Human Serum Albumin Binding of 2-[ ( Carboxymethyl ) sulfanyl ]-4-oxo-4-( 4-tert-butylphenyl ) butanoic Acid and its Mono-Me Ester

Interactions of 2-[(carboxymethyl)sulfanyl]-4-oxo-4-(4-tert-butylphenyl)butanoic acid (compound 1) and its mono-Me ester (compound 2) with the human serum albumin (HSA) have been studied by fluorescence spectroscopy. Comp. 1 exerts antiproliferative activity toward human tumor cells and significant selectivity (tumor vs. healthy cells) in vitro. Competitive binding study with warfarin and ibuprofen as binding site probes, revealed that one molecule of comp. 1 selectively binds to HSA Sudlow site I (warfarin site) with moderate binding constant (Kb = (2.8 ± 0.5)  10 4 M -1 at 37 ± 1 C), while comp. 2 binds to Sudlow site II (Kb = (3.2 ± 0.9)  10 4 M -1 at 37 ± 1 C). Fluorescence quenching at different temperatures was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. Energy resonance transfer between HSA and comp. 1 was examined according to Förster’s non-radiative energy transfer theory. Distance of about 10 Å between ligand and Trp214 (HSA) was obtained. Docking studies confirmed HSA Sudlow site I as a preferable comp. 1 binding site, and Sudlow site II as comp. 2 binding site. Molecular dynamics simulations proved the stability of comp. 1/HSA complex.


2-[(Carboxymethyl
)sulfanyl]-4-oxo-4-phenyl)butanoic acids (CSAB) exert antiproliferative activity toward human tumor cell lines in low micromolar to submicromolar concentrations and significant selectivity.Depending on substitution pattern on the aroyl ring, it was shown that some congeners are significantly less toxic toward healthy human cells, comparing to tumor cells, in vitro [1].Low compounds' toxicity tested on animal model (in vivo) was also proved [2].Within the frame of physico-chemical profiling of CSAB, we report human serum albumin binding of the derivative bearing 4-tert-Bu-group on aroyl ring (1) and its mono-Me ester (2), Figure 1.
Human serum albumin (HSA) is the most abundant protein in blood plasma and tissue fluids (concentration in serum approximately 0.6 mM) and plays an important role in osmotic pressure and pH regulation, transport of endogenous (fatty acids, hormones, bile acids, amino acids, etc.) and exogenous doi: 10.5599/admet.2.2.28 127 compounds (drug molecules and nutrients).HSA performs other functions such as sequestering oxygen free radicals and inactivating various toxic lipophilic metabolites [3].It is a monomeric polypeptide that folds into three structurally distinct and evolution-related helical domains (I-III).Each domain comprises two subdomains (A and B), possessing common structural motifs, connected by a random coil (Figure 2).Two out of six binding sites have been identified as primary sites for the binding of drugs and drug-like compounds [4,5].Sudlow site I is located in subdomain IIA.Low stereoselectivity of this site toward small organic molecules (SOM) might be ascribed to its flexibility.Ligands that strongly bind to site I are generally supposed to be dicarboxylic acids and/or bulky heterocyclic molecules with a negative charge localized in the middle of the molecule (warfarin, azapropazone, phenylbutazone, etc.).Sudlow site II is located in subdomain IIIA, and is smaller, but topologically similar to site I. Site II appears to be less flexible, since ligand binding to this site often shows stereoselectivity, and binding is strongly affected by small structural modifications of the ligand.Site II (also called the indole-benzodiazepine site) preferably accommodates aromatic carboxylic acids, with a negative charge located distant from the hydrophobic region of the molecule.It is specific for ibuprofen, 1-anilino-8-naphthalene sulfonic acid (ANS), diazepam, dansylsarcosine, etc.However, these structural features are not strict prerequisites for site I and site II binding, since numerous ligands are known to bind to both drug binding sites, though with different affinities [6].The SOM in blood circulation can exist as free (unbound) form, or bound to plasma proteins (PP).Weak PP binding leads to a short lifetime or poor tissue distribution of SOM, whereas strong binding decreases the concentrations of free SOM in plasma.Consequently, investigations of the lead compounds' affinity to serum albumin, and the corresponding interaction mechanisms, along with other important ADME/Tox properties (CYP450 transformation, hERG blockade, volume of distribution, etc.), may provide useful information about their pharmacokinetics [6,7].
Binding of SOM to proteins is studied by different instrumental techniques [8][9][10][11][12].Fluorescence measurements are often used to study SOM binding to proteins because information on binding mechanism, binding mode, binding constants, binding sites, intermolecular distances, etc., can be easily obtained.Fluorescence of albumins mostly originates from two types of fluorophores: tryptophanyl and tyrosyl [13].Tryptophan, tyrosine, and phenylalanine are three fluorophores in HSA (and in the majority of other proteins), but the fluorescence of HSA almost exclusively originates from tryptophan 214 (Trp214) alone.Phenylalanine has a very low quantum yield and the fluorescence of tyrosine is almost entirely quenched if it is near to an amino group, a carboxyl group, or a tryptophan.Upon binding of SOM to HSA, changes of intrinsic fluorescence intensity of HSA become induced by the microenvironment of Trp214 [14].
Computational methods, such as molecular dynamics (MD) simulations and molecular docking, are very useful tools for studying the protein-ligand interactions.MD is often used for simulations of protein conformational changes upon binding to ligand.If the position, or the orientation, of the ligand in protein In the present work, we used combination of experimental and computational techniques in order to find the location and to estimate the strength of compounds 1 and 2 binding to HSA.The number of binding sites per HSA molecule has also been determined.The fluorescence quenching mechanism was proposed according to the fluorescence measurements at different temperatures.Competitive binding experiments, using specific binding site probes (warfarin and ibuprofen), revealed the specific sites of comp. 1 and comp. 2 binding to HSA.Along with this, the possibility of resonance energy transfer between HSA as a donor, and comp. 1 as an acceptor of electron excitation were examined according to the Förster's resonance energy transfer (FRET) theory [15,16].Binding of both compounds to HSA was also studied by molecular docking, and the stability of comp.1/HSA complex evaluated by molecular dynamics simulations.

