Article pubs.acs.org/molecularpharmaceutics

Insight into the Binding Mechanism of Imipenem to Human Serum Albumin by Spectroscopic and Computational Approaches Md Tabish Rehman, Hira Shamsi, and Asad U. Khan* Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, Uttar Pradesh, 202 002, India S Supporting Information *

ABSTRACT: The mechanism of interaction between imipenem and HSA was investigated by various techniques like fluorescence, UV−vis absorbance, FRET, circular dichroism, urea denaturation, enzyme kinetics, ITC, and molecular docking. We found that imipenem binds to HSA at a high affinity site located in subdomain IIIA (Sudlow’s site I) and a low affinity site located in subdomain IIA−IIB. Electrostatic interactions played a vital role along with hydrogen bonding and hydrophobic interactions in stabilizing the imipenem−HSA complex at subdomain IIIA, while only electrostatic and hydrophobic interactions were present at subdomain IIA−IIB. The binding and thermodynamic parameters obtained by ITC showed that the binding of imipenem to HSA was a spontaneous process (ΔGD° = −32.31 kJ mol−1 for high affinity site and ΔG°D = −23.02 kJ mol−1 for low affinity site) with binding constants in the range of 104−105 M−1. Spectroscopic investigation revealed only one binding site of imipenem on HSA (Ka ∼ 104 M−1). FRET analysis showed that the binding distance between imipenem and HSA (Trp-214) was optimal (r = 4.32 nm) for quenching to occur. Decrease in esterase-like activity of HSA in the presence of imipenem showed that Arg-410 and Tyr-411 of subdomain IIIA (Sudlow’s site II) were directly involved in the binding process. CD spectral analysis showed altered conformation of HSA upon imipenem binding. Moreover, the binding of imipenem to subdomain IIIA (Sudlow’s site II) of HSA also affected its folding pathway as clear from urea-induced denaturation studies. KEYWORDS: fluorescence quenching, drug displacement, molecular docking, FRET, urea-induced unfolding, synchronous fluorescence, 3-D fluorescence

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INTRODUCTION Bacterial antibiotic resistance (BAR) is a global phenomenon that has spread rapidly over the past few decades. BAR is defined as the development of resistance in bacteria against an antibiotic toward which it was originally susceptible.1,2 The dissemination of disease by resistant bacteria is difficult to control with conventional antibiotics, which in turn results in prolonged illness, ineffective treatment, elevated mortality rate, and significantly increased cost of treatment.3 Bacteria develop resistance against antibiotics exclusively by the production of βlactamases, which cleave the β-lactam ring of various clinically significant β-lactam antibiotics.4 Other methods of acquiring antibiotic resistance include expression of drug efflux pumps, modification of the drug target, and change in the metabolic pathway.5,6 Extended spectrum β-lactamases (ESBLs) have been widely distributed in different parts of the world and are often associated with the members of Enterobacteraceae.7−9 ESBLs expressing bacteria are resistant toward penicillins, different generations of cephalosporins, aztreonam, and various antibiotic/inhibitor combinations.10 The antibiotics of the carbapenem group such as imipenem, meropenem, doripenem, and ertapenem are considered as future drugs of choice to control bacterial infections. The affinity of many drugs toward © 2014 American Chemical Society

serum albumin determines its overall distribution, efficacy, and metabolism.11−13 Earlier, the interaction of radiolabeled carbapenem and other β-lactam antibiotics with HSA has been explored, but a detailed mechanism of interaction is still elusive.14−17 Thus, the molecular characterization of HSA− imipenem interaction is indispensable as it could play a significant role in pharmacology and pharmacodynamic behavior of imipenem since only the free drug would be available to inhibit bacterial infections.18 HSA is the most abundant protein found in human plasma.19 It constitutes about half of the total serum protein, and its concentration varies in the 35−50 mg/mL range. It primarily functions as a carrier molecule to transport various exogenous and endogenous molecules such as hormones, drugs, and fatty acids.20,21 The X-ray crystal structure of HSA revealed it as a globular heart-shaped protein of 585 amino acid residues with a molecular weight of 66 kDa. The whole structure is stabilized by 17 disulfide bonds and is composed of three structurally Received: Revised: Accepted: Published: 1785

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following the above procedure at three different temperatures, i.e, 298, 303, and 308 K. All the fluorescence intensities were corrected for the inner filter effect. For 3D fluorescence measurements, a wavelength range of 220−500 nm was used for emission spectra by exciting the protein sample at 220 nm with an increment of 10 nm and 19 spectra were recorded.26 Equimolar concentrations (2 μM) of HSA and imipenem were used. The synchronous fluorescence spectra of HSA in the absence and presence of imipenem were recorded when the Δλ value between the excitation and emission wavelengths was stabilized at 15 and 60 nm, respectively. The concentrations of HSA and imipenem were 2 μM each. Thermodynamics and Binding Parameters by ITC Measurements. The energetics of the binding of imipenem to HSA at 298 K was measured using a ITC-200 microcalorimeter (MicroCal Inc., Northampton, MA). Prior to the titration experiment, all samples were degassed properly on a Thermo Vac supplied with the calorimeter. The sample and reference cells were loaded with HSA solution (15 μM) and 20 mM sodium phosphate buffer (pH 7.0), respectively. Multiple injections of 1.5 μL of imipenem solution (1.285 mM) were made into the sample cell containing HSA. Each injection was made over 3 s with an interval of 180 s between successive injections. The reference power and stirring speed were set at 7 μcal−1 and 307 rpm, respectively. Heat of dilution for the ligand was determined in the control experiment and was subtracted from the integrated data before curve fitting. The data were fitted and analyzed according to the sequential binding mode of two binding sites using Origin 7.0 software provided with the instrument. Drug Displacement Experiment. Site-specific marker displacement experiment was carried out by titrating complexes of HSA with diazepam, warfarin, and ibuprofen at molar ratios of 1:0, 1:0.5, and 1:1 with increasing imipenem concentration. Warfarin and diazepam are site specific markers that bind to HSA at Sudlow’s site I (subdomain IIA) and Sudlow’s site II (subdomain IIIA), respectively.23 On the other hand, ibuprofen binds primarily at Sudlow’s site II (subdomain IIIA) and has a secondary binding site at subdomain IIA−IIB.20 Recently, subdomain IB is reported as a third major drug binding site on HSA.27 The intrinsic fluorescence of HSA was recorded between 300 and 400 nm after excitation at 295 nm. Both the excitation and emission slits were set at 5 nm. Absorbance Spectra Measurements. The far-UV and near-UV absorption spectra of HSA alone or in the presence of imipenem were measured in the ranges 240−200 nm and 320− 240 nm, respectively. The concentrations of HSA and imipenem were 2 μM each. CD Spectra Measurements. Far-UV and near-UV CD spectra of HSA in the absence and presence of imipenem (1:1 molar ratio) were taken at protein concentrations of 5 and 20 μM in 0.1 and 1.0 cm path length cells, respectively. All the spectra were corrected for the appropriate blanks. The observed ellipticity is converted to mean residual ellipticity [θ] in deg· cm2·dmol−1 by using the following equation:28

