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Aug 17, 2015 - (CEL) and argpyrimidine (Arg-P), was studied on the basis ... albumin (BSA) for the formation of Arg-P and CEL followed by drug interac...
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Advanced Glycation End Products Modulate Structure and Drug Binding Properties of Albumin Saurabh Awasthi,† N. Arul Murugan,‡ and N. T. Saraswathi*,† †

Molecular Biophysics Lab, School of Chemical and Biotechnology, SASTRA University, Thanjavur-613401, Tamilnadu, India Division of Theoretical Chemistry and Biology, School of Biotechnology, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden

‡

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ABSTRACT: The extraordinary ligand binding properties of albumin makes it a key player in the pharmacokinetics and pharmacodynamics of many vital drugs. Albumin is highly susceptible for nonenzymatic glycation mediated structural modifications, and there is a need to determine structural and functional impact of specific AGEs modifications. The present study was aimed toward determining the AGE mediated structure and function changes, primarily looking into the effect on binding affinity of drugs in the two major drug binding sites of albumin. The impact of the two most predominant AGEs modifications, i.e., carboxyethyllysine (CEL) and argpyrimidine (Arg-P), was studied on the basis of the combination of in vitro and in silico experiments. In vitro studies were carried out by AGEs modification of bovine serum albumin (BSA) for the formation of Arg-P and CEL followed by drug interaction studies. In silico studies involved molecular dynamics (MD) simulations and docking studies for native and AGEs modified BSAs. In particular the side chain modification was specifically carried out for the residues in the drug binding sites, i.e., Arg-194, Arg-196, Arg-198, and Arg-217, and Lys-204 (site I) and Arg-409 and Lys-413 (site II). The equilibrated structures of native BSA (n-BSA) and glycated BSA (G-BSA) as obtained from MD were used for drug binding studies using molecular docking approach. It was evident from the results of both in vitro and in silico drug interaction studies that AGEs modification results in the reduced drug binding affinity for tolbutamide (TLB) and ibuprofen (IBP) in sites I and II. Moreover, the AGEs modification mediated conformational changes resulted in the shallow binding pockets with reduced accessibility for drugs. KEYWORDS: advanced glycation end products, albumin, argpyrimidine, carboxyethyllysine, drug binding affinity, diabetes, molecular dynamics simulation

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fluorescent AGEs, i.e., argpyrimidine (Arg-P).8 Recent studies based on chemical and spectroscopic methods have shown that the major fluorescent product of methylglyoxal modification is Nδ-(5-methylimidazolon-2-yl)-L-ornithine (5-methylimidazolone), which is identical to Nδ-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine).9−11 In another mass spectroscopic study of methylglyoxal modified albumin, the major product of arginine modification was found to be hydroimidazolone Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine with minor formation of argpyrimidine and methylglyoxal lysine dimer.12 Carboxyethyllysine (CEL) is the product of lysine modification by pyruvic acid (Figure 2a). Arg-P and CEL have been recognized as one of the most predominant AGEs formed under hyperglycemic conditions. 13 Arg-P accumulation has been found to be the key pathological factor

INTRODUCTION Formation of advanced glycation end products (AGEs) is a key factor in the development of various complications such as Alzheimer’s disease,1 cataract,2 Parkinson’s disease3 and chronic kidney disease.4 Hyperglycemic conditions where the blood glucose level goes above normal result in increased flux of glucose and its metabolic intermediates toward different pathways contributing to the formation of highly toxic pro-oxidants called AGEs.5 AGEs are highly heterogeneous compounds which have been classified as fluorescent, nonfluorescent, cross-linking, and non-cross-linking.6 Figure 1 presents a schematic representation for the nonenzymatic glycation reaction leading to the formation AGEs. Glyoxylic acid (GA), pyruvic acid (PA), and methylglyoxal (MG) are the metabolic intermediates of glucose formed through the polyol pathway. Hyperglycemic conditions result in the increased flux of glucose through this pathway leading to the formation and accumulation of AGEs at an accelerated rate.7 Methylglyoxal is the most reactive oxaldehyde formed in vivo from the glycolytic pathway, which leads to the modification of arginine residues, resulting in the formation of highly toxic © 2015 American Chemical Society

Received: Revised: Accepted: Published: 3312

April 23, 2015 July 21, 2015 August 17, 2015 August 17, 2015 DOI: 10.1021/acs.molpharmaceut.5b00318 Mol. Pharmaceutics 2015, 12, 3312−3322