Reagents and Apparatus
Human serum albumin (HSA) was kindly provided by Blood Transfusion Institute of Serbia and purified according to standard procedure [17].The concentration of purified HSA was determined using Bradford method [18].doi: 10.5599/admet.2.2.28 129 Phosphate buffered saline (PBS) was used to maintain the physiological pH value (pH 7.34).Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and potassium chloride used for PBS preparation, as well as warfarin and ibuprofen, were purchased from Sigma-Aldrich.All solutions were prepared with Millipore water.
Fluorescence spectra were recorded on Horiba Jobin Yvon Fluoromax-4 spectrometer, equipped with Peltier element and magnetic stirrer for cuvette, using quartz cell with 1 cm path length and 4 mL volume.An excitation wavelength was 280 nm, with 5 nm slits; emission spectra were recorded in 300-450 nm wavelength range, with 5 nm slits, and 0.1 s integration time.Background PBS signal was subtracted from the each spectrum.
UV/Vis spectra were recorded on GBC Cintra 6 spectrophotometer (GBC Dandenong, Australia) using quartz cell with 1 cm path length and 4 mL volume.All spectra were recorded against the corresponding blank (1 PBS) in the 220-450 nm wavelength range, with 500 nm/min scan speed.

Spectroscopy
Stock solutions of HSA (c = 1.08  10 -3 M), comp. 1 (c 1 = 1.12  10 -3 M), comp. 2 (c 2 = 1.62  10 -3 M) warfarin (c = 4.38  10 -4 M), and ibuprofen (c = 4.61  10 -4 M) were prepared in 1 PBS, pH 7.34, and kept in refrigerator.For comp. 1 and 2 /HSA interaction studies, HSA solution was freshly prepared from the stock, by dilution with a buffer (HSA concentration was kept constant, c = 1  10 -6 M), and titrated with comp. 1 or 2 stock solution to avoid large sample dilution.Upon addition of each aliquot, system was stirred and left to equilibrate for 15 min, before UV/Vis absorption and fluorescence emission spectra recording.For competitive binding experiments, solution containing HSA (c = 1  10 -6 M) and binding site probe (warfarin or ibuprofen, c = 1  10 -6 M) was stirred and left to equilibrate for 1 h.Afterwards, titration with compounds 1 or 2 was performed as described above.All experiments were performed in triplicates and values reported as mean values.
In order to remove the inner filter effect originating from HSA and studied compounds, the absorbancies were measured at the excitation (280 nm) and emission (340 nm) wavelengths, and fluorescence intensities corrected according to Lakowicz equation (1): where F corr and F obs are corrected and observed fluorescence intensities, and A ex and A em are absorbancies at the excitation and emission wavelengths, respectively [19].Corrected fluorescence intensities were used for further calculations.