similar domains (I, II, and III), each containing two subdomains (A and B).22 Generally, ligands bind to the two principal regions of HSA that are located in the hydrophobic cavities of subdomain IIA and IIIA, also known as Sudlow’s site I and Sudlow’s site II, respectively.23 The binding of antibiotics to HSA significantly affects many functions such as metabolism, membrane penetration, half-life, and other pharmacokinetic properties. 24 Thus, the detailed understanding of the interaction between antibiotics with HSA is of great importance for the better understanding of the protein structure−function and designing the drug therapy procedure. The present work was initiated to understand the structural and functional relationships of imipenem (drug of choice) interaction with HSA under physiological conditions by various spectroscopic, thermodynamic, and molecular modeling approaches. The outcome of this study will provide an insight into the molecular basis of the interaction between carbapenem imipenem and HSA.

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EXPERIMENTAL SECTION Reagents. Human serum albumin (fatty acid free), imipenem, urea, p-nitrophenyl acetate (p-NPA), and all buffers were from Sigma-Aldrich (St. Louis, MO, USA). Warfarin, ibuprofen, and diazepam were from Ranbaxy, India. Samples were prepared in 20 mM sodium phosphate buffer, pH 7.0. Protein concentration was determined spectrophotometrically by using molar extinction coefficients of 36,500 M−1 cm−1 at 280 nm.25 All other materials were of analytical grade, and double distilled water was used throughout the study. Apparatus. All fluorescence spectra were measured on a Shimadzu RF-5301PC spectrofluorometer (Shimadzu Corporation, Kyoto, Japan) equipped with a thermostatically controlled cell holder and attached to a water bath to maintain desired constant temperature. The excitation and emission slits were set at 5 nm. CD spectra were collected on a Jasco J-810 spectropolarimeter (Jasco International Co. Ltd., Tokyo, Japan) equipped with a Peltier-type temperature controller (PTC-423S/15) and attached to a water bath. The instrument was calibrated with (+)-10-camphorsulfonic acid. All the spectra were measured at 298 K using a scan speed of 100 nm/min and response time of 1 s. UV−visible absorption spectra and enzyme kinetics measurements were recorded on a Shimadzu UV-1800 double beam spectrophotometer (Shimadzu International Co. Ltd., Kyoto, Japan) at 298 K. ITC measurements were performed on ITC-200 microcalorimeter (MicroCal Inc., Northampton, MA) at 298 K. All the samples were properly degassed on the Thermo Vac unit of the instrument before measurement. All the mathematical equations are written in MathType, and the data was analyzed using Sigma Plot 10.

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METHODOLOGY

Fluorescence Spectra Measurements. Fluorescence quenching was monitored by measuring intrinsic fluorescence between 300 and 400 nm after selectively exciting the Trp-214 at 295 nm. Both the excitation and emission slits were set at 5 nm. To a 3 mL sample containing 2 μM HSA was successively added 0.2 μM imipenem in such a manner that the total volume added was not more than 30 μL. Moreover, the effect of temperature on HSA−imipenem interaction was determined by

[θ ] =

θobs (10ncl)

(1)

where θobs is the observed ellipticity in mdeg, n is the total number of amino acid residues (585) in the protein, c is the molar concentration of the protein, and l is the path length in 1786

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cm. The α-helical content of the protein was calculated by using the following formula:29 ⎛ [θ ] − 4000 ⎞ α ‐helix (%) = ⎜ 208 ⎟× 100 ⎝ 33000 − 4000 ⎠

The values of ΔG°I , ΔG°II, mI, mII, CmI, and CmII (the midpoint of transition I is ΔG°I /mI and of transition II is ΔG°II/mII) are given in Table 7. Esterase-like Activity Measurements. The esterase-like activity of HSA toward p-NPA has been attributed to the subdomain IIIA (Sudlow’s site II).31 The effect of imipenem binding on the catalytic activity of HSA toward p-NPA is determined by steady state kinetics at pH 7.0 and 298 K. The concentration of HSA was kept constant at 12 μM while the concentration of p-NPA was varied from 0 to 700 μM. The rate of p-NPA hydrolysis was determined by measuring the appearance of p-nitrophenol (yellow product) at 405 nm for 60 s. The concentration of p-nitrophenol was determined by absorbance measurements using a molar extinction coefficient value of 17,800 M−1 cm−1 at 405 nm. The kinetic parameters (kcat and Km) were determined according to the Michaelis− Menten method by fitting the data to the following equations:

(2)