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Molecular Pharmaceutics

can largely impact the pharmacokinetics and pharmacodynamics of many drugs and other ligands interacting with albumin.16 Arginine and lysine residues are the most common glycation sites in albumin both in vivo and in vitro. The drug binding sites in albumin are located in the subdomain IA (Sudlow’s site I), subdomain IIIA (Sudlow’s site II), and a newly studied subdomain IB as a major drug binding region.17 The seven fatty acid binding sites located in three different domains of albumin make it the major depot for the fatty acids and hormones in the body.18 Site I in albumin is preferred for binding of anionic heterocyclic drugs whereas site II is preferentially occupied by ligands having aromatic carboxylate groups with extended conformations.19,20 Recently, a number of studies have been carried out using both human serum albumin (HSA) and BSA to determine the effect of glycation on the structure and ligand binding properties of albumin,21 yet the impact of individual AGEs remains undefined. With this motivation the present study was aimed to determine the effect of two AGEs specifically Arg-P and CEL on the ligand interaction properties of albumin in sites I and II. Nonenzymatic glycation results in the formation of a highly heterogeneous group of end products, which has been the major limiting factor in using the X-ray crystallographic approach for elucidating the structure of glycated proteins. Computational modeling approaches, in particular, molecular dynamics (MD) simulations, can be used to obtain the structure of glycated end products given that the structure of native protein is available. Moreover, the glycation induced changes in the secondary structure and conformation and drug binding ability of the macromolecular molecular structure can be studied in detail. Currently, MD simulations are extensively used in the study of structure, dynamics, and function of many biomacromolecules.22 In the current study, we have used MD simulations to obtain the room temperature structures of both native and AGEs modified BSA, which furthermore have been used to study their drug binding ability. Two drugs, namely, tolbutamide and ibuprofen, were used to determine the changes in the drug binding properties of BSA on AGEs modification. Tolbutamide belongs to the sulfonylurea

Figure 1. Schematic representation depicting the nonenzymatic glycation and formation of advanced glycation end products (AGEs). Glucose reacts with the free amino groups in proteins and leads to the formation of intermediate Amadori product by a reversible reaction. Amadori product undergoes oxidative modification and gives rise to the formation of a range of heterogeneous compounds called AGEs. The increased AGEs levels in the body are fundamental to the structure function modification of proteins, amyloid aggregation, and severe complications such as Alzheimer’s disease, Parkinson’s disease, cataract, skin aging, and glycation of serum proteins.

in the induced apoptosis in the lens epithelial cells and development of familial amyloidotic polyneuropathies (FAPs).8,9 Both extracellular and intracellular proteins have been found to be prone to glycation induced modification in vivo both in normal and in hyperglycemic conditions.14 Serum albumin is the most abundant protein and major carrier for both endogenous and exogenous ligands in the blood. Albumin is a protein that is highly prone to glycation induced structural modifications which can directly impact its functions.15 Albumin being the major drug carrier in the body, its glycation

Figure 2. AGEs modification of (a) lysine and (b) arginine residue for the formation of CEL and Arg-P. Methylglyoxal reacts irreversibly with the arginine side chain and gives rise to the formation of argpyrimidine (Arg-P). Pyruvic acid (PA) specifically reacts with the lysine side chain leading to the formation of CEL modified BSA. 3313

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incubated with 250 μL of 0.01% TNBSA for 2 h at room temperature. After incubation, 250 μL of 10% SDS and 1 N HCl was added and absorbance was read at 335 nm using a Thermo Scientific Evolution-201 spectrophotometer. Freshly prepared BSA sample was used as a control. Molecular Dynamics Simulations. Amber28 was used to perform MD simulations for n-BSA and AGEs modified BSA. The structure for native BSA was based on the structure with reference ID 4F5S as reported in the protein database. To propose the starting structure for AGEs modified BSA, side chain modifications were carried out for Arg-194, Arg-196, Arg-198, Arg-217, Lys-204 (site I residues) and Arg-409 and Lys-413 (site II residues) in the structure of native BSA. The arginine residues were modified for the formation of argpyrimidine and lysine residues as carboxyethyllysine. Amber force field has been used for describing the interactions of amino acids while general Amber force field has been used for describing the modified residues. To describe the water solvent, we employed the TIP3P model. The simulation box for native and AGEs modified BSA included approximately 31900 water molecules. We have added 17 and 22 sodium ions to neutralize the native and AGEs modified BSAs, respectively. The simulation box dimensions were, approximately, 124, 94, and 106 Å. The simulations were carried out in isothermal−isobaric ensemble at 300 K and 1 atmospheric pressure. The time step for the integration of the equation of motion was 2 femtoseconds (fs), and the time scale for equilibration was 1 ns. The total time scale for production run was 50 ns, which was sufficient to capture the glycation induced changes in native BSA. The last configurations of the production run for both native and AGEs modified BSA have been used for the analysis of drug binding ability. The secondary structure analysis for both BSAs has been carried out for the last 4 ns trajectory from MD, which included 1000 configurations. In particular, this analysis has been carried out using the timeline module as implemented in VMD software. Drug Binding Pocket Analysis. Drug binding pocket analysis was carried out for native and modified form of the BSA using DoGSiteScorer developed at the University of Hamburg, Germany, in cooperation with Merck kgA and BioSolveIT.29 A grid based function method is used by DoGSiteScorer, which uses a difference of Gaussian filters to detect potential pockets on the protein surface. The other properties of protein such as volume, depth, and surface, as well as functional group present in the pocket, were also analyzed. In Vitro Drug Binding. Fluorometric titrations for drug binding studies were carried out using Jasco-FP8200 spectrofluorimeter. The fluorescence spectra were recorded with λex of 280 nm and λem in the range of 300 to 400 nm. The slit width for both the excitation and emission monochromator was set at 2.5 nm. Both native and modified BSA (3 mL) was titrated against the range of concentrations for both the drugs, and the fluorescence intensity was recorded after an equilibration period of 1 min. The concentration of BSA was kept constant at 1 mg mL−1 and drug concentration range from 20 to 500 μM. All the titrations were repeated thrice to obtain the average values for binding constants. Molecular Docking. Both native and AGEs modified forms of BSA were used for a comparative analysis for drug binding by employing the molecular docking approach. The sdf files for TLB and IBP were retrieved from PubChem database and converted into .pdb format using MarvinSketch Version 15.6.29.0 developed by ChemAxon. The preprocessing of both protein and ligands was carried out with AutoDock tools for protein and