Synthesis
The preparation and characterization of the comp. 1 were described earlier [1].Compound 2 was obtained by mixing 180 mg (7.75 × 10 -4 mol) of (E)-4-(4-tert-Bu-phenyl)-4-oxobut-2-enoic acid and 1.3 mol equivalents (107 mg) of thioglycolic acid Me-ester during 20 h in 15 mL of MeOH.The volume of the solvent was then reduced to approx.1/8 of the initial, the product precipitated with hexanes/Et 2 O (approx. 4 : 1; 8 mL), and collected by filtration.Crude product was rinsed with small amount of Et 2 O on the funnel, then dried on air.180 mg of the isolated product was obtained (69 % of the theoretical yield).Product is characterized by 1 H and 13 C NMR and LC/HR-MS, confirming > 98% purity.
Docking of both compounds was done by AutoDock Vina 1.1 [27].For docking studies, H atoms were added to the HSA structure (PDB ID 1BJ5 [28]), the system was neutralized (18 Na + ions added) and embedded in explicit solvent (water) sphere encompassing 35 Å from the protein boundary.Molecules of myristic acid, cocrystallized with the protein, were retained.Similar procedure was used to prepare comp.1/HSA complex for MD.CHARMm22 force field and Gasteiger charges were used for MD simulations.Solvated protein and protein/comp.1 complex were minimized at 300 K, during 30000 steps (30 ps).The last frame from the minimization was used for heating of the system to 300 K (10000 steps, 10 ps).The system prepared in this way was submitted to 3 ns of unconstrained MD simulation.Structures of compounds 1 and 2 were docked in the whole structure of HSA, using grid resolution of 1 Å (default in Vina).Exhaustiveness was set to 100.Compounds 1 and 2 were treated as flexible, while protein sidechains were treated as rigid.Best docking solutions of compounds 1 and 2 were optimized and rescored by Fred Rescore, the part of OEDocking suite [29][30][31].All MD simulations were performed in NAMD 2.9 [32] in Linux environment, on multi-node cluster [33] equipped with the dual Intel Xeon X5560 @ 2.8 GHz processors.
Computations in GRID [34] were performed with the carboxylate anion (COO -) probe in the box encompassing the whole protein, or with the water probe (OH2) in the boxes encompassing Sudlow sites I and II, separately, using grid resolution of 0.3 Å.The movement of protein side-chains during calculations was allowed in calculations with COO -probe, while the side chains were hold rigid in calculation with OH2 probe.Figures were prepared in PyMol v 0.99 [35], while Figure 10 is made by OE Docking Report.
Atomic charges of comp. 1 and Trp214 in excited states were obtained in MOPAC2012 [23,24], by singlepoint calculations with semiempirical molecular orbital PM6 method [25], using eigenvector-following optimizer and considering low dielectric constant of medium (4, characteristic for protein interior).MOPAC keywords OPEN(2,2), SINGLET, ROOT=2 (MECI calculations) have been used.The -NH-and -C(O)-termini of Trp214 were capped with Me groups.Exact coordinates of comp. 1 and Trp214, obtained from docking calculations and posterior MD minimization, as described above, were retained.

Results and Discussion
Intrinsic fluorescence of HSA originates from Trp214 residue, the only Trp residue in HSA.Addition of compound 1 to HSA in solution induces changes in HSA fluorescence emission spectrum (Figure 3).As it can be seen on Figure 3

