Please use this identifier to cite or link to this item: http://localhost:8081/xmlui/handle/123456789/1704
Authors: Bisht, Kushuma
Issue Date: 2009
Abstract: Palmatine belongs to the class of quaternary protoberberine alkaloid that has been utilized in Ayurvedic and Chinese traditional medicines since long time. It is found in the roots, rhizomes and stem bark of many species of berberidaceae, fumaraceae, menispermacea and other plant families. It exhibits a wide range of pharmacological effects including antimicrobial, antimalarial, anti-inflammatory, antipyretic, hepatoprotective and vasodilatory activity. The cytotoxic activity of palmatine to HL-60 leukemic cells has also been well documented. Palmatine has been reported to be effective against experimental tumors by inhibiting the activity of reverse transcriptase and was also found to exert sedative effect by decreasing the levels of catecholamines in rat brains. It acts as photosensitizer by generating singlet oxygen. The molecular basis for designing DNA binding drugs with improved specificity and affinity stems from the ability to identify the structural elements of the drug and DNA which are responsible for the specificity of the binding and the stabilization of the drug-DNA complex. An analytical technique to elucidate the mode of drug-DNA interaction could be essential for the design of new drugs. Nuclear Magnetic Resonance (NMR) spectroscopy, Absorption spectroscopy, Fluorescence Spectroscopy, and restrained Molecular Dynamics (rMD) are some of the analytical techniques which have been used in this study to investigate conformation of drug, DNA and drug-DNA complexes. The Ph.D. thesis work has been reported in the form of six chapters. Chapter 1 contains introduction of 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 techniques used, that is, - ID NMR, Double Quantum Filtered Correlation Spectroscopy (DQF-COSY), *H - lH Nuclear Overhauser Enhancement Spectroscopy (NOESY) for the proton assignment; 'H - 31P Heteronuclear Multiple Bond Correlation Spectroscopy (HMBC), 3IP - 31P NOESY and Diffusion Ordered Spectroscopy (DOSY) studies are discussed. The strategies used for restrained energy minimization, restrained Molecular Dynamics (rMD) simulations and quantum mechanical calculations involving GIAO method (for chemical shift calculation) and DFT method (for optimization) are discussed. Absorption and Fluorescence spectroscopy and Time-Correlated Single-Photon Counting methods (TCSPC) used to investigate drug-DNA interaction are also discussed. Chapter 3 deals with the structural and electronic properties of palmatine using density functional theory (DFT) employing B3LYP exchange correlation. The geometries of these molecules have been fully optimized at B3LYP/6-311G** level. The chemical shift of *H and l3C resonances phase Nuclear Magnetic Resonance (NMR) spectra of these molecules have been calculated in gaseous and solvent phase using the Gauge-invariant atomic model (GIAO) method as implemented in Gaussian 98 and 03. A restrained Molecular Dynamics approach was used to obtain the optimized solution structure of the drug. Comparison of the calculated NMR chemical shifts with the experimental values revealed that DFT methods produce good results for both proton and carbon chemical shifts. The importance of the basis sets with solvent effect to the calculated NMR parameters has been discussed. It has been found that calculated structure and chemical shifts in solvent phase predicted with B3LYP/6-311G** were in good agreement with the present experimental data and measured values reported earlier. Chapter 4 deals with the study of palmatine interaction with DNA using Absorption and Fluorescence spectroscopic techniques. Several DNA and oligonucleotides were used in this study to elucidate the sequence binding affinity of these drugs. Titration of these drugs with increasing amount of DNA showed that palmatine binds more effectively to AT rich sequences then to GC rich sequences Percentage hypochromicity, binding constant, extinction coefficient of bound palmatine, fluorescence-enhancement and relative fluorescence obtained from absorption and fluorescence spectroscopy were used to study the binding affinity. Chapter 5 deals with 31P, *H NMR and rMD studies on binding of palmatine with DNA sequence d-(CGATCG)2. The following experiments were performed on the palmatine-DNA complex - !H and 31P NMR titration studies at various drug (D)/DNA duplex (N) ratios up to 2.0 at 283 K, 298 K in 90% H20 and 10% D20, temperature dependence of P and H NMR of the palmatine-DNA complex having D/N = 1.0 and 2.0 in the range of 278 - 328 K; 2D 31P - 3IP exchange spectra of drug- DNA complex by phase sensitive NOESY; DOSY experiments of the palmatine-DNA complex and uncomplexed palmatine, rMD studies on the solution structure of palmatine - d-(CGATCG)2 complex using inter-proton distance restraints obtained from 2D NOESY. Results revealed that the addition of palmatine to the oligonucleotide did not induce significant chemical shift variation of the phosphate signals in the 31P NMR spectra. Absence of large downfield shift in 31P NMR spectra suggest that there is no characteristic unwinding of the DNA helix due to change in backbone torsional angle C,, C3'-03'-P-05' from gauche" to trans as observed with intercalators. Since, only one set of resonances are observed for the complexes at different D/N ratios for free hexamer and hexamer bound to palmatine, it is expected that 31P signals from the bound DNA are in fast exchange with the corresponding signals from free DNA to be followed individually on NMR time scale. The proton resonances of palmatine were broad even at low values of drug/DNA (D/N = 0.2) and move upfield with respect to the free drug. An upfield shift of -0.09-0.31 ppm was found for the drugs protons on binding with DNA up to D/N ratio of 2.0. Maximum upfield shift in the drug protons was found to be for H24 and H28. Thermal melting of imino shows that the drug is stabilizing the DNA. The thermal melting temperature of the palmatine-hexamer was found to be 298 ± 2 K which is 5 K higher than that observed for uncomplexed d-(CGATCG)2. The presence of all sequential NOE connectivities in the NOESY spectra at D/N = 2.0, as expected in standard B-DNA geometry confirmed that the DNA duplex is intact with apparently no opening of base pairs to accommodate drug chromophore as generally observed on intercalation. 2D NOESY experiment allowed the detection of several contacts between the protons of palmatine molecule and those of the double helix, specifically with protons of the CG base pairs at the terminal end. The drug protons H(36,37, 38), H(41,42,43) and H(46,47,48) shows intermolecular contacts with G6H1', C1H1' and C1H6 of the hexamer d-(CGATCG)2. H(36,37,38) and H(41,42,43) give close contacts with G6H8. Similarly, H10 proton show intermolecular peaks with C1H6 and C1H5. Zero intermolecular NOEs are found for imino residues and with protons of the central minor groove region of the hexamer. The result suggests that the drug is binding externally to the hexamer sequence in a specific orientation which gives rise to the observed NOEs. In contrast to the previous NMR characterized minor groove complex, the resonances of DNA protons located outside the minor groove binding site appear perturbed. The analysis of DNA chemical shift perturbation induced by palmatine suggests that the extremity of the hexamer is the site of binding. Chapter 6 summarizes the results obtained and their implications in understanding the molecular basis of action of palmatine.
Other Identifiers: Ph.D
Research Supervisor/ Guide: Barthwal, Ritu
metadata.dc.type: Doctoral Thesis
Appears in Collections:DOCTORAL THESES (Bio.)

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