Motilal Maiti

Interaction of sanguinarine iminium and alkanolamine form with calf thymus DNA

The interaction of sanguinarine iminium form (structure I) and sanguinarine alkanolamine form (structure II) with calf thymus DNA has been studied in buffer of pH 5.2 and pH 10.5, respectively, where the physicochemical properties of DNA remain unchanged. The binding of sanguinarine iminium form to DNA is characterized by hypochromism and bathochromism in the absorption band, quenching of fluorescence intensity, increase in fluorescence polarization anisotropy, increase in positive and negative ellipticity of DNA, sign and magnitude of the thermodynamic parameters and increase in contour length of sonicated rodlike duplex DNA indicating that it binds to DNA by a mechanism of intercalation. In contrast, sanguinarine alkanolamine form does not show (i) any significant change in fluorescence polarization anisotropy, (ii) alteration of B form structure of DNA and (iii) increase in contour length of DNA indicating that it does not bind to DNA. But at a very high concentration of DNA, the alkanolamine form is influenced to form an iminium-DNA complex.

Spectroscopic and Thermodynamic Studies on the Binding of Sanguinarine and Berberine to Triple and Double Helical DNA and RNA Structures

Abstract A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T·AxT and U·AxU) and duplexes (A·T and A·U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C·GxC+ triplex and G·C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.

Binding of sanguinarine to deoxyribonucleic acids of differing base composition

The binding of the alkaloid sanguinarine to natural DNAs of differing GC content has been studied by spectrophotometry and viscometry techniques. Binding parameters determined from spectrophotometric measurements by Scatchard analysis, according to an excluded-site model, indicate a very high specificity of sanguinarine binding to GC rich DNA. In the strong binding region, the increase of contour length of DNA depends strongly on its base composition, being larger with GC rich DNA than with AT rich DNA. It is concluded that the alkaloid binds preferentially to the GC pairs in DNA template.

Polymorphic Nucleic Acid Binding of Bioactive Isoquinoline Alkaloids and Their Role in Cancer

Bioactive alkaloids occupy an important position in applied chemistry and play an indispensable role in medicinal chemistry. Amongst them, isoquinoline alkaloids like berberine, palmatine and coralyne of protoberberine group, sanguinarine of the benzophenanthridine group, and their derivatives represent an important class of molecules for their broad range of clinical and pharmacological utility. In view of their extensive occurrence in various plant species and significantly low toxicities, prospective development and use of these alkaloids as effective anticancer agents are matters of great current interest. This review has focused on the interaction of these alkaloids with polymorphic nucleic acid structures (B-form, A-form, Z-form, HL-form, triple helical form, quadruplex form) and their topoisomerase inhibitory activity reported by several research groups using various biophysical techniques like spectrophotometry, spectrofluorimetry, thermal melting, circular dichroism, NMR spectroscopy, electrospray ionization mass spectroscopy, viscosity, isothermal titration calorimetry, differential scanning calorimetry, molecular modeling studies, and so forth, to elucidate their mode and mechanism of action for structure-activity relationships. The DNA binding of the planar sanguinarine and coralyne are found to be stronger and thermodynamically more favoured compared to the buckled structure of berberine and palmatine and correlate well with the intercalative mechanism of sanguinarine and coralyne and the partial intercalation by berberine and palmatine. Nucleic acid binding properties are also interpreted in relation to their anticancer activity.

Conversions of the left-handed form and the protonated form of DNA back to the bound right-handed form by sanguinarine and ethidium: a comparative study.