Quenching mechanism
Static and dynamic quenching can be distinguished on the basis of different temperature and viscosity dependence, as well as by lifetime measurements.Dynamic quenching is highly dependent upon diffusion.Higher temperatures result in faster diffusion and hence larger values for bimolecular quenching constant.On the other hand, higher temperatures will typically result in a dissociation of weakly bound complexes, and therefore decrease bimolecular quenching constant in static process.
Stern-Volmer equation ( 2) can be used to fit experimental data: where F 0 and F represent HSA fluorescence intensities in absence (F 0 ), and in presence (F) of the quencher; K sv is Stern-Volmer quenching constant (if the quenching is known to be dynamic, the Stern-Volmer constant is represented by K D, otherwise this constant is described as K SV .),k q is the bimolecular quenching constant, τ 0 is the lifetime of the fluorophore in the absence of quencher (in this case mean fluorescence lifetime for HSA, τ 0 = 7.085 ± 0.094 ns [36]), and [Q] is the concentration of quencher.The plot F 0 /F = f([Q]) at three temperatures is shown on Figure 4. Results of linear regression analysis are given in Table 1.2) at different temperatures (Figure 4); τ 0 = 7.085 ± 0.094 ns.
T (K) (5.9 ± 0.3)  10 12 0.999 Stern-Volmer plots (Figure 4) show good linearity, indicating single quenching type (static or dynamic).As it can be seen from Table 1, Stern-Volmer quenching constant, K sv , decreases as temperature is increased, and the quenching rate constant, k q , values are higher than the maximum scatter collision quenching constant value (2  10 10 M -1 s -1 ) [37].All these findings confirm that the quenching mechanism in comp.1/HSA interaction is static.

Analysis of binding equilibria -binding constant and the number of binding sites
In order to determine preferable binding site of comp. 1 to HSA, competitive binding experiments were performed.Warfarin was used as a Sudlow site I probe, and ibuprofen as a Sudlow site II probe.HSA-site probe complexes were titrated with comp. 1 and fluorescence emission spectra recorded.Results are shown on Figure 5.When small molecules bind independently to a set of equivalent sites of the protein (each binding site has the same capacity for binding of a quencher), the equilibrium between free and bound molecules for the static quenching process is given by modified Stern-Volmer equation ( 3 3) for the apparent binding constant (K b ) and the number of binding sites (n) determination in HSA/comp. 1 interaction; static quenching mechanism was assumed; t = 37±1 C.Binding constant for HSA -(R,S)-warfarin at 37±1 C was also experimentally determined (logK b = 3.4 ± 0.3).
In order to check how small structural variations influence binding affinity and selectivity, mono-Me ester of comp. 1 (Figure 1) was synthesized.Comparing to comp. 1, newly prepared compound (2) has only one ionisable (-COOH) group.Compounds 1 and 2 have similar molecular volumes and formally the same number of H-bond acceptors.The same set of competitive binding experiments was performed; results are shown on Figures 7 and 8, and in Table 3.  3) for the apparent binding constant (K b ) and the number of binding sites (n) determination in HSA-comp.2 interaction; static quenching mechanism was assumed; t = 37 ± 1 C.As it can be seen from experimental results, comp. 1 binds to warfarin binding site (Sudlow site I), while it seems that preferable site for comp. 2 binding is ibuprofen binding site (Sudlow site II).Molecular modeling studies are in agreement with experimental results.
Fluorescence data are often used to evaluate the enthalpy and entropy of complex formation.In order to study the thermodynamics of comp. 1 to HSA binding, fluorescence titration spectra were recorded and binding constant determined at several temperatures according to modified Stern-Volmer equation (3), (an example of linear fitting is shown on Figure 6).Results are shown in Table 4.
where K d stands for dissociation constants (reciprocal to K b values), produced unreliable results.As one of the best methods to study thermodynamics of binding is isothermal titration calorimetry (ITC) [12], we plan to use this method in future experiments.