Urea-Induced Unfolding Studies. The stability of HSA upon imipenem binding in a 1:1 molar ratio was determined by urea-induced unfolding of HSA at pH 7.0 and 298 K. The samples were prepared by adding 100 μL of HSA stock solution to different volumes of 20 mM sodium phosphate buffer, followed by the addition of urea stock solution (10 M) and imipenem to get the desired concentrations of denaturant and ligand. The final samples (3.0 mL) were incubated at room temperature for 8−10 h, and the unfolding process was followed by intrinsic fluorescence measurements at 341 nm after exciting the protein at 295 nm.30 Data Analysis. The unfolding curves of HSA alone and HSA−imipenem complex followed two-step transitions, indicating the presence of at least three different conformational states. The unfolding process is represented as follows:

N↔I↔D

v=

kcat =

(3)

where N, I, and D are the native, intermediate, and denatured states of the protein, respectively. Assuming that the processes N ↔ I (transition I) and I ↔ D (transition II) follow two-state mechanisms, the fraction of molecules in intermediate state ( f I) and denatured state ( f II) and the associated Gibbs free energy changes (ΔGI and ΔGII) can be calculated by using the following equations: fI =

fII =

(4)

(5)

⎡ (y − y ) ⎤ N ⎥ ΔG I = −RT ln⎢ ⎢⎣ (yI − y) ⎥⎦

(6)

⎡ (y′ − y ) ⎤ I ⎥ ΔG II = −RT ln⎢ ⎢⎣ (yD − y) ⎥⎦

(7)

(8)

ΔG II = ΔG II° − mII[urea]

(9)

(12)

where Km is the Michaelis−Menten constant and K′m is the apparent Km at an inhibitor concentration of Io. Molecular Docking Studies. Protein and Ligand Preparation. The molecular docking studies were performed as described previously.33 The three-dimensional structure of HSA (1AO6) was used for docking study using Autodock 4.2.34 The SDF file of imipenem was retrieved from the DrugBank database (Accession number: DB01598) and converted into a PDB file using OpenBabel software.35 The energy of the ligand (imipenem) was minimized by MMFF94 using OpenBabel.35 Gasteiger partial charges were added to the ligand atoms. Nonpolar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on the protein model. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of ADT. Affinity grid maps of 50 × 50 × 50 Å grid points and 0.375 Å spacing were generated using AutoGrid program.34 The AutoDock parameters were set to default, and distancedependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Docking Simulations. Docking simulations were performed using LGA and the Solis and Wets local search methods.36 Initial positions, orientations, and torsions of the ligand molecules were set randomly. All rotatable torsions were released during docking. Each run of the docking experiment was set to terminate after a maximum of 250000 energy

where R is the universal gas constant, T is the temperature (in K), and y and y′ are the observed fluorescence intensities corresponding to transitions I and II, respectively. yN, yI, and yD are the fluorescence intensities of the N, I, and D states of HSA, respectively, and were obtained by considering a linear dependence of the optical property in pretransition, intermediate, and post-transition regions of the unfolding curves, respectively. Values of f I and ΔGI and f II and ΔGII were plotted as a function of [urea] as shown in Figure 12. The values of ΔGI and ΔGII were plotted as a function of [urea], and the plots were analyzed for ΔG°I and ΔG°II (ΔG values at 0 M urea). The values of mI and mII were calculated as the slope (δΔG/ δ[urea]) using the relations ΔGI = ΔGIo − mI [Urea]

(11)

⎞ ⎛ Km Ki = ⎜ ⎟Io ⎝ K ′m − K m ⎠

(y′ − yI ) (yD − yI )

Vmax [E]

(10)

where v and Vmax are the initial and maximum velocity of hydrolysis, respectively, [S] is the substrate concentration used, [E] is the enzyme concentration, and kcat and Km are the kinetic parameters. The effect of imipenem binding on the esterase-like activity of HSA was determined by monitoring the rate of p-NPA hydrolysis as described above at different imipenem:HSA ratios (0:1, 0.25:1, 0.50:1, 0.75:1, and 1:1), and the result was analyzed by Lineweaver−Burk plot. Moreover, the inhibition constant (Ki) was calculated from the following equation:32

(y − yN ) (yI − yN )

Vmax[S] K m + [S]

1787

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evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å and quaternion and torsion steps of 5 were applied. The final docking figures were generated using Discovery Studio2.5 (Accelrys).

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RESULTS AND DISCUSSION Characterization of Imipenem Binding Site on HSA. HSA Fluorescence and Mechanism of Quenching by Imipenem. The phenomenon of fluorescence is observed as a result of photon emission when an electron in a higher energy level is returned back to a lower energy level. In this process, fluorescence quenching is observed due to various molecular interactions such as reactions in the excited state, molecular rearrangements, energy transfer, and static and dynamic quenching. The measurement of intrinsic fluorescence quenching of protein has been widely used to elucidate the mechanism of its interaction with a ligand or drug molecule.37,38 The present study showed the effect of imipenem binding on the fluorescence property of HSA (Figure 1). A progressive

Figure 2. Imipenem-induced fluorescence quenching of HSA at 298 K (▲), 303 K (●), and 308 K (▼). Panel A shows the decrease in relative fluorescence intensity, and panel B shows the Stern−Volmer plot for HSA−imipenem interactions. The concentration of HSA was 2 μM in 20 mM sodium phosphate buffer at pH 7.0.

