AA (MA) to Plasma Proteins: UVâvisible

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Article Cite This: ACS Omega 2019, 4, 4592−4603

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Binding of Mammea A/AA (MA) to Plasma Proteins: UV−visible Spectroscopy and Molecular Dynamics Simulations Study Baruch A. Ateba,†,‡ Daniel Lissouck,† Luc M. Mbaze,́ ‡ Mathieu. J. M. Tjegbe,‡ Anatole G. B. Azeb́ aze,́ ‡ and Cyril A. Kenfack*,† †

ACS Omega 2019.4:4592-4603. Downloaded from pubs.acs.org by 79.110.18.177 on 03/05/19. For personal use only.

Laboratoire Optique et Applications, Centre de Physique Atomique Moléculaire et Optique Quantique, Faculté des Sciences, Université de Douala, B.P. 8580 Douala, Cameroon ‡ Laboratoire de Chimie analytique, structurale et des matériaux, Département de Chimie, Faculté des Sciences, Université de Douala, B.P. 24157 Douala, Cameroon ABSTRACT: Mammea A/AA (MA) is the active compound of Mammea africana stem bark extract, exhibiting anticancer, antimicrobial, and antioxidant properties. To further prospect the usage of MA as a drug, its unusual ratiometric absorbance was exploited in this work to monitor its binding to plasma proteins. Bovine serum albumin (BSA) and human serum albumin (HSA) were considered as models of biological targets. From this process, the binding constant and thermodynamic parameters were evaluated. These binding parameters were similar to those obtained from the conventional fluorescence quenching, thus validating our approach. To further understand the difference of the binding parameters in BSA and HSA, accelerated molecular dynamics simulations for 60 ns were performed, using the restrained electrostatic potential procedure to derive realistic charge distribution on MA, which takes into account multipole contributions. This revealed that MA was bound to a site in the subdomain IB and surrounded by more charged and polar residues in HSA as compared to BSA, thus explaining the different solvatochromism observed for the two proteins. This study proves that MA as a drug can be transported by blood albumin. In addition, due to its ratiometric response in absorbance upon binding to a biological target, MA can be applied as a molecular probe to follow biomolecular interactions.

1. INTRODUCTION Mammea A/AA (MA) (Figure 1) is a phenyl coumarin extracted from the stem bark of Mammea africana, a plant

this compound to be protected by encapsulation inside the hydrophobic cavity of β-CD, which is expected to improve its solubility and reduce its toxicity while preserving its biologically relevant activities. The mechanism that governs the significant and selective antimicrobial activity of MA against certain bacteria is still to be elucidated. A thorough understanding of the modes of action of MA requires the study of its interaction with all relevant targets, such as carrier proteins, nucleic acids, and enzymes. The action of many biologically active compounds is often related to their affinities toward serum albumin,6−11 which is the abundant carrier protein of the blood plasma. It is well known that the nature and magnitude of the drug−protein interaction play a key role in the biological activity of a drug; therefore, weak binding leads to short lifetimes or poor distribution of ligands, whereas strong binding decreases the concentration of free ligands in the plasma.12 Moreover, binding is a system-dependent process that is influenced by the features of the binding site, the extent of protein and ligand conformational relaxation upon association, and the protein and ligand charge distribution.13,14 Thus, the investigation of the interaction of MA with albumin could significantly

Figure 1. Chemical structure of Mammea A/AA.

widely distributed in the equatorial rain forest region of Cameroon and used in local traditional medicine for the treatment of malaria, microbial infections, stomach pains, and skin diseases. This compound was proven to have antimicrobial, anticancer, and antioxidant properties,1−4 but it presents a low solubility in water and a toxicity against normal cell lines.4 The ability of MA to form an inclusion complex with βcyclodextrin (CD) was recently demonstrated,5 thus allowing © 2019 American Chemical Society

Received: December 3, 2018 Accepted: January 30, 2019 Published: March 1, 2019 4592

