The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.     

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.     

Investigations on the interactions between naphthalimide‐based anti‐tumor drugs and human serum albumin by spectroscopic and molecular modeling methods

The interactions between the three kinds of naphthalimide‐based anti‐tumor drugs (NADA, NADB, NADC) and human serum albumin (HSA) under simulated physiological conditions were investigated by fluorescence spectroscopy, circular dichroism spectroscopy and molecular modeling. The results of the fluorescence quenching spectroscopy showed that the quenching mechanisms for different drugs were static and their affinity was in a descending order of NADA > NADB > NADC. The relative thermodynamic parameters indicated that hydrophobic force was the predominant intermolecular force in the binding of NAD to HSA, while van der Waals interactions and hydrogen bonds could not be ignored. The results of site marker competitive experiment confirmed that the binding site of HSA primarily took place in site I. Furthermore, the molecular modeling study was consistent with these results. The study of circular dichroism spectra demonstrated that the presence of NADs decreased the α‐helical content of HSA and induced the change of the secondary structure of HSA. Copyright © 2015 John Wiley & Sons, Ltd.     

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin—azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.     

3ircular dichroism simulation shows a site-II-to-site-I displacement of human serum albumin-bound diclofenac by ibuprofen

Purpose: The purpose of this study was to confirm the hypothesis that a site-II-to-site-I displacement takes place when some nonsteroidal anti-inflammatory drugs are displaced by another drug from their high-affinity binding site to a site of lower affinity on human serum albumin (HSA).Methods: Diclofenac, sodium salt, was used as a representative example because of its prominent reversal of the Cotton effect. Effects of site-specific drugs on the free fraction of diclofenac were determined by equilibrium dialysis, and effects on induced circular dichroism (CD) of diclifenac bound to HSA were studied by CD and CD simulation techniques.Results: Ibuprofen, a site-II-specific drug, altered the CD spectrum of the diclofenac-HSA complex at a molar ratio of 0.5∶1 to that obtained at a higher ratio (5∶1) without ibuprofen. The induced CD spectrum obtained in the presence of ibuprofen was very similar to one that assumed that all diclofenac displaced from its high-affinity binding site (site II) became rebound to a lower-affinity site (site I). The rebinding could be influenced by a free energy linkage between the two sites which would make site I (or parts thereof) more suitable for diclofenac binding.Conclusion: We have confirmed the existence of a site II-to-site displacement, which is very striking and pharmacologically important, because the concentration of unbound drug being displaced is much lower than expected for a competitive mechanism.     

Species-dependent stereoselective drug binding to albumin: a circular dichroism study

Drug binding to albumins from different mammalian species was investigated to disclose evidence of species-dependent stereoselectivity in drug-binding processes and affinities. This aspect is important for evaluating the reliability of extrapolating distribution data among species. The circular dichroism (CD) signal induced by drug binding to the albumins [human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), and dog serum albumin (DSA)] were measured and analyzed. The binding of selected drugs and metabolites to HSA significantly differed from the binding to the other albumins in terms of affinity and conformation of the bound ligands. In particular, phenylbutazone, a marker of site one on HSA, showed a higher affinity for binding to BSA with respect to RSA, HSA, and DSA, respectively. In the case of diazepam, a marker of site two on HSA, the affinity decreased in order from HSA to DSA, RSA, and BSA. The induced CD spectra were similar in terms of energy and band signs, suggesting almost the same conformation for the bound drug to the different albumins. Stereoselectivity was high for the binding of ketoprofen to HSA and RSA. A different sign was observed for the CD spectra induced by the drug to the two albumins because of the prevalence of a different conformation of the bound drug. Interestingly, the same induced CD spectra were obtained using either the racemic form or the (S)-enantiomer. Finally, significant differences were observed in the affinity of bilirubin, being highest for BSA, then decreasing for RSA, HSA, and DSA. A more complex conformational equilibrium was observed for bound bilirubin.     