Table 1. Stern−Volmer Quenching Constants and Binding Parameters for HSA−Imipenem Interactions at Different Temperatures

Figure 1. Imipenem-induced fluorescence quenching of HSA. The concentration of HSA was 2 μM, and the concentration of imipenem was varied from 0 to 2 μM (a−k) in a successive increment of 0.2 μM. The intrinsic fluorescence of the protein was measured in 20 mM sodium phosphate buffer, pH 7.0 at 298 K upon excitation at 295 nm. Inset shows the structure of imipenem.

decrease in the fluorescence intensity along with 1 nm blue shift was observed due to quenching of HSA fluorescence. The results indicated that the microenvironment of Trp-214 was relatively less hydrophobic upon imipenem binding. Quenching experiments were performed at different temperatures (298, 303, and 308 K) to explore the mechanism and thermodynamics of imipenem binding (Figure 2). A decrease in relative fluorescence intensities (RFI) was observed, and the data were analyzed according to the Stern−Volmer equation38 (Figure 2 and Table 1).

temp (K)

KSV × 104 (M−1)

kq × 1013 (M−1 s−1)

Ka × 104 (M−1)

n

R2

298 303 308

8.83 7.63 6.33

1.55 1.34 1.16

9.43 8.85 7.73

1.01 1.01 1.02

0.9925 0.9984 0.9983

the lifetime of the protein fluorescence in the absence of quencher, respectively. It is clear from Table 1 that KSV values for HSA−imipenem interactions at different temperature were of the order of 104 M−1, suggesting that a significant interaction between HSA and imipenem was responsible for the quenching mechanism. The linear dependence of quenching at different temperatures suggested that only one type of quenching mechanism (either static or dynamic) dominated, and there was only one type of equivalent imipenem binding site on HSA. The kq values were determined from the ratios of KSV/τo at different temperatures (Table 1) after taking τo for HSA = 5.71 × 10−9 s.39 The kq values (∼ 1013 M−1 s−1) were found to be considerably larger than the maximum dynamic quenching constant (∼1010 M−1 s−1),40 which indicated that the imipenem-induced quenching

Fo = 1 + KSV[Q] = 1 + kqτo[Q] (13) F where Fo and F are the fluorescence intensities in the absence and presence of imipenem (quencher), KSV is the Stern− Volmer constant, [Q] is the molar concentration of quencher, and kq and τo are the bimolecular quenching rate constant and 1788

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of HSA fluorescence was due to complex formation. The nature of the quenching process can also be determined by closely evaluating its dependence on temperature. At elevated temperatures, dynamic quenching is increased due to faster diffusion. On the other hand, static quenching decreases at higher temperatures due to the dissociation of weakly bound complexes. Thus, the decrease in KSV and kq with increasing temperature also suggested that the quenching of HSA fluorescence by imipenem was initiated by complex formation rather than by dynamic quenching. Binding and Thermodynamics of Imipenem−HSA Interaction. So far, it is clear that imipenem quenches HSA fluorescence by forming a stable complex and it has one set of equivalent binding sites on HSA. We determined the binding constant (Ka) and the number of binding sites (n) by using the following modified Stern−Volmer equation:40

Figure 4 shows the dependence of binding constant (Ka) on 1/T, the slope of which is equal to −ΔH/R, and the intercept

(Fo − F ) = log K a + n log[Q] (14) F Figure 3 and Table 1 show a linear temperature dependence of log[(Fo − F)/F] versus log [Q] plots, the slopes and log

Figure 4. van’t Hoff plot for the binding of imipenem to HSA in 20 mM sodium phosphate buffer pH 7.0.

gives an estimate of ΔS/R. The negative ΔH and positive ΔS values indicated that electrostatic interactions were predominantly involved in stabilizing HSA−imipenem complex (Table 2). At pH 7.0, HSA was negatively charged (isoelectric point of Table 2. Thermodynamic Parameters for the Binding of Imipenem to HSA As Estimated from Fluorescemce Quenching at Different Temperatures

intercepts of which were equal to n and log Ka values, respectively. It has been reported earlier that several ligands bind to serum albumin with binding constants between 103 and 105 M−1.41,42 In our case, the binding constant for HSA− imipenem complex was 9.43 × 104 M−1 at 298 K and there was only one binding site of imipenem on HSA. The major forces involved in protein−ligand interactions include electrostatic interactions, hydrogen bonds, van der Waals interactions, and hydrophobic interactions. Assuming that the change in enthalpy (ΔH) was not significant over the studied temperature range, the following van’t Hoff equation (eq 15) and thermodynamic equation (eq 16) were used to determine the enthalpy change (ΔH), entropy change (ΔS), and free-energy change (ΔG). ΔS ΔH − R RT

ΔG = ΔH − ΤΔS

ΔH (kJ mol−1)

ΔS (J K−1 mol−1)

TΔS (kJ mol−1)

ΔG (kJ mol−1)

298 303 308

−15.16

44.40

13.23 13.46 13.68

−28.39 −28.62 −28.84

HSA = 4.7−4.9) while the net charge on imipenem was zero as pKa of the carboxyl group is 3.63 and that of amino group is 10.88. Thus, the opposite charges of imipipenem and HSA were involved in electrostatic interactions. Moreover, the negative charge of ΔG indicated that the binding of imipenem to HSA was a spontaneous process (Table 2). The residence time (τ) of protein−drug complex is widely considered as a benchmark for determining the dose response behavior of a drug and the rate of its elimination.30,42 It is defined as the reciprocal of the dissociation rate constant (τ = 1/kb). The residence time (τ) of HSA−imipenem complex can be determined by the kinetic model using eq 17.30 (17) P + L ↔ PL

Figure 3. Modified Stern−Volmer plots for the quenching of HSA by imipenem at 298 K (▲), 303 K (●), and 308 K (▼). The concentration of HSA was 2 μM in 20 mM sodium phosphate buffer at pH 7.0.

ln K a =

temp (K)

Let us assume that the forward reaction is diffusioncontrolled; then the rate of HSA−imipenem complex formation (kf) will be 4 × 109 M−1 s−1. For the reverse reaction, the rate of HSA−imipenem dissociation (kb) was calculated from the binding constant (Ka =kf/kb = 9.43 × 104 M−1) and was found to be 4.24 × 104 s−1. Hence, the residence time (τ) of HSA− imipenem complex was calculated as 23.6 μs at 298 K, which was significantly higher and an indication of a stable HSA− imipenem complex. Fluorescence Resonance Energy Transfer (FRET). FRET has been widely used to measure molecular distances between donor and acceptor molecules. It occurs only when the