DOI: 10.1021/acsomega.8b03385 ACS Omega 2019, 4, 4592−4603

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contribute to a better comprehension of its pronounced biological activities in comparison to other coumarin derivatives. Fluorescence titration assay is the most used method in the investigation of drug binding to albumin. This method is based on the tryptophan residue fluorescence quenching upon binding of a small molecule, from which the binding constant and thermodynamic parameters are evaluated. However, this method gives less information on the physicochemical pattern of the drug−protein interface, as tryptophan in most of the cases is far from the binding site. This information is often obtained theoretically from molecular modeling tools. Recently, the absorbance of MA was investigated by us in various organic solvents.5 We found that the absorbance of this compound between 300 and 360 nm shows significant peaks at 340 and 317 nm, each of which comprises two transitions, one being an locally-excited (LE) and the other a charge-transfer (CT) state. The CT and LE were, respectively, sensitive to polarity and proticity. The wavelength positions of these two transitions separated by energy gaps of 452 and 1369 cm−1, respectively, for the long- and short-wavelength bands in hexane, were thought to coincide in polar-protic media at a position depending on the donor potency. This results in an increase of the absorbance, which was more accentuated on the long-wavelength band, thus conferring MA with a ratiometric absorbance, which can be exploited as an alternative to fluorescence method in the study of its binding to a biological target. This method has an advantage to unveil the characteristics of the binding site through MA solvatochromism. In this work, this approach was tested by considering BSA and HSA as biological targets. The binding of MA to these two proteins was monitored by recording its absorbance with increasing concentration of the proteins at 298, 295, and 292 K. From this process, the binding constant and the thermodynamic parameters were estimated. Our results were similar to those of the fluorescence quenching assay taken as a control method. The residues in the vicinity of MA in the two complexes were investigated by molecular dynamics (MD) simulation and allowed to rationalize the difference in the binding thermodynamic parameters for the considered proteins.

Figure 2. Normalized UV absorption spectra of MA (50 μM) alone (blue) and in the presence of increasing concentrations of (A) BSA and (B) HSA (0−120 μM) at 298 K. The red curve corresponds to saturation.

1.03 in BSA and 0.80 to 0.97 in HSA at room temperature. Reporting these data in the equation 1/R = 0.78α + 0.58 (see ref 5) describing the variation of the absorbance ratio R with Abrahams H-bond donor acidity α yields respectively 0.5 and 0.58 for α, thus suggesting that the binding site of MA is highly protic and can be ranged between methanol (α = 0.43) and formamide (α = 0.62). The binding constants obtained by fitting the experimental data to eq 1 (see Materials and Methods) at 298, 295, and 292 K, taking 1 as n value, are reported in Table 1. These values are in the range of micro molar and increase with the increasing temperature, thus showing that MA forms a complex with BSA and HSA in the ground state, which is less stable when the temperature increases. The corresponding binding thermodynamic parameters were further evaluated from the knowledge of the dependence of k on temperature. Using eq 5, the binding free energies of −7.0 and −6.28 kcal mol−1 were obtained for BSA and HSA, respectively, thus showing that the binding is spontaneous. To elucidate the forces that govern the binding mechanism, the enthalpy and entropy were evaluated from Van’t Hoff eq eq 4, by plotting lnK versus 1/T (Figure 4). The yields for the enthalpy, ΔH, were −34.9 and −45.8 kcal mol−1 and for the entropy, ΔS, were −392.7 and −529.1 cal K−1

2. RESULTS AND DISCUSSION 2.1. MA Absorbance in the Presence of BSA and HSA. To prove the binding of BSA and HSA to MA, the absorption spectra of MA were recorded in the presence of the increasing concentration of these proteins. The spectra are displayed in Figure 2A,B, showing two bands at 354 and 317 nm for free MA. Upon addition of proteins, a hyperchromic effect is observed for both proteins, accompanied by additional 8 and 5 nm blue shifts of the band located at 354 nm. These observations indicate that MA is moving from a highly polarprotic water environment to low polar-protic media, thus evidencing its binding to BSA and HSA. The significant blue shift of the long-wavelength band in BSA in comparison to HSA suggests that the dielectric constant and H-bond donor potency of the corresponding binding site are relatively small. To estimate the binding constant, the ratio R of the short- and long-wavelength absorption bands was evaluated at different protein concentrations. The resulting data were plotted and displayed in Figure 3A,B, showing that the ratio R increases with the concentration of the protein. R varies from 0.80 to 4593


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Figure 4. Van’t Hoff plot for the interaction of MA with BSA (black) and HSA (red) deduced from absorbance titration.

appreciate the reliability of our results, the fluorescence quenching assay was undertaken as the control method.

