Human serum albumin binding studies of a new platinum(IV) complex containing the drug pregabalin: experimental and computational methods

Article abstract of DOI:10.1080/00958972.2019.1568419

A new platinum(IV) complex, [Pt(en)(Cl)₂(Pregabalin)₂], containing the drug pregabalin was synthesized and characterized by elemental analysis, FT-IR, ¹H NMR, mass spectrum, thermogravimetric analysis (TGA), molecular docking and RHF/PM6 method. Also, the interaction of Pt(IV) complex with human serum albumin (HSA) was studied by using UV–vis, fluorometric, circular dichroism (CD) spectroscopies and molecular docking techniques. The results demonstrated that the binding of the complex to HSA caused strong fluorescence quenching of HSA through static quenching mechanism. Hydrogen bonds and van der Waals contacts are the major forces in the stability of protein-Pt(IV) complex and the process of the binding of complex with HSA was enthalpy driven (ΔH = –105.8 kJ·mol^?1). The results of CD and UV–vis spectroscopy indicated that the binding of the complex to HSA caused conformational changes in HSA. In addition, the study of molecular docking and RHF/PM6 method confirm the experimental results with respect to the mechanism of binding. (Figure presented.).

Full text of DOI:10.1080/00958972.2019.1568419

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Human serum albumin binding studies of a
new platinum(IV) complex containing the drug
pregabalin: Experimental and computational
methods
Nahid Shahabadi, Sara Amiri & Avat (Arman) Taherpour
To cite this article: Nahid Shahabadi, Sara Amiri & Avat (Arman) Taherpour (2019):
Human serum albumin binding studies of a new platinum(IV) complex containing the drug
pregabalin: Experimental and computational methods, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2019.1568419
To link to this article: https://doi.org/10.1080/00958972.2019.1568419
Accepted author version posted online: 23
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Human serum albumin binding studies of a new platinum(IV) complex
containing the drug pregabalin: Experimental and computational methods
NAHID SHAHABADI*†‡, SARA AMIRI† and AVAT (ARMAN) TAHERPOUR‡§
†Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran
‡Medical Biology Research Center (MBRC) Kermanshah University of Medical Sciences, Kermanshah, Iran
§Organic Chemistry Department, Chemistry Faculty, Razi University, Kermanshah, Iran
A new platinum(IV) complex, [Pt(en)(Cl)2(Pregabalin)2], containing the drug pregabalin was
1
synthesized and characterized by elemental analysis, FT-IR, H NMR, mass spectrum,
thermogravimetric analysis (TGA), molecular docking and RHF/PM6 method. Also, the
interaction of Pt(IV) complex with human serum albumin (HSA) was studied by using UV-vis,
fluorometric, circular dichroism (CD) spectroscopies and molecular docking techniques. The
results demonstrated that the binding of the complex to HSA caused strong fluorescence
quenching of HSA through static quenching mechanism. Hydrogen bonds and van der Waals
contacts are the major forces in the stability of protein-Pt(IV) complex and the process of the
binding of complex with HSA was enthalpy driven (ΔH = -105.8 kJ∙mol-1). The results of CD
and UV-vis spectroscopy indicated that the binding of the complex to HSA caused
conformational changes in HSA. In addition, the study of molecular docking and RHF/PM6
method confirm the experimental results with respect to the mechanism of binding.
Keywords: Pt(IV) Complex; Human serum albumin (HSA); Fluorescence quenching; Molecular
docking; Hydrogen interaction
1. Introduction
Pregabalin (figure 1) is a structural analogue of the inhibitory neurotransmitter γ-amino butyric
acid (GABA), but it is not functionally related to it. Pregabalin is structurally related to
gabapentin and has a similar pharmacological profile and anticonvulsant and analgesic activity
[1]. The predominant mechanism of action is thought to be through its presynaptic binding to the
α2δ subunit of voltage-gated calcium channels which in turns leads to reduced release of
neurotransmitters, e.g., glutamate, substance P, and calcitonin gene-related peptide [2].
*Corresponding author. Email: nahidshahabadi@yahoo.com
1

