Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 10⁴ M^?1implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.     

Deciphering the binding of carbendazim (fungicide) with human serum albumin: A multi-spectroscopic and molecular modelling studies

Carbendazim is a benzimidazole fungicide used to control the fungal invasion. However, its exposure might lead to potential health problems. The present study evaluates the interaction of carbendazim (CAR) with human serum albumin (HSA) which is an important drug carrier protein and plays a very crucial role in the transportation of small molecules. A number of biophysical techniques were employed to investigate the binding of CAR with HSA. The increased UV-absorption of HSA on titrating with CAR suggests the formation of HSA–CAR complex and it could be due to the exposure of aromatic residues. The fluorescence study confirmed that CAR quenches the fluorescence of HSA and showed the static mode of quenching. CAR (50 µM) quenches around 56.14% of the HSA fluorescence. The quenching constant, binding constant, number of binding site and free energy change was calculated by fluorescence quenching experiment. Competitive displacement assay showed Sudlow’s site I as the primary binding site of CAR on HSA. The synchronous fluorescence study revealed the perturbation in the microenvironment around tyrosine and tryptophan residues upon binding of CAR to HSA. The circular dichroism results suggested that the binding of CAR to HSA altered its secondary structure. Molecular docking experiment demonstrated the binding of CAR to Sudlow’s site I of HSA. Docking studies suggested that the hydrogen bonding, van der Waals and pi-alkyl are playing role in the interaction of CAR with HSA. The study confirmed the conformational changes within HSA upon binding of CAR.     

Study the interactions between human serum albumin and two antifungal drugs: Fluconazole and its analogue DTP

Binding affinities of fluconazole and its analogue 2-(2,4-dichlorophenyl)-1,3-di(1H-1,2,4-triazol-yl)-2-propanol (DTP) to human serum albumin (HSA) were investigated under approximately human physiological conditions. The obtained result indicated that HSA could generate fluorescent quenching by fluconazole and DTP because of the formation of non-fluorescent ground-state complexes. Binding parameters calculated from the Stern–Volmer and the Scatchard equations showed that fluconazole and DTP bind to HSA with binding affinities of the order 10⁴ L/mol. The thermodynamic parameters revealed that the binding was characterized by negative enthalpy and positive entropy changes, suggesting that the binding reaction was exothermic. Hydrogen bonds and hydrophobic interaction were found to be the predominant intermolecular forces stabilizing the drug–protein. The effect of metal ions on the binding constants of fluconazole–HSA complex suggested that the presence of Mg²⁺ and Zn²⁺ ions could decrease the free drug level and extend the half-life in the systematic circulation. Docking experiments revealed that fluconazole and DTP binds in HSA mainly by hydrophobic interaction with the possibility of hydrogen bonds formation between the drugs and the residues Arg 222, Lys 199 and Lys 195 in HSA.     

The effect of non-enzymatic glycation on the unfolding of human serum albumin

We monitored the unfolding of human serum albumin (HSA) and glycated human serum albumin (gHSA) subjected to guanidine hydrochloride (GndHCl) by using fluorescence and circular dichroism (CD) spectroscopy. A two-state model with sloping baselines best described the Trp-214 fluorescence unfolding measurements, while a three-state model best described the far-UV CD unfolding data. Glycation of HSA increased the [D](50%) point by approximately 0.20M. This corresponded to an increase in the free energy of unfolding of gHSA relative to HSA of 2.6kJ/mol. The intrinsic fluorescence of Trp-214 in gHSA is 0.72 of that of HSA and the far-UV CD spectrum of gHSA is nearly identical to that of HSA. These results showed that glycation altered the local structure around Trp-214 while not significantly impacting the secondary structure, and this alteration translated into an overall change in the stability of gHSA compared to HSA.     

Probing the Interaction of Human Serum Albumin With Bilirubin in the Presence of Aspirin by Multi-Spectroscopic,Molecular Modeling and Zeta Potential Techniques: Insight on Binary and Ternary Systems

Abstract

Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (K_a) and number of binding site (n) obtained were 1.6 × 10⁶ M^?1 and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 10⁶ M^?1 and 0.84, respectively, in the presence of ASA (ternary complex) at δ_ex = 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (C_CIAC) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.     