class of antidiabetic drug which binds in the drug site I of BSA whereas ibuprofen is an anti-inflammatory drug based its inhibition activity for COX-1 and COX-223 and binds in site II.24 Experiments based on fluorescence measurements were carried out to determine the drug binding properties for native and AGEs modified BSA. This also has been validated through in silico molecular modeling. To determine the structural changes in BSA due to site specific AGEs modification of lysine and arginine residues, molecular dynamics simulations were carried out. Furthermore, the changes in the secondary structure due to AGEs modification have been investigated by comparing the secondary structure of both native and AGEs modified BSAs. The structural properties analyzed for AGEs modification of the BSA include the binding pocket volume, accessible surface area, pocket depth, and hydrophobic surface. Molecular docking studies were carried out to determine the changes in the molecular interactions of drugs in sites I and II of native and modified forms. The results suggest asymmetric conformational changes in BSA due to AGEs modification and decrease in the drug binding affinity for both sites I and II. Arg-P modification of BSA led to comparatively poorer drug binding than CEL.

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MATERIALS AND METHODS Materials. BSA, IBP, TLB, PA, sodium cyanoborohydride, MG, 2,4,6-trinitrobenzenesulfonic acid (TNBSA), and 9,10phenanthrenequinone (PNQ) were purchased from Sigma, USA. All other chemicals of AR grade for the preparation of buffer were obtained from Merck, India. AGEs Modification of BSA. The AGEs modified forms of BSA were prepared following the method described earlier.25 Briefly Arg-P and CEL modified BSA were prepared using MG and PA. Native BSA (20 mg/mL) prepared in sodium phosphate buffer (50 mM, pH 7.5) was incubated with 100 μM MG and PA under sterilized conditions. CEL-BSA was prepared by using sodium cyanoborohydride as reducing agent. Incubations were carried out for 24 h (at 37 °C) in capped glass vials followed by extensive dialysis against PBS (pH, 7.4) at 4 °C. The AGEs modified BSA was stored in the deep freezer (−20 °C) and used for further studies. Native PAGE Gel. Gel electrophoresis for the AGEs modified BSA was carried out by using 10% separating gel. 4 μg of protein sample was loaded in each well. Staining was carried out using 0.1% (w/v) Coomassie brilliant blue (CBB), and images were recorded using UVi-Tec gel documentation system. Circular Dichroism. The circular dichroism analysis for native and AGEs modified BSA was carried out on a Jasco J-810 spectropolarimeter equipped with a temperature control system using a quartz cell with a path length of 0.2 cm. Three scans were accumulated at a scan speed of 100 nm min−1, with data being collected at every nanometer from 190 to 260 nm. The concentration of BSA was 1 μM. The drug concentration for TLB and IBP was kept at 100 μM for molecular interaction studies with both native and AGEs modified BSA. Determination of Arginine and Lysine Modification. The modification of arginine and lysine was determined using TNBSA and 9,10-phenathroquinone as described earlier.26 Briefly the MG modified BSA (1 mg mL−1) was incubated with 9,10-phenanthrenequinone and 2 M NaOH for a duration of 3 h at 60 °C followed. This was followed by the addition of 1 N HCl, and further incubation was carried out in dark at room temperature. Fluorescence measurements were carried out at λex = 312 and λem = 395 nm. The lysine modification was determined using TNBSA assay.27 The CEL-BSA (1 mg mL−1) sample was 3314

