Ankur Bikash Pradhan

Deciphering the Positional Influence of the Hydroxyl Group in the Cinnamoyl Part of 3-Hydroxy Flavonoids for Structural Modification and Their Interaction with the Protonated and B Form of Calf Thymus DNA Using Spectroscopic and Molecular Modeling Studies

Studies on the interaction of naturally occurring flavonoids with different polymorphic forms of nucleic acid are helpful for understanding the molecular aspects of binding mode and providing direction for the use and design of new efficient therapeutic agents. However, much less information is available on the interactions of these compounds with different polymorphic forms of DNA at the molecular level. In this report we investigated the interaction of two widely abundant dietary flavonoids quercetin (Q) and morin (M) with calf thymus (CT) DNA. Spectrophotometric, spectropolarimetric, viscosity measurement, and molecular docking simulation methods are used as tools to delineate the binding mode and probable location of the flavonoids and their effects on the stability and conformation of DNA. It is observed that in the presence of the protonated form of DNA the dual fluorescence of Q and M resulting from the excited-state intramolecular proton transfer (ESIPT) is modified significantly. Structural analysis showed Q and M binds weakly to the B form (groove binding) compared to the protonated form of CT DNA (electrostatic interaction). In both cases, Q binds strongly to both forms of DNA compared to M.

Molecular Aspects of the Interaction of Iminium and Alkanolamine Forms of the Anticancer Alkaloid Chelerythrine with Plasma Protein Bovine Serum Albumin

The interaction between a quaternary benzophenanthridine alkaloid chelerythrine (herein after, CHL) and bovine serum albumin (herein after, BSA) was probed by employing various spectroscopic tools and isothermal titration calorimetry (ITC). Fluorescence studies revealed that the binding affinity of the alkanolamine form of the CHL is higher compared to the iminium counterpart. This was further established by fluorescence polarization anisotropy measurement and ITC. Fluorescence quenching study along with time-resolved fluorescence measurements establish that both forms of CHL quenched the fluorescence intensity of BSA through the mechanism of static quenching. Site selective binding and molecular modeling studies revealed that the alkaloid binds predominantly in the BSA subdomain IIA by electrostatic and hydrophobic forces. From Forster resonance energy transfer (FRET) studies, the average distances between the protein donor and the alkaloid acceptor were found to be 2.71 and 2.30 nm between tryptophan (Trp) 212 (donor) and iminium and alkanolamine forms (acceptor), respectively. Circular dichroism (CD) study demonstrated that the α-helical organization of the protein is reduced due to binding with CHL along with an increase in the coiled structure. This is indicative of a small but definitive partial unfolding of the protein. Thermodynamic parameters obtained from ITC experiments revealed that the interaction is favored by negative enthalpy change and positive entropy change.

Exploring the mode of binding of the bioflavonoid kaempferol with B and protonated forms of DNA using spectroscopic and molecular docking studies

Protonation-induced conformational changes in natural DNAs under the conditions of low pH at low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH 3.4, 10 mM [Na+], and at 10 °C, natural DNAs adopt an unusual and stable conformation remarkably different from the canonical B-form conformation. The protonated conformation of naturally occurring calf thymus (CT) DNA has been characterized by UV-Vis absorption and circular dichroism (CD) studies. Binding interaction of kaempferol (KMP), a bioactive flavonoid, with B-form and the protonated form of CT DNA has been explored using various spectroscopic techniques. The determined binding constant, fluorescence quenching experiment, viscosity measurement, CD study, helix melting study and molecular docking simulation confirm the groove binding of KMP with B-form and external stacking interaction with the protonated form of CT DNA. The dual fluorescence of KMP resulting from the excited state intramolecular proton transfer is modified remarkably upon binding with the protonated form of DNA. This is the first report so far where a naturally occurring flavonoid has been shown to bind to the protonated form of DNA.

An insight into the interaction of phenanthridine dyes with polyriboadenylic acid: Spectroscopic and thermodynamic approach

Interaction of two phenanthridine dyes, namely ethidium bromide (EB) and propidium iodide (PI) with polyriboadenylic acid was investigated using various spectroscopic techniques. They were found to bind only with the single stranded form of the polymer, while no affinity was observed for the double stranded form. Enhanced binding observed for PI compared to EB may be attributed to the presence of external alkyl chain in PI. Thermodynamic studies showed negative enthalpy and negative entropy changes for the binding of both the dyes. Salt dependent studies revealed a lesser electrolytic contribution compared to the nonelectrolytic contribution to the total Gibbs free energy change in each case. This indicated importance of hydrophobic and van der Waals interaction for the binding process. Overall, the binding data and detail energetics of interaction presented here would be helpful in the design of phenanthridine based molecules that interact with specific RNA structure.

Binding of Phenazinium Dye Safranin T to Polyriboadenylic Acid: Spectroscopic and Thermodynamic Study

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na+] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.

Induction of self-structure in polyriboadenylic acid by the benzophenanthridine plant alkaloid chelerythrine: a spectroscopic approach

The naturally occurring benzophenanthridine plant alkaloid chelerythrine (CHL) was found to bind strongly to single-stranded polyriboadenylic acid (poly-A) with a high association constant of the order of 107 M−1. The association was monitored by various spectroscopic and viscometric techniques. Binding of the alkaloid induced self-structure formation of a poly-A helix that showed cooperative melting transition in circular dichroism. The mode of binding of CHL to poly-A was intercalation, as revealed by fluorescence quenching, sensitization of fluorescence experiment and viscosity measurement. Transfer of fluorescence energy from RNA bases to CHL has been demonstrated from fluorimetric studies. Thermodynamic data obtained from temperature dependence of the binding constant revealed that association was driven by a negative enthalpy change and opposed by a negative entropy change. Since the interaction of naturally occurring small molecules with RNA is an active area of research, this study renders the scope of exploring chelerythrine as RNA targeted therapeutic agent.