Interaction of a tyrosine kinase inhibitor,vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking

Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB–HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92–6.89?×?10^3?M^?1 at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB–HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J?mol^?1 K^?1) and negative ΔH (?6.57?kJ?mol^?1) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB–HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow’s site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca²⁺, Zn²⁺, Cu²⁺, Ba²⁺, Mg²⁺, and Mn²⁺ in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.     

Characterizing the interaction between pyrogallol and human serum albumin by spectroscopic and molecular docking methods

In the present study, the interaction of Pyrogallol (PG) with human serum albumin (HSA) was investigated by UV, fluorescence, Circular dichroism (CD), and molecular docking methods. The results of fluorescence experiments showed that the quenching of intrinsic fluorescence of HSA by PG was due to a static quenching. The calculated binding constants (K) for PG-HSA at different temperatures were in the order of 10⁴?M ^?1, and the corresponding numbers of binding sites, n were approximately equal to unity. The thermodynamic parameters, ΔH and ΔS were calculated to be negative, which indicated that the interaction of PG with HSA was driven mainly by van der Waals forces and hydrogen bonds. The negative value was obtained for ΔG showed that the reaction was spontaneous. In addition, the effect of PG on the secondary structure of HSA was analyzed by performing UV–vis, synchronous fluorescence, and CD experiments. The results indicated that PG induced conformational changes in the structure of HSA. According to Förster no-radiation energy transfer theory, the binding distance of HSA to PG was calculated to be 1.93?nm. The results of molecular docking calculations clarified the binding mode and the binding sites which were in good agreement with the results of experiments.

Communicated by Ramaswamy H. Sarma     

Comparative study of the interactions between bisphenol-A and its endocrine disrupting analogues with bovine serum albumin using multi-spectroscopic and molecular docking studies

Interaction studies of bisphenol analogues; biphenol-A (BPA), bisphenol-B (BPB), and bisphenol-F (BPF) with bovine serum albumin (BSA) were performed using multi-spectroscopic and molecular docking studies at the protein level. The mechanism of binding of bisphenols with BSA was dynamic in nature. SDS refolding experiments demonstrated no stabilization of BSA structure denatured by BPB, however, BSA denatured by BPA and BPF was found to get stabilized. Also, CD spectra and molecular docking studies revealed that BPB bound more strongly and induced more conformational changes in BSA in comparison to BPA. Hence, this study throws light on the replacement of BPA by its analogues and whether the replacement is associated with a possible risk, raising a doubt that perhaps BPB is not a good substitute of BPA.     

Saffron carotenoids (crocin and crocetin) binding to human serum albumin as investigated by different spectroscopic methods and molecular docking

Therapeutic effects of saffron ingredients were studied in some diseases. The pharmacokinetics and pharmacodynamics of these ingredients were also studied, but their transport mechanism is not clearly known. Serum albumin has been known as the most important transporter of many drugs in the body that affects their disposition, transportation, and bioavailability. Here, we investigated the interaction of crocin (Cro) with HSA, for the first time, and compared with the crocetin (Crt)–HSA interaction. UV and fluorescence spectroscopy, circular dichroism (CD), and molecular docking was applied to investigate the possibility and mechanism of binding of HSA with these natural carotenoids. The gradually addition of Cro increased HSA absorbency at 278 nm, while Crt decreased it. Both of these changes induced HSA unfolding that was confirmed by the decreased α-helix content, as determined by the CD. Both carotenoids quenched HSA fluorescence emission, but with different mechanisms. The Stern–Volmer plots indicated a dynamic quenching of intrinsic emission of HSA due to Cro addition, while Crt quenching followed both static and dynamic quenching mechanisms. Docking results indicated binding of Cro/Crt in sub-domain IIA, Sudlow site I of HSA, which accompanied with the hydrogen bonding of Cro/Crt with Tyr138. The interaction of these ligands (Cro/Crt) caused HSA unfolding and affects the hydrophobic environment of Trp241, which result in the quenching of Trp fluorescence. The UV spectroscopy and fluorescence quenching data indicated the differences in the mechanisms of interaction of Cro/Crt with HSA, which is due to the differences in the structure and hydrophobicity of these ligands.     