3. ALBUMIN INTRINSIC FLUORESCENCE IN THE PRESENCE OF MA 3.1. BSA and HSA Fluorescence Quenching by MA. In this process, reverse titration of BSA and HSA solution with MA was undertaken. The increasing concentration effects of MA (0 to 120 μM) on BSA (resp. HSA) fluorescence intensity at 298, 295, and 292 K are presented in Figure 5, showing a strong fluorescence emission peak at 353 nm after being excited at 295 nm (resp. 285 nm) under the experimental conditions, whereas MA had no intrinsic fluorescence. A noticeable intrinsic fluorescence decrease was observed. This finding shows that MA binding caused microenvironment changes and produced BSA−MA and HSA−MA complexes. This fluorescence quenching may be induced by a dynamic process due to intermolecular collision between MA and the protein in the excited state, which triggers the de-excitation through a nonradiative channel, or by a static process due to the formation of a nonfluorescent complex in the ground state.15−17 To distinguish between the dynamic and static types of fluorescence quenching, the temperature effects on the interaction between MA and the plasma proteins were further examined. In the interaction between MA and BSA or HSA at different temperatures, a strong linear relationship between F0/ F and the concentration [Q] of the quencher was obtained (Figure 6). The Stern−Volmer quenching constants (KSV) for MA presented in Table 2 appreciably change between 292 and 298 K, and their value was varying from 11 822 to 4356 mol−1 in BSA and from 25 424 to 24 250 mol−1 in HSA. The decrease of the Stern−Volmer quenching constant KSV with the increasing temperature is consistent with a static quenching.15 This is supported by the values of the Kq that were far larger

Figure 3. Binding titration of MA (50 μM) with (A) BSA and (B) HSA at 298 K (black), 295 K (red) and 292 K (blue). The solid line corresponds to the fit of the experimental points with eq 1.

mol−1, respectively, for BSA and HSA. The negative values of ΔH and ΔS suggest that the binding of MA to the proteins is driven by Van der Waals forces and H-bonding interaction and is mainly enthalpy driven. This study proves that the binding of MA with BSA is energetically more favorable in comparison to that with HSA. The remarkable favorable enthalpy in an HSA complex may prove the fact that MA in its binding site was in close proximity with more aromatics and polar (or charged) residues that are thought to promote nonbonding interaction, which is attractive, whereas the unfavorable entropy contribution is probably due to the presence of protic residues that can establish H-bonding interaction with MA inside the protein. This point will be examined in the theoretical part. To

Table 1. Binding Parameters at Different Temperatures Obtained from MA Absorbance BSA

HSA

T (K) K × 104 mol−1 ΔG (kcal mol−1) ΔH (kcal mol−1) ΔS (cal K−1 mol−1) K × 104 mol−1 ΔG (kcal mol−1) ΔH (kcal mol−1) ΔS (cal K−1 mol−1) 298 295 292

11.1 21.2 37.5

−7.0 −7.2 −7.45

−34.9

−392.7

4.0 11.01 19.5 4594

−6.28 −6.81 −7.27

−45.82

−554.0


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Figure 5. Fluorescence emission spectra of (A) BSA and (B) HSA at a concentration of 50.0 μM in Tris−HCl buffer, pH 7.40, 298 K in the absence and presence of MA (0−120 μM) after excitation at 295 and 285 nm, respectively.