Biophysical insight into the interaction of the bioflavonoid kaempferol with triple and double helical RNA and the dual fluorescence behaviour of kaempferol

The interaction of the naturally occurring flavonoid, kaempferol (KMP) with single, double and triple helical forms of RNA has been investigated by different spectroscopic and viscometric techniques. It was found that KMP binds with triple helical [poly(U).poly(A)*poly(U), hereafter U.A*U, the dot represents the Watson–Crick and asterisk represents Hoogsteen base pairing respectively] and double helical [poly(A).poly(U), hereafter A.U] forms of RNA, whereas no interaction was observed with single stranded polyuridylic acid [poly-U] under identical experimental conditions. The binding of KMP was found to be stronger with U.A*U (20 × 104 M−1) compared to that of the parent duplex A.U (9.5 × 104 M−1). From the Stern–Volmer quenching constant, viscosity measurement and perturbation of CD spectra of RNA revealed that KMP binds to the U.A*U structure by intercalation while partial intercalation has been proposed for the binding to the duplex RNA structure. Thermodynamic data obtained from the temperature dependence study showed that the association was favoured by negative enthalpy and positive entropy changes. Experimental observations indicated that KMP binds and stabilizes the RNA-triplex more than its parent duplex counterpart.

Role of hydroxyl groups in the B-ring of flavonoids in stabilization of the Hoogsteen paired third strand of Poly(U).Poly(A)*Poly(U) triplex

We have reported the interaction of two flavonoids namely quercetin (Q) and morin (M) with double stranded poly(A).poly(U) (herein after A.U) and triple stranded poly(U).poly(A)*poly(U) (herein after U.A*U, dot represents the Watson-Crick and asterisk represents Hoogsteen base pairing respectively) in this article. It has been observed that relative positions of hydroxyl groups on the B-ring of the flavonoids affect the stabilization of RNA. The double strand as well as the triple strand of RNA-polymers become more stabilized in presence of Q, however both the duplex and triplex remain unaffected in presence of M. The presence of catechol moiety on the B-ring of Q is supposed to be responsible for the stabilization. Moreover, after exploiting a series of biophysical experiments, it has been found that, triple helical RNA becomes more stabilized over its parent duplex in presence of Q. Fluorescence quenching, viscosity measurement and helix melting results establish the fact that Q binds with both forms of RNA through the mode of intercalation while M does not bind at all to either forms of RNA.

Micelle assisted structural conversion with fluorescence modulation of benzophenanthridine alkaloids.

In this study we have reported the anionic surfactant (Sodium dodecyl sulfate, SDS) driven structural conversion of two benzophenanthridine plant alkaloids namely Chelerythrine (herein after CHL) and Sanguinarine (herein after SANG). Both the alkaloids exist in two forms: the charged iminium and the neutral alkanolamine form. The iminium form is stable at low pH (<6.5) and the alkanolamine form exists at higher pH (>10.1). The fluorescence intensity of the alkanolamine form is much stronger than the iminium form. The iminium form of both the alkaloids remains stable whereas the alkanolamine form gets converted to the iminium form in the SDS micelle environment. The iminium form possesses positive charge and it seems that electrostatic interaction between the positively charged iminium and negatively charged surfactant leads to the stabilization of the iminium form in the Stern layer of the anionic micelle. Whereas the conversion of the alkanolamine form into the iminium form takes place and that can be monitored in naked eye since the iminium form is orange in colour and the alkanolamine form has blue violet emission. Such a detail insight about the photophysical properties of the benzophenanthridine alkaloids would be a valuable addition in the field of alkaloid-surfactant interaction.

Inhibitory effects of the dietary flavonoid quercetin on the enzyme activity of zinc(II)-dependent yeast alcohol dehydrogenase: Spectroscopic and molecular docking studies.

A multispectroscopic exploration was employed to investigate the interaction between the metallo-enzyme alcohol dehydrogenase (ADH) from yeast with bioflavonoid quercetin (QTN). Here, we have characterized the complex formation between QTN and Zn2+ in aqueous solution and then examined the effect of such complex formation on the enzymatic activity of a zinc(II)-dependent enzyme alcohol dehydrogenase from yeast. We have observed an inhibition of enzymatic activity of ADH in presence of QTN. Enzyme inhibition kinetic experiments revealed QTN as a non-competitive inhibitor of yeast ADH. Perturbation of Circular dichroic (CD) spectrum of ADH in presence of QTN is observed due to the structural changes of ADH on complexation with the above flavonoid. Our results indicate a conformational change of ADH due to removal of Zn2+ present in the enzyme by QTN. This was further established by molecular modeling study which shows that the flavonoid binds to the Zn2+ ion which maintains the tertiary structure of the metallo-enzyme. So, QTN abstracts only half of the Zn2+ ions present in the enzyme i.e. one Zn2+ ion per monomer. From the present study, the structural alteration and loss of enzymatic activity of ADH are attributed to the complex formation between QTN and Zn2+.