2D 31P NMR exchange spectrum give a direct proof of the existence of DNA bound to drug molecule. Presence of large downfield shift of ~ 1.3 ppm indicates that drug intercalates between the base pairs of DNA and induce opening of base pairs to 6.8 A by change in backbone torsional angles. Besides this, linewidth and spin-lattice relaxation studies (Ti) are also done to see the change in the phosphodiester backbone of DNA. Chapters 6 deals with a detailed proton NMR study of binding of adriamycin with DNA hexamer sequence d-(TGATCA)2. Titration studies are performed upto drug to DNA duplex ratio of 2.0 at 275 K. 2D NOESY experiments on 1:1, 1.5:1 and 2.0:1 drug-DNA complexes yields several intra-molecular and inter-molecular contacts. The two sets of resonance protons of DNA for T4NH, G2NH, T1NH, T1CH3 andT4CH3 protons show that the bound complex is in slow exchange with uncomplexed DNA. The absence of sequential connectivities at the intercalation site, that is, TlpG2/C5pA6 base pairs steps, has been found. Along with it, the fluorescence life time measurement, Diffusion Ordered Spectroscopy (DOSY) studies are also done to see the formation of intercalated complex. The restrained Molecular Dynamics Studies of complex of adriamycin with DNA Hexamer Sequence d-(TGATCA)2 is done using the inter-proton distance obtained from 2D NOESY spectra. The helical parameters and backbone torsional angles etc. have been analyzed using CURVES software version 5.1. Chapter 7 deals with Phosphorus-31 NMR studies on binding of 4'-epiadriamycin with DNA hexamer sequence d-(CGATCG)2. Titration studies are performed by adding increasing amounts of drug to a fixed concentration ofDNA at 275, 298 and 318 K. Besides, the 5 phosphate group resonances observed in d-(CGATCG)2, additional peaks are seen, which are in slow exchange with those in the uncomplexed DNA at the low temperature on NMR time scale. 2D 31P NMR exchange spectrum give a direct proof of the existence of DNA bound to drug molecule. Presence of large downfield shift of ~ 1.7 ppm indicates that drug intercalates between the base pairs ofDNA and induce opening of base pairs to 6.8 Aby change in backbone torsional angles. Chapter 8 deals with a detailed proton NMR study of binding of 4'-epiadriamycin with DNA hexamer sequence d- (CGATCG)2. Titration studies are performed upto drug to DNA duplex ratio of 2.0 at 275, 298, 318 K. 2D NOESY experiments on 1:1, 1.5:1 and 2.0:1 drug-DNA complexes yields several intra-molecular and inter-molecular contacts. The two sets of resonance protons of DNA for T4NH, G2NH, G6NH, and T4CH3 protons show that the bound complex is in slow exchange with uncomplexed DNA. The drug is intercalated to the DNA and is placed close to ClpG2/C5pG6 base pairs. Intermolecular peaks of drug and DNA is present. Further, the fluorescence life time measurement, Diffusion Ordered Spectroscopy (DOSY) studies are also show the formation of intercalated complex. Besides this, the model is built using the inter-proton distance obtained from NMR by rMD simulations on complex of 4'- epiadriamycin complexed with d-(CGATCG)2 Sequence dependent variations have been observed. The elucidations of the inter-proton distances obtained from NMR experiments and exchange of bound and free DNA by 31P NMR experiments, along with rMD simulations of the structure of drug-DNA complex show that these drugs intercalate between the base pairs of DNA and stabilize the complex. Deviation is found at the base pairs where the drug intercalates from the canonical B-DNA conformation. Finally the conclusion is summarized.