Molecular modeling
Interaction of comp. 1 with HSA was examined by molecular docking and MD.For docking and MD simulations both carboxylic groups of 1 were deprotonated to mimic the ionization state of the molecule comparable with its ionization under assay conditions.For docking, the whole structure of HSA cocrystallized with myristic acid was used.Protonation states of ionizable residues were ascribed by empirical function [38], and H atoms were added.The protein was neutralized with Na + counter-ions, and then embedded in sphere of explicit water molecules.System was minimized without any constraint in NAMD 2.9.In this way obtained HSA structure was used for docking.
Two out of three best docking solutions found 1 in warfarin binding site (Sudlow site I, Figure 9a); this is in agreement with experimental results.As it can be seen on Figure 9a, carboxylates of the studied compound make salt bridges with Lys195, Lys199, Arg218, and Arg257; the tert-Bu group makes hydrophobic interactions with Leu219, Leu238, and Ile264, but interactions of the phenyl ring, or the aroyl carbonyl group, with HSA residues were not observed.The first three docking poses found comp. 2 in Sudlow site 2 (Figure 9b).Carboxylate moiety makes polar contacts with Arg410 and Tyr411, while ester carbonyl forms H-bond with the backbone NH of the Cys392.The Me-group of the COOMe moiety makes hydrophobic contacts with Ala449.The nonpolar part of the ligand is involved in hydrophobic contacts with Val344, Leu453 and Ala449.As AutoDock Vina provides only estimated overall energy of binding of SOM to respective biological targets, we optimized and rescored binding poses of comp. 1 and 2 by Chemgauss4 scoring function.According to results obtained, highest contribution to ligand-HSA binding should be ascribed to steric interactions.Because the cavity of Sudlow site II is more tight than the cavity of Sudlow site I, comp. 2 make more van der Waals contacts with the amino-acid residues in the Sudlow site II, comparing with the same type of contacts of the comp. 1 with the Sudlow site I (Figure 10, Table 5).More apolar residues in Sudlow site II, comparing to Sudlow site I, causes less favorable protein desolvation term for the comp. 1 binding to Sudlow site I, comparing to the same term for the binding of comp. 2 to Sudlow site II.In this respect, it should be noted that in the frame of the Sudlow site II, global minimum (-11.4kcal/mol) of GRID water probe was found at the very bottom of this site (Figure 13 b).This position is flanked with Ser342, Arg348, and Met446.This is in agreement with the structural features of Sudlow site II binders, bearing H-bond donors or H-bond acceptors at the termini of the hydrophobic part of molecules (naproxen, 6-MNA, iopanoic acid, etc.).As expected, ligand desolvation term is less favorable for the comp. 1 comparing to comp.2, as two carboxylates vs. one, respectively, should be desolvated.On the other hand, comp. 1 bound to the Sudlow site I makes more polar contacts (salt bridges) comparing to comp. 2 bound to Sudlow site II (polar contacts with Arg410 and Tyr411, Figure 9b), thus H-bonding term of the bound ligand is more favorable for the comp.2. As ionic bridges that tightly anchor comp. 1 to Sudlow site I were observed from docking solutions, we searched PDB database for structures of HSA cocrystallized with SOM, having similar structural features.Three such structures were found: 2BXA -CMPF (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid) bound to Sudlow site I. Carboxylates of the ligand make salt bridges with Arg222, Lys199 and Arg257 [6].doi: 10.5599/admet.2.2.28 137 2XSI -Dansyl-L-glutamate bound to Sudlow site I.One carboxylate makes the salt bridge with Lys199, the second carboxylate is in close vicinity of Arg's 218 and 222 [39]; 2XVW -Dansyl-L-arginine bound to Sudlow site I [39].The guanidino moiety of the ligand makes the salt bridge with the Glu153, carboxylate of the ligand is in the close vicinity of Arg218.In all three structures the two salt bridges between the ligand and side-chains of the protein are highly probable.
Afterwards, we performed molecular dynamics simulation of the comp.1/HSA complex, starting from the structure obtained from docking.System was set in a way similar to settings for docking.Protein with docked ligand and myristic acid molecules was neutralized, embedded in water sphere, then minimized, and heated.System prepared in this way was submitted to 3 ns of unconstrained MD simulations.Analyzing the MD trajectory, we found that examined comp. 1 is tightly bound to Sudlow site I: it was anchored by salt bridges of two carboxylates with Arg -NH(NH 2 ) 2 and Lys terminal -NH 3 moieties; Lys199, Lys219, Arg218, and Arg257 were involved in salt bridges.
Distances between Arg guanidino moiety and comp. 1 carboxylates were defined as -NHC(NH 2 ) 2 to -COO -atoms; while distances with Lys were defined as terminal (Nξ) -N +  Fluctuation of the 4-tert-Bu-Ph moiety is illustrated by the distance between centroids defined on the planes of Trp214 phenyl ring and the phenyl ring of comp. 1, as well as by the torsion angles between those two planes (Figure 12).Trp214 is chosen because of its spatial vicinity to 4-tert-Bu-Ph moiety of the compound, in docking solution used for MD simulation.Along with docking and MD simulations, we examined HSA structure by GRID software.Interaction energies of the GRID COO -probe (deprotonated carboxyl group) with the whole structure of HSA were calculated.Overall energy minimum (-25 kcal/mol) of the probe interaction energy was found bellow Sudlow site II (Figure 13) i.e. relatively far from the part of HSA where comp. 1 is bound (as concluded from experiments and modeling).Most probably, compound is too bulky to be accommodated in Sudlow site II.