Figure 6. Stern−Volmer plots for quenching of (A) BSA and (B) HSA with MA in buffer solution at different temperatures of 298 K (black), 295 K (red), and 292 K (green).

than 2.0 × 1010 mol L−1.16,17 The static quenching mode for MA is consistent with the formation of a nonfluorescent complex in the ground state. This result shows that the modifications observed in both MA absorbance and albumin fluorescence are the consequence of the formation of a complex between MA and serum proteins in the ground state. As a consequence, the binding constant and binding thermodynamic parameters associated with each of these measurements are expected to have the same values. 3.1.1. Binding Parameters. The binding of biomolecules is well characterized in fluorescence titration by the binding site number and binding constant. Figure 7 shows the plots of log[(F0 − F)/F] versus the concentration of MA log[Q] at different temperatures (298, 295, and 292 K). The binding constant Kb and binding site n of MA to BSA or HSA were calculated using eq 3. The values of Kb and n were calculated as the intercept and slopes of the double-logarithm curves as depicted in Figure 7. These results are presented in Table 3. The values of n were approximately equal to 1 corresponding to the presence of a single binding site and thus supporting the value used in R ratio fitting. The Kb values are on the same scale as those obtained from absorbance titration and increase with the decreasing temperature. 3.1.2. Binding Mode. Using the binding constants that were found at three temperatures above (298, 295, and 292 K), the

changes in enthalpy (ΔH) and entropy (ΔS) values were obtained from linear Van’t Hoff plots (Figure 8) with eq 4, and the results are presented in Table 3. The free-energy change ΔG was calculated with eq 5. Its values were −6.54 and −6.45 kcal mol −1 for BSA and HSA, respectively, at room temperature. The corresponding ΔH values were −33.53 and −44.47 kcal mol−1 and ΔS values were −379.36 and −533.84 cal K−1 mol−1 for BSA and HSA, respectively. These values are again very close to those obtained from absorbance titration. 3.1.3. Binding Distances. As the maximum absorption of MA and the fluorescence of BSA and HSA coincide, there is a high probability of fluorescence resonant energy transfer. This was exploited here to accurately describe the position of the binding site of MA with respect to Trp residues. For this purpose, the distances between the ligands of MA and the tryptophan residues in BSA and HSA are estimated from the calculation of the spectral overlap integral between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor (J), the critical distance when the efficiency of energy transfer is 50% (R0), and the efficiency of energy transfer (E), using eqs 6−8, respectively, and are reported in Table 4. The distances between MA and the Trp residues of BSA and HSA were estimated to be 2.48 4595


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Table 2. Stern−Volmer Quenching Constants (KSV) and Bimolecular Quenching Rate Constants (Kq) of the Interaction of MA with BSA and HSA at Different Temperatures BSA

HSA

T (K)

KSV (mol−1)

Kq (×1012 mol−1 s−1)

R*

K (mol−1)

Kq (×1013 mol−1 s−1)

R

298 295 292

5394 11 990 12 523

0.53 1.19 1.25

0.99 0.99 0.99

24 445 25 250 25 424

0.240 0.252 0.254

0.99 0.96 0.97

Figure 8. Van’t Hoff plot for the interaction of MA with BSA (black) and HSA (red) deduced from fluorescence titration.

Table 4. Distances among BSA, HSA, and MA BSA HSA

E

J (cm3 L mol−1)

R0 (nm)

r (nm)

0.59 0.384

1.552 × 10−14 2.379 × 10−14

2.64 2.79

2.48 3.02

to the values obtained from MA absorbance measurements, thus supporting the efficiency of the MA ratiometric absorbance as a tool for monitoring its binding to biological targets. To further understand the relative stability of BSA− MA with respect to HSA−MA complexes, a docking of MA on these proteins followed by 60 ns of MD simulations was undertaken.

4. DOCKING AND MOLECULAR DYNAMICS SIMULATIONS 4.1. Docking and Molecular Dynamics Simulations of MA Binding to BSA and HSA. To obtain preliminary information about the binding site, we first searched for the best pose between the proteins and MA using the Schrödinger program. This preliminary docking showed that MA binds to BSA and HSA at a pocket in the subdomain IB (Figure 9). To make a refinement of the geometry given by molecular docking, we performed MD simulations of BSA−MA and

Figure 7. Double-log plots of MA quenching effects on (A) BSA and (B) HSA fluorescence at 298 K (black), 295 K (red), and 292 K (green).

and 3.02 nm, respectively. The donor-to-acceptor distance, r < 7 nm,18 shows that the energy transfer from BSA and HSA to MA occurs with high probability. This fluorescence titration study confirms that MA binds to BSA and HSA. The retrieved binding parameters are very close

Table 3. Binding Thermodynamic Parameters Obtained from Fluorescence Quenching Measurements at Different Temperatures BSA T (K)

Kb × 104 mol−1

298 295 292

6.23 7.92 19.87

HSA

n

ΔG (kcal mol−1)

ΔH (kcal mol−1)

1.24 1.17 1.26

−6.54 −6.61 −7.08

−33.53

ΔS (cal K−1 mol−1) Kb × 104 mol−1 −379.36

4596

5.36 9.11 25.02

n

ΔG (kcal mol−1)

ΔH (kcal mol−1)

ΔS (cal K−1 mol−1)

1.06 1.18 1.23

−6.45 −6.7 −7.22

−44.47

−533.84


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Figure 9. Structures of (A) BSA and (B) HSA showing MA in its binding site.

HSA−MA complexes for 60 ns, starting with the best docked structures of the two complexes. For comparative purposes, MD simulations for 60 ns were also performed on the protein alone in buffer. The stability of our systems was examined by means of rootmean-square deviations (RMSDs), root-mean-square fluctuations (RMSFs), and the radius of gyration (Rg) with respect to the initial structure. The RMSDs of the full proteins and complexes with respect to initial structures are shown in Figure 10. The systems are well equilibrated as the RMSD values fluctuate around 2.5 Å for both the BSA alone and BSA−MA complex and around 2.5 and 3 Å for HSA and HSA−MA, respectively. These results showed that the insertion of MA did not appreciably affect the amplitude of the bonds’ deformation and hence the geometry of BSA and HSA. This is further supported by the radius of gyration (Rg) (Figure 11), which fluctuates around 27 Å for BSA and 26.3 Å for HSA, thus remaining very close to the experimental value of 29.9 ± 0.08 Å19 recorded for BSA alone. The geometry corresponding to the frame with the lowest RMSD with respect to the initial structure in the complexes (Figure 12) was retrieved for structural analysis. This shows that the amino acid residues at a distance of less than 4 Å from MA comprised Leu122, Leu178, Ile181, His145, Glu182, Pro117, Pro119, Tyr137, Tyr160, Arg185, Met184, Ile141, Leu115, Lys114, Val188, Arg144, Pro113, Leu189, and Leu112 in BSA, whereas HSA was surrounded by Tyr161, Val116, Tyr138, Leu115, Arg186, Met123, Arg117, Arg114, Arg145, Asp183, Glu520, Leu514, Ser517, and Thr515. It appears that the vicinity of MA in HSA contains more polar and charged residues. In the HSA-MA complex, the proximity of Glu520,Leu514, Ser517, and Thr515 with the binding pocket is probably the consequence of the local volume inflation consecutive to MA binding, as these residues belong to subdomain IIIB. Interestingly, the calculated center-of-mass distances between MA and Trp213 and Tr134 (resp. Trp214) of BSA (resp. HSA) were estimated to be 23.88 and 17.28 Å (resp. 28.51 Å) (Figure 13), respectively, very close to the experimental distance r = 24.8 Å (resp. r = 30.2 Å), thus demonstrating that MA was bound to these proteins in the subdomain IB. Furthermore, the fluctuations of a dynamical system (e.g., protein) about some average position or the local protein mobility were analyzed by the time-averaged RMSF values of the free protein and the complexes, plotted against residue

Figure 10. Time dependence of root-mean-square deviations (RMSD’s). Cα RMSD values for free BSA (in black) and the BSA−MA complex (in red) during a 60 ns MD simulation.

numbers, on the basis of the 60 ns trajectory data (Figure 14). The resulting RMSF profile indicates that BSA and HSA dynamical motion is reduced upon MA binding as the residue displacement decreases. This behavior is more evident in the loop region and for residues that are in close contact with MA, namely, Leu115, His145, and Glu182 in BSA and Leu115 and Asp183 in HSA, which were H-bonded to MA (Figure 15). 4597


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the binding free energy. The negative (unfavorable) entropy ΔS may be explained by the H-bonding interaction of MA with residues in binding sites.