The interaction of sanguinarine and ethidium with right-handed (B-form), left-handed (Z-form) and left-handed protonated (designated as H(L)-form) structures of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) was investigated by measuring the circular dichroism and UV absorption spectral analysis. Both sanguinarine and ethidium bind strongly to the B-form DNA and convert the Z-form and the H(L)-form back to the bound right-handed form. Circular dichroic data also show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation either in Z-form or in H(L)-form. Both the rate and extent of B-form to Z-form transition were decreased by sanguinarine and ethidium under ionic conditions that otherwise favour the left-handed conformation of the polynucleotides. The rate of decrease is faster in the case of ethidium as compared to that of sanguinarine. Scatchard analysis of the spectrophotometric data shows that sanguinarine binds strongly to both the polynucleotides in a non-cooperative manner under B-form conditions, in sharp contrast to the highly-cooperative binding under Z-form and H(L)-form conditions. Correlation of binding isotherms with circular dichroism data indicates that the cooperative binding of sanguinarine under the Z-form and the H(L)-form conditions is associated with a sequential conversion of the polymer from a left-handed to a bound right-handed conformation. Determination of bound alkaloid concentration by spectroscopic titration technique and the measurement of circular dichroic spectra have enabled us to calculate the number of base pairs of Z-form and H(L)-form that adopt a right-handed conformation for each bound alkaloid. Analysis reveals that 2-3 base pairs (bp) of Z-form of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) switch to the right-handed form for each bound sanguinarine, while approximately same number of base pairs switch to the bound right-handed form in complexes with H(L)-form of these polynucleotides. Comparative binding analysis shows that ethidium also converts approximately 2 bp of Z-form or H(L)-form to bound right-handed form under same experimental conditions. Since sanguinarine binds preferentially to alternating GC sequences, which are capable of undergoing the B to Z or B to H(L) transition, these effects may be an important part in understanding its extensive biological activities.

Influence of DNA Structures on the Conversion of Sanguinarine Alkanolamine Form to Iminium Form

Abstract Sanguinarine exhibits pH dependent structural equilibrium between iminium form (structure I) and alkanolamine form (structure II) with a pKa of 7.4 as revealed from spectrophoto-metric titration. The titration data show that the compound exists almost exclusively as structure I and structure II in the pH range 1 to 6 and 8.5 to 11, respectively. The interaction of structure I and structure II to several B-form natural and synthetic double and single stranded DNAs has been studied by spectrophotometric, spectrofluorimetric and circular dichroic measurements in buffers of pH 5.2 and pH 10.4 where the physicochemical properties of DNA remain in B-form structure. The results show that structure I bind strongly to all B-form DNA structures showing typical hypochromism and bathochromism of the alkaloids absorption maximum, quenching of steady-state fluorescence intensity and perturbations in circular dichroic spectrum. The structure II does not bind to DNA, but in presence of large amount of DNA significant population of structure I is generated, which binds to DNA and forms a structure I-DNA intercalated complex. The nature and magnitude of the spectral pattern are very much dependent on the structure as well as base composition of each DNA. The generation of the structure I from structure II is significantly affected by increasing ionic strength of the medium. The conversion of structure II to structure I in presence of high concentration of DNA in solution is explained through formation of a binding equilibrium process between structure II and structure I-DNA intercalated complex.

Thermodynamics of the interactions of sanguinarine with DNA: influence of ionic strength and base composition

Using a combination of spectrophotometric and spectrofluorimetric techniques, we report the first thermodynamic characterization of sanguinarine binding to a series of natural and synthetic host DNA duplexes over a wide range of temperature and sodium concentration. The binding isotherms fit reasonably well to the neighbour exclusion model. The salt and temperature dependence of the binding constants is used to estimate the thermodynamic parameters involved in the interaction of the alkaloid with DNA. The resulting binding data are found to be sensitive to the ionic strength of the medium, base composition and sequence of base pairs. When the sodium ion concentration is increased from 0.005 M to 0.5 M, the binding free energy changes vary in a range from -8.47 to -7.1 kcal mol-1, which corresponds to a binding constant range from 1.85 x 10(6) to 1.8 x 10(5) M-1 at 20 degrees C. More distinct is the spread in the binding enthalpy changes which range from -6.35 to -2.62 kcal mol-1 corresponding to binding entropy changes from +7.22 to +15.3 cal K-1 mol-1 at 20 degrees C. On the other hand when the GC content of the host DNA duplexes is increased, the binding free energy varies in a range from -7.28 to -8.58 kcal mol-1 with the binding enthalpy changes ranging from -0.46 to -14.31 kcal mol-1, while corresponding binding entropy changes range from +23.3 to -19.56 cal K-1 mol-1 at 20 degrees C. Sanguinarine binding to natural DNAs and homo- and heteropolymers of AT is characterized by negative enthalpy changes and positive entropy changes, while binding to homo- and heteropolymers of GC is reflected by both negative enthalpy changes and entropy changes. Possible molecular contributions towards sign and magnitude of the thermodynamic parameters and their dependence on ionic strength, base composition and sequences, are discussed.

