Trypsin inhibition by Ligupurpuroside B as studied using spectroscopic,CD, and molecular docking techniques

It is well known that Ligupurpuroside B is a water-soluble polyphenolic compound and used to brew bitter tea with antioxidant activities. It acted as a stimulant to the central nervous system and a diuretic (increase the excretion of urine), was used to treat painful throat and high blood pressure, and also exerted weight-loss function. In this regard, a detailed investigation on the mechanism of interaction between Ligupurpuroside B and trypsin could be of great interest to know the pharmacokinetic behavior of Ligupurpuroside B and for the design of new analogues with effective pharmacological properties. Ligupurpuroside B successfully quenched the intrinsic fluorescence of trypsin via static quenching mechanism. The binding constants (K_a) at three temperatures (288, 298, and 308 K) were 1.7841?×?10⁴, 1.6251?×?10⁴ and 1.5483?×?10⁴ L mol^?1, respectively. Binding constants revealed the stronger binding interaction between Ligupurpuroside B and trypsin. The number of binding sites approximated to one, indicating a single class of binding for Ligupurpuroside B in trypsin. The enzyme activity result suggested that Ligupurpuroside B can inhibit trypsin activity. Thermodynamic results revealed that both hydrogen bonds and hydrophobic interactions play main roles in stabilization of Ligupurpuroside B-trypsin complex. Circular dichroism (CD) results showed that the conformation of trypsin changed after bound to ligupurpuroside B. Molecular docking indicated that Ligupurpuroside B can enter the hydrophobic cavity of trypsin and was located near Trp215 and Tyr228 of trypsin.

Communicated by Ramaswamy H. Sarma     

Interaction of two flavonols with fat mass and obesity-associated protein investigated by fluorescence quenching and molecular docking

The binding of two flavonols with fat mass and obesity-associated protein (FTO) was studied using fluorescence spectroscopy, Stern-Volmer kinetics, UV-Vis absorption, and molecular docking. The quenching of FTO fluorescence was determined to be static with binding constants on the order of 10⁴ M^?1. The interaction was studied over three temperatures, and the binding was found to be exothermic with a positive change in entropy. Thermodynamic analysis and molecular modeling suggest that hydrophobic interaction and hydrogen bonding interaction are the main binding force in stabilizing the flavonol–FTO complex.     

The study on interactions between levofloxacin and model proteins by using multi-spectroscopic and molecular docking methods

The interactions of levofloxacin (LEV) with lysozyme (LYZ), trypsin and bovine hemoglobin (BHb) were investigated, respectively, by using multi-spectral techniques and molecular docking in vitro. Fluorescence studies showed that LEV quenched LYZ/trypsin fluorescence in a combined quenching ways and BHb fluorescence in a static quenching with binding constants of .14, .51 and .20 × 10⁵ L mol^?1 at 298 K, respectively. The thermodynamic parameters demonstrated that hydrophobic forces, hydrogen bonds, and van der Waals forces played the major role in the binding process. The binding distances between LEV and the inner tryptophan residues of LYZ, trypsin, and BHb were calculated to be 4.04, 3.38, and 4.52 nm, respectively. Furthermore, the results of circular dichroism spectra (CD), UV–vis, and three-dimensional fluorescence spectra indicated that the secondary structures of LYZ, trypsin, and BHb were partially changed by LEV with the α-helix percentage of LYZ-LEV system increased while that of BHb-LEV system was decreased, the β-sheet percentage of trypsin-LEV system increased from 41.3 to 42.9%. UV–vis spectral results showed that the binding interactions could cause conformational and some micro-environmental changes of LYZ, trypsin, and BHb. The results of molecular docking revealed that in LYZ and trypsin systems, LEV bound to the active sites residues GLU 35 and ASP 52 of LYZ and trypsin at the active site SER 195, and in BHb system, LEV was located in the central cavity, which was consistent with the results of synchronous fluorescence experiment. Besides, LEV made the activity of LYZ decrease while the activity of trypsin increased.     