Determining binding sites of drugs on human serum albumin using FIA-QCM

A simple and effective method was developed for determining binding sites of drugs on human serum albumin (HSA) by independent binding or competitive displacement of bilirubin using flow injection analysis-quartz crystal microbalance (FIA-QCM) system. Both independent and competitive bindings were entirely monitored in real time. Bilirubin as a site I-binding ligand was pre-bound to HSA sensor so as to occupy the drug-binding site I. When the model site II-binding drugs (ibuprofen, ketoprofen and flurbiprofen) were injected into the bilirubin pre-bound HSA system, the frequency continuously decreased by 6Hz, 4Hz and 5Hz, respectively, which was the same as that of their individual binding to HSA sensor. It indicated that the drug binding to site II was independent and did not interfere with bilirubin binding. However, when the model site I-binding drugs (iodipamide and magnesium salicylate) were introduced into the system, the frequency remained unchanged in the initial several minutes and then rapidly decreased by 4Hz for iodipamide and increased by 4Hz for magnesium salicylate. This phenomenon revealed site I-binding drugs competitively bound to HSA against bilirubin and displaced the pre-bound bilirubin. The results demonstrate FIA-QCM can be a valid approach for monitoring the dynamic interaction between drugs and HSA in real time further identifying drug-binding sites without the need of labels.     

Influence of quercetin on the interaction of gliclazide with human serum albumin – spectroscopic and docking approaches

Protein‐binding interactions are displacement reactions which have been implicated as the causative mechanisms in many drug–drug interactions. Thus, the aim of presented study was to analyse human serum albumin‐binding displacement interaction between two ligands, hypoglycaemic drug gliclazide and widely distributed plant flavonoid quercetin. Fluorescence analysis was used in order to investigate the effect of substances on intrinsic fluorescence of human serum albumin (HSA) and to define binding and quenching properties of ligand–albumin complexes in binary and ternary systems, respectively. Both ligands showed the ability to bind to HSA, although to a different extent. The displacement effect of one ligand from HSA by the other one has been described on the basis of the quenching curves and binding constants comparison for the binary and ternary systems. According to the fluorescence data analysis, gliclazide presents a substance with a lower binding capacity towards HSA compared with quercetin. Results also showed that the presence of quercetin hindered the interaction between HSA and gliclazide, as the binding constant for gliclazide in the ternary system was remarkably lower compared with the binary system. This finding indicates a possibility for an increase in the non‐bound fraction of gliclazide which can lead to its more significant hypoglycaemic effect. Additionally, secondary and tertiary structure conformational alterations of HSA upon binding of both ligands were investigated using synchronous fluorescence, circular dichroism and FT‐IR. Experimental data were complemented with molecular docking studies. Obtained results provide beneficial information about possible interference upon simultaneous co‐administration of the food/dietary supplement and drug.     

Enhancement of the binding affinity of methylene blue to site I in human serum albumin by cupric and ferric ions

In this work, the binding characteristics of methylene blue (MB) to human serum albumin (HSA) and the influence of Cu²⁺ and Fe³⁺ on the binding affinity of MB to HSA were investigated using fluorescence, absorption, circular dichroism (CD) spectroscopy and molecular modelling. The results of competitive binding experiments using the site probes ketoprofen and ibuprofen as specific markers suggested that MB was located in site I within sub‐domain IIA of HSA. The molecular modelling results agreed with the results of competitive site marker experiments and the results of CD spectra indicated that the interaction between MB and HSA caused the conformational changes in HSA. The binding affinity of MB to HSA was enhanced but to a different extent in the presence of Cu²⁺ and Fe³⁺, respectively, which indicated that the influence of different metal ions varied. Enhancement of the binding affinity of MB to HSA in the presence of Cu²⁺ is due to the formation of Cu²⁺–HSA complex leading to the conformational changes in HSA, whereas in the presence of Fe³⁺, enhancement of the binding affinity is due to the greater stability of the Fe³⁺–HSA–MB complex compared with the MB–HSA complex. Copyright © 2015 John Wiley & Sons, Ltd.     

Determining molecular binding sites on human serum albumin by displacement of oleic acid

An NMR method was developed for determining binding sites of small molecules on human serum albumin (HSA) by competitive displacement of (13)C-labeled oleic acid. This method is based on the observation that in the crystal structure of HSA complexed with oleic acid, two principal drug-binding sites, Sudlow's sites I (warfarin) and II (ibuprofen), are also occupied by fatty acids. In two-dimensional [(1)H,(13)C]heteronuclear single quantum coherence NMR spectra, seven distinct resonances were observed for the (13)C-methyl-labeled oleic acid as a result of its binding to HSA. Resonances corresponding to the major drug-binding sites were identified through competitive displacement of molecules that bind specifically to each site. Thus, binding of molecules to these sites can be followed by their displacement of oleic acids. Furthermore, the amount of bound ligand at each site can be determined from changes in resonance intensities. For molecules containing fluorine, binding results were further validated by direct observations of the bound ligands using (19)F NMR. Identifying the binding sites for drug molecules on HSA can aid in determining the structure-activity relationship of albumin binding and assist in the design of molecules with altered albumin binding.     