(15) (16) 1789

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absorption spectrum of the acceptor molecule overlaps the fluorescence emission spectrum of the donor molecule.43 According to Förster nonradiation energy transfer theory, the distance (r) between imipenem and HSA (Trp-214) can be calculated by using the following equations.44−46 The efficiency of energy transfer (E) can be calculated as E=

R o6 R o6

+r

6

=1−

F Fo

(18)

where Fo and F are the fluorescence intensities of HSA in the absence and presence of imipenem, respectively, r is the distance between donor and acceptor molecules, and Ro is the distance at which the transfer efficiency becomes equal to 50%. Ro can be determined as R o6 = 8.79 × 10−25K 2n−4 ΦJ

(19)

Figure 5. Normalized overlap of the HSA absorption spectra (- - -) with the fluorescence spectra of HSA () at 298 K. The shaded part of the graph represents the overlap integral (J). The concentrations of both HSA and imipenem were 2 μM in 20 mM sodium phosphate buffer, pH 7.0.

2

where K is the factor related to the geometry of the donor and acceptor dipoles, n is the refractive index of the medium, Φ is the fluorescence quantum yield of the donor in the absence of acceptor, and J is the overlap integral of donor fluorescence emission and the acceptor absorption spectra, which can be calculated as

Table 4. Thermodynamic and Binding Parameters of HSA− Imipenem Obtained by ITC

∞

J=

∫0 Fλελλ 4 dλ ∞

∫0 Fλ dλ

(20)

where Fλ is the fluorescence intensity of the donor at wavelength λ and ελ is the molar extinction coefficient of acceptor at wavelength λ. For HSA, K2, Φ, and n were taken as 2/3, 0.118, and 1.33, respectively46 and the results are presented in Table 3. The normalized spectral overlap between

Ro (nm)

r (nm)

EFRET

8.47 × 10−14

3.23

4.32

0.15

Kb × 104 (M−1)

ΔH (kJ mol−1)

TΔS (kJ mol−1)

ΔG (kJ mol−1)

high affinity low affinity

10.1 0.18

−26.75 −20.09

5.56 2.93

32.31 23.02

(ΔG) for both high and low affinity sites suggested that the binding of imipenem to HSA was a spontaneous process.32 It was interesting to note that the binding and thermodynamic parameters obtained by fluorescence spectroscopy (Table 2) differ from those obtained by ITC (Table 4). Similar results were also observed previously and were argued to be due to the assumption made in noncalorimetric approaches that ΔH does not depend on temperature.31,32 Moreover, the difference in the magnitude of binding and thermodynamic parmeters obtained by ITC and fluorescence spectroscopy was due to the fact that ITC measured a global change in the property, whereas the fluorescence spectroscopy measured only local changes around the Trp-214.31,32 Molecular Docking of Imipenem on HSA. To describe the binding site and the residues involved in interaction of imipenem with HSA, molecular docking studies were performed using AutoDock 4.2. The docking results clearly showed that imipenem binds to HSA principally at subdomain IIIA (Sudlow’s sites II) and to a lesser extent at subdomain IIA−IIB (Figure 8). At subdomain IIIA, imipenem specifically interacted with Leu-387, Ile-388, Asn-391, Cys-392, Phe-403, Leu-407, Arg-410, Lys-414, Leu-430, Val-433, Gly-434, Cys438, Ala-449, Leu-453, and Ser-489 (Figure 8A,B). We also found that the negatively charged amino group of imipenem interacted electrostatically with Arg-410 and Lys-414 and to a lesser extent with Asn-391, Leu-430, and Ser-489 (Figure 8B). The docking results are in excellent agreement with our observation of the thermodynamic data. In addition to electrostatic interactions, docking results also showed that other forces such as hydrogen bonds (with Ser-489) and hydrophobic interactions (with Leu-387, Ile-388, Cys-392, Phe403, Leu-407, Val-433, Gly-434, Cys-438, Ala-449, Leu-453) were also involved in HSA−imipenem interaction, but the electrostatic forces played a significant role in stabilizing HSA−

Table 3. FRET Parameters for the Binding of Imipenem to HSA J (M−1 cm−1)

binding sites

the absorption spectrum of acceptor (imipenem) and the fluorescence emission spectrum of donor (Trp-214 of HSA) is shown in Figure 5. In the present study, the values of J, Ro, r, and E were 8.47 × 10−14 M−1 cm−1, 3.23 nm, 4.32 nm, and 0.15, respectively. The values of Ro and r were within 2−8 nm range, which is a prerequisite for FRET to take place. Moreover, we found that 0.5Ro < r < 1.5Ro, indicating the existence of static quenching due to complex formation between imipenem and HSA (Trp-214). Mode of Binding and Thermodynamic Parameters by ITC. In order to measure more reliable binding and thermodynamic parameters of imipenem binding to HSA, we performed ITC experiment at 298 K. Data were best fitted after assuming the sequential binding of imipenem at two sites on HSA, and the results are presented in Table 4 and Figure 7. We found that imipenem had two sets of binding sites with affinities of the order of 104 and 105 for low and high affinity binding sites, respectively. The negative enthalpic change (ΔH) for the binding of imipenem to HSA at low as well as high affinity sites indicated that the binding process was exothermic and involved electrostatic interactions.32 Moreover, positive entropic change (ΔS) indicated that hydrophobic interactions were also involved in the binding process at both high and low affinity sites. Further, the negative values of Gibbs free energy change 1790

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Figure 7. ITC of HSA−imipenem interaction for determining binding and thermodynamic parameters. Each downward peak in the upper panel shows changes in heat accompanying each titration. Lower panel represents an integrated plot of the amount of heat liberated per injection as a function of the molar ratio of imipenem to HSA.