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measure to confirm the AGEs modification of BSA. TNBSA and PNQ were used for the determination of lysine and arginine modification, respectively. Figure 3a presents the results for the determination of arginine and lysine modification at different concentrations of MG and PA. The level of arginine and lysine modification at 100 μM MG and PA was found to be 10−12% and 5−6% respectively. Arginine (Arg-194, Arg-196, Arg-198, Arg-217, and Arg-409) and lysine (Lys-204 and Lys-413) residues have been recognized as the interacting residues with several drugs,35,36 and thus glycation of these residues can also eventually influence their affinity and binding pattern. The Arg-P and CEL modified BSA was used to carry out drug interaction studies using site specific drugs. The nonenzymatic glycation of albumin leads to changes in its overall charge and influences the migration under the influence of electric field. Native PAGE was used to analyze both the native and AGEs modified BSA, and the results are shown as inset in Figure 3a. CEL and Arg-P modified BSA exhibited comparatively faster movement toward the anode than native BSA. Glycation modification of lysine and arginine residues in BSA leads to the changes in the overall charge of the protein, leading to the differential movement under the influence of electric field. The results from native PAGE confirmed the AGEs modification of BSA. Glycation is known to induce the conformational transition in albumin from its native helical structure to a beta sheeted form.17 To determine the AGEs modification induced conformational change in BSA, CD scans were performed, and results are presented in Figure 3b. The native BSA was found to exhibit helical conformation, which was evident from the prominent dips, at 208 nm and at 222 nm. The CD spectra for Arg-P and CEL modified BSA exhibited structural features similar to those of the BSA, but with less pronounced dips at 208 and 222 nm. This change in the CD spectra for the AGEs modified BSA is attributed to the loss of helical content due to AGEs modification and increase in β sheet. Molecular Dynamics Simulations and Structural Analysis. Molecular dynamics simulations serve as an important tool toward understanding the physical basis of the structure and function of biological macromolecules. To elucidate the effect of such AGEs modification of albumin, molecular dynamics (MD) simulations were carried for AGEs modified BSA, and only those drug interacting lysine and arginine residues in site I and site II were modified. The structural impact of such site specific modifications was determined by carrying out a comparative analysis of native and modified BSA. The structural features such as pocket volume, depth, solvent accessible area, and hydrophobic surface were analyzed using DoGSiteScorer. Pocket predictions with DoGSiteScorer are based on calculated size, shape, and chemical features of automatically predicted pockets, incorporated into a support vector machine for druggability estimation. DoGSite employs a grid-based function prediction method which uses a difference of Gaussian filter to detect potential pockets on the protein surface and splits them into subpockets. Subsequently global properties, describing the volume, depth, surface, and functional groups present in the pockets, are determined. It was evident from the results that ArgP and CEL modification of arginine and lysine residues in sites I and II results in the asymmetric conformational change in the albumin structure (Table 1). There was a 35% and 40% decrease in pocket volume for sites I and III respectively, for AGEs modified BSA. In contrast to sites I and III, site II showed an increase of 40% in the pocket volume for the AGEs modified

ligand preparation. Polar hydrogen atoms were added to the protein, and energy minimization was carried out for both protein and ligands. AutoDock Vina30 was used to carry out molecular docking studies for both TLB and IBP with native and modified forms of BSA. AutoDock Vina is an open-source program for molecular docking designed by Dr. Oleg Trott at The Scripps Research Institute, San Diego, CA, US.

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RESULTS AND DISCUSSION AGEs Modification of BSA. Investigations were carried out to determine both structural and functional changes in albumin

Figure 3. (a) Determination of lysine and arginine modification in AGEs modified form of BSA using TNBSA and PNQ respectively. The level of modification was increasing with the increasing concentrations of methylglyoxal and pyruvic acid, and at 100 μM the arginine and lysine modification was found to be 10−12% and 5−6% respectively. The native PAGE gel for the AGEs modified BSA in shown in the inset, which demonstrates that CEL and Arg-P modified BSA move closer to anode when compared to the native BSA. (b) Effect of AGEs modification on the conformation of BSA. Far-UV CD spectra of native BSA (normal control), Arg-P modified BSA, and CEL modified BSA were recorded in the wavelength range of 190−260 nm. The CD data were expressed as molar ellipticity (CD mdeg).

