Binding Sites Of Human Serum Albumin Research Articles

Human Serum Albumin Structure in Presence of Different Concentrations of Cortisol and Glucose: An In Vitro Modeling Under Normal and Hyperglycemic Conditions

Background: Glucose is an essential element in the supply of body’s energy. In diseases such as diabetes, glucose increase is associated with disturbance in metabolism. Cortisol is an important hormone in the regulation of glucose metabolism, and human serum albumin (HSA) is one of the most important glucose and cortisol transmitters in blood. Interaction between albumin and these ligands could affect HSA secondary structure and its stability. Objectives: The aim of this study was to investigate HSA secondary structure in the presence of different concentrations of glucose and cortisol. Methods: This was an in vitro (analytical/descriptive) study in which, completely randomized design was used to study the interaction between human serum albumin at 37°C at different glucose concentrations of 0, 80, 180, 240, and 400 mg/dL and cortisol at concentrations of 0, 10, 20, and 40 µg/dL. Intrinsic fluorescence spectroscopy and Circular dichroism (CD) were performed to obtain data. Results: HSA secondary structure underwent changes in the presence of different concentrations of cortisol and glucose. P values less than 0.01 were considered to be statistically significant. Fluorescence spectroscopy and CD results showed that at normal glucose concentrations, HSA was very flexible, beta-sheet content reduced, and the maximum increase in fluorescence and blue-shift happened. At higher concentrations of glucose, HSA became rigid. Also, HSA in presence of 10 µg/dL cortisol was very flexible; but a cortisol concentration of 40 µg/dL caused stability in HSA structure in presence of different glucose concentrations. Conclusions: Under normal glucose conditions, very low cortisol concentrations create large changes in HSA secondary structure. At normal glucose concentrations, some of the binding sites of HSA that are all occupied by glucose at higher concentrations become available to cortisol. Cortisol structure is very hydrophobic, which causes large changes in HSA secondary structure and significant increases in quenching and blue shift. In conclusion, binding of compounds such as medicines to HSA sites may be affected by competitive bindings of glucose, depending on its concentration in the blood.

Interaction of lafutidine in binding to human serum albumin in gastric ulcer therapy: STD-NMR, WaterLOGSY-NMR, NMR relaxation times, Tr-NOESY, molecule docking, and spectroscopic studies

In this study, lafutidine (LAF) was used as a model compound to investigate the binding mechanism between antiulcer drugs and human serum albumin (HSA) through various techniques, including STD-NMR, WaterLOGSY-NMR, 1H NMR relaxation times, tr-NOESY, molecule docking calculation, FT-IR spectroscopy, and CD spectroscopy. The analyses of STD-NMR, which derived relative STD (%) intensities, and WaterLOGSY-NMR, determined that LAF bound to HSA. In particular, the pyridyl group of LAF was in close contact with HSA binding pocket, whereas furyl group had a secondary binding. Competitive STD-NMR and WaterLOGSY-NMR experiments, with warifarin and ibuprofen as site-selective probes, indicated that LAF preferentially bound to site II in the hydrophobic subdomains IIIA of HSA. The bound conformation of LAF at the HSA binding site was further elucidated by transferred NOE effect (tr-NOESY) experiment. Relaxation experiments provided quantitative information about the relationship between the affinity and structure of LAF. The molecule docking simulations conducted with AutoDock and the restraints derived from STD results led to three-dimensional models that were consistent with the NMR spectroscopic data. The presence of hydrophobic forces and hydrogen interactions was also determined. Additionally, FT-IR and CD spectroscopies showed that LAF induced secondary structure changes of HSA.

Quantitative parameters of complexes of tris(1-alkylindol-3-yl)methylium salts with serum albumin: Relevance for the design of drug candidates