Energy transfer
Fluorescence energy transfer can occur between the donor molecule in excited state (Trp214 from HSA) and the acceptor molecule in the ground state (comp.1).Distance between the donor and the acceptor can be calculated according to the Förster's theory of non-radiation energy transfer [40].The energy transfer depends upon the distance between donor and acceptor, relative orientation of their dipoles, the extent to which donor emission and acceptor absorption spectra overlap, and a donor quantum yield.The energy doi: 10.5599/admet.2.2.28 139 transfer efficiency, E, represents the fraction of photons absorbed by donor and transferred to the acceptor [19], and can be calculated according to the following equation ( 6): where r represents the distance between donor and acceptor; R 0 is called the Förster's distance, and represents the distance at which energy transfer is 50% efficient (it is typically in the range of 20 to 60 Å for protein-protein interactions).R 0 is calculated according to equation ( 7): where κ 2 stands for the spatial orientation factor of the dipole, n is a refractive index of the medium, Φ is the donor fluorescence quantum yield, and J is the overlap integral of the donor emission and the acceptor absorption spectra (Figure 14).The overlap integral, J, can be calculated according to equation ( 8): where F(λ) represents normalized fluorescence intensity at wavelength λ, ε(λ) is molar absorption coefficient of the acceptor at wavelength λ.In the present study, we have used k 2 = 2/3 (for randomly oriented dipoles), n = 1.336, and Φ = 0.118 [41].Using previous equations, we have calculated the overlap integral (J = 3.26  10 -15 M -1 cm 3 ), the energy transfer efficiency (E = 0.058), the Förster's distance (R 0 = 21.6 Å), and the distance between donor (Trp214 in HSA) and acceptor (comp.1) (r = 36.6Å).Although such results are comparable with many literature reports on the fluorescence energy transfer between HSA and ligand bound to it, obtained distance between the donor and the acceptor (36.6 Å) is inadequately high.Experimental fluorescence data showed that comp. 1 is bound to Sudlow site I. Docking study also found comp. 1 in Sudlow site I, and revealed distance of 8.5 Å between centroids defined on aromatic moiety of Trp214 and aroyl moiety of comp. 1.The attention was paid to κ 2 term (7), which stands for spatial orientation factor of the dipoles.κ 2 Accounts for the angle between donor and acceptor transition dipole moments, and the angle between donor/acceptor transition dipole moments and the vector joining those two dipoles.If all of these angles are 0°, κ 2 = 4; if all angles are 90°, κ 2 = 0.The κ 2 value of 2/3 is commonly used, and accounts for random distribution of the vector orientation, i.e. fast and complete rotation of vectors during donor excited state lifetime.For ligand bound to the protein, such assumption obviously does not hold.Movement of both Trp214 of HSA and ligand bound to HSA was constrained by the rest of the protein, and both entities could only experience rotations and the tumbling of the whole protein-ligand complex, which, for sure, did not influence spatial orientation of the dipoles.If a very small κ 2 value (0.001) is used, and all other values kept as described above, reasonable value for r (approx.10 Å) is obtained.Angle between dipoles of Trp214 and comp. 1 in excited states, calculated on semiempirical level of theory, retaining spatial orientation of Trp214 and comp. 1 exactly as found by molecular docking and posterior minimization by MD, is close to 90°, as is shown on Figure 15.The possibility of energy resonance transfer is examined according to Förster's non-radiative energy transfer theory.According to calculated distance between HSA Trp214 and comp. 1 it was concluded that energy resonance transfer between HSA and comp. 1 is feasible process.Somewhat more detailed

Figure 2 .
Figure 2. a) Structure of HSA (PDB ID 1BJ5) and the location of major binding sites, Trp214 is depicted in stick representation.b) Sudlow site I, with bound warfarin, is depicted from the PDB entry 2BXD.c) Sudlow site II, with bound ibuprofen is depicted from the PDB entry 2BGX.