Table 1. Binding Pocket Analysis for Native and AGEs Modified BSA Using DoGSitescorera protein native BSA

modified BSA

pocket

vol (Å3)

surface (Å2)

liposurface (Å2)

depth (Å)

1 2 3 1 2 3

2347 772 609 1518 1083 900

2379 1246 648 1413 1379 1275

1631 986 415 964 1009 921

39 22 17 21 18 24

a The major structural features analyzed were pocket volume, accessible surface, liposurface, and pocket depth. The data represent the mean values (±SD) of at least three independent experiments.

upon specific AGEs modifications. The AGEs modification of proteins leads to the changes in the total charge, helicity, and their aggregational properties.31−34 The present study involved in vitro AGEs modifications of BSA for the formation Arg-P (the product of arginine modification) and CEL (the product of lysine modification) to understand the effect of glycation induced structural changes in albumin on its ligand carrying abilities. In vitro AGEs modification of BSA was done for the formation of Arg-P and CEL in place of arginine and lysine residues, respectively. BSA contains 59 lysine residues, and 34 of these have been identified to be sites for glycation modification.17 Determination of lysine and arginine modification was used as a 3315

DOI: 10.1021/acs.molpharmaceut.5b00318 Mol. Pharmaceutics 2015, 12, 3312−3322

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Figure 4. Structural superimposition of native (blue) and AGEs modified (yellow) BSAs. Dotted arrows in black color indicate the major deviations in the modified structure from the native state. The AGEs modified arginine (R 194, 196, 198, 217, and 409 as Arg-P) and lysine (K 204 and 413 as CEL) residues have been represented as the ball and stick models in magenta color. The conformational shifts in two ligand binding sites of AGEs modified BSA. The red arrow indicates the conformational shift in the helix from native to modified BSA form. Native structure is shown in blue whereas modified form is represented with yellow color.

modification of arginine and lysine residues in sites I and II leads to the overall changes in the three-dimensional structure. Also analysis was carried out to determine the AGEs mediated structural changes in sites I and II, which can directly influence the ligand binding properties of albumin. The superimposed view of the two drug binding sites for native and modified BSA which shows the asymmetric conformational changes based on shift in the α-helices is presented as an inset in Figure 4. The arrow indicates the shift of a specific helix from native (blue) to the AGEs modified form of BSA (yellow). These results suggested that such AGEs modifications resulted in the large conformation changes in both drug binding sites of BSA. This also suggests that under hyperglycemia conditions where nonenzymatic glycation takes place in a nonspecific manner and at an accelerated rate such AGEs modification could lead to more severe impact on the structure and function of albumin. The Arg-194, Arg-196, Arg-198, Arg-217, and Arg-409 and Lys-204 and Lys-413 form the crucial ligand interacting residues in the two drug binding sites of BSA, i.e., sites I and II, respectively.35,36 Surface representations for the drug binding pockets, i.e., sites I and II, clearly showed the changes in AGEs modified form of BSA (Figure 5). Based on the observations, it is

form when compared with native BSA. A similar pattern was observed for the other structural features such as accessible surface area, hydrophobic surface, and the pocket depths which were decreased for AGEs modified BSA in the case of sites I and III whereas there was an increase in the case of site II. These findings led us to the understanding that AGEs modifications in the drug binding sites I and II lead to the overall conformational change in BSA structure. In order to furthermore determine the AGEs mediated conformational changes in the BSA, structural superimposition was carried out for AGEs modified and native BSA. Structural superimpositions were carried out with respect to Cα atom using “Superpose v1.0”. Superpose is a structural superposition server, which calculates the superimpositions using a modified quaternion approach.37 The results suggest the large conformational change in BSA on AGEs modification which was evident from the RMSD value of 2.8 Å. Figure 4 shows the superimposed structures of native and AGEs-BSA where the AGEs modified residues are represented in the form of ball and stick model. The arrows in the form of black dashed line represent the conformational shift in the native and AGEs-BSA. It was evident from the superimposition of modified and native BSA that AGEs 3316

DOI: 10.1021/acs.molpharmaceut.5b00318 Mol. Pharmaceutics 2015, 12, 3312−3322

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Figure 5. Surface view of the drug binding site I (a, b) and site II (c, d) for native and AGEs modified form of BSA. Modified lysine and arginine residues are shown with stick representation. The modification of arginine and lysine residues to Arg-P and CEL respectively led to the conformational changes in the drug binding sites and resulted in the reduced accessibility and shallow pockets. The coloring of the surface represents the subdomains involved in the formation of the pocket.

Figure 6. Plot for Stern−Volmer constant (Ksv) and association constant (Ka) of native and AGEs modified form of BSA with tolbutamide and ibuprofen. Panels a and b represent the graph for Ksv and Ka for binding of IBP whereas panels c and d represent the graph for TLB interaction of BSA.