Binding of NDGA and morin with phospholipase A2: experimental and computational evidences

The effects of morin and nordihydroguaiaretic acid (NDGA), two plant secondary metabolites, on porcine pancreatic phospholipase A₂ (PLA₂) were investigated by isothermal titration calorimetry (ITC) and in silico docking analyses. The binding energies obtained for NDGA and morin from the ITC studies are ? 6.36 and ? 5.91 kcal mol^? 1, respectively. Similarly, the glide scores obtained for NDGA and morin towards PLA₂ were ? 7.32 and ? 7.23 kcal mol^? 1, respectively. Further the docked complexes were subjected to MD simulation in the presence of explicit water molecules to check the binding stability of the ligands in the active site of PLA₂. The bound ligands make hydrogen bonds with the active site residues of the enzyme and coordinate bonds with catalytically important Ca²⁺ ion. The binding of ligands at the active site of PLA₂ may also contribute to the reported anti-inflammatory properties of NDGA and morin.     

Dissection of binding of trypsin to its natural inhibitor Gensenoside-Rg1 using spectroscopic methods and molecular modeling

Abstract

The interaction of trypsin with Gensenoside-Rg1 (G-Rg1) was studied using fluorescence, ultraviolet–visible (UV–vis), and circular dichroism (CD) spectroscopies along with enzyme activity assay and molecular docking. The enzyme activity assays showed that G-Rg1 inhibited the activity of trypsin effectively. The fluorescence experiments indicated that a complex of G-Rg1–trypsin was formed and that the fluorescence of trypsin was quenched by G-Rg1 via a mixed-quenching mechanism (both static and dynamic quenching). The thermodynamic analysis suggested that hydrophobic interaction and hydrogen bond were the major forces between G-Rg1 and trypsin. According to the theory of Förster’s non-radiation energy transfer, the binding distance between trypsin and G-Rg1 was calculated to be 2.01?nm, which implies that energy transfer occurred within the complex. The experimental results obtained from UV–vis absorption spectra, synchronous fluorescence spectra, and CD spectra indicated that G-Rg1 was mainly located on tryptophan moiety and that the interaction between G-Rg1 and trypsin led to conformational changes of trypsin with some α-helix and unordered coil structures being transformed into β-sheet structures. In addition, docking results supported the above experimental findings and suggested the possible binding location of G-Rg1 on trypsin along with the possible hydrogen bonds and hydrophobic interactions between G-Rg1 and trypsin. The experimental results from this study should be useful to minimize the antinutritional effects and make full use of Genseng extracts in the food industry and also be helpful to the design of the drugs for the diseases related to overexpression of trypsin.

Communicated by Ramaswamy H. Sarma     

Capecitabine as a minor groove binder of DNA: molecular docking,molecular dynamics,and multi-spectroscopic studies

The interaction mechanism and binding mode of capecitabine with ctDNA was extensively investigated using docking and molecular dynamics simulations, fluorescence and circular dichroism (CD) spectroscopy, DNA thermal denaturation studies, and viscosity measurements. The possible binding mode and acting forces on the combination between capecitabine and DNA had been predicted through molecular simulation. Results indicated that capecitabine could relatively locate stably in the G-C base-pairs-rich DNA minor groove by hydrogen bond and several weaker nonbonding forces. Fluorescence spectroscopy and fluorescence lifetime measurements confirmed that the quenching was static caused by ground state complex formation. This phenomenon indicated the formation of a complex between capecitabine and ctDNA. Fluorescence data showed that the binding constants of the complex were approximately 2 × 10⁴ M^?1. Calculated thermodynamic parameters suggested that hydrogen bond was the main force during binding, which were consistent with theoretical results. Moreover, CD spectroscopy, DNA melting studies, and viscosity measurements corroborated a groove binding mode of capecitabine with ctDNA. This binding had no effect on B-DNA conformation.     