Study on the interaction of fisetholz with BSA/HSA by multi-spectroscopic,cyclic voltammetric,and molecular docking technique

The interaction of fisetholz with bovine serum albumin (BSA) and human serum albumin (HSA) was investigated by multi-spectroscopic, cyclic voltammetric, and molecular docking technique. The results revealed that there was a static quenching of BSA/HSA induced by fisetholz. The binding constants (K_a) and binding sites (n) were calculated at different temperatures (293, 303, and 311?K). The enthalpy change (ΔH) were calculated to be –17.20?kJ mol^?1 (BSA) and –18.28?kJ mol^?1 (HSA) and the entropy change (ΔS) were calculated to be 35.41?J mol^?1 (BSA) and 24.02?J mol^?1 (HSA), respectively, which indicated that the interaction between fisetholz and BSA/HSA was mainly by electrostatic attraction. Based on displacement experiments using site probes, indomethacin and ibuprofen, the binding site of fisetholz to BSA/HSA was identified as sub-domain IIIA, which was further confirmed by molecular docking method. There was little effect of K⁺, Ca²⁺, Cu²⁺, Zn²⁺, and Fe³⁺ on fisetholz-BSA or fisetholz-HSA complex. The spectra of synchronous fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) all showed that fisetholz binding to BSA/HSA leads to secondary structures change of the two serum albumins. According to the Förster non-radiation energy transfer theory, the binding distance between fisetholz and BSA/HSA was 2.94/4.68?nm. The cyclic voltammetry as a supporting tool also indicated that fisetholz interacted with protein.

Communicated by Ramaswamy H. Sarma     

Paclitaxel binding to the fatty acid-induced conformation of human serum albumin--automated docking studies

Paclitaxel (Taxol^®) binding to the conformation of human serum albumin assumed in the presence of long-chain fatty acids was studied by automated docking. Reduced binding affinities at both the primary and secondary sites were predicted, compared to those characterizing the interaction with the fatty acid-free protein. The baccatin core of paclitaxel was found to play a more important role than its C13 side chain in determining the ligand binding mode as well as in contributing to the overall binding energy at the primary site.     

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     

Exploring the interaction between tyrphostin 9 and human serum albumin using biophysical and computational methods

Abstract

Tyrphostin 9 (Tyr 9) is a potent platelet-derived growth factor receptor (PDGFR) inhibitor, which induces apoptosis in various cancer cell types. The binding of Tyr 9 to the major transport protein, human serum albumin (HSA) was investigated using several spectroscopic techniques and molecular docking method. Fluorescence quenching titration results showed progressive decrease in the protein fluorescence with increasing drug concentrations. A decreasing trend of the Stern-Volmer constant, K _sv with increasing temperature characterized the drug-induced quenching as static quenching, thus pointed towards the formation of Tyr 9–HSA complex. The binding constant of Tyr 9–HSA interaction was found to lie within the range 3.48–1.69?×?10⁵ M^?1 at three different temperatures, i.e. 15 °C, 25 °C and 35?°C, respectively and suggested intermediate binding affinity between Tyr 9 and HSA. The drug–HSA complex seems to be stabilized by hydrophobic forces, van der Waals forces and hydrogen bonds, as suggested from the thermodynamic data as well as molecular docking results. The far-UV and the near-UV CD spectral results showed slight alteration in the secondary and tertiary structures, respectively, of the protein upon Tyr 9 binding. Interaction of Tyr 9 with HSA also produced microenvironmental perturbations around protein fluorophores, as evident from the three-dimensional fluorescence spectral results but increased protein’s thermal stability. Both competitive drug binding results and molecular docking analysis suggested Sudlow’s Site I of HSA as the preferred Tyr 9 binding site.

Communicated by Ramaswamy H. Sarma     

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (K_a), ΔH, ΔS and ΔG values to be 15.5×10⁴M^?1, ?116.2±18.1 Kcal mol^?1, ?366 cal mol^?1K^?1 and ?7.13 Kcal mol^?1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.     

