STRUCTURAL STUDIES OF BERBERINE AND BERBERRUBINE AND THEIR INTERACTION WITH DNA

Abstract

Medicinal plants are the most valuable assets nowadays due to presence of small doses of active compounds which produce physiological actions in human and animal body. Some of the important bioactive compounds are alkaloids, glycosides, resins, gums and mucilages which possess anticancer, antiviral, antidiabetic and antimicrobial activity. The medicinal value of berberine, a protoberberine alkaloid found in root, rhizome and stem bark of Berberis vulgaris L. plant has been recognized since ancient times. Apart from its anti-cancerous property, it also displays a wide variety of biological and pharmacological activities e.g. antimicrobial, antiplasmodial, antidiarrhoeal, cardiovascular, etc. Replacement of OCH3 group at position 7 (Figure 1) in berberine with OH group, resulting in a derivative called berberrubine, induces a large change in its antitumor activity. Several studies have proved that Berberine acts as an anticancerous agent by binding to DNA, but actual mode of binding is still not clear. 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 seven 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), Total Correlation Spectroscopy (TOCSY), 'H - [H 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 also discussed. Absorption and Fluorescence spectroscopy methods used to investigate drug-DNA interaction are discussed -followed by Time-Correlated Single-Photon Counting method (TCSPC) are discussed. Chapter 3 deals with the structural and electronic properties of protoberberine alkaloids, berberine and berberrubine, 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 13C 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. One- and twodimensional HSQC ('H-13C), HMBC ('H-13C) and ROESY ('H-'H) spectra have been recorded at 500 MHz for berberine and berberrubine molecules in D2O solution. All proton and carbon resonances for berberine and berberrubine were unambiguously assigned and inter proton distances have been obtained from their respective NOESY spectra. A restrained Molecular Dynamics approach was used to obtain the optimized solution structure of both drugs. 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 berberine and berberrubine interaction with DNA using Absorption and Fluorescence spectroscopic techniques. The spectral properties of berberrubine molecules was also obtained and reported for the first time. 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 berberine binds effectively to CT DNA, Poly dA-dT, Poly dG-dC and small oligonucleotides, while berberubine does not show any binding towards alone DNA. It indicates that berberrubine binds to DNA in presence of topoisomerase. Percentage hypochromicity, binding constant, extinction coefficient of bound berberine, fluorescence-enhancement and relative fluorescence obtained from absorption and fluorescence spectroscopy were used to study the binding affinity. Maximum binding affinity of berberine was found to occur towards octamer sequence d-(CCAATTGG)2, having an intrinsic promoter site as CCAAT. Sequence specificity of berberine has been proved towards AATT rich sequences. Our results also suggest minor groove mode of binding of berberine, as it needs more than two base pairs for binding. The importance of the neighbour base to the binding site has also been discussed. It has been found that presence of guanine at +1 position to the binding site is an inhibitory factor for the interaction of berberine. TCSPC analysis of berberine-d-(CCAATTGG)2 complex also substantiates effective binding. Chapter 5 deals with the solution structure and thermal stability studies of a promoter site containing octamer d-(CCAATTGG)2 and comparison of results the with the crystal structure available in literature. The CCAAT box is one of the most common elements in eukaryotic promoters. The frequency of CCAAT box appears to be relatively higher in TATA less promoters and found to be present in several of oncogenes like MCT-1 and TLX-3. Solution structure of self complementary octamer d-(CCAATTGG)2, has been investigated using NMR spectroscopy and rMD calculations. Complete resonance assignment for all the protons (except H5', 5") has been obtained following standard procedures based on DQF-COSY and 2D NOESY. *H -ID NMR has been used to study the thermal stability of the sequence d- > (CCAATTGG)2. Melting temperature has been found to be 308 ± 2 K by analyzing the denaturation in the base pair stacking using imino and methyl protons. Inter proton distance restraints were derived from two dimensional Nuclear Overhauser Enhancement (NOE) spectra. Constraints for torsion angles have been calculated by quantitative analysis of3IP-'H HMBC and coupling patterns inDQF COSY spectrum. Structural refinement has been carried out using rMD with B-DNA as starting structure. Final rMD model, obtained with a 0.25 RMSD value, has been compared with crystal structure and canonical B-DNA. All the helicoidal parameters showed a deviation from B-DNA, particularly at central AATT region. This was characterized by the more negative value of propeller twist indicating occurrence of deep and narrow minor groove occurrence, which can be used for designing of new minor groove binders with better activity Chapter 6 deals with 31P, *H NMR and rMD studies on binding ofberberine with DNA octamer sequence d-(CCAATTGG)2. The following experiments were performed on the berberine-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 31P and *H NMR of the berberine-DNA complex having D/N = 1.0 and 2.0 in the range of278 - 328 K; 2D 31P - 31P exchange spectra of drug-DNA complex by a phase sensitive NOESY; DOSY experiments of the beberine-DNA complex and uncomplexed berberine, rMD studies on the solution structure of berberine - d-(CCAATTGG)2 complex using inter-proton distance restraints obtained from 2D NOESY. Results revealed that the addition of berberine to the oligonucleotide did not induce significant chemical shift variation of the phosphate signals in the 31P NMR spectra indicating that the phosphodiester conformation remains unaffected. The proton resonances of berberine were broad even at low values of drug/DNA (D/N = 0.5) and move upfield with respect to the free drug. An upfield shift of -0.1-0.6 ppm was found for the drugs protons on binding with DNA up to D/N ratio = 2. Maximum upfield shift in the drug protons was found to be for H28 and H24, which are located on the concave and convex side of berberine, respectively. Thermal melting temperature calculated by change in chemical shift of methyl protons chemical shift change with temperature is found be 312 ± 2 K, which is 5 K higher than that observed for uncomplexed d- (CCAATTGG)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. 22 intermolecular peaks obtained suggested that the dug protons, H10, H14, H28 and H24 located at ring D and ring B were giving cross peaks with the sugar protons of DNA at A3, A4, T5, T6 region, while H5, H3, H36, 37, 38, H41, 42, 43 protons gives cross peaks with the C1-G8 base pair in promoter sequence. rMD structure suggests that drug binds to the minor groove of the central part of the oligonucleotides (AATT) which clearly excludes the intercalation mode for berberine and also supports its sequential preference to AATT. Chapter 7 summarizes the results obtained and their implications in understanding the molecular basis of action of berberine.