Paclitaxel-HSA interaction. Binding sites on HSA molecule

Paclitaxel (trade name Taxol) is one of the world's most effective anticancer drugs. It is used to treat several cancers including tumours of the breast, ovary and lung. In the present work the interaction of paclitaxel with human serum albumin (HSA) in aqueous solution at physiological pH has been investigated through CD, fluorescence spectroscopy and by the antibody precipitate test. Binding of paclitaxel to albumin impact on protein structure and it influences considerably albumin binding of other molecules like warfarin, heme or bilirubin. The paclitaxel-HSA interaction causes the conformational changes with the loss of helical stability of protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the paclitaxel-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilization of the warfarin binding site, which includes Trp 214, and high affinity bilirubin binding site located in subdomain IIA.     

Proton nuclear magnetic resonance study on the multimode interactions of human serum albumin with drug molecules

The interactions of human serum albumin (HSA) with a number of ligands (mostly drugs) were examined by proton nuclear magnetic resonance spectroscopy. Ligand presence-absence difference spectra of HSA solutions were measured. Nonspecifically bound drugs such as tiaramide showed difference spectrum patterns which were similar to the spectra of the drugs themselves but were broadened as to the line-widths of signals. Thus, the difference spectra of these drugs reflect only the changes in the surroundings of the drug molecules, that is, between the bound and free states. In contrast, specifically bound drugs like ibuprofen and warfarin showed difference spectra in which signals from the HSA molecule only were observed. Furthermore, according to the characteristic peaks in these difference spectrum patterns, specifically bound drugs may be classified into several groups; the drugs in the first group bind to the ibuprofen binding site, those in the second group to the warfarin binding site, and those in the third group to sites other than the warfarin and ibuprofen sites. These findings suggest that the specific binding of drugs to HSA brings about a conformational change of this protein which is specifically correlated to the binding site.     

Binding of the Promen fluorescent probe to human serum albumin: a fluorescence spectroscopic study.

The binding of Promen (6-propionyl-2-methoxynapthalene) to human serum albumin (HSA) was measured by fluorescence spectroscopy, finding only one class of binding sites on the protein. Hydrophobic interactions play an important role to stabilize the complex. Attempts were made to characterize its binding site using as competitors warfarin, phenylbutazone and diazepam, which bind in a specific site or region on the HSA. Fluorescence polarization measurements and spectrofluorimetric results suggest that diazepam and Promen bind at different but interacting binding sites on the HSA. The changes in the fluorescence emission of the bound Promen in the presence of these drugs, allow to use Promen to detect unspecific interactions with the site II on the 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.     

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.     

Familial dysalbuminemic byperthyroxinemia may result in altered warfarin pharmacokinetics

Two distinct genotypes that result in the amino acid substitutions R218P and R218H in subdomain 2A of human serum albumin (HSA) have been identified as the cause of familial dysalbuminemic hyperthyroxinemia (FDH). These substitutions increase the affinity of subdomain 2A for thyroxine by approximately 10-fold elevating plasma thyroxine levels in affected individuals. While many studies have examined the binding of thyroxine to FDH HSA, the binding of FDH HSA to drugs has not been widely investigated. The widely administered drug warfarin was selected as a model compound to study FDH HSA/drug interactions since it binds to subdomain 2A and its pharmacokinetics are dramatically influenced by HSA binding. Using two independent methods, fluorescence spectroscopy and equilibrium dialysis with radioactive warfarin, the binding of recombinant R218P, R218H, R218M and wild type HSA to warfarin was measured. Both methods showed an approximately 5-fold decrease in the affinity of R218P, R218H and R218M HSA for warfarin relative to wild type HSA. The Kd values determined by fluorescence spectroscopy for wild type, R218H, R218P and R218M HSA binding to warfarin were 1.35, 5.38, 5.61, and 8.34 microM, respectively. The values determined by equilibrium dialysis were 5.36, 29.5, 14.5, and 23.4 microM, respectively. Based on the above findings one would expect the free serum warfarin concentration in homozygous R218P and R218H FDH patients to be elevated about 5-fold, resulting in about a 5-fold reduction in the serum half-life of the drug.     

Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study.

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-haem) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem-NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules--buried in close proximity to the haem binding pocket--induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1H-NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, are 5.4 +/- 1.1 x 10(-4) m and 2.1 +/- 0.4 x 10(-5) m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA-haem is lower than that reported for drug binding to haem-free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) m in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA-haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA.     

Crystal structure analysis of warfarin binding to human serum albumin: anatomy of drug site I

Human serum albumin (HSA) is an abundant transport protein found in plasma that binds a wide variety of drugs in two primary binding sites (I and II) and can have a significant impact on their pharmacokinetics. We have determined the crystal structures at 2.5 A-resolution of HSA-myristate complexed with the R-(+) and S-(-) enantiomers of warfarin, a widely used anticoagulant that binds to the protein with high affinity. The structures confirm that warfarin binds to drug site I (in subdomain IIA) in the presence of fatty acids and reveal the molecular details of the protein-drug interaction. The two enantiomers of warfarin adopt very similar conformations when bound to the protein and make many of the same specific contacts with amino acid side chains at the binding site, thus accounting for the relative lack of stereospecificity of the HSA-warfarin interaction. The conformation of the warfarin binding pocket is significantly altered upon binding of fatty acids, and this can explain the observed enhancement of warfarin binding to HSA at low levels of fatty acid.     

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.     

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.     

Studies on the interactions between human serum albumin and imidazolium [trans-tetrachlorobis(imidazol)ruthenate(III)].

The interactions between imidazolium [trans-tetrachlorobis(imidazol) ruthenate(III)] (Ru-im) and human serum albumin (HSA) have been investigated through UV-Vis, CD, fluorescence spectroscopy and by the antibody precipitation test. Binding of Ru(III)-imidazole species to albumin has a strong impact on the protein structure and influences considerably the albumin binding of other molecules such as warfarin or heme. The metal complex-HSA interactions cause conformational changes with the loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the ruthenium-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilisation of the warfarin binding site which includes Trp 214, observed in the metal-bound HSA.     

High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE

Characterizing how chemical compounds bind to human serum albumin (HSA) is essential in evaluating drug candidates. Using warfarin as a test system, we validate the application of BIACORE SPR biosensors to reliably determine binding constants for drug/HSA interactions. The binding responses for warfarin over HSA surfaces were extremely reproducible even though warfarin is small compared to the size of the immobilized protein. At high concentrations, warfarin bound at more than one site on HSA, which is consistent with its known binding properties. The affinity we determined for the high-affinity site (K(25 degrees C)(d) = 3.7 +/- 1.2 microM), as well as the dissociation rate constant (k(25 degrees C)(d) = 1.2 s(-1)), are also consistent with binding constants determined previously. These results validate the biosensor technology and illustrate how BIACORE can be used to study drug/HSA interactions in a high-resolution mode. Using a set of 10 test compounds, we present a protocol for determining equilibrium dissociation constants for HSA in a high-throughput mode. Our method involves working at low compound concentrations and fitting the equilibrium data for all compounds simultaneously. We show that the % bound values determined by SPR correlate with the values determined by solution-based methods. The ability to examine directly the binding of small molecules (130-800 Da), coupled with minimal sample requirements and automated instrumentation, makes BIACORE technology applicable for evaluating drug/HSA interactions.     

Site I on human albumin: differences in the binding of (R)- and (S)-warfarin.

The binding of drugs known to interact with area I on human serum albumin (HSA) was investigated using a chiral stationary phase obtained by anchoring HSA to a silica matrix. In particular, this high-pressure affinity chromatography selector was employed to study the binding properties of the individual enantiomers of warfarin. The pH and composition of the mobile phase modulate the enantioselective binding of warfarin. Displacement chromatography experiments evidenced significant differences in the binding of the warfarin enantiomers to site I. The (S)-enantiomer was shown to be a direct competitor for (R)-warfarin, while (R)-warfarin was an indirect competitor for the (S)-enantiomer. Salicylate directly competed with (R)-warfarin and indirectly with (S)-warfarin. This behavior was confirmed by difference CD experiments, carried out with the same [HSA]/[drug] system in solution.     