Fluorescence spectroscopic investigation of competitive interactions between ochratoxin A and 13 drug molecules for binding to human serum albumin

Ochratoxin A (OTA) is a highly toxic mycotoxin found worldwide in cereals, foods, animal feeds and different drinks. Based on previous studies, OTA is one of the major causes of the chronic tubulointerstitial nephropathy known as Balkan endemic nephropathy (BEN) and exerts several other adverse effects shown by cell and/or animal models. It is a well‐known fact that OTA binds to various albumins with very high affinity. Recently, a few studies suggested that reducing the bound fraction of OTA might reduce its toxicity. Hypothetically, certain drugs can be effective competitors displacing OTA from its albumin complex. Therefore, we examined 13 different drug molecules to determine their competing abilities to displace OTA from human serum albumin (HSA). Competitors and ineffective chemicals were identified with a steady‐state fluorescence polarization‐based method. After characterization the competitive abilities of individual drugs, drug pairs were formed and their displacing activity were tested in OTA‐HSA system. Indometacin, phenylbutazone, warfarin and furosemide showed the highest competing capacity but ibuprofen, glipizide and simvastatin represented detectable interaction too. Investigations of drug pairs raised the possibility of the presence of diverse binding sites of competing drugs. Apart from the chemical information obtained in our model, this explorative research might initiate future designs for epidemiologic studies to gain further in vivo evidence of long‐term (potentially protective) effects of competing drugs administered to human patients. Copyright © 2012 John Wiley & Sons, Ltd.     

In vitro plasma protein binding and aqueous aggregation behavior of astaxanthin dilysinate tetrahydrochloride

The tetrahydrochloride salt of astaxanthin di-L-lysinate (lys(2)AST) is a highly water-dispersible astaxanthin-amino acid conjugate, with an aqueous dispersibility of > or = 181.6 mg/mL. The statistical mixture of stereoisomers has been well characterized as an aqueous-phase superoxide anion scavenger, effective at micromolar (microM) concentrations. In the current study, the aqueous aggregation behavior and in vitro plasma protein binding [with fatty-acid-free human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP)] were investigated with a suite of techniques, including circular dichroism (CD) and UV-vis spectroscopy, ultrafiltration, competitive ligand displacement, and fluorescence quenching. Induced CD bands obtained in Ringer buffer solution of HSA demonstrated high affinity monomeric binding of the compound at low ligand per protein (L/P) ratios (in aqueous solution alone the carotenoid molecules formed card-pack aggregates). The binding constant ( approximately 10(6)M(-1)) and the binding stoichiometry (approximately 0.2 per albumin molecule) were calculated from CD titration data. CD displacement and ultrafiltration experiments performed with marker ligands of HSA indicated that the ligand binding occurred at a site distinct from the main drug binding sites of HSA (i.e., Sites I and II). At intermediate L/P ratios, both monomeric and aggregated ("chirally complexed") binding occurred simultaneously at distinct sites of the protein. At high L/P ratios, chiral complexation predominantly occurred on the asymmetric protein template. The tentative location of the chirally-complexed aggregation on the HSA template was identified as the large interdomain cleft of HSA, where carotenoid derivatives have been found to bind previously. Only weak binding to AGP was observed. These results suggest that parenteral use of this highly potent, water-dispersible astaxanthin-amino acid conjugate will result in plasma protein association, and plasma protein binding at sites unlikely to displace fatty acids and drugs bound at well-characterized binding sites on the albumin molecule.     

Combination mode of antimalarial drug mefloquine and human serum albumin: Insights from spectroscopic and docking approaches

