Characterization of the interaction between acotiamide hydrochloride and human serum albumin: ¹H STD NMR spectroscopy, electrochemical measurement, and docking investigations

Abstract

The interaction between acotiamide hydrochloride (Z-338) and human serum albumin (HSA) was investigated by multiple spectroscopic analyses, electrochemical approaches, and computer-aided molecular docking studies. ¹H nuclear magnetic resonance (NMR) spectroscopy and saturation transfer difference (STD) data indicated that Z-338 weakly interacted with HSA. STD signals showed that the benzene and five-membered rings of Z-338 were responsible for the binding efficiency. Fluorescence lifetime measurements implied that Z-338 quenched the intrinsic fluorescence of HSA with a new complex formation via static mode. Key parameters regarding this interaction were calculated from differential pulse voltammetry and fluorescence spectroscopy. Results obtained from the two methods above ascertained the static mechanism and revealed that hydrogen bonding combined with van der Waals forces played a major role in HSA–Z-338 binding. Although the displacement of probes from both sites I and II was observed from competitive STD-NMR experiments, molecular docking results suggested that Z-338 was preferentially bound to site II of HSA. This finding was supported by the esterase-like activity result. A decrease in the esterase-like activity of HSA after Z-338 binding showed that the Arg-410 and Tyr-411 of subdomain IIIA were directly involved in the binding process, which corroborated the accuracy of docking studies. Furthermore, circular dichroism spectra, Fourier transform infrared spectroscopy, and 3D fluorescence demonstrated that Z-338 slightly disturbed the microenvironment of amino residues and affected the secondary structure of HSA. Overall, this study provides valuable information to further understand the use of Z-338.

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1. Introduction

Acotiamide hydrochloride (N-[2-[bis(1-methylethyl) amino]ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl) amino] thiazole-4-carboxamide monohydrochloride trihydrate, Z-338) is a new drug currently being developed for the treatment of functional dyspepsia (FD).¹ Several studies have demonstrated that Z-338 can inhibit the activity of acetylcholinesterase (AChE),² which degrades the neurotransmitter acetylcholine (ACh). The beneficial effects of Z-338 on FD treatment in clinical studies have been proven across Europe, Japan, and the USA.³ As an AChE inhibitor, Z-338 is also used to treat myasthenia gravis.⁴

Human serum albumin (HSA), as the most abundant carrier protein in the circulatory system, plays a major role in the transport and deposition of many endogenous and exogenous ligands.^5,6 Given the availability of multiple interior hydrophobic pockets and the flexibility to adapt to the drug's shape,⁷ HSA can increase the apparent solubility of hydrophobic drugs and modulate drug delivery to cells in vivo. Therefore, the interaction between drugs and HSA has long been focused in many important research fields.^8–10

Several methods have been employed to explore the binding of drugs to protein, such as equilibrium dialysis,¹¹ isothermal titration calorimetry,¹² capillary electrophoresis,¹³ and spectroscopic analysis.¹⁴ Another rising technique used for the system is nuclear magnetic resonance (NMR), which is a powerful tool for understanding weak binding processes at the molecular level and analyzing biological systems in real time under the systems' working conditions.¹⁵ Accordingly, the saturation transfer difference (STD) is an appropriate tool; it offers several advantages over normal methods to detect binding activity. First, the low-binding affinity ligands can usually be directly identified with high efficiency, even from a substance mixture. Second, the ligand possessing the strongest contact with the protein shows the most intense NMR signals, enabling the mapping of the ligand's binding epitope. And third, its high sensitivity allows using little protein with a molecular weight greater than 10 kDa. That is a crucial factor in NMR-based detection system.^16–19 Given the advantages described above, STD-NMR methods have been widely used to characterize ligand binding in recent years.^20–22 In the present study, the binding affinity of the H proton in Z-338 with HSA and possible binding site were explored by ¹H NMR spectroscopy with the STD technique. To distinguish the quenching mechanism, fluorescence lifetime methods were utilized. The dominant binding parameters were obtained from differential pulse voltammetry (DPV) and fluorescence analyses. Molecular docking techniques were employed to visualize the main binding site of Z-338 on HSA, and the esterase-like activity of HSA after Z-338 binding was assessed. Moreover, the circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), and 3D fluorescence spectra were obtained to analyze whether the secondary structure of HSA changed after Z-338 addition. Overall, this study will provide insights into the molecule-based interaction between Z-338 and HSA.

2. Materials and methods

2.1 Reagents and chemicals

Essential fatty acid free HSA was purchased from Sigma-Aldrich (Milwaukee, USA) and used without further purification. The stock solution of HSA (2.0 × 10⁻⁵ M) was prepared by dissolving solid HSA in 0.05 M PBS at pH 7.4 and stored at 0–4 °C. Acotiamide hydrochloride (Z-338) (≥99.8%) was purchased from Shandong RuiYue Biotechnology Co., Ltd. A stock solution of the drug (2.0 × 10⁻³ M) was prepared by dissolution in anhydrous ethanol. Warfarin and ibuprofen were supplied by Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). Analytical-grade deuterium oxide (D₂O, 99.9% purity), dimethyl sulfoxide-d₆ (DMSO-d₆), and p-nitrophenyl acetate (p-NPA) were obtained from J&K Scientific Ltd. (Beijing, China). All other solutions employed in this work were diluted to the required volume with PBS prepared from triple-distilled water, and all other reagents used were of analytical grade.

2.2 NMR measurements

All NMR experiments were conducted on a Varian Inova 700 MHz spectrometer operating at 298 K by using VNMRJ software (version 2.1B). Stock solutions of Z-338 (4.0 × 10⁻² M), warfarin (8.0 × 10⁻² M), and ibuprofen (8.0 × 10⁻² M) were prepared in DMSO-d₆ and diluted with 0.05 M PBS (50% [v/v] D₂O and H₂O mixture) for further use. Sample volume was 600 μL in a 5 mm NMR tube. The final concentrations of HSA and Z-338 were 1.0 × 10⁻⁵ and 4.0 × 10⁻⁴ M (ratio of [HSA]/[drug] = 1 : 40), respectively. Competition studies were performed using warfarin and ibuprofen, the two primary binding agents to sites I and II in HSA. Samples, which included identical HSA and Z-338 concentrations containing four different concentrations of warfarin (2.0 × 10⁻⁴ M, 4.0 × 10⁻⁴ M, 6.0 × 10⁻⁴ M and 8.0 × 10⁻⁴ M, respectively) or ibuprofen (2.0 × 10⁻⁴ M, 4.0 × 10⁻⁴ M, 6.0 × 10⁻⁴ M and 8.0 × 10⁻⁴ M, respectively), were analyzed. For the STD experiments, the selective saturation of HSA was achieved by a train of 50 ms Gaussian pulses. These pulses were applied at an on-resonance saturation of −0.5 ppm and an off-resonance saturation of −45 ppm. All the spectra were acquired using a sweep width of 8389.26 Hz, 256 transients, and acquisition time of 1 s. The total scan number in the STD experiments was 1024 with 16 ppm spectral widths in typical cases. Each sample was recorded with Watergate solvent suppression to obtain a standard ¹H spectrum. All the spectra obtained were processed and analyzed using ACD/CNMR software (Advanced Chemistry Development, Inc., version 11.0).

2.3 Fluorescence spectroscopy

The fluorescence lifetimes of the HSA and HSA–Z-338 system were measured on a Horiba Jobin Yvon FluoroLog-TCSPC spectrofluorometer (HORIBA, France). The HSA concentration was fixed at 1.0 × 10⁻⁵ M, and the Z-338 concentration varied from 0 M to 2.0 × 10⁻⁵ M. Samples were recorded by fixing 280 nm as the excitation wavelength and 345 nm as the emission wavelength at room temperature. Experimental data were analyzed using the tail-fitting method, and the qualities of the fits were assessed by χ² values and residuals.

Fluorescence measurements were executed using a Cary Eclipse fluorescence spectrophotometer (Varian, USA) equipped with 1.0 cm quartz cells. Prior to the fluorescence measurements, an appropriate volume of Z-338 solution was added to HSA (2.0 × 10⁻⁶ M) that ranged from 0 M to 2.4 × 10⁻⁵ M and mixed for 30 min in a thermostat water bath at 298 K, 305 K, and 310 K. An excitation wavelength of 280 nm was employed, and the emission spectra were recorded from 300 nm to 500 nm, with widths of excitation and emission slits set at 5 and 10 nm, respectively. All fluorescence intensities were corrected to decrease the inner filter effect by using a previously reported method.²³

The 3D fluorescence spectra of the HSA (2.0 × 10⁻⁶ M) and HSA–Z-338 system (molar ratio, 1 : 10) were obtained at an excitation wavelength range of 200–400 nm at 5 nm increments. The emission spectra were monitored from 200 nm to 500 nm.

2.4 Electrochemical investigation

Electrochemical studies were performed on a CHI660E electrochemical workstation (Shanghai Chenhua Instruments Co., China), which was coupled with a conventional three-electrode cell composed of a platinum wire auxiliary electrode, a saturated calomel reference electrode, and a working electrode. The cyclic voltammetry (CV) and DPV behavioral patterns of Z-338 alone and after HSA addition in PBS at pH 7.4 were measured. After each addition of HSA to Z-338, an interaction time of 20 min was maintained, and a voltammogram was recorded. The CV measurements were recorded from −1.0 V to 1.0 V at a scan rate of 0.05 V s⁻¹; the DPV was recorded from 0.2 V to 0.8 V with an amplitude of 50 mV and a pulse period of 0.5 s. The oxidative peak of Z-338 was selected for further analysis. To avoid the competitive adsorption between Z-338 and HSA at the electrode surface, a two-step procedure was developed as described in a ref. 24.

2.5 Molecular docking

Docking studies were performed with the AutoDock 4.2 suite of programs, which utilizes the Lamarckian Genetic Algorithm (LGA) implemented therein. The native structure of HSA obtained from the Protein Data Bank (PDB ID: 1H9Z) was used as template. Receptor (HSA) and ligand (Z-338) files were prepared using AutoDock Tools. Grid maps of 126 × 126 × 126 points in the x, y, and z directions were calculated using AUTOGRID. During the calculation of grid maps, we applied a grid-point spacing of 0.896 Å. The LGA approach was employed to search the global optimum binding position. All the other parameters were in default settings. A subsequent round of dockings with the number of independent runs set to 150 was performed to locate the optimum binding site. The docked conformation with the lowest energy based on the AutoDock scoring function was selected as the binding mode. The output from AutoDock was rendered using Discovery Studio 3.1 software package (State Key Laboratory of Biotherapy, Sichuan University, China).

2.6 Esterase activity assay

The effect of Z-338 on the esterase activity of HSA toward p-NPA was analyzed on a TU-1901 UV-vis spectrophotometer (Persee, Beijing, China) by steady-state kinetics at 298 K. The concentration of HSA was kept constant at 1.2 × 10⁻⁵ M, whereas the concentration of p-NPA was varied from 0 M to 7.0 × 10⁻⁴ M. The rate of p-NPA hydrolysis was determined by measuring the appearance of p-nitrophenol, a yellow product, at 405 nm for 60 s.²⁵ The concentration of p-nitrophenol was determined from the observed absorbance and a standard curve (y = 0.01274x + 0.00444; R = 0.9999).

2.7 CD spectroscopy

The CD spectra of HSA in the absence and presence of Z-338 were measured using Jasco J-815 Circular Dichroism Spectropolarimeter (Jasco, Japan) at 298 K. The spectra of both HSA and the HSA–Z-338 complex with a molar ratio of 1 : 10 were recorded in the range of 200–250 nm. Data were collected with an interval of 1 nm and a scan speed of 100 nm min⁻¹ at 298 K. The corresponding buffer solution was used for baseline correction. All the reported spectra were the average of three consecutive scans for each sample.

2.8 FT-IR spectroscopy

FT-IR measurements were conducted on a Nicolet-6700 FT-IR (Thermo, USA) spectrometer with a smart OMNI-sampler accessory. The infrared (IR) spectra were obtained using the ATR method at a resolution of 4 cm⁻¹ and with 64 scans at room temperature. The absorption spectra of both the HSA and HSA–Z-338 complexes with a molar ratio of 1 : 1 were recorded in the range of 4000–600 cm⁻¹. The corresponding absorbance contributions of the PBS and free Z-338 solutions were recorded and digitally subtracted from the same instrumental parameters.

3. Results and discussion

3.1 NMR spectroscopy for HSA–Z-338 system

NMR technique, as a well-established powerful method, is extensively used to study the interaction between small molecular compounds and biological macromolecules, particularly for screening applications in the pharmaceutical industry.²⁶ The ligand-observed screening is attractive specifically because of its few limitations, such as protein size, non-requirement for isotope labeling, and performance under low protein concentrations.²⁷ In recent years, STD-NMR spectroscopy, as a new NMR-based screening method, was applied to characterize the drug–protein binding activity and has attracted considerable interest among researchers.^21,28 We report herein the binding interaction between Z-338 and HSA, based on ¹H NMR spectroscopy using STD technique. This technique exploited the nuclear Overhauser effect (NOE) transference from the macromolecule to the ligand, under physiological conditions of temperature and pH, and without labeling requirements. When a macromolecule was saturated by a selective radio-frequency irradiation, this saturation will spread within the entire macromolecule via spin diffusion, and will subsequently be transferred to the bound ligand through intermolecular NOEs.²⁶ The most tightly bound ligand ¹H with the macromolecule will receive the most intense magnetization transfer and provide the most intense NMR signals.²⁷

Fig. 1 displays the reference ¹H NMR spectrum Z-338 with HSA in a 40 : 1 ratio (a) and the corresponding STD-NMR spectrum (b). The full STD-NMR spectrum of HSA–Z-338 system is depicted in the ESI as Fig. S1.† As shown in Fig. 1, similar signals of Z-338 can be observed between the ¹H NMR and STD-NMR spectra. Protons of the Z-338 molecule nearest to the HSA can easily be identified from the STD spectrum because these components are saturated to the highest degree.²⁹ The STD effects on the H-5, H-20, H-27, H-16, H-10, and H-13 of the Z-338 molecule were observed despite the weak STD signals. Therefore, the benzene and five-membered rings of Z-338 may assume the most intimate contact with HSA. This phenomenon proved that the Z-338 bound to the HSA receptor slightly, and the ¹H STD-NMR technique can effectively detect the binding process between Z-338 and HSA. Detailed information on the weak interaction was discussed as follows.

Fig. 1 ¹H NMR spectrum of Z-338 and HSA in 40 : 1 ratio obtained with a Watergate scheme for solvent suppression (a) and the corresponding STD spectrum (b).

3.2 Analysis of fluorescence quenching of HSA by Z-338

Fluorescence techniques were used to provide information on the binding properties of small molecules to a protein.³⁰ In the present work, the interaction between Z-338 and HSA was investigated by measuring the intrinsic fluorescence intensity of HSA in the absence and presence of Z-338. The fluorescence spectra are illustrated in Fig. 2. A significant decrease in the fluorescence intensity of HSA was observed when the Z-338 was titrated into a fixed concentration of HSA. Generally, the intrinsic fluorescence of HSA originates from the tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues, with the last residue contributing only slightly.³¹ Some small molecules can change the microenvironment of a fluorophore and decrease the intrinsic fluorescence intensity of HSA by interaction. Thus, the observed decrease in fluorescence intensity can be ascribed to the binding of Z-338 with HSA, which confirmed the ¹H NMR results.

Fig. 2 Fluorescence spectra of HSA in the presence of Z-338: (a) [HSA] = 2.0 × 10⁻⁶ M, (b–g) [Z-338] = 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 × 10⁻⁵ M; (inset) fluorescence lifetime spectra of HSA in the absence and presence of Z-338. [HSA] = 1.0 × 10⁻⁵ M. [Z-338] = 0, 1.0 × 10⁻⁵ M and 2.0 × 10⁻⁵ M (a–c, respectively).

A decrease in fluorescence intensity is known as quenching.³² Fluorescence quenching can generally be classified as static when caused by ground-state complex formation and dynamic when arising from diffusion. The quenching mechanisms can be distinguished by the differences in temperature-dependent behavior and calculated by the well-known Stern–Volmer equation.³³

where F₀ and F are the fluorescence intensities in the absence and presence of a quencher, respectively; [Q] is the concentration of the quencher; and K_sv represents the Stern–Volmer quenching constant. The obtained K_sv values were 3.271 × 10⁴ (298 K, R = 0.9969), 2.538 × 10⁴ (305 K, R = 0.9934), and 1.997 × 10⁴ (310 K, R = 0.9975) M⁻¹ for HSA–Z-338 system. Usually, the quenching constants of dynamic fluorescence quenching increase with temperature because of their dependence on diffusion. By contrast, the stability of a formed complex decreases at high temperatures; hence, a low static quenching constant is produced. In the present study, the values of K_sv gradually decreased with increasing temperature, which demonstrated an observed quenching following a static mechanism dependent on complex formation.

To further distinguish static quenching from dynamic quenching, we measured the fluorescence lifetimes of HSA and the HSA–Z-338 system. The average fluorescence lifetime (〈τ〉) was calculated from the decay times and the relative amplitude (α) by using the following relation:³⁴

〈τ〉 = τ₁α₁ + τ₂α₂ (2)

The fluorescence decay of HSA in the absence and presence of Z-338 is depicted in Fig. 2 (inset, a–c) and Table 1. Fig. 2 and Table 1 reveal that the fluorescence decay curve of HSA is a single exponential with a lifetime value of 5.689 ns. After Z-338 addition, the average lifetime of the HSA fluorophore marginally decreased to 5.514 ns. Generally, static quenching with the formation of static ground-state complexes does not decrease the decay time of the uncomplexed fluorophores.³⁵ A decrease in the mean decay time of the entire excited-state population should be attributed to dynamic quenching, which is a rate process acting on the entire excited-state population. The bound Z-338 may directly influence the fluorophore lifetime, and the observed fluorescence originates from the uncomplexed fluorophores. A minimal decrease in the average lifetime of uncomplexed fluorophores was noted with increasing Z-338 concentration. This alteration of the average fluorescence lifetime in the tested systems suggests that a new complex (HSA–Z-338) was formed by the static quenching process, a result consistent with the fluorescence quenching analysis findings. By forming a complex with the HSA, the Z-338 molecule can be temporarily stored in the plasma and employed to prolong the action time.

Table 1 Lifetimes of fluorescence decay of HSA at different concentrations of Z-338

System	CZ-338 (μM)	τ1 (ns)	τ2 (ns)	α1	α2	〈τ〉 (ns)	χ^2
HSA–Z-338	0	2.451	6.633	0.226	0.774	5.689	0.993
	10	2.510	6.644	0.239	0.761	5.656	1.049
	20	2.047	6.326	0.190	0.810	5.514	1.033

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3.3 Binding constant and number of binding sites

Considering that the quenching mechanism is static, we determined the binding constant and the number of binding sites by electrochemical approaches and fluorescence spectroscopy. Fig. S2† shows the CV curves of Z-338 bound and unbound to HSA. As shown in Fig. S2,† an oxidative peak appeared close to 0.5 V. This peak has been assigned to the hydroxyl in the benzene ring of the Z-338 molecule. After HSA was added, the oxidative peak slightly decreased. Such phenomenon might be attributed to the formation of an electrochemical non-active complex.³⁶ To further analyze the binding property, DPV was conducted. Fig. 3A presents the DPV curves of Z-338 at different concentrations without HSA. Fig. 3B displays the DPV of Z-338 (0.5 × 10⁻³ M) in the absence (curve a) and presence of HSA (curves b and c). As shown in Fig. 3, the oxidative peak increased with increasing concentration of Z-338. However, the oxidative peak of Z-338 decreased when HSA was present. These results confirmed the binding process between Z-338 and HSA. To quantitatively analyze the DPV data, we determined the binding constant β and the number of binding sites m by using the following equation:³⁷where ΔI is the peak current change of the same concentration of Z-338 without and with HSA, ΔI_max is the maximum peak current change, and C_drug is the concentration of Z-338. As another important technique, fluorescence spectra were employed in our study. The relationship between the fluorescence intensity and the quenching medium can be deduced from the following formula:³⁵where F₀ and F are the fluorescence intensities before and after the addition of the quencher, respectively, and [Q] represents the concentration of the quencher. K is the binding constant, and n is the number of binding sites.

Fig. 3 DPVs of Z-338 at the concentration of 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 × 10⁻⁴ M (a–f) respectively (A); DPVs for (a): 5.0 × 10⁻⁴ M Z-338; (b): 5.0 × 10⁻⁴ M Z-338 with 1.0 × 10⁻⁵ M HSA; (c): 5.0 × 10⁻⁴ M Z-338 with 3.0 × 10⁻⁵ M HSA (B).

From the equations above, the binding constant β (values obtained from the DPV method) and K (present values obtained from fluorescence analysis) at three temperatures are summarized in Table 2. The number of bound sites m/n was also calculated. Where m = 1.429, 1.446, and 1.496 at 298 K, 305 K, and 310 K, respectively and n = 0.991, 0.987, and 0.954 at 298 K, 305 K, and 310 K respectively. The binding parameters obtained by the two approaches above were consistent.

Table 2 The binding constant β^a(K^b) and thermodynamic parameters of the HSA–Z-338 system at three different temperatures

System	T/K	β^a(K^b) (10^4 M^−1)	ΔG^a(ΔG^b) (kJ mol^−1)	ΔH^a(ΔH^b) (kJ mol^−1)	ΔS^a(ΔS^b) (J mol^−1 K^−1)
a The binding constants and thermodynamic parameters obtained from differential pulse voltammetry analysis.b Binding constants and thermodynamic parameters obtained from fluorescence analysis. The values within parentheses were gotten from fluorescence method.
HSA–Z-338	298	2.606(2.985)	−25.18(−25.61)
	305	1.337(1.888)	−24.19(−24.76)	−67.42(−61.56)	−141.75(−120.64)
	310	0.914(1.125)	−23.48(−24.16)

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The β/K values decreased with increasing temperatures, further confirming that the quenching was static. Compared with other strong protein–drug systems with binding constants over 10⁶ M⁻¹, HSA–Z-338 exhibited more moderate binding constants. This conclusion was in line with the STD-NMR results indicating that the binding affinity between HSA and Z-338 was weak. The weak affinity between Z-338 and HSA may promote the diffusion of Z-338 from the circulatory system to the target organ (stomach).³⁸ In addition, the number of binding sites m/n was approximately close to one. Therefore, at least one binding site with high affinity of Z-338 in HSA was present.

3.4 Thermodynamic analysis and the nature of binding forces

Given the binding constants of different temperatures, thermodynamic parameters such as ΔG, ΔH, and ΔS can be obtained using the van't Hoff equation and Gibbs–Helmholtz equation.³⁹ The nature of binding forces can be well explained by these parameters. The negative values for ΔG in Table 2 indicate that the binding process of HSA with Z-338 occurred spontaneously. Four acting forces, namely, hydrogen bonds, van der Waals forces, electrostatic forces, and hydrophobic interaction forces, usually exist between small molecules and macromolecules. In Table 2, the negative values of ΔH and ΔS denote that both hydrogen bonds and van der Waals forces contributed dominantly in the formation and stabilization of the HSA–Z-338 complex.⁴⁰

3.5 Identification of the HSA main binding site

3.5.1 Analysis of the competitive STD-NMR experiments. HSA, as an α-helix rich protein, comprises 585 amino-acid residues in three homologous α-helical domains (I–III). Each domain contains 10 helices and is divided into antiparallel six-helix and four-helix subdomains (A and B, respectively). The model transport protein HSA is well known for its high conformational adaptability to a surprisingly broad spectrum of ligands.⁴¹ Sudlow et al. suggested that HSA contained two main distinct binding sites, namely, sites I and II.⁴² Other binding sites were regarded as secondary sites. Warfarin can bind specifically to site I, which is located in subdomain IIA, and ibuprofen binds to site II, which is located in subdomain IIIA.⁴³ Although few ligands are found to be bound in one or several of these secondary binding sites, these substances are usually bound to site I, II, or both sites.⁴⁴

To identify the binding sites of Z-338 on HSA, we regarded competitive STD-NMR experiments as a potential way of comparing the relative affinities of the competing compounds (Z-338 competed with warfarin/ibuprofen and the ratio of [Z-338]/[warfarin/ibuprofen] = 1 : 2) for HSA. The site marker was titrated in a solution containing fixed amounts of Z-338 and HSA. The competitive STD-NMR spectra are presented in Fig. 4. As shown in Fig. 4, the signals of Z-338 were completely abolished after the two site markers added. To further obtain the accurate information about the binding sites, we conducted another STD competition studies by altering the concentration of site markers to confirm whether Z-338 and the site markers compete for the same binding site on the protein or not. The results are depicted in the ESI as Fig. S3 and S4.† By close observation of these spectra, we can draw the conclusion that Z-338 competed with warfarin/ibuprofen in both sites I and II. That is, Z-338 can bind with HSA in site I or site II.⁴⁵ In addition, molecular docking studies were conducted to determine the preferable binding site and obtain a detailed structural representation.

Fig. 4 STD spectra of a mixture solution containing 4.0 × 10⁻⁴ M Z-338 and 1.0 × 10⁻⁵ M HSA (A); STD spectra of a mixture solution containing 8.0 × 10⁻⁴ M warfarin, 4.0 × 10⁻⁴ M Z-338 and 1.0 × 10⁻⁵ M HSA (B); STD spectra of a mixture solution containing 8.0 × 10⁻⁴ M ibuprofen, 4.0 × 10⁻⁴ M Z-338 and 1.0 × 10⁻⁵ M HSA (C). The signals of the competitor are indicated by ※ (warfarin) and # (ibuprofen).

3.5.2 Molecular docking of the HSA–Z-338 interaction. A comprehensive understanding of the probable binding locus of the Z-338 within a complex protein environment is essential. To explore the binding site, the strategy of AutoDock-based blind docking simulation has been adopted. Particular emphasis is rendered toward evaluating unbiased results in this context. The AutoDock strategy was used to search over the entire surface of the protein for the binding sites that simultaneously optimize the conformations of the peptides.⁴⁶ A total of 10 multimember conformational clusters were gathered from 150 docking calculations (Fig. 5). As shown in Fig. 5, the highest populated cluster contained over half of the obtained conformations and found to be on the lowest energy scale. In this cluster, the most favorable conformation of Z-338 binding with HSA was selected for analysis.

Fig. 5 Cluster analyses of the AutoDock docking runs of HSA–Z-338 system.

The stereo view of the favorable docked configuration (Fig. 6A) displays that the Z-338 molecule is well inserted into the hydrophobic cavity of subdomain IIIA, i.e., drug site II of HSA. The predicted binding model results indicated that Z-338 is a higher affinity site II binder. In addition, the optimum interaction between Z-338 and HSA is found to be characterized by a favorable binding energy of −5.28 kcal mol⁻¹, with an inhibition constant of 7.06 × 10⁻⁵ M. At this juncture, the docking simulation results are necessary for comparison with those obtained from the experiments, i.e., the results from DPV and fluorescence methods. For this purpose, we adopted the assumption that the predicted binding constant is the reciprocal of the inhibition constant acquired from docking analysis. With this assumption, the binding constant of the HSA–Z-338 interaction predicted from the docking result was found to be 1.42 × 10⁴ M⁻¹. The free energy change can be calculated from ΔG = −RT ln K, that is, −23.69 kJ mol⁻¹ at 298 K. These above findings revealed by the employed blind-docking simulation are in line with experimental results (Sections 3.3 and 3.4), establishing the reliability of the findings, and may substantiate the practical applicability and feasibility of the methods employed to estimate the parameters.

Fig. 6 (A) Portrait of optimal HSA–Z-338 conformation generated by AutoDock version; (B) 2D docking representation of the interaction between HSA and Z-338, which was rendered using Discovery Studio 3.1 software package. Residues involved in van der Waals interactions are represented by green circles. Hydrogen bonding interactions with amino acid side chains are represented by a green line with the arrows directed toward the electron donor.

To better visualize the binding mode, the 2D ligand interaction diagram is generated and shown in Fig. 6B. The Z-338 molecule is mainly surrounded by Asn-391, Cys-392, Cys-438, Ile-388, Leu-457, Arg-485, Ser-489, Phe-488, Val-473, Val-426, Leu-491, Val-415, Arg-410, Leu-460, Tyr-411, Leu-453, Leu-430, Leu-407, Gly-431, and Phe-403, which are active amino acids for the HSA–ligand interaction. Two hydrogen bonds were found to exist in the HSA–Z-338 system. The hydroxyl in the benzene ring of the Z-338 molecule came in contact with Leu-430 through one hydrogen bond with a distance of 2.094 Å. Tyr-411 was in a suitable position to form another hydrogen bond with the O atom in carbonyl at 2.159 Å. The newly formed hydrogen bonds may make the Z-338 ligand adapt to the shape of a pocket in subdomain IIIA more easily, thereby stabilizing the docking conformation. In addition, when the ligand entered into the receptor, both molecules underwent changes to minimize the space between the two molecules under the influence of van der Waals forces.⁴⁷ The term “van der Waals forces” is generally used to denote a variety of nonspecific interactions, such as dipole/dipole, multipole/multipole, induced dipole/induced dipole, and even cation/dipole interactions. However, recent studies attempted to reveal the details underlying these distinct interactions.⁴⁸ For instance, the interactions involving π electron systems have been shown to include cations and CH groups. Cation/π interactions usually occur between a positively charged molecule and a π system, i.e., aromatic rings, alkenes, and alkynes. Fig. 6B shows that cation/π interactions occur within the HSA–Z-338 system. The residue Arg-410 acts as a cation participating in the cation/π interactions with two rings (benzene and five-membered rings). In conclusion, this docking conformation reconfirmed that the binding forces were mainly governed by hydrogen bonds and van der Waals forces.

3.5.3 Effect of Z-338 binding on the esterase-like activity of HSA. Besides the well-known ligand-binding capacity of HSA, it exhibits an interesting enzymatic property usually described in terms of esterase-like activity. Means et al.^49,50 suggested that HSA can act as an esterase of p-NPA, and this activity leads to the acetylation of a tyrosine residue. Site II has been suggested to be the primary reactive site, especially the residues Arg-410 and Tyr-411 in subdomain IIIA. By contrast, site I is not involved in this interaction, and drugs bound to site I do not inhibit the reaction at low drug-to-HSA ratios.⁵¹ Thus, the catalytic activity of HSA on p-NPA was investigated to ascertain the involvement of Arg-410 and Tyr-411 in Z-338 binding. The kinetic parameters (k_cat and K_m) were determined in accordance with the Michaelis–Menten method by fitting the data to the following equations:where ν and V_max are the initial and maximum velocities of hydrolysis, respectively; [S] is the substrate concentration used; [E] is the enzyme concentration; and k_cat and K_m are kinetic parameters.

The effect of Z-338 binding on the esterase-like activity of HSA was analyzed by monitoring the rate of p-NPA hydrolysis as described above at different Z-338 : HSA ratios (0 : 1, 0.25 : 1, 0.50 : 1, 0.75 : 1, and 1 : 1). By measuring the concentration of p-nitrophenol, the rate of p-NPA hydrolysis was determined. The values of k_cat and K_m are presented in Table 3. The K_m values for the hydrolysis of p-NPA by HSA clearly increased from 58.27 μM to 127.31 μM, whereas the k_cat values were similar at different HSA : Z-338 molar ratios. Ultimately, the progressively increased K_m values and similar k_cat values diminished the catalytic efficiency (k_cat/K_m) of HSA toward p-NPA with increasing Z-338 concentrations. Furthermore, the kinetic data were plotted as Lineweaver–Burk plots (Fig. 7). The increased slope with constant intercept at a series of Z-338 concentrations indicated competitive inhibition of HSA's esterase-like activity by Z-338. On the basis of the above results, we conclude that Z-338 inhibited the esterase-like activity of HSA by binding to the active site residues Arg-410 and Tyr 411 at subdomain IIIA of HSA, which corroborates the accuracy of the docking results. In particular, Z-338 binding to site II was confirmed.

Table 3 Steady-state kinetic parameters for esterase-like activity of HSA

HSA : drug	Km (μM)	kcat (s^−1)	kcat/Km (M^−1 s^−1)
1 : 0	58.27	0.0092	157.89
1 : 0.25	75.69	0.0094	124.19
1 : 0.50	92.32	0.0096	103.99
1 : 0.75	105.85	0.0097	91.64
1 : 1	127.31	0.0101	79.33

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Fig. 7 Lineweaver–Burk plot for the steady-state kinetics of p-NPA hydrolysis by HSA in the absence and presence of varying concentrations of Z-338 at 298 K. The p-NPA concentration was varied from 0 to 7.0 × 10⁻⁴ M. The concentration of HSA was 1.2 × 10⁻⁵ M while the concentration of Z-338 was 0 M (■), 3.0 × 10⁻⁶ M (●), 6.0 × 10⁻⁶ M (▲), 9.0 × 10⁻⁶ M (▼), and 12.0 × 10⁻⁶ M (◀) in PBS, pH 7.0.

3.6 Conformational investigations

3.6.1 CD spectral study. CD, as a powerful and sophisticated tool, is often used to quantitatively analyze the secondary structural changes of protein after small-molecule binding. The far-UV CD spectrum of native HSA (Fig. 8A) exhibits two negative bands in the region at about 208 (π–π*) and 222 nm (n–π*), which indicated an α-helix rich secondary structure. After Z-338 addition, the CD spectral profile of HSA was found to undergo a decrease in CD signal with no significant shift in peak wavelengths. This result implied the occurrence of Z-338-induced conformational change of the native protein by a decrease in the number of α-helices in the native HSA. The α-helical content of HSA is estimated from the following equations:⁵²where C_p is the molar concentration of protein, n is the number of amino-acid residues in the protein, and l is the path length. MRE₂₀₈ is the observed MRE value at 208 nm. Meanwhile, 4000 and 33 000 are the MRE values of β-form with random coil conformation and a pure α-helix at 208 nm, respectively.

Fig. 8 (A) Circular dichroism spectra of free HSA (2.0 × 10⁻⁶ M) and the HSA complex with Z-338 ([HSA]/[Z-338] = 1 : 10) at pH 7.40; (B) FT-IR spectra of HSA (2.0 × 10⁻⁴ M) and HSA–Z-338 system ([HSA]/[Z-338] = 1 : 1) in the region of 1400–1800 cm⁻¹.

By applying the preceding equations, we noted that the percentage of α-helices in free HSA (∼63.8%) agrees closely with literature reports.⁴¹ A loss in the α-helical content from 63.8% to 59.6% after binding with 2.0 × 10⁻⁵ M Z-338 suggested minor peptide strand unfolding, thus apparently demonstrating a drug-induced perturbation of the secondary structure of HSA. This binding process may partially destroy the original hydrogen bonding network of HSA, thus making the polypeptide chain become tender to accommodate the Z-338 molecule in a specific manner inside the protein. Hence, this minor decrease in α-helical content may not affect the physiological function of this carrier protein during the transport of the Z-338 molecule.

3.6.2 FT-IR spectral study. FT-IR spectroscopy, as another efficient tool in the study of the structural characterization of proteins, was used to detect the interaction between Z-338 and HSA. In the IR region, proteins exhibit a number of amide bands. Among these amide bands, the amide I (1600–1700 cm⁻¹, mainly C=O stretching vibrations) and amide II (1500–1600 cm⁻¹, C–N stretch coupled with N–H bending modes) bands, which are sensitive to protein secondary structure, were investigated frequently.⁵³

As shown in Fig. 8B, the FT-IR spectra of free and Z-338-bound forms of HSA were obtained. The characteristic amide I and II absorption peak positions of free HSA were at 1652.70 and 1550.48 cm⁻¹, correspondingly. After Z-338 addition, the two amide bands apparently decreased with a peak shift (amide I: from 1652.70 cm⁻¹ to 1650.78 cm⁻¹; amide II: from 1550.48 cm⁻¹ to 1548.56 cm⁻¹) in absorbance intensity. The peak position shift of amide I, along with the decrease in peak intensity, implies that the α-helical content of HSA has been changed. All these changes demonstrated that the C=O and C–N groups in the protein polypeptides were perturbed, leading to conformational changes in the secondary structure of HSA.

3.6.3 Three-dimensional fluorescence spectroscopy. Further evidence of the conformational change of HSA upon the addition of Z-338 was provided by 3D spectra. 3D fluorescence spectroscopy is an emerging fluorescence analysis technique in recent years and can provide additional information or evidence regarding the conformational changes of protein in the presence of drugs.⁵⁴ As shown in Fig. 9, the 3D fluorescence spectra of HSA and HSA–Z-338 were investigated. The related characteristic parameters are listed in Table 4.

Fig. 9 3D fluorescence spectra of HSA alone (2.0 × 10⁻⁶ M) and in the presence of Z-338 ([HSA]/[Z-338] = 1 : 10).

Table 4 Three-dimensional fluorescence spectral parameters of HSA alone and in the presence of Z-338

System	Peak no.	Peak position [λex/λem (nm nm^−1)]	Intensity F
HSA	a	280/280 → 350/350	230.48 → 372.87
	I	280/338	565.19
	II	225/338	543.28
HSA–Z-338	a	280/280 → 350/350	246.58 → 408.20
	I	280/342	472.71
	II	225/344	337.74

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Peak a, which refers to the Rayleigh scattering peak (λ_ex = λ_em), increased with the addition of Z-338.⁵⁵ This phenomenon may be attributed to the formation of HSA–Z-338 complexes, which induced the change in macromolecular diameter and enhanced the intensity of peak a. Peak I refers to the spectral characteristic of the Trp and Tyr residues, and peak II reveals the fluorescence behavior of the polypeptide backbone structures.⁵⁶ After Z-338 addition, the HSA solution produced a decrease in intensity along with a slight red shift of the maximum emission wavelength in peaks I and II. Table 4 shows that the peak I of the HSA–Z-338 complex decreased by 16% along with a minor red shift of 4 nm. Peak II of the HSA–Z-338 complex red shifted by 6 nm with an 38% decrease in intensity. Hydrogen bonding is indispensable in the formation of HSA secondary and tertiary structures because the structures and functions of several biological molecules are highly dependent on intramolecular hydrogen bonding. From the analysis of intensity changes and peak shifts, we conclude that Z-338 presents minimal effects on the conformation of HSA. As such, the formed hydrogen bond between Z-338 and HSA does not show significant influences on the original bonding, and this condition may be beneficial for HSA.

4. Conclusions

This work first demonstrated a detailed binding mechanism of Z-338 with HSA. The protons in benzene and five-membered rings of Z-338 bound tightly with the hydrophobic cavity of HSA in site II. Hydrogen bonding and van der Waals forces were the main elements to drive the HSA–Z-338 binding reaction. This interaction did not affect the stability of HSA, though the newly formed hydrogen bonds disturbed the original H-network of this protein. However, the esterase-like activity of HSA decreased and the secondary structure of HSA changed after Z-338 addition. Namely, the binding of Z-338 on HSA influenced the catalytic activity and conformation of the latter biomolecule. STD-NMR techniques employed in our study provided the binding affinity of H proton in Z-338 with HSA and ascertained the binding site. The static quenching mechanism was confirmed by fluorescence lifetime measurements. The results obtained from the experiments were in line with molecular docking simulation. Overall, this study can provide some important biophysical insights into the binding mechanisms between drugs and proteins.

Acknowledgements

This work was supported by the Applied Basic Research Project of Sichuan Province (Grant No. 2014JY0042), the National Development and Reform Commission and Education of China (Grant No. 2014BW011) and the Large-scale Science Instrument Shareable Platform Construction of Sichuan Province (Grant No. 2015JCPT0005-15010102).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra08310b

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