BSA shows a comparatively narrowed and shallow pocket. When compared to site II, drug site I was found to be largely affected due to the presence of four arginine residues (Arg-194, Arg-196,

evident that the AGEs modification resulted in the change of shape and the depth of binding pockets. In contrast to the native BSA where both binding pockets are wide and deep, modified 3317

DOI: 10.1021/acs.molpharmaceut.5b00318 Mol. Pharmaceutics 2015, 12, 3312−3322

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Table 2. Stern−Volmer Quenching Constant (Ksv) and Association Constant (Ka) for Native and AGEs Modified Forms (Arg-P BSA and CEL BSA) with Tolbutaimde (TLB) and Ibuprofen (IBP)a Native BSA

a

Arg-P BSA

CEL BSA

drugs

Ksv × 10−4 (M−1)

Ka × 105 (M−1)

Ksv × 10−4 (M−1)

Ka × 105 (M−1)

Ksv × 10−4 (M−1)

Ka × 105 (M−1)

TLB IBP

2.2 (±0.20) 2.7 (±0.18)

2.9 (±0.50) 2.6 (±0.26)

1.5 (±0.12) 1.6 (±0.16)

1.4 (±0.14) 1.2 (±0.08)

1.7 (±0.16) 1.7 (±0.12)

1.5 (±0.12) 1.7 (±0.08)

The data represent the mean values (±SD) of at least three independent experiments.

Figure 7. Conformational changes in native BSA and AGEs modified BSA as determined by CD analysis. There were changes in the conformation on binding of TLB and IBP to native and AGEs modified BSA.

secondary structure for native BSA exhibits 17.3%, 70%, and 12.3% of turn, helix, and random coil structure, respectively, while the AGEs modified BSA exhibits 19.9%, 67.2%, and 12.9% of turn, helix, and random coil structures, respectively. The analysis shows that the AGEs modification leads to increased turn-like and random coil structures while it decreases the helixlike structures. This is consistent with the glycation induced structural changes, i.e., decrease in helical contents of BSA as obtained from CD measurements as discussed above. In Vitro Drug Interaction Studies. The nonenzymatic glycation of albumin has been proposed to be associated with the loss of its carrier properties.38 Studies in the past have been focused to determine the effect of nonenzymatic glycation using different reducing sugars on the drug binding affinity of albumin using both BSA and HSA for tolbutamide,39 warfarin, and ketoprofen.22 In the current study we attempted to understand the impact of two specific AGEs on the drug binding properties of albumin in sites I and II. Both in vitro and in silico studies were carried out to determine the effect of AGEs modifications on drug interaction with BSA. Drug interaction studies were mainly carried out for the two major sites as determine by Sudlow et al. (1975),40 i.e., sites I and II. Fluorescence quenching studies provide the best means to determine the ligand affinities for proteins with intrinsic fluorescence.41 The quenching experiments showed comparatively lower drug binding affinity of Arg-P and CEL-BSA, which was evident from estimates of the drug binding constants (Ka and Ksv). There are two possible ways for fluorescence quenching to occur, i.e., static or dynamic quenching, which can be explained based on the Stern−Volmer equation, eq 1.

Figure 8. Binding energy of TLB and IBP for native and AGEs modified BSA as determined by molecular docking using AutoDock. AGEs modified BSA exhibited comparatively less affinity for both TLB and IBP when compared to its native form.

Arg-198, and Arg-217) in the modified form as Arg-P. Such reduced accessibility of drug binding pockets was mainly due to the presence of bulky groups in the form of Arg-P in place of arginine residues and CEL in place of lysine residues. These AGEs modified residues could lead to the increased steric hindrance and thus impaired drug binding in these pockets. These results provide insights about the possible effect of glycation mediated structural changes in albumin, which can directly affect its ligand carrier properties. Also the size and shape of binding pocket can largely influence their ligand carrier functions, and these observations suggest possible AGEs mediated structural modulations which can directly impact drug binding function of albumin. We have also investigated the secondary structure of both native and AGEs modified BSAs in order to see how the glycation leads to the structural changes in BSA. The secondary structure analysis has been carried out for 1000 configurations corresponding to 4 ns simulation time scale. The average

F0/F = 1 + Kqτ0K[Q] = 1 + K sv[Q]

(1)

where F0 and F are the fluorescence intensities before and after the addition of quencher, Kq is the quenching rate constant, τ0 is the lifetime of the fluorophore in the absence of the quencher 3318