hydrogen bonds were involved in the binding of imipenem to subdomain IIA−IIB of HSA (Figure 8D). The ASA of imipenem was estimated to be 421.16 Å2, which was reduced to 56.06 Å2 upon binding to HSA at Sudlow’s site II located at subdomain IIIA (Table S1 in the Supporting Information). A similar observation was also reported in the case of diazepam, a well-known Sudlow’s site II (subdomain IIIA) binding drug (Table S1 in the Supporting Information). On the other hand, the ASA of imipenem bound only at subdomain IIA−IIB of HSA was much more (142.20 Å) as compared to that in the case of burial of ibuprofen (45.62 Å), a known subdomain IIA−IIB binding drug. Thus, a partial burial of imipenem at subdomain IIA−IIB indicated only a superfacial binding mode. Moreover, the change in accessible surface area (ΔASA) of HSA in the absence and presence of imipenem bound at subdomain IIIA (Sudlow’s site II) and subdomain IIA−IIB showed that the residues that were involved in diazepam−HSA and ibuprofen−HSA interactions (taken as references) were also involved in the binding of imipenem to HSA at subdomain IIIA (Sudlow’s site II) and subdomain IIA− IIB, respectively (Table S2 in the Supporting Information). Identification of Imipenem Binding Site by Drug Displacement. A majority of ligands and drugs bind to HSA at two primary sites (known as Sudlow’s sites I and II), which are located deep inside the subdomains IIA and IIIA, respectively. Warfarin and diazepam are considered as marker drug molecules that exclusively bind to HSA at Sudlow’s sites I

Figure 6. Identification of imipenem binding site on HSA by sitespecific drug displacement method. Stern−Volmer plots of imipeneminduced fluorescence quenching of HSA:diazepam (panel A), HSA:warfarin (panel B), and HSA−ibuprofen (panel C) preincubated at 1:0 (●), 1:0.5 (▲), and 1:1 (▼) molar ratios. The concentration of HSA was 2 μM at pH 7.0 and 298 K.

imipenem complex at Sudlow’s site II located in subdomain IIIA (Figure 8B). On the other hand, Ala-213, Lys-323, Asp324, Leu-327, Leu-347, Ala-350, Lys-351, Glu-354, and Val-482 were involved in the interaction between HSA and imipenem at subdomain IIA−IIB interface (Figure 8C,D). Moreover, the negatively charged amino group of imipenem was found to interact electrostatically with Lys-351, Leu-327, Leu-347, Asp324, and Glu-354 residues (Figure 8D). Further, Ala-213, Lys323, Ala-350, and Val-482 residues of HSA made hydrophobic interactions with imipenem. It was interesting to note that no 1791

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Figure 8. Molecular docking of imipenem on the three-dimensional structure of HSA (1AO6). Panels A and C show the residues of HSA interacting with imipenem at subdomain IIIA (Sudlow’s site II) and subdomain IIA−IIB, respectively. Panels B and D show the residues of HSA involved in electrostatic (pink) and hydrophobic (light green) interactions. The hydrogen bond is represented as red and blue short dashes in panels A and B, respectively.

Figure 9. Far-UV (panel A) and near-UV (panel B) CD spectra of HSA alone () or in complex with imipenem (−•−) at a 1:1 molar ratio. The concentration of HSA and imipenem was 2 μM in 20 mM sodium phosphate buffer at pH 7.0 and 298 K.

and II, respectively.23 In order to locate the binding site of imipenem on HSA, fluorescence quenching was measured at different HSA-marker molar ratios (1:0, 1:0.5, and 1:1) as a function of varying imipenem concentration (Figure 6). We found that the binding of imipenem (as reflected by slope of the regression line) decreased at a 1:0.5 HSA:diazepam ratio and was completely abolished at a 1:1 HSA:diazepam ratio. We also found that imipenem did not compete with warfarin for binding to Sudlow’s site I (subdomain IIA) and hence no fluorescence quenching was observed in the case of HSA:warfarin complex. ITC experiments together with molecular docking studies showed that imipenem had a high affinity and a low affinity binding site on HSA, located at subdomain IIIA (Sudlow’s site II) and at subdomain IIA−IIB, respectively. We further confirmed the binding of imipenem to subdomain IIA−IIB by performing drug displacement experi-

ments using ibuprofen. Ibuprofen binds specifically to Sudlow’s site II and has a secondary binding site at subdomain IIA− IIB.23 The quenching of HSA fluorescence with increasing imipenem concentration decreased with increasing ibuprofen concentrations, thus showing that the two drugs shared a common binding site. We also found that the quenching of HSA fluorescence by imipenem was more in HSA−ibuprofen complex than that observed in HSA−diazepam complex. Conformational Changes in HSA upon Imipenem Binding. Far-UV and near-UV CD Spectra. The possible effect of imipenem binding on the overall structure of HSA was monitored by CD spectroscopy, and the results are presented in Figure 9. The far-UV CD spectra of HSA in the absence of imipenem showed two negative bands at 208 and 222 nm, a characteristic of the α-helix protein.30 Upon imipenem binding at a 1:1 molar ratio, loss in the overall secondary structure of 1792

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Figure 10. Synchronous fluorescence spectra (panel A is Δ15; panel B is Δ60) of HSA alone () or in complex (1:1) with imipenem (- - -). The concentrations of HSA and imipenem were 2 μM in 20 mM sodium phosphate buffer, pH 7.0 at 298 K.