Triarylmethane derivatives are extensively investigated as antitumor and antibacterial drug candidates alone and as photoactivatable compounds. In the series of tris(1-alkylindol-3-yl)methylium salts (TIMs) these two activities differed depending on the length of N-alkyl chain, with C4–5 derivatives being the most potent compared to the shorter or longer chain analogs and to the natural compound turbomycin A (no N-substituent). Given that the human serum albumin (HSA) is a major transporter protein with which TIMs can form stable complexes, and that the formation of these complexes might be advantageous for phototoxicity of TIMs we determined the quantitative parameters of TIMs-HSA binding using spectroscopic methods and molecular docking. TIMs bound to HSA (1:1 stoichiometry) altered the protein's secondary structure by changing the α-helix/β-turn ratio. The IIa subdomain (Sudlow site I) is the preferred TIM binding site in HSA as determined in competition experiments with reference drugs ibuprofen and warfarin. The values of binding constants increased with the number of CH2 groups from 0 to 6 and then dropped down for C10 compound, a dependence similar to the one observed for cytocidal potency of TIMs. We tend to attribute this non-linear dependence to an interplay between hydrophobicity and steric hindrance, the two key characteristics of TIMs-HSA complexes calculated in the molecular docking procedure. These structure-activity relationships provide evidence for rational design of TIMs-based antitumor and antimicrobial drugs.

Investigation of the binding of cis/trans-[MCl4(1H-indazole)(NO)]− (M = Ru, Os) complexes to human serum albumin

Overall binding affinity of sodium or indazolium cis/trans-[MCl4(1H-indazole)(NO)] (M = Ru, Os) complexes towards human serum albumin (HSA) and high molecular mass components of the blood serum was monitored by ultrafiltration. HSA was found to be mainly responsible for the binding of the studied ruthenium and osmium complexes. In other words, this protein can provide a depot for the compounds and can affect their biodistribution and transport processes. In order to elucidate the HSA binding sites tryptophan fluorescence quenching studies and displacement reactions with the established site markers warfarin and dansylglycine were performed. Conditional stability constants for the binding to sites I and II on HSA were computed showing that the studied ruthenium and osmium complexes are able to bind into both sites with moderately strong affinity (logK’ = 4.4–5.1). Site I is slightly more favored over site II for all complexes. No significant differences in the HSA binding properties were found for these metal complexes demonstrating negligible influence of the type of counterion (sodium vs indazolium), the metal ion center identity (Ru vs. Os) or the position of the nitrosyl group on the binding event. Electron paramagnetic resonance spin labeling of HSA revealed that indazolium trans-[RuCl4(1H-indazole)(NO)] and long-chain fatty acids show competitive binding to HSA. Moreover, this complex has a higher affinity for site I, but when present in excess, it is able to bind to site II as well, and displace fatty acids.

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.

Interaction with Serum Albumin As a Factor of the Photodynamic Efficacy of Novel Bacteriopurpurinimide Derivatives

Optimization of the chemical structure of antitumor photosensitizers (PSs) is aimed at increasing their affinity to a transport protein, albumin and irreversible light-induced tumor cell damage. Bacteriopurpurinimide derivatives are promising PSs thanks to their ability to absorb light in the near infrared spectral region. Using spectrophotometry, we show that two new bacteriopurpurinimide derivatives with different substituents at the N atoms of the imide exocycle and the pyrrole ring A are capable of forming non-covalent complexes with human serum albumin (HSA). The association constant (calculated with the Benesi-Hildebrand equation) for N-ethoxybacteriopurpurinimide ethyloxime (compound 1) is higher than that for the methyl ether of methoxybacteriopurpurinimide (compound 2) (1.18×10(5) M-1 vs. 1.26×10(4) M(-1), respectively). Molecular modeling provides details of the atomic interactions between 1 and 2 and amino acid residues in the FA1 binding site of HSA. The ethoxy group stabilizes the position of 1 within this site due to hydrophobic interaction with the protein. The higher affinity of 1 for HSA makes this compound more potent than 2 in photodynamic therapy for cultured human colon carcinoma cells. Photoactivation of 1 and 2 in cells induces rapid (within a few minutes of irradiation) necrosis. This mechanism of cell death may be efficient for eliminating tumors resistant to other therapies.

Interaction between Human Serum Albumin and antidiabetic compounds and its influence on the O2(1Δg)-mediated degradation of the protein