Interaction of Prussian blue nanoparticles with bovine serum albumin: a multi-spectroscopic approach

Owning to their exceptional properties, Prussian blue nanoparticles (PBNPs) are promising in a variety of biomedical applications. In this scenario, understanding of how PBNPs interact and behave in biological systems is essential. Herein, the interaction of PBNPs with protein was investigated. Specifically, the citric acid stabilized PBNPs with a size of 10 nm were synthesized and characterized. The interactions of these PBNPs with the model protein, bovine serum albumin (BSA), were then probed by spectroscopic methods. It was found that the BSA intrinsic fluorescence was quenched upon addition of PBNPs due to the static interaction, suggesting the binding of PBNPs with BSA. Moreover, the synchronous fluorescence and circular dichroism spectra indicated the conformational change of BSA due to the presence of PBNPs.     

Binding of Janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach

In this report, we have investigated the binding affinity of tofacitinib with human serum albumin (HSA) under simulated physiological conditions by using UV–visible spectroscopy, fluorescence quenching measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC) and molecular docking methods. The obtained results demonstrate that fluorescence intensity of HSA gets quenched by tofacitinib and quenching occurs in static manner. Binding parameters calculated from modified Stern–Volmer equation shows that the drug binds to HSA with a binding constant in the order of 10⁵. Synchronous fluorescence data deciphered the change in the microenvironment of tryptophan residue in HSA. UV spectroscopy and DLS measurements deciphered complex formation and reduction in hydrodynamic radii of the protein, respectively. Further DSC results show that tofacitinib increases the thermo stability of HSA. Hydrogen bonding and hydrophobic interaction are the main binding forces between HSA and tofacitinib as revealed by docking results.     

Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein

Interaction of tetracycline (TC) derivatives viz. oxytetracycline, doxycycline, demeclocycline and chlorotetracycline with bovine serum albumin (BSA) and concomitant changes in protein conformation were studied using fluorescence quenching and circular dichroism measurements. Fluorescence data revealed the presence of one to three binding sites on BSA for different TC derivatives. Binding studies with the marker ligands, warfarin and bilirubin, elucidated site-I as a primary binding site for TCs on albumin. Scatchard analysis revealed the binding affinity (K_a) and capacity (n) for these derivatives vary in the range from 0.8 to 3.2×10⁶ l/mole and 1.3–3.4, respectively. Significant reduction (60–45%) in secondary structure (-helical content) of BSA was noticed upon interaction with different TC derivatives in presence of Cu (II) ions. High affinity binding of TCs with BSA signifies drug stability. However, excessive binding at higher TC concentrations in combination with Cu (II) induces conformational change in protein structure, which may exert detrimental effect on cellular protein.     

Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV–vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV–vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.     

Investigation of the molecular interactions of triclocarban with human serum albumin using multispectroscopies and molecular modeling

Triclocarban (TCC), as a broad spectrum antibacterial agent widely used in personal care products, has recently been recognized as environmental pollutant with the potential of adversely affecting wildlife and human health. However, the behavior of TCC in blood circulatory system and the potential toxicity of TCC at the molecular level have been poorly investigated. In this study, the effect of TCC on human serum albumin (HSA) and the binding mechanism of TCC to HSA were examined using spectroscopic techniques and molecular modeling methods. The fluorescence results suggested that the fluorescence of HSA was quenched by TCC through a static quenching mechanism and nonradiation energy transfer, and TCC was bound to HSA with moderately strong binding affinity via hydrophobic interaction based on the analysis of the thermodynamic parameters. The site marker competitive experiments revealed that TCC bound into subdomain IIA (site I) of HSA. In addition, the results obtained from the circular dichroism, Fourier transform infrared (FT-IR), 8-anilino-1-naphthalenesulfonic acid fluorescence, synchronous fluorescence, three-dimensional fluorescence spectra and dynamic light scattering suggested the change in the microenvironment and conformation of HSA during the binding reaction. Finally, the best binding mode of TCC and specific interaction of TCC with amino acid residues were determined using molecular docking and molecular dynamics simulations. In a word, the present studies can provide a way to help us well understand the transport, distribution and toxicity effect of TCC when it diffused in the human body.

Communicated by Ramaswamy H. Sarma