Exploring the binding mode of donepezil with calf thymus DNA using spectroscopic and molecular docking methods

Donepezil (DNP) is one of approved drugs to treat Alzheimer's disease (AD). However, the potential effect of DNP on DNA is still unclear. Therefore, the interaction of DNP with calf thymus DNA (DNA) was studied in vitro using spectroscopic and molecular docking methods. Steady‐state and transient fluorescence experiments showed that there was a clear binding interaction between DNP and DNA, resulting from DNP fluorescence being quenched using DNA. DNP and DNA have one binding site between them, and the binding constant (K_b) was 0.78 × 10⁴ L·mol^?1 at 298 K. In this binding process, hydrophobic force was the main interaction force, because enthalpy change (ΔH) and entropy change (ΔS) of DNP–DNA were 67.92 kJ·mol^?1 and 302.96 J·mol^?1·K^?1, respectively. DNP bound to DNA in a groove‐binding mode, which was verified using a competition displacement study and other typical spectroscopic methods. Fourier transform infrared (FTIR) spectrum results showed that DNP interacted with guanine (G) and cytosine (C) bases of DNA. The molecular docking results further supported the results of spectroscopic experiments, and suggested that both Pi‐Sigma force and Pi‐Alkyl force were the major hydrophobic force functioning between DNP and DNA.     

Pharmacophore generation,atom-based 3D-QSAR,molecular docking and molecular dynamics simulation studies on benzamide analogues as FtsZ inhibitors

FtsZ is an appealing target for the design of antimicrobial agent that can be used to defeat the multidrug-resistant bacterial pathogens. Pharmacophore modelling, molecular docking and molecular dynamics (MD) simulation studies were performed on a series of three-substituted benzamide derivatives. In the present study a five-featured pharmacophore model with one hydrogen bond acceptors, one hydrogen bond donors, one hydrophobic and two aromatic rings was developed using 97 molecules having MIC values ranging from .07 to 957 μM. A statistically significant 3D-QSAR model was obtained using this pharmacophore hypothesis with a good correlation coefficient (R² = .8319), cross validated coefficient (Q² = .6213) and a high Fisher ratio (F = 103.9) with three component PLS factor. A good correlation between experimental and predicted activity of the training (R² = .83) and test set (R² = .67) molecules were displayed by ADHRR.1682 model. The generated model was further validated by enrichment studies using the decoy test and MAE-based criteria to measure the efficiency of the model. The docking studies of all selected inhibitors in the active site of FtsZ protein showed crucial hydrogen bond interactions with Val 207, Asn 263, Leu 209, Gly 205 and Asn-299 residues. The binding free energies of these inhibitors were calculated by the molecular mechanics/generalized born surface area VSGB 2.0 method. Finally, a 15 ns MD simulation was done to confirm the stability of the 4DXD–ligand complex. On a wider scope, the prospect of present work provides insight in designing molecules with better selective FtsZ inhibitory potential.     

An investigation into the altered binding mode of green tea polyphenols with human serum albumin on complexation with copper

Green tea is rich in several polyphenols, such as (?)-epicatechin-3-gallate (ECG), (?)-epigallocatechin (EGC), and (?)-epigallocatechin-3-gallate (EGCG). The biological importance of these polyphenols led us to study the major polyphenol EGCG with human serum albumin (HSA) in an earlier study. In this report, we have compared the binding of ECG, EGC, and EGCG and the Cu(II) complexes of EGCG and ECG with HSA. We observe that the gallate moiety of the polyphenols plays a crucial role in determining the mode of interaction with HSA. The binding constants obtained for the different systems are 5.86?±?0.72?×?10⁴ M^?1 (K _ECG-HSA), 4.22?±?0.15?×?10⁴ M^?1 (K _{ECG-Cu(II)-HSA}), and 9.51?±?0.31?×?10⁴ M^?1 (K _{EGCG-Cu(II)-HSA}) at 293?K. Thermodynamic parameters thus obtained suggest that apart from an initial hydrophobic association, van der Waals interactions and hydrogen bonding are the major interactions which held together the polyphenols and HSA. However, thermodynamic parameters obtained from the interactions of the copper complexes with HSA are indicative of the involvement of the hydrophobic forces. Circular dichroism and the Fourier transform infrared spectroscopic measurements reveal changes in α-helical content of HSA after binding with the ligands. Data obtained by fluorescence spectroscopy, displacement experiments along with the docking studies suggested that the ligands bind to the residues located in site 1 (subdomains IIA), whereas EGC, that lacks the gallate moiety, binds to the other hydrophobic site 2 (subdomain IIIA) of the protein.     