DOI: 10.1021/acs.molpharmaceut.5b00318 Mol. Pharmaceutics 2015, 12, 3312−3322

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Figure 9. Molecular interaction pattern of tolbutamide (TLB) in drug binding site I of native and AGEs modified BSAs. (a) TLB bound in site I of native BSA. (b) TLB bound in site I of AGEs modified BSA. TLB interacts in site I of BSA by forming hydrogen bonds with the Arg-198 and His-241. The other arginine and lysine residues forming site I include Arg-194, Arg-196, and Lys-204. The interaction pattern TLB in the case of AGE-BSA showed no Hbonds due to the presence of Arg-P in the place of normal arginine residues.

which equals 10−8 s,42 [Q] is the concentration of the quencher, and Ksv is the Stern−Volmer quenching constant. Figure 6a,c shows the Stern−Volmer plot for the native and AGEs modified BSA. There was a decrease in Ksv in the case of AGEs modified BSA compared to its native form for the interaction of both TLB and IBP. The Arg-P modified BSA exhibited comparatively lower Ksv value than its CEL modified form. The value of Ksv for TLB and IBP in the case of native BSA was found to be 2.2 and 2.7 (×10−4), which is comparatively higher when compared to the Arg-P (1.5 and 1.6 (×10−4) and CEL (1.7 and 1.7 (×10−4)) modified BSA respectively. Such decrease in the Ksv could be attributed to the conformational changes due to glycation, and also a similar effect has been observed in the case of glycated HSA earlier.32 From this drug binding data the association constant (Ka) can be calculated using the following equation:

Log(F0 − F /F ) = n log K a − n log(1/[Q t] − (F0 − F )[Pt ]/F0) (2)

where F0 and F are the fluorescence intensities in the absence and presence of the quencher, and [Qt] and [Pt] are the total quencher concentration and the total protein concentration, respectively. Figure 6b,d presents the plots for association constant Ka for native and modified BSA. The values for both Ksv and Ka have been tabulated (Table 2). The value of Ka for TLB and IBP in the case of native BSA was found to be 2.9 and 2.6 (×105), which is comparatively higher when compared to the Arg-P (1.4 and 1.2 (×105) and CEL (1.5 and 1.7 (×105) modified BSA respectively. Based on the determination of the association constant (Ka) it was evident that the AGEs modified form of BSA exhibited comparatively less affinity for both site I and II specific drugs, i.e., TLB and IBP. It was also observed that Arg-P modified 3319

DOI: 10.1021/acs.molpharmaceut.5b00318 Mol. Pharmaceutics 2015, 12, 3312−3322

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Molecular Pharmaceutics

Figure 10. Molecular interaction pattern of ibuprofen (IBP) in site II of native and AGEs modified BSA. (a) IBP bound in drug site II of native BSA. (b) IBP bound in drug site II of AGEs modified BSA. IBP interacts in site II of BSA by forming hydrogen bonds with the Arg-412. The other lysine and arginine residues forming site II include Arg-409 and Lys-413.

BSA showed comparatively lower affinity for both drugs when compared to CEL-BSA. Subdomains IB and IIA of albumin form the drug site I of BSA, which serves as a main binding pocket for anionic heterocyclic molecules, whereas site II can accommodate drugs with aromatic rings and extended conformations. The reduced drug binding affinity of AGEs modified BSA can be mainly attributed to the modification of arginine residues (Arg194, Arg-196, Arg-198, Arg-217, and Arg-409), which are the major ligand interacting residues. CEL modified BSA also exhibited comparatively reduced affinity for both TLB and IBP compared to native BSA. The lysine residues which are involved in drug binding in BSA include Lys-204, Lys-411, and Lys-413 in sites I and II. The formation of AGEs modifications on BSA also leads to a reduced accessibility of binding pockets due to the presence of bulky groups in the form of argpyrimdine and carboxyethyllysine. Overall the results from in vitro drug interaction studies showed a reduced affinity of the drug for AGEs modified BSA while Arg-P modification was found to have a greater effect on drug binding than CEL.

In order to better understand the molecular interaction and conformational changes on drug binding to native and AGEs modified BSA, circular dichroism (CD) spectropolarimetry was used, and results are presented in Figure 7. Native protein exhibits helical conformation, which was evident from the minima at 208 and 222 nm. CD spectra for both TLB and IBP interaction with native BSA suggest the conformational changes leading to the loss of helicity. Similar conformational changes were seen in the case of both Arg-P and CEL-BSA. Molecular Docking Approach. In order to determine the effect of AGEs modifications on biomolecular interaction of drug in both sites and changes in their binding pattern, molecular docking studies were carried out. Both native and AGEs modified BSA were used for docking by using AutoDock Vina. The results from the docking studies for drug were analyzed based on their binding energy (kcal/mol) and the interaction pattern in sites I and II. Docking studies showed comparatively lower binding affinity of TLB and IBP for AGEs modified. Figure 8 presents the binding energies for TLB and IBP in the case of native and 3320