5. CONCLUSIONS The interaction between MA and BSA and HSA was studied by exploiting the MA ratiometric absorbance and using the conventional tryptophan fluorescence quenching as a control method. This work demonstrates that the variation of MA absorbance under the addition of protein solution at different temperatures can provide all of the binding parameters such as the binding constant, binding free energy, enthalpy, and entropy. The MA absorption bands’ solvatochromism upon the addition of proteins is evidence of the interaction between MA and its biological target, which is driven by van der Waals forces and hydrogen bonding formation. Molecular dynamics simulation used as a complementary method suggests that MA interacts with the subdomain IB of BSA and HSA. The work providing information on the binding parameters of BSA−MA and HSA−MA complexes has implications in the pharmaceutical usage of this compound. The ratiometric response of MA in absorbance upon binding to a biological target suggest that MA can be used as a molecular probe to follow biomolecular interactions or in surface science. The potency of MA to discriminate BSA and HSA, for which the binding sites are different by only a few residues, may guarantee the extreme sensitivity of MA. 6. MATERIALS AND METHODS 6.1. Materials. The UV−visible absorption and fluorescence titration were recorded using a fiber-optic spectrometer Avantes 2048 (Avantes, Netherlands) with spectral sensitivity within the 250−1100 nm range. The operating solution was held in a 1.0 cm quartz cell and placed in a sample compartment equipped with a thermostatic bath (HUBER, Germany). MA samples were isolated and purity was checked as described in ref.1 BSA and HSA were obtained from SigmaAldrich. 6.2. Methods. 6.2.1. Absorbance Titration. Titrations were performed by adding increasing concentrations of BSA/ HSA to a 50 μM solution of MA. The binding was monitored by the displacement of the absorption peak and the variation of the absorbance. The complex binding constant k was obtained as a fit parameter by plotting the ratio R = AS/AL of the shortand long-wavelength optical densities as a function of the total protein concentration and fitting to the following equation

Figure 11. Time evolution of the gyration radius (Rg). Rg values during 60 ns of MD simulations of HSA (in black) and HSA−MA (in red).

To appreciate the deformation of MA upon binding, the variation of the dihedral angles a1, a2, and a3 describing, respectively, the orientations of the phenyl, but-2-enyl, and oxobutyl groups5 in the protein with respect to free MA was evaluated. The calculated variations were −32, +10, and +128° in BSA and +82, +24, and +111° in HSA, respectively, thus providing an evidence that albumin protein interacts specifically with MA through conformational adjustments of the protein and ligand structure, along with the adaptation of ligand conformation to this site. The MD simulations thus reveal that MA binds to the same site in BSA and HSA. These two sites differ by the presence of more polar and charged residues in HSA, which confers the MA environment in this protein with a very high dielectric constant. This could explain the limited solvatochromism of the longer-wavelength absorption band at 354 nm in comparison to BSA. The negative value of ΔH is likely due to nonbonding interactions of MA with residues in the closer vicinity. Indeed, the aromatic Tyr residues adopt a π−π stacking conformation with respect to MA (Figure 15), thus favoring the mixing of their molecular orbitals, which results in attractive van der Waals forces. In addition, the charged amino acids inside the binding pockets such as Arg, Asp, and Glu are likely to establish nonelectrostatic π−cation or π−anion interactions, which are also attractive. These forces are probably the origin of the favorable enthalpy contribution to

R = R0 +

(R ∞ − R 0) [Lt]

(1 + k([Mt] + n[Lt])) −

(1 + k([Mt] + n[Lt]))2 + nk 2[Mt][Lt] 2k

(1)

where R0 and R∞ are, respectively, the ratio R without the protein and at protein saturation. [Lt] and [Mt] are the total MA and protein concentrations, respectively; n is the number of proteins in the complex. Parameters were recovered from nonlinear fit of the above equation to the experimental data set using MATLAB software. 6.2.2. Fluorometric Titration. In the fluorometric titration experiments, 800 μL samples of each protein were prepared. MA was prepared in an acetone/water solvent (20:80%) and kept in the dark for three days. The fluorescence intensity was measured at 298, 295, and 292 K. Excitations were fixed at 295 4598


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Figure 12. Two-dimensional schematic representation of the frame with the lowest RMSD with respect to the initial structure in the (A) MA−BSA and (B) MA−HSA complexes plotted using the LIGPLOT program.