INTERACTIONS OF BERBERINE WITH POLY(A) AND TRNA

The interaction of berberine chloride with poly(A) and tRNA has been studied by various spectroscopic techniques. Binding parameters determined from spectrophotometric and spectrofluorimetric measurements by Scatchard analysis indicate a very high effective binding capacity of berberine to poly(A) as compared to DNA or tRNA. The circular dichroism studies show that binding of berberine to poly(A) causes a significant change in the circular dichroic spectrum of poly(A) itself, as manifested by (i) a decrease of both positive and negative bands and (ii) appearance of a conservative type of extrinsic circular dichroic spectrum in the wavelength range of 300-400 nm, while it does not cause any significant alteration to the A form structure of tRNA. It is concluded that berberine interacts stronger with poly(A) than DNA or tRNA. The results are interpreted in terms of its reported biological activities.

DNA-binding cytotoxic alkaloids: comparative study of the energetics of binding of berberine, palmatine, and coralyne.

Deoxyribonucleic acid is the site of storage and retrieval of genetic information through interaction with proteins and other small molecules. In the present study, the interaction of two natural cytotoxic protoberberine plant alkaloids, berberine and palmatine, and a synthetic derivative, coralyne, with mammalian herring testis DNA was investigated using a combination of isothermal titration calorimetry, differential scanning calorimetry, and optical melting experiments to characterize the energetics of their binding. The binding constants of these alkaloids to DNA under identical conditions were evaluated from the UV melting data, and the enthalpy of binding was elucidated from isothermal titration studies. The binding constants of berberine, palmatine, and coralyne to DNA were found to be 1.15 x 10(4), 2.84 x 10(4), and 3.5 x 10(6) M(-1) at 20 degrees C in buffer of 20 mM [Na+]. Parsing of the free energy change of the interaction observed into polyelectrolytic and nonpolyelectrolytic components suggested that although these alkaloids are charged, the major contributor of about 75% of the binding free energy arises from the nonpolyelectrolytic forces. The binding in case of palmatine and coralyne was predominantly enthalpy driven with favoring smaller entropy terms, while that of berberine was favored by both negative enthalpy and positive entropy changes. Temperature dependence of the binding enthalpies determined from ITC studies in the range 20-40 degrees C was used to calculate the binding-induced change in heat capacity (DeltaC(o)(p)) values as -117, -135, and -157 cal/mol K, respectively, for berberine, palmatine, and coralyne. Taken together, the results suggest that the DNA binding of the planar synthetic coralyne is stronger and thermodynamically more favored compared to the buckled natural berberine and palmatine.

DNA Polymorphism Under the Influence of Low pH and Low Temperature

The polymorphic behaviour on the conformation of a alternating GC polymer and its methylated analogue has been studied under the influence of low pH, low temperature and low ionic strength from the measurements of UV-absorption and circular dichroic spectroscopy. Studies indicate that both the polymers isomerize to a stable left handed type conformations. The duplex nature of these conformations were inferred from thermal denaturation curves and the temperature dependence of the CD spectra. In natural DNA, the influence of low pH and low temperature also shows a defined conformational change, characterized by two positive CD bands. This conformational status is achieved in all DNAs irrespective of base composition or sequence of base pairs. Further evidence to this altered polymorphic state of natural DNAs is inferred from ethidium binding study.