The interaction between mefloquine (MEF), the antimalarial drug, and human serum albumin (HSA), the main carrier protein in blood circulation, was explored using fluorescence, absorption, and circular dichroism spectroscopic techniques. Quenching of HSA fluorescence with MEF was characterized as static quenching and thus confirmed the complex formation between MEF and HSA. Association constant values for MEF-HSA interaction were found to fall within the range of 3.79-5.73 × 10⁴ M^˗1 at various temperatures (288, 298, and 308 K), which revealed moderate binding affinity. Hydrogen bonds and hydrophobic interactions were predicted to connect MEF and HSA together in the MEF-HSA complex, as deduced from the thermodynamic data (ΔS = +133.52 J mol⁻¹ K⁻¹ and ΔH = +13.09 kJ mol⁻¹) of the binding reaction and molecular docking analysis. Three-dimensional fluorescence spectral analysis pointed out alterations in the microenvironment around aromatic amino acid (tryptophan and tyrosine) residues of HSA consequent to the addition of MEF. Circular dichroic spectra of HSA in the wavelength ranges of 200-250 and 250-300 nm hinted smaller changes in the protein's secondary and tertiary structures, respectively, induced by MEF binding. Noncovalent conjugation of MEF to HSA bettered protein thermostability. Site marker competitive drug displacement results suggested HSA Sudlow's site I as the MEF binding site, which was also supported by molecular docking analysis.     

Use of an immobilised human serum albumin HPLC column as a probe of drug-protein interactions: the reversible binding of valproate

The reversible binding of valproate to human serum albumin determines a decrease of the binding of ligands that selectively bind to site I, site II, and bilirubin binding site. The binding inhibition was followed by displacement chromatography methodology using increasing concentrations of the competitor, i.e. valproate, in the mobile phase. Significant binding inhibition was observed for drugs binding at site I and site II. The greater displacement was observed for the more retained enantiomer of benzodiazepines and profens. A reduction of the affinity was observed also in the case of phenol red, this compound being selected as representative of bilirubin binding site. Difference circular dichroism spectroscopy was also used to characterise the binding of valproate to human serum albumin. This antiepilectic drug was proved to affect the binding at site I, II, and bilirubin binding site. The data have physiological relevance because significant inhibition of the binding resulted at clinic concentrations of valproate.     

Understanding the mode of binding mechanism of doripenem to human serum albumin: Spectroscopic and molecular docking approaches

The infections caused by multidrug resistant bacteria are widely treated with carabapenem antibiotics as a drug of choice, and human serum albumin (HSA) plays a vital role in binding with drugs and affecting its rate of delivery and efficacy. So, we have initiated this study to characterize the mechanism of doripenem binding and to locate its site of binding on HSA by using spectroscopic and docking approaches. The binding of doripenem leads to alteration of the environment surrounding Trp‐214 residue of HSA as observed by UV spectroscopic study. Fluorescence spectroscopic study revealed considerable interaction and complex formation of doripenem and HSA as indicated by K_sv and K_q values of the order of 10⁴ M^?1 and 10¹² M^?1 s^?1, respectively. Furthermore, doripenem quenches the fluorescence of HSA spontaneously on a single binding site with binding constant of the order of 10³ M^?1, through an exothermic process. Van der Waals forces and hydrogen bonding are the major forces operating to stabilize HSA‐doripenem complex. Circular dichroism spectroscopic study showed changes in the structure of HSA upon doripenem binding. Drug displacement and molecular docking studies revealed that the binding site of doripenem on HSA is located on subdomain IB and III A. This study concludes that, due to significant interaction of doripenem on either subdomain IB or IIIA of HSA, the availability of doripenem on the target site may be compromised. Hence, there is a possibility of unavailability of threshold amount of drug to be reached to the target; consequently, resistance may develop in the bacterial population.     

Investigating the antagonistic action between aspirin and tamoxifen with HSA: identification of binding sites in binary and ternary drug-protein systems by spectroscopic and molecular modeling approaches

The combination of several drugs is often necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease of the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. The aim of this study has been to analyze the interactions of tamoxifen (TMX) and aspirin (ASA) with human serum albumin (HSA) and to evaluate the mechanism of a simultaneous binding of TMX and ASA to the protein. Fluorescence analysis was used to estimate the effect of the drugs on the protein fluorescence and to define the binding and quenching properties of drug-HSA complexes. The binding sites for TMX and ASA were identified in ternary structures of HSA by means of spectrofluroscence. The analysis of the fluorescence quenching of HSA in binary and ternary systems pointed at TMX and ASA having an effect on the HSA-ASA and HSA-TMX complexes. Furthermore, the results of synchronous fluorescence, resonance light scattering and circular dichroism of the binary and ternary systems showed that the binding of TMX and ASA to HSA could induce conformational changes in HSA. Moreover, the simultaneous presence of TMX and ASA during binding to HSA should be taken into account in multi-drug therapy, as it induces the necessity of a monitoring therapy owing to the possible increase of uncontrolled toxic effects. Competitive site marker experiments demonstrated that the binding site of ASA and TMX to HSA differed in the binary system as opposed to in its ternary counterpart. Finally, molecular modeling of the possible binding sites of TMX and ASA in binary and ternary systems to HSA confirmed the experimental results.     