1 H and 13 C
NMR spectra were recorded in CDCl 3 on a Bruker Avance 500/125 MHz instrument.ESI-MS spectra were recorded on Agilent Technologies 6210-1210 TOF-LC-ESI-HR/MS instrument in positive mode.
F 0 and F are corrected fluorescence intensities, in the absence and in the presence of a quencher, respectively; K b is binding constant, and n is the number of binding sites.Linear dependence log(F 0 -F)/F = f(log[Q]), shown on Figure 6, is used to obtain apparent binding constant values and the number of binding sites.Results are shown in

Figure 9 .
Figure 9. a) Comp. 1 docked to HSA.The system is depicted after energy minimization of the neutralized and solvated system.Compound is bound to Sudlow site I. Salt bridges between -COO -groups of the studied compound and residues Lys195, Lys199, Arg218, and Arg257, observed during MD simulations, are marked by two-headed arrows.b) Comp. 2 docked to HSA.The system is depicted as obtained from the best-ranked docking solution.Polar contacts are marked by two-headed arrows.

Figure 10 .
Figure 10.Schematic depiction of steric contacts between comp. 1 (a), and comp. 2 (b) with the Sudlow site I and the Sudlow site II, respectively.Solid lines represent protein contacts, notch lines -protein cavity.

3 H
of lysine to -COO -distance.Fluctuations of these distances are illustrated on Figure11.Hydrophobic part appeared fairly more mobile, as compared to polar parts of 1. Distances between Leu219, Leu238, Ile264, and tert-Bu group of 1 significantly increase (comparing to docked solution (3.3-3.6 Å)) during first 0.5 ns of simulations; at the end of the simulation tert-Bu group appeared close to Leu234 and Leu260 (3.5 Å).

Figure 12 .
Figure 12. a) Distance between centroids defined on Trp214 phenyl ring and the phenyl ring of comp. 1, b) torsion angle between planes defined on Trp214 phenyl ring and the phenyl ring of comp.1; both during 3 ns of MD simulations.

Figure 13 .
Figure 13.a) Position of GRID COO -probe global minimum (-25 kcal/mol, red spheres) bellow Sudlow site II.Residues of the Sudlow site II, Arg410, Tyr411, Leu453, and Ser489, are marked.b) Position of water GRID probe global minimum (-11.4kcal/mol, light blue sphere) in the frame of the Sudlow site II.

Figure 15 .
Figure 15.Dipoles (values in Debye) of the Trp214 and the comp. 1 in excited states, calculated on semiempirical level of theory.
Interactions between 2-[(carboxymethyl)sulfanyl]-4-oxo-4-(4-tert-butylphenyl)butanoic acid (comp.1) and its mono-Me-ester (comp.2) with the human serum albumin (HSA) have been studied by fluorescence spectroscopy and molecular modeling.Examined compounds have the ability to quench the intrinsic fluorescence of HSA through the static quenching mechanism.Competitive binding experiments at t = 37 ± 1 C with specific binding site probes (warfarin for Sudlow site I and ibuprofen for Sudlow site II) showed that comp. 1 selectively binds to Sudlow site I with moderate binding constant K b = (2.8 ± 0.5)  10 4 M -1 , while comp. 2 preferably bind to Sudlow site II with binding constant K b = (3.2± 0.9)  10 4 M -1 .These results, along with the estimated effective quenching constants suggest that the binding constant between comp. 1 or 2 and HSA is a moderate one.Hence, both comp. 1 and 2 can be stored and carried by HSA in the human body.

Table 4 .
Binding constant for comp.1/HSA interaction measured at different temperatures; log K b values given as mean values of three times performed measurements.
. . .   As it can be seen from Table 4, determined binding constant (K b ) values have high error values, thus data fit to either linear van΄t Hoff equation (4): doi: 10.

Table 5 .
The AutoDock Vina and the Chemgauss4 scores for the best-ranked docking solutions of the comp. 1 and the comp. 2 with the HSA.In kcal/mol; **Components of score, and the total score obtained by Chemgauss scoring function are dimensionless. *