The complexity depicted by disease scenarios as diabetes mellitus, constitutes a very interesting field of study when drugs and biologically relevant components may be affected by such environments. In this report, the interaction between the protein Human Serum Albumin (HSA) and two antidiabetics (Andb), Gliclazide (Gli) and Glipizide (Glip) was studied through fluorescence and docking assays, in order to characterize these systems. On the basis that HSA and Andb can be exposed in vivo at high Reactive Oxygen Species (ROS) concentrations in diabetic patients, the degradative process of the protein free and bound to Andb, in presence of the species singlet molecular oxygen (O2(1Δg)), was evaluated. Fluorescence and docking assays indicated that Gli, as well as Glip bind to HSA on two sites, with binding constants values in the order of 104–105M−1. Likewise, docking assays revealed that the location of Gli or Glip on the protein may be the HSA binding sites II and III. Thermodynamic parameters showed that the interaction between HSA and Glip is a favored, enthalpically-controlled process. Oxygen uptake experiments indicated that Glip is less photooxidizable than Gli through a O2(1Δg)-mediated process. Besides, the protein–Andb binding produced a decrease in the overall rate constant for O2(1Δg) quenching as compared to the value for the free protein. This fact could be interpreted in terms of a reduction in the availability of Tyrosine residues in the bonded protein, with a concomitant decrease in the physical quenching deactivation of the oxidative species.

Spectroscopic analysis of the impact of oxidative stress on the structure of human serum albumin (HSA) in terms of its binding properties

Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) are produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative bursts, exceed the physiological buffering capacity and result in oxidative stress. ROS result in oxidation of serum albumin, which causes a number of structural changes in the spatial structure, may influence the binding and cause significant drug interactions, particularly in polytherapy. During the oxidation modification of amino acid residues, particularly cysteine and methionine may occur.The aim of the study was to investigate the influence of oxidative stress on human serum albumin (HSA) structure and evaluate of possible alterations in the binding of the drug to oxidized human serum albumin (oHSA). HSA was oxidized by a chloramine-T (CT). CT reacts rapidly with sulfhydryl groups and at pH 7.4 the reaction was monitored by spectroscopic techniques. Modification of free thiol group in the Cys residue in HSA was quantitatively determined by the use of Ellman’s reagent. Changes of albumin conformation were examined by comparison of modified (oHSA) and nonmodified human serum albumin (HSA) absorption spectra, emission spectra, red-edge shift (REES) and synchronous spectroscopy. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region of 200–250nm involve structural alterations in peptide backbone conformation. Synchronous fluorescence spectroscopy technique confirmed changes of position of tryptophanyl and tyrosyl residues fluorescent band caused by CT. Moreover analysis of REES effect allowed to observe structural changes caused by CT in the region of the hydrophobic pocket containing the tryptophanyl residue.Effect of oxidative stress on binding of anti-rheumatic drugs, sulfasalazine (SSZ) and sulindac (SLD) in the high and low affinity binding sites was investigated by spectrofluorescence, ITC and 1H NMR spectroscopy, respectively. SSZ and SLD change the affinity of each other to the binding site in non- and modified human serum albumin. The presence of SLD causes the increase of association constant (Ka) of SSZ–oHSA system and the strength of binding and the stability of the complexes has been observed while in the presence of SSZ a displacement of SLD from the SLD–HSA has been recorded. The analysis of 1H NMR spectral parameters i.e. changes of chemical shifts of the drug indicate that the presence of SSZ and SLD have a mutual influence on changes in the affinity of human serum albumin binding site and this competition takes place not only due to the additional drug but also to the oxidation of HSA.

Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein

Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in blood system which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we studied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperine caused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching of proteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The binding constants obtained from fluorescence emission were found to be Kpiperine = 5.7 ± .2 × 105 M−1 and Kpiperine = 9.3± .25 × 104 M−1 which correspond to the free energy of −7.8 and −6.71 kcal M−1at 25 °C for HSA and AGP, respectively. Further, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA due to partial destabilization of HSA–piperine complexes. Consequently, inference drawn from the site-specific markers (phenylbutazone, site I marker) studies to identify the binding site of HSA noticed that piperine binds at site I (IIA), which was further authenticated by molecular docking and molecular dynamic (MD) studies. The binding constants and free energy corresponding to experimental and computational analysis suggest that there are hydrophobic and hydrophilic interactions when piperine binds to HSA. Additionally, the MD studies have showed that HSA–piperine complex reaches equilibration state at around 3 ns, which prove that the HSA–piperine complex is stable in nature.

Study of the binding interaction between fluorinated matrix metalloproteinase inhibitors and Human Serum Albumin

Fluorinated, arylsulfone-based inhibitors of Matrix Metalloproteinases (MMP) have been used, in the [18F]-radiolabelled version, as radiotracers targeted to MMP-2/9 for Positron Emission Tomography (PET). Although they showed acceptable tumour uptake, specificity was rather low. To get further insights into the reason of low specificity, the binding interaction of these compounds with Human Serum Albumin (HSA) has been investigated. 19F NMR spectroscopy showed that all compounds considered partition between multiple HSA binding sites, being characterized by either slow-exchange kinetics (with Ka in the order of 105 M−1) and fast-exchange kinetics (with Ka in the order of 104 M-1). For 2-(2-(4′-(2-fluoroethoxy)biphenyl-4-ylsulfonyl)phenyl)acetic acid (1a) and 2-(2-(4′-(2-fluoroacetamido)biphenyl-4-ylsulfonyl)phenyl)acetic acid (1c), these slow and fast-exchanging binding sites could be mapped to Sudlow's site I and II, respectively. It is shown that high affinity albumin binding constitutes a theoretical limitation for the specificity achievable by MMP-inhibitors as MMP-targeted PET tracers in cancer imaging, because albumin accumulating aspecifically in tumours lowers the binding potential of radiotracers.

A principal component analysis of the dynamics of subdomains and binding sites in human serum albumin

The conformational dynamics of human serum albumin (HSA) was investigated by principal component analysis (PCA) applied to three molecular dynamics trajectories of 200 ns each. The overlap of the essential subspaces spanned by the first 10 principal components (PC) of different trajectories was about 0.3 showing that the PCA based on a trajectory length of 200 ns is not completely convergent for this protein. The contributions of the relative motion of subdomains and of the subdomains (internal) distortion to the first 10 PCs were found to be comparable. Based on the distribution of the first 3 PC, 10 protein conformers are identified showing relative root mean square deviations (RMSD) between 2.3 and 4.6 Å. The main PCs are found to be delocalized over the whole protein structure indicating that the motions of different protein subdomains are coupled. This coupling is considered as being related to the allosteric effects observed upon ligand binding to HSA. On the other hand, the first PC of one of the three trajectories describes a conformational transition of the protein domain I that is close to that experimentally observed upon myristate binding. This is a theoretical support for the older hypothesis stating that changes of the protein onformation favorable to binding can precede the ligand complexation. A detailed all atoms PCA performed on the primary Sites 1 and 2 confirms the multiconformational character of the HSA binding sites as well as the significant coupling of their motions.

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.

Spectroscopic study of interaction between osthole and human serum albumin: Identification of possible binding site of the compound

The studies on the interaction between human serum albumin (HSA) and drugs have been an interesting research field in life science, chemistry and clinical medicine. Osthole possesses a variety of pharmacological activities including anti-tumor, anti-inflammation, anti-seizure, anti-hyperlipidemic and anti-osteoporosis effects. The interaction of osthole with HSA and its binding site in HSA by spectroscopic methods is the subject of this work. By monitoring the intrinsic fluorescence of the single Trp214 residue and performing site markers displacement measurements, the specific binding of osthole in the vicinity of Sudlow's site I of HSA has been clarified. The changes in the secondary structure of HSA after its complexation with ligand were studied with CD spectroscopy, which indicate that osthole induced only a slight decrease in the helix structural content of the protein. In addition, the mean distance between osthole and HSA fluorophores is estimated to be 4.96nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Osthole can quench the intrinsic fluorescence of HSA by dynamic quenching, and analysis of the thermodynamic parameters of binding showed that hydrophobic interactions play an important role in the stabilizing of the complex. Increase of protein surface hydrophobicity (PSH) was also observed upon the osthole binding.

Binding of Hydroxyquinoline Probes to Human Serum Albumin: Combining Molecular Modeling and Förster’s Resonance Energy Transfer Spectroscopy to Understand Flexible Ligand Binding

Human serum albumin (HSA) is the most abundant protein in blood plasma. It has high relevance for the lipid metabolism, and its ability to bind a large variety of natural and pharmaceutical compounds makes it a crucial determinant of drug pharmaco-kinetics and -dynamics. The drug binding properties of HSA can be characterized by spectroscopic analysis of bound probe molecules. We have recently characterized the subdomain IIA binding site of HSA using three hydroxyquinoline derivatives. In this work, we extend our study by combining data from energy transfer experiments, ligand docking, and long molecular dynamics (MD) simulations. Multiple possible binding locations are found within the subdomain IIA site, and their solvent accessibility and interactions with ligands are analyzed in detail. Binding pockets appear well hydrated during simulations, with ligands in direct contact to water molecules at all times. Binding free energies in good agreement to experiment are calculated. The HSA apoprotein is found to exhibit significant conformational flexibility over 250 ns of simulation time, but individual domains remain structurally stable. Two rotamers of Trp214 were observed on a time scale longer than 50 ns in the MD simulations, supporting the experimental observation of two fluorescence lifetime components. The flexible protein structure and heterogeneous nature of its binding sites explain the ability of HSA to act as a versatile molecular transporter. The combination of experimental and computational molecular distance information allows the conclusion that hydroxyquinoline probes bind in a binding mode similar to the anticoagulant drug warfarin.

Pharmacokinetic Alteration of 99mTc-MAG3 using Serum Protein Binding Displacement Method

When a radiopharmaceutical is simultaneously administered with a medicine that has high affinity for the same plasma protein, the radiopharmaceutical is released at higher concentrations in blood, leading to enhanced transfer into target tissues. This is known as the serum protein binding displacement method. In this study, we investigated the pharmacokinetic alteration of technetium-99m-labeled mercaptoacetylglycylglycylglycine ((99m)Tc-MAG3) using the serum protein binding displacement method. Rat and human serum protein binding rates of (99m)Tc-MAG3 were measured by ultrafiltration with or without displacers of human serum albumin (HSA) binding sites I and II (200μM and 400μM loading). Male Wistar rats were injected with (99m)Tc-MAG3 (740kBq/0.3mL saline) via the tail vein, and biodistribution was assessed at 2, 5, 10 and 15min. Dynamic whole-body images were obtained for (99m)Tc-MAG3 (11.1MBq/0.3mL saline)-injected rats, with or without HSA displacers. (99m)Tc-MAG3 strongly bound to HSA (87.37%±2.13%). Using HSA site I displacers, the free fraction of (99m)Tc-MAG3 increased significantly (1.20 to 1.47 times) when compared with controls. For biodistribution and imaging, rapid blood clearance was observed with bucolome (BCL) loading, which is an HSA site I displacer. With BCL loading, peak times for rat renograms were respectively shifted from 240s to 110s, and from 170s to 120s. We found that (99m)Tc-MAG3 bound to the HSA binding site I. It was confirmed that pharmacokinetic distribution of (99m)Tc-MAG3 is altered by presence of BCL, which leads to increases in the free fraction of (99m)Tc-MAG3, and BCL produced rapid blood clearance and fast peak times on rat renograms. The serum protein binding displacement method using (99m)Tc-MAG3 and BCL, a safe displacer for humans, may be applicable to clinical study and lead to better diagnostic images with shorter waiting times and lower radiation doses for patients.

Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies

Indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) and its Na(+) analogue (KP1339) are two of the most prominent non-platinum antitumor metal complexes currently undergoing clinical trials. After intravenous administration, they are known to bind to human serum albumin (HSA) in a noncovalent manner. To elucidate their HSA binding sites, displacement reactions with the established site markers warfarin and dansylglycine as well as bilirubin were monitored by spectrofluorimetry, ultrafiltration-UV-vis spectrophotometry, and/or capillary zone electrophoresis. Conditional stability constants for the binding of KP1019 and KP1339 to sites I and II of HSA were determined, indicating that both Ru(III) compounds bind to both sites with moderately strong affinity (log K(1)' = 5.3-5.8). No preference for either binding site was found, and similar results were obtained for both metal complexes, demonstrating low influence of the counter ion on the binding event.

Analysis of drug interactions with modified proteins by high-performance affinity chromatography: Binding of glibenclamide to normal and glycated human serum albumin

High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, Ka, 1.4–1.9×106M−1 at pH 7.4 and 37°C) and lower affinity sites (Ka, 4.4–7.2×104M−1). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood.

PH-Dependent Complexation of Histamine H1 Receptor Antagonists and Human Serum Albumin Studied by UV Resonance Raman Spectroscopy

UV resonance Raman spectroscopy was used to characterize the binding of three first-generation histamine H(1) receptor antagonists-tripelennamine (TRP), mepyramine (MEP), and brompheniramine (BPA)-to human serum albumin (HSA) at pH 7.2 and pH 9.0. Binding constants differ at these pH values, which can be ascribed to the different extent of protonation of the ethylamino side chain of the ligands. We have recently shown [Tardioli et al. J. Raman Spectrosc. 2011, 42, 1016-1024] that for the solution conformation of TRP and MEP the side chain plays an important role by allowing an internal hydrogen bond with the aminopyridine nitrogen in TRP and MEP. Results presented in this paper suggest that the existence of such molecular structures has serious biological significance on the binding affinity of those ligands to HSA. At pH 7.2, only the stretched conformers of protonated TRP and MEP bind in HSA binding site I. Using UV absorption data, we derived binding constants for the neutral and protonated forms of TRP to HSA. The neutral species seems to be conjugated to a positive group of the protein, affecting both the tryptophan W214 and some of the tyrosine (Y) vibrations. BPA, for which the structure with an intramolecular hydrogen bonded side chain is not possible, is H bound to the indole ring nitrogen of W214, of which the side chain rotates over a certain angle to accommodate the drug in site I. We propose that the protonated BPA is also bound in site I, where the Y150 residue stabilizes the presence of this compound in the binding pocket. No spectroscopic evidence was found for conformational changes of the protein affecting the spectroscopic properties of W and Y in this pH range.

5-Imino-1,2-4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors: Determination of blood–brain barrier penetration and binding to human serum albumin

5-Imino-1,2,4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors were characterized for their ability to pass the blood–brain barrier (BBB) together with their human serum albumin (HSA) binding using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). To study the blood–brain barrier penetration, a parallel artificial membrane permeability assay (PAMPA) using a porcine brain lipid was employed. For the HPLAC investigation, HSA was previously covalently immobilized to the silica matrix of the HPLC column. This HSA-based column was used to characterize the high affinity binding sites of 5-imino-1,2,4-thiadiazoles and quinazolines derivatives to HSA. Displacement experiments in the presence of increasing concentrations of competitors known to bind selectively to the main binding sites of HSA were carried out to determine their possible binding site. The same drug–protein system was studied by CD.The analysed compounds were able to pass BBB, they present good drug-like properties and they showed a high affinity to HSA. Competition experiments showed an anticooperative interaction at sites I and II, and an independent binding at bilirubin binding site on HSA.

Evaluation of the binding of oxovanadium(iv) to human serum albumin

The understanding of the biotransformations of insulin mimetic vanadium complexes in human blood and its transport to target cells is an essential issue in the development of more effective drugs. We present the study of the interaction of oxovanadium(iv) with human serum albumin (HSA) by electron paramagnetic resonance (EPR), circular dichroism (CD) and visible absorption spectroscopy. Metal competition studies were done using Cu(II) and Zn(II) as metal probes. The results show that V(IV)O occupies two types of binding sites in albumin, which compete not only with each other, but also with hydrolysis of the metal ion. In one of the sites the resulting V(IV)O-HSA complex has a weak visible CD signal and its X-band EPR spectrum may be easily measured. This was assigned to amino acid side chains of the ATCUN site. The other binding site shows stronger signals in the CD in the visible range, but has a hardly measurable EPR signal; it is assigned to the multi metal binding site (MBS) of HSA. Studies with fatted and defatted albumin show the complexity of the system since conformational changes, induced by the binding of fatty acids, decrease the ability of V(IV)O to bind albumin. The possibility and importance of ternary complex formation between V(IV)O, HSA and several drug candidates - maltol (mal), picolinic acid (pic), 2-hydroxypyridine-N-oxide (hpno) and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone (dhp) was also evaluated. In the presence of maltol the CD and EPR spectra significantly change, indicating the formation of ternary VO-HSA-maltol complexes. Modeling studies with amino acids and peptides were used to propose binding modes. Based on quantitative RT EPR measurements and CD data, it was concluded that in the systems with mal, pic, hpno, and dhp (V(IV)OL(2))(n)(HSA) species form, where the maximum value for n is at least 6 (mal, pic). The degree of formation of the ternary species, corresponding to the reaction V(IV)OL(2) + HSA -->/<-- V(IV)OL(2)(HSA) is hpno > pic ≥ mal > dhp. (V(IV)OL)(n)(HSA) type complexes are detected exclusively with pic. Based on the spectroscopic studies we propose that in the (V(IV)OL(2))(n)(HSA) species the protein bounds to vanadium through the histidine side chains.