Competition of antibacterial drugs for binding sites of human serum albumin

Simultaneous binding of two drugs to human serum albumin (HSA) was studied by flow microcalorimetry. The following drug pairs were used: sulfadimethoxine and cefazolin. Sulfadimethoxine and dicloxacillin, sulfadimethoxine and chlortetracycline. A procedure for estimating the calorimetric titration curves in competing binding of the drugs to the HSA homogeneous active site is described. Comparison of the theoretical and experimental titration curves enabled detection of the ligand competition for the biopolymer binding site. It was shown that sulfadimethoxine displaced cefazolin in the HSA active site, the nature of the HSA association with dicloxacillin and sulfadimethoxine was independent and binding of doxycycline or chlortetracycline to HSA had no influence on sulfadimethoxine interaction with protein.     

Impact of Potential Blockers on Ru(III) Complex Binding to Human Serum Albumin

The effects of aspirin, vitamin B2 and warfarin as potential blockers of the ruthenium binding sites in HSA were investigated through UV/visible, circular dichroism (CD), fluorescence spectroscopy and the inductively coupled plasma-atomic emission spectroscopy ICP(AES). The studies on the interactions of several biologically relevant molecules with HSA have shown that drugs like aspirin or warfarin may strongly influence the interaction of serum protein with anticancer drugs. It can derive from the influence of the drug on protein conformation or binding close to binding site of anticancer drug. Aspirin, vitB2 and warfarin bind to IIA subdomain leading to partial blocking of the ruthenium binding site in HSA.     

Human serum albumin binding of novel antiretroviral nucleoside derivatives of AZT.

The binding of novel nucleoside derivatives (2-7) to the Human Serum Albumin (HSA) was studied using zidovudine (AZT), as standard compound. The applicability of two different techniques to separate unbound drug from drug-protein complex was analyzed: the gel filtration and ultrafiltration methods. Ultrafiltration was found to be an adequate procedure for the separation of unbounded drug from the drug-protein complex. Incubation temperature ranging from 0 to 37 degrees C did not modify considerably the bound fractions. The same effects were observed as HSA concentration was modified. Binding assays of studied compounds to purified 1% (w/v) HSA at 0 degrees C, indicate that bound fraction of 2-7 ranges from 13 to 47%, exhibiting a higher affinity to HSA than AZT (12%), which would introduce some interesting improvements in their pharmacokinetic properties. In addition, by means of displacement studies using HSA site specific drugs such as diazepam and salicylate, it was determined that AZT binds to site I of the HSA molecule, by a mainly entropy driven process (DeltaS = 10.834 cal/mol degrees K), being these observations extensive to 2-7. Some structural basis to explain enhanced affinity of these novel derivatives was also established.     

Study on the molecular recognition action of lamivudine by human serum albumin

In this work, the interaction of an anti‐HIV drug lamivudine and human serum albumin (HSA) was studied by multispectroscopic and molecular modeling methods. The fluorescence emission spectra showed that the fluorescence of HSA was quenched by lamivudine through static mechanism with HSA‐lamivudine complex produced at ground state. According to the binding equilibriums observed at 4 different temperatures, the number of binding site, binding constant, enthalpy change, entropy change, and Gibbs free energy change of the interaction were calculated. The results indicated that there was only 1 main binding site under present concentration condition, and then, the location of this binding site was ascertained by molecular probe experiments using warfarin and ibuprofen as site markers. The interaction was a spontaneous and exothermic process. Hydrogen bonds and van der Waals force were the major power that fixed lamivudine on Sudlow's site I in subdomain IIA of HSA molecule. The distance between donor and acceptor was determined by Förster's nonradiative fluorescence resonance energy transfer theory. Circular dichroism spectra exhibited the alteration of HSA's secondary structures. Molecular modeling investigation revealed the structure of HSA‐lamivudine complex, including the conformation of lamivudine in binding site, the amino residues close to lamivudine, and the interaction forces between receptor and ligand. The study may be beneficial to therapists in understanding the distribution of lamivudine in vivo and explaining its drug‐resistant mechanism in clinical diagnosis.