Figure 13. Relative orientation and calculated distances between the Trp residues and MA in (A) BSA and (B) HSA.

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Figure 14. RMSF values as a function of residue numbers. (A) BSA, (B) HSA, protein alone (black), and complexes (red) with MA. Figure 15. (A) MA binding site on BSA, showing three hydrogen bonds (Leu115, His145, and Glu182). (B) MA binding site on HSA, showing two hydrogen bonds (Leu115 and Asp183).

and 285 nm for both free proteins and in the presence of the increasing concentration of MA. 6.2.3. Fluorescence Quenching. Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore that is induced by a variety of molecular interactions with a quencher molecule.20 Quenching process is described by the Stern−Volmer equation.15

i F − F yz zz = log Kb + n log[Q] logjjj 0 k F {

(3)

where Kb is the binding constant for a site and n is the number

(

F0 = 1 + KSV[Q] = 1 + Kqτ0[Q] (2) F where F0 and F are the relative fluorescence intensities in the absence and presence of the quencher, respectively; τ0 is the lifetime of the fluorophore in the absence of a quencher with a value of 10−8 s;21 and Kq is the quenching constant of a biomolecule. KSV is the Stern−Volmer quenching constant and [Q] is the quencher concentration. KSV was obtained by plotting F0/F versus [Q], and if Kq is much larger than 2.6 × 1010, the maximum diffusion collision quenching rate constant of various quenchers with the biopolymer,16 then the quenching is static in nature.22,23 MA could bind independently to a set of equivalent sites; the number of binding sites and the binding constant were found with the following equation8

of bindings per albumin. The plot of log

F0 − F F

) versus log[Q]

yields log Kb as the intercept and n as the slope. Very often, the interaction between a quencher and biomolecules is mediated by van der Waals forces, hydrogen bonds, and electrostatic forces.15 To describe the interaction of MA with BSA and HSA, the thermodynamic parameters were calculated using the Van’t Hoff equation24 ln K = −

ΔH ΔS + RT R

(4)

where K is the binding constant to a site, R is the universal gas constant (8.314 J mol−1 K−1), and ΔH° and ΔS° are the changes in enthalpy and entropy, respectively, during the quenching process. 4600


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program (http://www.schrodinger.com). MA was docked with these structures of HSA and BSA by default parameters of standard precision methods. The best poses of the molecular docking for the binding of drug−protein were considered from the Glide score (G-score) value.26 Structural figures were generated with PyMol. 6.2.5.2. Molecular Dynamics Simulations. Given the importance of drug−protein interactions in the biological activity of a drug, here classical MD simulations were carried out on free BSA and BSA−MA complexes, as well as free HSA and HSA−MA complexes. The starting structure of MA−BSA and MA−HSA was taken from the docking pose. A series of accelerated MD calculations were performed using the GPU version of pmemd (pmemd.cuda) implemented in the AMBER14 package.27 The LEaP module was used to construct a model of the solvated BSA−MA and HSA−MA complexes in the presence of counterions. The sander and particle-mesh Ewald molecular dynamics (pmemd) modules were used for energy minimization and MD calculations, respectively. The AMBER force field ff14SB28 was used for modeling the HSA and BSA systems. The force field parameters of MA were generated by the antechamber module, based on the general AMBER force field.29 The spatial charges of MA were retrieved from quantum mechanics calculations using the RESP30 procedure, following ab initio optimization of MA molecules at the HF/6-31G* level by Gaussian 09 (Gaussian Inc., Pittsburgh).31 Each system (free BSA, free HSA, and BSA− MA and HSA−MA complexes) was solvated with a 12 Å cubic box of explicit TIP3P water molecules32 to neutralize the whole charge; the required number of Na+ counterions was then added. All simulations were carried out under periodic boundary conditions using a 2 fs time step and SHAKE algorithm33 to constrain all bonds between hydrogen and heavy atoms. Periodic boundary conditions were also applied. A cutoff of 10 Å was applied to nonbonding interactions. Water molecules were minimized [500 steps with the steepest descendent (SD) algorithm and additional 1000 steps with the conjugate gradient (CG) algorithm], whereas the protein was frozen. Then, the whole systems were minimized by means of 1000 steps of SD and 2500 steps of CG. Each system was then heated at a constant volume (NVT ensemble) up to 298 K for 200 ps using the Langevin thermostat34,35 and additionally equilibrated at a constant pressure (NPT ensemble, Berendsen barostat) for 1000 ps to achieve convergence of the density. A production run of 60 ns MD simulations at a constant pressure and 298 K temperature was performed for structural and binding thermodynamic data collection and analysis. 6.2.5.3. Structural Analysis. The structural analysis (RMSD, RMSF, and Rg) was carried out using the cpptraj program of AMBER14.36 Next, the frame with the lowest RMSD, with respect to the initial structure, was taken as the reference output structure.

The free-energy change (ΔG) associated with the interaction of MA with BSA and HSA can be calculated from the following equation24 ΔG = −RT ln Kb = ΔH − T ΔS

(5)

The energy transfer between MA and BSA or HSA was examined in the framework of Föster’s nonradiative energy transfer theory.16 In this theory, the energy transfer efficiency is related to the distance between the donor (protein) and acceptor (MA) by the following equation15 E=1−

R6 F = 6 0 6 F0 R0 + r

(6)

where E represents the energy transfer efficiency between the donor and the acceptor, r is the distance between the donor and acceptor, and R0 is the critical distance when the energy transfer efficiency is 50%. The value of R60 can be calculated using the following relation15 R 06 = 8.79 × 10−25k 2n−4ϕJ

(7)

where k2 is the spatial orientation factor related to the geometry of the donor and acceptor of dipoles, ϕ is the fluorescence quantum yield of the donor, n is the averaged refractive index of the medium, and J is the spectral overlap integral between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. The value of J can be calculated using the following equation15 J=

∑ F(λ)ε(λ)λ 4Δλ ∑ F(λ)Δλ

(8)

where F is the corrected fluorescence intensity of the donor in the wavelength range from λ to λ + Δλ, ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ, and Δλ is the span of the wavelength. In the present case, k2 = 2/3, n = 1.360, and ϕ = 0.150 for BSA and k2 = 2/3, n = 1.336, and ϕ = 0.118 for HSA. 6.2.4. Inner Filter Effect Correction. MA absorbs strongly at Trp excitation and emission wavelengths. At higher concentrations, this can introduce an inner filter effect that decreases the fluorescence emission of BSA/HSA and influences the quenching process. The fluorescence intensities were corrected for the inner filter effect using the following relationship25 Fcor = Fobs × e(Aex + Aem)/2

(9)

where Fcor and Fobs are the corrected and observed fluorescence intensities and Aex and Aem are the absorbances of MA at the excitation and emission wavelengths of albumin, respectively. 6.2.5. In Silico Simulations. To explain the experimental observations of MA binding to BSA or HSA, we employed docking studies of MA with these proteins, followed by MD simulations study. 6.2.5.1. Structure Preparation. The structures of free HSA (chain A: PDB entry 1AO6, structure resolution 2.5 Å) and free BSA (chain A: PDB entry 4F5S, structure resolution 2.47 Å) were obtained from the protein data bank, where nonstandard residues were removed from their structures. The geometry of MA was optimized with density functional theory method, using the B3LYP functional in combination with the 6-31G basis set for all atoms. These structures were used as an input in docking using the Maestro 9.7 of Schrödinger Small Molecule Drug Discovery Suite 2014-1

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Cyril A. Kenfack: 0000-0002-2795-2576 Notes

The authors declare no competing financial interest. 4601


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ACKNOWLEDGMENTS C.A.K. acknowledges the Abdus Salam International Centre for Theoretical Physics (ICTP) for its support to CEPAMOQ through the OEA-AC-71 project and the High Performance Computing Center of the University of Strasbourg for supporting this work by providing scientific support and access to computing resources. A part of the computing resources was funded by the Equipex Equip@Meso project (Programme Investissements d’Avenir). C.A.K. also acknowledges Prof. Dr. Yves Mély and Dr. Guy Duportail for the technical support.

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AA (MA) to Plasma Proteins: UVâvisible

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