Pregabalin has no plasma protein binding consistent with the lack of interactions with other
anticonvulsants, certain antidiabetics, and oral contraceptives [2], thus readily penetrates the
blood-brain barrier [3]. Analysis of pregabalin for structural alerts using the in silico predictive
systems DEREK and CASE/Multicase suggests that pregabalin contains no biologically relevant
molecular features associated with genotoxicity or carcinogenicity (S.K. Duddy, unpublished
data) [4].
Platinum-based antineoplastic drugs (informally called platins) are chemotherapeutic
agents used to treat cancer. In this kind of chemotherapy, famous drugs include cis-platin and
carboplatin, but several others have been proposed or are under development [5]. Despite the
great efficacy of cis-platin, it shows a high cytotoxicity also for normal tissues and causes several
side-effects as neuro- and nephrotoxicity. Moreover, the effectiveness of cis-platin as a drug is
limited by the development of drug resistance [6]. These disadvantages in turn led to the
development of the next generation of platinum analogues. Octahedral platinum(IV) complexes,
which are now under development, can be described by the general formula cis-[PtA2X2Y2] with
Y as a monodentate anionic leaving ligand. These octahedral complexes display some
advantages compared to the square-planar Pt(II) complexes. They can be administered orally due
to their increased stability and solubility in the gastrointestinal tract. Adverse effects are reduced
due to their lower reactivity. New opportunities of synthesis and more possibilities of structural
variations are opened. In the body, they are reduced to more reactive Pt(II) complexes which
form DNA adducts. Satraplatin [bis-(acetato)-ammine dichloro-(cyclohexylamine) platinum(IV)
(figure 2a), formerly known as JM-216] is a third-generation orally available platinum analogue.
Satraplatin is not recognized by the DNA-repair proteins, so DNA remains damaged and cannot
be replicated, the cell dies, and the problem of resistance is solved [7].
Satraplatin has potent growth inhibition on several human cancers both in vitro and in
small-animal models. In small cell lung cancer cell lines, satraplatin has a 50% inhibitory
concentration in the submicromolar range, equivalent to or less than that of cis-platin [8]. That
shares some structural similarities with cis-platin but also has important differences. Satraplatin
inhibited the growth of cervical cancer cell lines, including those resistant to cis-platin [9]. The
oral route of administration and the intermittent schedule makes it very convenient for clinical
use [10].
2

It is obvious that structural similarity of platinum complexes synthesized with satraplatin
can have a therapeutic effect on cancer. On the other hand, the structural similarity of the
pregabalin drug with plasma amino acids have caused that bioavailability property of the
synthesized complex on the base of platinum strengthens the body. Accordingly, the synthesis of
platinum complex containing pregabalin with therapeutic properties in the field of cancer was
studied.
Human serum albumin (HSA) is the most abundant protein constituent of blood plasma
and serves as a protein stowing component. The proteins generally involved with drug delivery
are serum albumin, lipoproteins, and al-glycoprotein. Serum albumin is the most plentiful protein
present in the circulatory system of a wide variety of organisms.
HSA can bind and carry many drugs through the bloodstream, which are poorly soluble
in water. It has been exposed that the distribution, free concentration and the metabolism of
various drugs can be significantly changed as a result of their binding to HSA [11]. Different
groups of drugs and other bioactive small molecules bind reversibly to albumin and other serum
ingredients, which is an action as carriers. Therefore, detailed investigation of drug-protein
interaction assumes significance for thorough understanding of the pharmacokinetic behavior of
a drug and for the design of analogues with effective pharmacological properties [12].
Since pregabalin cannot connect to plasma proteins [3], the aim of this study was to
design a platinum complex similar to anticancer drugs such as satraplatin and to investigate the
effects of platinum ion on the mode of interaction of pregabalin drug with HSA using a
combined computational and experimental approach. The quenching mechanism, binding
constants and the number of binding sites were evaluated by the fluorescence quenching method.
The nature of forces involved in the interaction was further discussed based on thermodynamic
parameters. The identification of the binding site was evaluated by the competitive fluorescence
method. The effect of Pt(IV) complex on the conformation of HSA was studied based on the
UV-vis and CD spectra. In addition, theoretical calculations (molecular docking and RHF/PM6
method) were used to improve the understanding of the interaction of the Pt(IV) complex with
HSA.
3

2. Materials and methods
2.1. