Species differences in the interaction of serum albumin with antibiotics

Interaction of oxacillin, cloxacillin, dicloxacillin, phenoxymethylpenicillin, methicillin, nafcillin and benzylpenicillin with human serum albumin (HSA) was studied with flow microcalorimetry and differential scanning calorimetry. The measured thermodynamic parameters of complex formation between the penicillins and HSA were compared with similar characteristics of their binding to bovine serum albumin. It was shown that there were species differences between these two globular proteins in their interaction with the above antibiotics in relation to both the number of the biopolymer active sites and the nature of the molecular forces in the complex formation. The effect of the first bound molecule of oxacillin, cloxacillin, dicloxacillin, nafcillin, phenoxymethylpenicillin and benzylpenicillin on HSA conformation was observed. It was demonstrated that there was thermostabilization of HSA on its interaction with the above drugs with preserving cooperative nature of thermal denaturation of the complexes in relation to HSA melting.     

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.     

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.     

Structural basis of the drug-binding specificity of human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that binds a remarkably wide range of drugs, thereby restricting their free, active concentrations. The problem of overcoming the binding affinity of lead compounds for HSA represents a major challenge in drug development. Crystallographic analysis of 17 different complexes of HSA with a wide variety of drugs and small-molecule toxins reveals the precise architecture of the two primary drug-binding sites on the protein, identifying residues that are key determinants of binding specificity and illuminating the capacity of both pockets for flexible accommodation. Numerous secondary binding sites for drugs distributed across the protein have also been identified. The binding of fatty acids, the primary physiological ligand for the protein, is shown to alter the polarity and increase the volume of drug site 1. These results clarify the interpretation of accumulated drug binding data and provide a valuable template for design efforts to modulate the interaction with HSA.     

Allosteric modulation of anti-HIV drug and ferric heme binding to human serum albumin

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional capacity to bind ligands (e.g. heme and drugs). Here, binding of the anti-HIV drugs abacavir, atazanavir, didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, and zidovudine to HSA and ferric heme-HSA is reported. Ferric heme binding to HSA in the absence and presence of anti-HIV drugs was also investigated. The association equilibrium constant and second-order rate constant for the binding of anti-HIV drugs to Sudlow's site I of ferric heme-HSA are lower by one order of magnitude than those for the binding of anti-HIV drugs to HSA. Accordingly, the association equilibrium constant and the second-order rate constant for heme binding to HSA are decreased by one order of magnitude in the presence of anti-HIV drugs. In contrast, the first-order rate constant for ligand dissociation from HSA is insensitive to anti-HIV drugs and ferric heme. These findings represent clear-cut evidence for the allosteric inhibition of anti-HIV drug binding to HSA by the heme. In turn, anti-HIV drugs allosterically impair heme binding to HSA. Therefore, Sudlow's site I and the heme cleft must be functionally linked.     

Competitive binding of fluoroquinolone antibiotics and some other drugs to human serum albumin: a luminescence spectroscopic study

Co‐administration of several drugs in multidrug therapy may alter the binding of each to human serum albumin (HSA) and hence their pharmacological activity. Thirty‐two frequently prescribed drug combinations, consisting of four fluoroquinolone antibiotics and eight competing drugs, have been studied using fluorescence and circular dichroism spectroscopic techniques. Competitive binding studies on the drug combinations are not available in the literature. In most cases, the presence of competing drug decreased the binding affinity of fluoroquinolone, resulting in an increase in the concentration of free pharmacologically active drug. The competitive binding mechanism involved could be interpreted in terms of the site specificity of the binding and competing drugs. For levofloxacin, the change in the binding affinity was small because in the presence of site II‐specific competing drugs, levofloxacin mainly occupied site I. A competitive interference mechanism was operative for sparfloxacin, whereas competitive interference as well as site‐to‐site displacement of competing drugs was observed in the case of ciprofloxacin hydrochloride. For enrofloxacin, a different behavior was observed for different combinations; site‐to‐site displacement and conformational changes as well as independent binding has been observed for various drug combinations. Circular dichroism spectral studies showed that competitive binding did not cause any major structural changes in the HSA molecule. Copyright © 2013 John Wiley & Sons, Ltd.     

Drug–drug plasma protein binding interactions of ivacaftor

Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR‐protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co‐administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co‐administered CF drugs for human serum albumin (HSA) and α₁‐acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site‐selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug–drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug–drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug–drug interactions of ivacaftor with co‐administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of drug-human serum albumin binding interactions with support vector machine aided online automated docking

MOTIVATION: Human serum albumin (HSA), the most abundant plasma protein is well known for its extraordinary binding capacity for both endogenous and exogenous substances, including a wide range of drugs. Interaction with the two principal binding sites of HSA in subdomain IIA (site 1) and in subdomain IIIA (site 2) controls the free, active concentration of a drug, provides a reservoir for a long duration of action and ultimately affects the ADME (absorption, distribution, metabolism, and excretion) profile. Due to the continuous demand to investigate HSA binding properties of novel drugs, drug candidates and drug-like compounds, a support vector machine (SVM) model was developed that efficiently predicts albumin binding. Our SVM model was integrated to a free, web-based prediction platform (http://albumin.althotas.com). Automated molecular docking calculations for prediction of complex geometry are also integrated into the web service. The platform enables the users (i) to predict if albumin binds the query ligand, (ii) to determine the probable ligand binding site (site 1 or site 2), (iii) to select the albumin X-ray structure which is complexed with the most similar ligand and (iv) to calculate complex geometry using molecular docking calculations. Our SVM model and the potential offered by the combined use of in silico calculation methods and experimental binding data is illustrated.     

Binding analysis of glycyrrhetinic acid to human serum albumin: fluorescence spectroscopy, FTIR, and molecular modeling

Fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), and molecular modeling methods were employed to analyze the binding of glycyrrhetinic acid (GEA) to human serum albumin (HSA) under physiological conditions with GEA concentrations from 4.0x10(-6) to 4.5x10(-5) mol L(-1). The binding of GEA to HSA was via two types of sites: the numbers of binding site for the first type was near 0.45 and for the second type it was approximately 0.75. The binding constants of the second type binding site were lower than those of the first type binding site at corresponding temperatures, the results suggesting that the first type of binding site had high affinity and the second binding site involved other sites with lower binding affinity and selectivity. The fluorescence titration results indicated that GEA quenched the fluorescence intensity of HSA through static mechanism. The FTIR spectra evidence showed that the protein secondary structure changed with reduction of alpha-helices about 26.2% at the drug to protein molar ratio of 3. Thermodynamic analysis showed that hydrogen bonds were the mainly binding force in the first type of binding site, and hydrophobic interactions might play a main role in the second type of binding site. Furthermore, the study of computational modeling indicated that GEA could bind to the site I of HSA and hydrophobic interaction was the major acting force for the second type of binding site, which was in agreement with the thermodynamic analysis.     

Analysis of binding interaction between the natural apocarotenoid bixin and human serum albumin by circular dichroism and fluorescence spectroscopy

Bixin is an important, pharmacologically active dietary cis-carotenoid, but its interaction with potential macromolecular targets is completely unexplored. This work was aimed to study the binding of bixin to human serum albumin (HSA), the most abundant protein in blood plasma. Circular dichroism (CD) spectroscopy in combination with UV/VIS absorption spectroscopy and fluorescence quenching techniques were applied. Appearance of induced CD bands in the UV- and VIS-absorption spectral regions indicated the formation of non-covalent carotenoid-albumin complexes. Shape and spectral position of the extrinsic Cotton effects suggested the binding of a single bixin molecule to HSA in chiral conformation. Scatchard and non-linear regression analyses of CD titration data resulted in similar values for the association constant (Ka = 6.6 and 4.6x10(5) M(-1), resp.) and for the number of binding sites (n = 1). The binding interaction was independently confirmed by fluorescence-quenching experiment from which the binding parameters were also calculated. CD Displacement measurements performed with marker ligands established that the main drug binding sites of HSA are not involved in binding of bixin. Palmitic acid decreased the amplitude of the induced CD bands suggesting a common albumin binding site for bixin and long-chain fatty acids. The above data indicate that HSA plays a significant role in the plasma transportation of bixin and related dietary carboxylic acid carotenoids.     

Affinity and Specificity of Levamlodipine-Human Serum Albumin Interactions: Insights into Its Carrier Function

The affinity and specificity of drugs with human serum albumin (HSA) are crucial factors influencing the bioactivity of drugs. To gain insight into the carrier function of HSA, the binding of levamlodipine with HSA has been investigated as a model system by a combined experimental and theoretical/computational approach. The fluorescence properties of HSA and the binding parameters of levamlodipine indicate that the binding is characterized by one binding site with static quenching mechanism, which is related to the energy transfer. As indicated by the thermodynamic analysis, hydrophobic interaction is the predominant force in levamlodipine-HSA complex, which is in agreement with the computational results. And the hydrogen bonds can be confirmed by computational approach between levamlodipine and HSA. Compared to predicted binding energies and binding energy spectra at seven sites on HSA, levamlodipine binding HSA at site I has a high affinity regime and the highest specificity characterized by the largest intrinsic specificity ratio (ISR). The binding characteristics at site I guarantee that drugs can be carried and released from HSA to carry out their specific bioactivity. Our concept and quantification of specificity is general and can be applied to other drug-target binding as well as molecular recognition of peptide-protein, protein-protein, and protein-DNA interactions.     

In vitro binding of leukotriene B4 (LTB4) to human serum albumin: evidence from spectroscopic, molecular modeling, and competitive displacement studies

Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B4 (LTB4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB4 was enhanced fivefold in the presence of HSA. The red-shifted, hypochromic, and reduced vibrational fine structure of the ligand/protein UV absorption spectrum indicated complexation of the two molecules in solution. Results obtained from CD titration experiments were subjected to non-linear regression analysis to estimate the binding parameters (Ka = 6.7 x 10(4) M(-1), n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB4/HSA complex, suggesting the role of a high-affinity fatty acid HSA binding site in the leukotriene complexation. Molecular modeling calculations based on the crystal structure of HSA predicted that the long-chain fatty acid site that overlaps with drug binding site II in subdomain IIIA was the most likely binding location for LTB4. Using the drug site II-specific marker ligand rac-ibuprofen, this prediction was confirmed with induced-CD displacement measurements. To the authors' knowledge, the current study represents the first demonstration of binding of LTB4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.     

Probing the interaction of thionine with human serum albumin by multispectroscopic studies and its in vitro cytotoxic activity toward MCF-7 breast cancer cells

The studies on protein–dye interactions are important in biological process and it is regarded as vital step in rational drug design. The interaction of thionine (TH) with human serum albumin (HSA) was analyzed using isothermal titration calorimetry (ITC), spectroscopic, and molecular docking technique. The emission spectral titration of HSA with TH revealed the formation of HSA–TH complex via static quenching process. The results obtained from absorption, synchronous emission, circular dichroism, and three-dimensional (3D) emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure of HSA. Results from ITC experiments suggested that the binding of TH dye was favored by negative enthalpy and a favorable entropy contribution. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of HSA. Molecular docking study further substantiates that TH binds to the hydrophobic cavity of subdomain IIA (Sudlow site I) of HSA. Further, we have studied the cytotoxic activity of TH and TH–HSA complex on breast cancer cell lines (MCF-7) by MTT assay and LDH assay. These studies revealed that TH–HSA complex showed the higher level of cytotoxicity in cancer cells than TH dye-treated MCF-7 cells and the significant adverse effect did not found in the normal HBL-100 cells. Fluorescence microscopy analyses of nuclear fragmentation studies validate the significant reduction of viability of TH–HSA-treated human MCF-7 breast cancer cells through activation of apoptotic-mediated pathways.     

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.     

Multi-spectroscopic and molecular modelling approach to investigate the interaction of riboflavin with human serum albumin

Riboflavin (RF) plays an important role in various metabolic redox reactions in the form of flavin adenine dinucleotide and flavin mononucleotide. Human serum albumin (HSA) is an important protein involved in the transportation of drugs, hormones, fatty acid and other molecules which determine the biodistribution and physiological fate of these molecules. In this study, we have investigated the interaction of riboflavin RF with HSA under simulative physiological conditions using various biophysical, calorimetric and molecular docking techniques. Results demonstrate the formation of riboflavin–HSA complex with binding constant in the order of 10⁴ M^?1. Fluorescence spectroscopy confirms intermediate strength having a static mode of quenching with stoichiometry of 1:1. Experimental results suggest that the binding site of riboflavin mainly resides in sub-domain IIA of HSA and that ligand interaction increases the α-helical content of HSA. These parameters were further verified by isothermal titration calorimetry ITC which confirms the thermodynamic parameters obtained by fluorescence spectroscopy. Molecular docking was employed to suggest a binding model. Based on thermodynamic, spectroscopic and computational observations it can be concluded that HSA-riboflavin complex is mainly stabilized by various non-covalent forces with binding energy of ?7.2 kcal mol^?1.     

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.     

Structural interrogation of phosphoproteome identified by mass spectrometry reveals allowed and disallowed regions of phosphoconformation

Background

Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.

Results

The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.

Conclusions

The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.     

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.