HSA was observed (Figure 9A). The α-helical contents (calculated using eq 2) of HSA in the absence and presence of imipenem (1:1) were found to be 63.6% and 60.5%, respectively. Near-UV CD spectra of HSA in the absence and presence of imipenem (1:1) were also measured to monitor the change in tertiary structure (Figure 9B). The near-UV CD spectra of HSA in the absence of imipenem was characterized by the presence of two negative bands around 262 and 268 nm and the presence of two positive peaks around 275 and 292 nm.47 In HSA−imipenem complex (1:1), partial disruption of the tertiary structure was observed as evident by the loss of peaks at 262 and 268 nm. The CD-spectral analysis showed that the overall conformation of HSA was disrupted in the presence of imipenem. Our results are in agreement with previous studies that advocate a partial loss in the overall conformation of HSA upon ligand binding. The binding of ligands such as genistein, palmitine, bis(2-benzimidazolyl)-1,2-ethanediol, tetracycline, 7hydroxycoumarin derivatives, and ibuprofen to HSA has resulted in the loss of α-helical content at various ligand:HSA molar ratios.30,48−52 Synchronous Fluorescence. Synchronous fluorescence is a popular technique to study the microenvironment of aromatic amino acid residues, particularly Tyr and Trp. The synchronous fluorescence spectrum was obtained by simultaneously scanning the excitation and emission monochromators while maintaining a constant wavelength interval between them to get information in the vicinity of Tyr (Δλ = 15) or Trp (Δλ = 60). It was clear from Figure 10A that the emission peak of HSA did not change significantly in the presence of imipenem (1:1), indicating that the microenvironment of Try residues remained unaltered. On the other hand, the emission peak of Trp-214 of HSA was red-shifted by 1 nm, indicating a modified microenvironment around Trp residue (Figure 10B). It is inferred that, upon imipenem binding, the conformation of HSA was changed in such a manner that the polarity around Trp-214 was increased marginally. UV−Vis Absorption Spectroscopy. UV−vis absorption spectroscopy has been widely used to study the conformation of protein in different experimental conditions including protein−ligand binding. The absorption spectra of HSA in the absence and presence of imipenem are shown in Figure S1 in the Supporting Information. The absorption spectrum of HSA was characterized by two prominent peaks at 205 and 278 nm, which reflected the conformations of protein backbone and

aromatic amino acids, respectively. Upon imipenem binding, the peak at 205 was red-shifted by 3 nm along with an increase in its intensity. Also, the intensity of peak at 278 nm was increased with no apparent shift in wavelength. The change in the conformation of HSA on imipenem binding was also evident from the difference spectrum (Figure S1A,B in the Supporting Information). Three-Dimensional Fluorescence. Three-dimensional fluorescence has been widely used to simultaneously monitor the change in molecular environment of peptide backbone and aromatic amino acids of a protein upon ligand binding.52 Figure S2A,B in the Supporting Information shows the threedimensional fluorescence spectra of HSA alone and in complex with imipenem, respectively. Peak a is the Rayleigh scattering peak (λem = λex), and peak b is the second-order scattering peak (λem = 2λex). On the other hand, peaks 1 and 2 represent the fluorescence spectral characteristics of Trp/Tyr residues and polypeptide backbone, respectively. Table 5 shows that the Table 5. Three-Dimensional Fluorescence Characteristics of HSA−Imipenem Complex conditions HSA only HSA + imipenem (1:1)

peak no.

peak position [λex/λem (nm/nm)]

intensity of the peak

1 2 1 2

280/335 230/332 280/336 230/335

1001 205 880 224

peak 1 of HSA−imipenem complex was decreased by 20% along with a minor red shift of 1 nm. On the other hand, peak 2 of HSA−imipenem complex was red-shifted by 3 nm along with 9% increase in its intensity. These results together with CD, intrinsic fluorescence, synchronous fluorescence, and absorbance spectroscopy data demonstrate that the binding of imipenem to HSA induces conformational changes in the overall structure of HSA. Such conformational changes are crucial for the physiological function of serum albumin as the altered conformation may have distorted binding site and modified affinity for other drugs and metabolites. Effect of Imipenem Binding on the Esterase-like Activity of HSA. HSA is primarily a transport protein, yet it possesses catalytic functions also like hydrolytic activity, esterase-like activity, etc.31,53 The residues Arg-410 and Tyr411, which are located in the subdomain IIIA or drug binding Sudlow’s site II of HSA, play a crucial role in esterase-like 1793

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Figure 11. Steady-state kinetics of p-NPA hydrolysis by HSA in the absence and presence of varying concentrations of imipenem at 298 K. The concentration of HSA was 12 μM while the concentration of imipenem was 0 μM (●), 3 μM (Δ), 6 μM (▲), 9 μM (▽), and 12 μM (▼) in 20 mM sodium phosphate buffer, pH 7.0. Panel A shows the Michaelis−Menten plot, while panel B shows the Lineweaver−Burk plot.

[urea] at HSA:imipenem molar ratios of 1:0 and 1:1, respectively. We found that HSA started to unfold at 2.50 M urea and followed a two-step transition with the accumulation of an intermediate state (I) around 4.75−5.25 M urea in the absence of any ligand.25,42 It was reported earlier that the unfolding of domain III was responsible for the formation of intermediate state in HSA upon urea denaturation.47,54,55 The binding of imipenem to HSA at a 1:1 molar ratio resulted in a shift of the N ↔ I transition toward higher [urea] without affecting the I ↔ D transition, indicating the stabilization of subdomain III in the presence of imipenem. The Cm value for the N ↔ I transition in 1:1 HSA:imipenem molar ratio was marginally higher (by 0.16 M) than in HSA alone, while that for the I ↔ D transition remained similar in both cases. The change in free energy of stabilization (ΔΔG°D) of HSA for the N ↔ I transition in the presence of imipenem (1:1) was found to be 3.5 kJ/mol, whereas the I ↔ D transition remained unaffected (ΔΔG°D = 0.3 kJ/mol) (Table 7). Thus, the native state of HSA was stabilized by 3.5 kJ/mol as compared to the intermediate state, while the N ↔ I transition remained unaffected upon imipenem binding. It is known from previous studies that the binding of small ligands to the native state is known to increase the overall stability of the protein.30,54 These results together with conformational studies clearly indicated that the binding of imipenem to the native HSA at subdomain IIIA induces conformational changes in domain I and/or domain II in such a way that the Trp-214 is now placed more closely to the bound imipenem. Our findings are well supported by other studies that have reported ligand-induced conformational changes between domains.24,30,33,50,52

activity. Thus, the catalytic activity of HSA was investigated on p-nitrophenyl acetate (p-NPA) to ascertain the involvement of Arg-410 and Tyr-411 in imipenem binding. The Michaelis− Menten plots of p-NPA hydrolysis at different imipenem:HSA concentrations (0:1, 0.25:1, 0.50:1, 0.75:1, and 1:1 molar ratios) were analyzed to deduce kcat and Km values (Figure 11A and Table 6). We found progressively increased Km values and Table 6. Steady-State Kinetic Parameters for Esterase-like Activity of HSA HSA:imipenem

Km (μM)

kcat (s−1)

kcat/Km (M−1 s−1)

1.00:0.00 1.00:0.25 1.00:0.50 1.00:0.75 1.00:1.00

73.8 92.8 126.1 153.4 198.3

0.0161 0.0161 0.0165 0.0164 0.0167

218.4 173.5 131.1 106.9 84.2

similar kcat values, which ultimately diminished the catalytic efficiency (kcat/Km) of HSA toward p-NPA with increasing imipenem concentrations. It was clear that the Km values for the hydrolysis of p-NPA by HSA were increased from 73.8 μM to 198.3 μM at imipenem:HSA molar ratios of 1:0 and 1:1, respectively. In addition, we found that the kcat values were similar at different HSA:imipenem molar ratios (Table 6). The kinetic data were also plotted as Lineweaver−Burk plots to deduce the mechanism by which imipenem inhibited the esterase-like activity of HSA (Figure 11B), and the inhibition constant (Ki) was calculated using eq 12. The increased slope with constant intercept at different imipenem concentrations was an indication of competitive inhibition of HSA’s esteraselike activity by imipenem, and the inhibition constant Ki was found to be 6.44 μM. The above results led us to infer that imipenem competitively inhibited the esterase-like activity of HSA by binding to the active site residues Arg-410 and Tyr 411 at subdomain IIIA (Sudlow’s site II) of HSA. Effect of Imipenem Binding on Stability and Unfolding of HSA. The effect of imipenem binding on the stability and the unfolding properties of HSA was investigated by urea-induced denaturation monitored by fluorescence intensity measurements as already explained in Methodology. Figure 12 shows the relative fluorescence intensity (RFI) at 341 nm, fraction denatured ( f D), and stability plots as a function of

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CONCLUSIONS This is the first study that demonstrates a detailed binding mechanism of imipenem on HSA and its influence on conformation, catalytic activity, and stability of HSA. Our results showed that imipenem binds to HSA at a high affinity site (Sudlow’s site II located in subdomain IIIA) and a low affinity site (located in subdomain IIA−IIB). It is well established in the literature that various ligands, including other β-lactam antibiotics (e.g., ibuprofen), bind to subdomain IIIA of HSA and quench its fluorescence, although it is smaller 1794

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is brought near to ligand bound at subdomain III. Moreover, from our FRET experiment, it is clear that the distance between ligand and Trp-214 was optimum for quenching to take place.56−58 The structure, function, and folding of HSA is disrupted upon imipenem binding, which in turn will affect the physiological function of HSA. Moreover, the binding of imipenem on HSA suggests that its bioavailability to the site of infection would be compromised. Further, a nonoptimal dose of the drug will put a positive selection pressure on pathogenic bacteria to develop resistance against imipenem (carbapenems) also, thereby augmenting the bacterial antibiotic resistance problem.

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ASSOCIATED CONTENT

S Supporting Information *

UV−vis absorption spectra (Figure S1) and three-dimensional fluorescence spectra (Figure S2) of HSA and HSA−imipenem complex. Accessible surface area (ASA) of drug before and after binding to HSA (Table S1) and change in accessible surface area (ΔASA) of HSA upon imipenem binding. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 00919837021912. Fax: 0091-571-2721776. E-mail: asad.k@rediffmail.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.T.R. acknowledges University Grants Commission (New Delhi, India) for Dr. D. S. Kothari Postdoctoral Fellowship. H.S. is thankful to the Department of Biotechnology (New Delhi, India) for financial assistance in the form of a studentship.

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ABBREVIATIONS USED BAR, bacterial antibiotic resistance; ESBLs, extended spectrum β-lactamases; HSA, human serum albumin; p-NPA, p-nitrophenyl acetate; CD, circular dichroism; FRET, fluorescence resonance energy transfer; ITC, isothermal titration calorimetry; ADT, AutoDockTools; MMFF, Merck molecular force field; LGA, Lamarckian genetic algorithm; ASA, accessible surface area

Figure 12. Equilibrium unfolding process and stability of HSA− imipenem complex at a molar ratio of 1:0 (●) and 1:1 (▲). Panel A shows urea-induced unfolding of HSA and HSA−imipenem complex as monitored by intrinsic fluorescence. Panel B shows a plot of fraction denatured ( f D) versus [urea] for N ↔ I and I ↔ D transitions. Panel C shows the stability curve as a function of [urea] for the transitions depicted in panel B. The concentrations of HSA and imipenem were 2 μM in 20 mM sodium phosphate buffer, pH 7.0 at 298 K.

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Table 7. Urea-Induced Unfolding of HSA in the Absence and Presence of Imipenem HSA:imipenem

transitions

m (kJ/ mol/M)

Cm (M)

ΔGD° (kJ/ mol)

ΔΔGD° (kJ/ mol)

1:0

N↔I I↔D N↔I I↔D

−5.65 −5.01 −6.16 −5.39

3.68 6.52 3.84 6.61

20.1 12.3 23.6 12.0

3.5 0.3

1:1

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Molecular Pharmaceutics

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dx.doi.org/10.1021/mp500116c | Mol. Pharmaceutics 2014, 11, 1785−1797