DOI: 10.1021/acs.molpharmaceut.5b00318 Mol. Pharmaceutics 2015, 12, 3312−3322

Molecular Pharmaceutics

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modified BSA. The binding energy for AGEs modified BSA was found to be −4.7 kcal/mol and −4.2 kcal/mol for TLB and IBP, which is comparatively lower (in terms of absolute free energy values) than that for the native BSA (TLB, −6.7, and IBP, −6.8 kcal/mol). The low binding affinity for AGEs modified BSA could be mainly due to the loss of hydrogen bonding. The scoring function for AutoDock Vina includes evaluations for dispersion/repulsion, hydrogen bonding, electrostatics interactions, and desolvation energy and thus provides explanation for the relative stability of various protein−ligand complexes. In order to obtain further insights regarding the molecular interaction pattern of TLB and IBP in native and AGEs modified BSA, docked structures were visualized with Pymol. Figures 9 and 10 show the ligand interaction pattern for TLB and IBP in drug binding sites I and II respectively. Based on these observations, it was found that AGEs modification mediated reduction in the affinity for TLB and IBP could have been due to the loss of hydrogen bonding and reduced accessibility of the drug binding site. Figure 9a,b shows the molecular interaction and the binding site of TLB in the case of native and modified BSA. Based on the observations from the crystallographic studies, drug binding site I of albumin has been further divided into three subsites, i.e., 3′azido-3′-deoxythymidine (AZT) subsite (PDB code 3B9L), indomethacin (IMN) subsite (PDB code 2BXM), and salicylic acid (2I30). It was observed that TLB binds in the AZT subsite of drug site I in BSA by forming two H-bonds with Arg-198 and His-247 with Trp-213 in close proximity. In contrast to this there was no such hydrogen bonding in the case of AGEs modified BSA. Similarly, Figure 10a,b presents the molecular interaction pattern of IBP in drug binding site II of native and modified BSA. IBP binds in site II of BSA with high affinity by forming H-bonds with Arg-412 while Arg-409 and Lys-413 are the other arginine and lysine residues present in close proximity, whereas in the case of modified BSA there was no such H-bond formation with Arg412 which was modified as argpyrimidine. These observations suggested that AGEs modification of arginine and lysine residues in sites I and II resulted in the loss of hydrogen bonding interaction in turn leading to the reduced affinity of TLB and IBP for BSA. The presence of bulky groups in the form of Arg-P contributed toward increased steric hindrance and thus reduced accessibility for drugs.

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00318. Fluorescence spectra for native and AGEs modified BSA in the presence of increasing concentrations of IBP and TLB (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. Phone: 04362-264101, ext 3681. Address: Molecular Biophysics lab, School of Chemical and Biotechnology, SASTRA University, Thanjavur-613401, Tamilnadu, India. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank SASTRA University for providing the financial assistance (teaching assistant fellowship for S.A.) and infrastructure for carrying out the research work. A. Veerappan and K. B. A. Ahmed are thanked for providing the facility for fluorescence measurements.

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ABBREVIATIONS USED AGEs, advanced glycation end products; CML, carboxymethyllysine; CEL, carboxyethyllysine; Arg-P, argpyrimidine; PA, pyruvic acid; MG, methylglyoxal; FAP, familial amyloidotic polyneuropathy; BSA, bovine serum albumin; MD, molecular dynamics; IBP, ibuprofen; TLB, tolbutamide; FAPs, familial amyloidotic polyneuropathies; PBS, phosphate buffer saline; fs, femtosecond; TNBSA, 2,4,6-trinitrobenzenesulfonic acid

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REFERENCES

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CONCLUSIONS

In summary, this paper presents the first demonstration of the structural change mediated functional impact of specific AGEs modification on BSA. The key observations from studies with AGEs modified BSA were as follows: (1) AGEs modification leads to arrays of asymmetric conformational changes in BSA; (2) modified form of BSA exhibited reduced pocket volume, depth and accessible surface area; (3) the formation of Arg-P and CEL in the drug binding sites I and II resulted in comparatively narrow and shallow binding pockets; and (4) modified BSA exhibited lower affinity of drugs in sites I and II. Under physiological conditions the nonenzymatic glycation reaction occurs in a highly nonspecific way, resulting in the formation of heterogeneous AGEs which could perhaps lead toward the development of a crippled drug carrier with impaired drug binding abilities. Based on these results we are able to conclude that advanced glycation modification of drug interacting lysine and arginine residues modulates structure and the affinity of ligands for both sites I and II of BSA. 3321

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