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.     

Interaction of virstatin with human serum albumin: spectroscopic analysis and molecular modeling

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ～pH 7.2, B form ～pH 9.0 and F form ～pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants K(a) for N and B isomers were found to be 6.09×10(5 )M(-1) and 4.47×10(5) M(-1), respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1:1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site.     

Surface-enhanced Raman spectroscopy study of the interaction of the antitumoral drug emodin with human serum albumin

Surface-enhanced Raman spectroscopy was employed in this work to study the interaction between the antitumoral drug emodin and human serum albumin (HSA), as well as the influence of fatty acids in this interaction. We demonstrated that the drug/protein interaction can take place through two different binding sites which are probably localized in the IIA and IIIA hydrophobic pockets of HSA and which correspond to Sudlow's I and II binding sites, respectively. The primary interaction site of this drug seems to be site II in the defatted albumin. Fatty acids seem to displace the drug from site II to site I in nondefatted HSA, due to the high affinity of fatty acids for site II. The drug interacts with the protein through its dianionic form in defatted HSA (when placed in the site II) and through its neutral form in the site I of nondefatted albumins.     

Human serum albumin: spectroscopic studies of the paclitaxel binding and proximity relationships with cisplatin and adriamycin

The interactions of anti-cancer drugs with blood constituents, particularly with serum albumin (HSA) may have a major influence on drug pharmacology and efficacy. In the present work the binding of paclitaxel (trade name Taxol) to human serum albumin and its effect on cisplatin and adriamycin interactions has been investigated through UV/visible, CD, fluorescence spectroscopy and the inductively couplet plasma atomic emission spectroscopy method. Displacement studies with use of bilirubin, as a competitive agent provided relevant information about the location of the binding site in HSA as well as the possible multidrug interactions.     

Influence of aspirin and iron(III) tetrasulfonated phthalocyanine on bilirubin binding by human serum albumin

The interaction of bilirubin with aspirin-modified human serum albumin (HSA) and the influence of iron tetrasulfonated phthalocyanine on bilirubin binding by the native protein has been studied by difference spectroscopy and circular dichroism measurements. Spectroscopic studies of the systems containing bilirubin and aspirin-modified HSA compared to the analogous systems with the native protein have shown that selective acetylation of albumin at lysine 199 inhibits bilirubin binding by this protein. In both cases, interaction between bilirubin and albumin leads to complex formation at a molar ratio of ligand to protein of 2:1. The studies of the reaction of bilirubin with fragments of albumin produced by reaction with CNBr have demonstrated that one of the strong bilirubin binding sites is located in the M fragment and is close to the high-affinity binding site of aspirin. The other one was found in fragment C. Acetylation of albumin brings about marked conformational change in the protein, which probably accounts for the decrease in its ability to react with anti-HSA antibody. Bilirubin does not change the secondary structure of albumin but, like aspirin, lowers its antigenicity. It has been suggested that the decrease in antigenic properties in this case results from cooperation of the closely neighboring antigenic and bilirubin-binding sites. The studies of the influence of iron(III) tetrasulfonated phthalocyanine on bilirubin binding by HSA suggest that there is no competition between strong sites for iron(III) tetrasulfonated phthalocyanine and bilirubin, but these compounds compete for some of the weaker sites.     

Studies on the interactions between human serum albumin and trans-indazolium (bisindazole) tetrachlororuthenate(III)

The interactions between HInd[RuInd2Cl4] and human serum albumin have been investigated through UV-Vis, circular dichroism (CD), fluorescence spectroscopy and the inductively coupled plasma-atomic emission spectroscopy (ICP(AES)) method. Binding of Ru(III)-indazole species to albumin has strong impact on protein structure and it influences considerably albumin binding of other molecules like warfarin, heme or metal ions. The metal complex-human serum albumin (HAS) interactions cause conformational changes with loss of helical stability of the protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the ruthenium-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilization of the warfarin-binding site, which includes Trp 214, observed in the metal-bound HSA.