Biophysical and computational comparison on the binding affinity of three important nutrients to β-lactoglobulin: folic acid,ascorbic acid and vitamin K3

Small globular protein, β-lactoglobulin (βLG), which has significant affinity toward many drugs, is the most abundant whey protein in milk. In this study, the interaction of βLG with three important nutrients, ascorbic acid (ASC), folic acid (FOL), and vitamin K3 (VK3) was investigated by spectroscopic methods (UV–visible and fluorescence) along with molecular docking technique. The results of fluorescence measurements showed that studied nutrients strongly quenched βLG fluorescence in static (FOL and ACS) or static–dynamic combined quenching (VK3) mode. The values of binding constants (K_βLG-ASC ~ 4.34 × 10⁴ M^?1, K_βLG-FOL ~ 1.67 × 10⁴ M^?1and K_βLG-VK3 ~ 13.49 × 10⁴ M^?1 at 310 K) suggested that VK3 and FOL had stronger binding affinity toward βLG than ASC. Thermodynamic analysis indicated that hydrophobic interactions are the major forces in the stability of FOL–βLG complex with enthalpy- and entropy-driving mode while, hydrogen bonds and van der Waals interactions play a major role for βLG–ASC and βLG–VK3 associations. The results of 3D fluorescence FT-IR and UV–Visible measurements indicated that the binding of above nutrients to βLG may induce conformational and micro-environmental changes of protein. Also, there is a reciprocal complement between spectroscopic techniques and molecular docking modeling. The docking results indicate that the ASC, FOL, and VK3 bind to residues located in the subdomain B of βLG. Finally, this report suggests that βLG could be used as an effective carrier of above nutrients in functional foods.     

Exploration of potential RSK2 inhibitors by pharmacophore modelling,structure-based 3D-QSAR,molecular docking study and molecular dynamics simulation

Abstract

The p90 ribosomal s6 kinase 2 (RSK2) is a promising target because of its over expression and activation in human cancer cells and tissues. Over the last few years, significant efforts have been made in order to develop RSK2 inhibitors to treat myeloma, prostatic cancer, skin cancer and etc., but with limited success so far. In this paper, pharmacophore modelling, molecular docking study and molecular dynamics (MD) simulation have been performed to explore the novel inhibitors of RSK2. Pharmacophore models were developed by 95 molecules having pIC₅₀ ranging from 4.577 to 9.000. The pharmacophore model includes one hydrogen bond acceptor (A), one hydrogen bond donor (D), one hydrophobic feature (H) and one aromatic ring (R). It is the best pharmacophore hypothesis that has the highest correlation coefficient (R² = 0.91) and cross validation coefficient (Q² = 0.71) at 5 component PLS factor. It was evaluated using enrichment analysis and the best model was used for virtual screening. The constraints used in this study were docking score, ADME properties, binding free energy estimates and IFD Score to screen the database. Ultimately, 12 hits were identified as potent and novel RSK2 inhibitors. A 15 ns molecular dynamics (MD) simulation was further employed to validate the reliability of the docking results.     

Probing the intermolecular interactions into serum albumin and anthraquinone systems: a spectroscopic and docking approach

Intermolecular interaction study of human serum albumin (HSA) with two anthraquinones i.e. danthron and quinizarin has been performed through fluorescence, UV-vis and CD spectroscopy along with docking analysis. The titration of drugs into HSA solution brought about the quenching of fluorescence emission by way of complex formation. The binding constants were found to be 1.51 × 10⁴ L mol^?1 and 1.70 × 10⁴ L mol^?1 at λ_exc = 280 nm while at λ_exc = 295 nm, the values of binding constants were 1.81 × 10⁴ L mol^?1 and 1.90 × 10⁴ L mol^?1 which hinted toward binding of both the drugs in the vicinity of subdomain IIA. Different temperature study revealed the presence of static quenching mechanism. Moreover, more effective quenching of the fluorescence emission was observed at λ_exc = 295 nm which also suggested that both the drug molecule bind nearer to Trp-214. Thermodynamic parameters showed that hydrophobic interaction was the major force behind the binding of drugs. The UV-vis spectroscopy testified the formation of complex in both the systems and primary quenching mechanism as static one. The changes in secondary structure and α-helicity in both the systems were observed by circular dichroism spectroscopy. Furthermore, molecular docking analysis predicted the probable binding site of drugs in subdomain IIA of HSA molecule. The types of amino acid residues surrounding the drug molecule advocated that van der Waals forces, hydrophobic forces and electrostatic forces played a vital role in the stabilization of drug-protein complex formed.     

Multi-spectroscopic studies on the interaction between traditional Chinese herb,helicid with pepsin

Study on the binding properties of helicid by pepsin systematically using multi-spectroscopic techniques and molecular docking method, and these interactions comprise biological recognition at molecular level and backbone of biological significance in medicine concerned with the uses, effects, and modes of action of drugs. We investigated the mechanism of interaction between helicid and pepsin by using various spectroscopic techniques viz., fluorescence spectra, UV–Vis absorption spectra, circular dichroism (CD), 3D spectra, synchronous fluorescence spectra and molecular docking methods. The quenching mechanism associated with the helicid–pepsin interaction was determined by performing fluorescence measurements at different temperatures. From the experimental results show that helicid quenched the fluorescence intensity of pepsin via a combination of static and dynamic quenching process. The binding constants (K_a) at three temperatures (288, 298, and 308 K) were 7.940?×?10⁷, 2.082?×?10⁵ and 3.199?×?10⁵ L mol^?1, respectively, and the number of binding sites (n) were 1.44, 1.14, and 1.18, respectively. The n value is close to unity, which means that there is only one independent class of binding site on pepsin for helicid. Thermodynamic parameters at 298 K were calculated as follows: ΔH^o (??83.85 kJ mol^?1), ΔG^o (??33.279 kJ mol^?1), and ΔS^o (??169.72 J K^?1 mol^?1). Based on thermodynamic analysis, the interaction of helicid with pepsin is driven by enthalpy, and Van der Waals’ forces and hydrogen bonds are the main forces between helicid and pepsin. A molecular docking study further confirmed the binding mode obtained by the experimental studies. The conformational changes in the structure of pepsin was confirmed by 3D fluorescence spectra and circular dichroism.     

Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 Å) and biochemical basis

Change in specificity, caused by the mutations at P1 site, of the serine protease inhibitors of different families is reported in the literature, but Kunitz (STI) family inhibitors are almost unexplored in this regard. In this paper, we present the crystal structure of a P1 variant of winged bean chymotrypsin inhibitor (WCI) belonging to Kunitz (STI) family, supplemented by biochemical, phylogenetic and docking studies on the mutant. A single mutation (Leu → Arg) at P1 converted WCI to a strong inhibitor of trypsin with an association constant of 4.8 × 10¹⁰ M^?1 which is comparable to other potent trypsin inhibitors of the family. The crystal structure (2.15 Å) of this mutant (L65R) shows that its reactive site loop conformation deviates from that of WCI and adopts a structure similar to that of Erythrina caffra trypsin inhibitor (ETI) belonging to the same family. Mutation induced structural changes have also been propagated in a concerted manner to the neighboring conserved scaffolding residue Asn14, such that the side chain of this residue took an orientation similar to that of ETI and optimized the hydrogen bonds with the loop residues. While docking studies provide information about the accommodation of non-specific residues in the active site groove of trypsin, the basis of the directional alteration of the reactive site loop conformation has been understood through sequence analysis and related phylogenetic studies.     

Bioprospecting of brown seaweed (Ochrophyta) from the Yucatan Peninsula: cytotoxic,antiproliferative, and antiprotozoal activities

In the Yucatan Peninsula coast, a large diversity of seaweed species are found, and recent studies have reported the presence of metabolites with pharmaceutical importance. In this study, a biological screening of brown seaweed extracts from Dictyota ciliolata, Padina sanctae-crucis, Sargassum fluitans, and Turbinaria tricostata was carried out. Their cytotoxicity and antiproliferative activities were evaluated by the sulforhodamine B assay on human embryonic kidney (HEK 293), human breast cancer (MCF-7), human prostate cancer (LNCaP), and human hepatic cancer (Hep-G2) cell lines. Seaweed extracts were also tested for their anti-trichomonal (Trichomonas vaginalis) and anti-giardicidal (Giardia lamblia) properties. Fucan fractions were extracted using successive maceration with ethanol/water and freeze-dried. Organics extracts were obtained from ethanol residue from liquid–liquid fractionation. A total of four ethanol extracts, four fucan-rich fractions, four ethanolic extracts, and 12 organic fractions were obtained. Only the ethanolic extracts from Turbinaria tricostata and D. ciliolata were active against LNCaP (CC₅₀ of 24.4 and 29.3 μg mL^?1, respectively). Interestingly, the activity found in the extracts from D. ciliolata and Turbinaria tricostata was maintained when both extracts were subjected to a liquid–liquid fractionation with hexane on the LNCaP cell line (CC₅₀ of 24.4 and 25.2 μg mL^?1, respectively). The antiproliferative assays showed that both dichloromethane and ethanolic fractions from P. sanctae-crucis were active against MCF-7, with IC₅₀ of 26.1 and 29.8 μg mL^?1, respectively. These species have been selected for further bio-guided fractionation and isolation of active compounds.     

Detection of interaction between lysionotin and bovine serum albumin using spectroscopic techniques combined with molecular modeling

A combination of fluorescence, UV–Vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) and molecular modeling approaches were employed to determine the interaction between lysionotin and bovine serum albumin (BSA) at physiological pH. The fluorescence titration suggested that the fluorescence quenching of BSA by lysionotin was a static procedure. The binding constant at 298 K was in the order of 10⁵ L mol^?1, indicating that a high affinity existed between lysionotin and BSA. The thermodynamic parameters obtained at different temperatures (292, 298, 304 and 310 K) showed that the binding process was primarily driven by hydrogen bond and van der Waals forces, as the values of the enthalpy change (ΔH°) and entropy change (ΔS°) were found to be ?40.81 ± 0.08 kJ mol^?1 and ?35.93 ± 0.27 J mol^?1 K^?1, respectively. The surface hydrophobicity of BSA increased upon interaction with lysionotin. The site markers competitive experiments revealed that the binding site of lysionotin was in the sub-domain IIA (site I) of BSA. Furthermore, the molecular docking results corroborated the binding site and clarified the specific binding mode. The results of UV–Vis absorption, CD and FT-IR spectra demonstrated that the secondary structure of BSA was altered in the presence of lysionotin.     

Investigation on the interaction between myricetin and dihydromyricetin with trypsin, α-chymotrypsin,lysozyme by spectroscopy and molecular docking methods

The interaction between myricetin and dihydromyricetin with trypsin, α-chymotrypsin and lysozyme was investigated using multispectral and molecular docking methods. The results of fluorescence quenching revealed that myricetin and dihydromyricetin could quench the intrinsic fluorescence of three different proteinases through a static quenching procedure. The binding constant and number of binding sites at different temperatures were measured. The thermodynamic parameters obtained at different temperatures showed van der Waals interactions and hydrogen bonds played the main roles in the interaction of myricetin with trypsin and lysozyme, hydrophobic force was dominant both in myricetin with α-chymotrypsin interaction and dihydromyricetin with trypsin and lysozyme interaction, as for the electrostatic forces, it was mainly the driving force in dihydromyricetin binding to α-chymotrypsin. There was non-radiative energy transfer between three proteinases and myricetin or dihydromyricetin with high probability. The microenvironment of trypsin, α-chymotrypsin and lysozyme is changed. The docking studies revealed that myricetin and dihydromyricetin entered the hydrophobic cavity of three proteinases and formed hydrogen bonds. The binding affinity of myricetin or dihydromyricetin is different with the trypsin, α-chymotrypsin and lysozyme due to the different molecular structure.     

Investigation of the interaction of aurantio‐obtusin with human serum albumin by spectroscopic and molecular docking methods

The interaction between human serum albumin (HSA) and aurantio‐obtusin was investigated by spectroscopic techniques combined with molecular docking. The Stern–Volmer quenching constants (K_SV) decreased from 8.56 × 10⁵ M^?1 to 5.13 × 10⁵ M^?1 with a rise in temperatures from 289 to 310 K, indicating that aurantio‐obtusin produced a static quenching of the intrinsic fluorescence of HSA. Time‐resolved fluorescence studies proved again that the static quenching mechanism was involved in the interaction. The sign and magnitude of the enthalpy change as well as the entropy change suggested involvement of hydrogen bonding and hydrophobic interaction in aurantio‐obtusin–HSA complex formation. Aurantio‐obtusin binding to HSA produced significant alterations in secondary structures of HSA, as revealed from the time‐resolved fluorescence, Fourier transform infrared (FT‐IR) spectroscopy, three‐dimensional (3D) fluorescence and circular dichroism (CD) spectral results. Molecular docking study and site marker competitive experiment confirmed aurantio‐obtusin bound to HSA at site I (subdomain IIA).     

Binding affinity of pyrano[3, 2-f]quinoline and DNA: spectroscopic and docking approach

The interaction between pyrano[3, 2-f]quinoline (PQ) and calf thymus DNA (CTDNA) using spectroscopic and molecular modeling approach has been presented here. Apparent association constant (1.05×10⁵ L/mol) calculated from UV-vis specta, indicates a moderate complex formation between CTDNA and PQ. The quenching phenomena as obtained from emission spectra of ethidium bromide (EB)–CTDNA by PQ was found to be a dynamic one and the binding constants found to be 8.64, 9.25, 11.17, 12.03 × 10⁴ L/mol at 293, 300, 308, and 315 K. Thermodynamic parameter enthalpy change (ΔH) and entropy change (ΔS), indicates weak force like van der Walls force and hydrogen bonds having the key role in this binding process. The results of circular dichroism (CD) demonstrate that PQ has not induced characteristic changed in CTDNA. Results achieved from UV absorption and fluorescence spectroscopy indicating the binding mode of PQ with DNA seems to be a nonintercalative binding. The theoretical results as originating from molecular modeling showed that PQ possibly will bind into the hydrophobic region of DNA having docking binding energy = ?10.03 kcal/mol and the obtained results are in consonance with the inferences obtained from experimental data. This result is important for the better understanding of pharmaceutical aspects of binding affinity of PQ and CTDNA.     

Preferential binding of fisetin to the native state of bovine serum albumin: spectroscopic and docking studies

We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10⁴ M^?1 and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (?G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol^?1 K^?1). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.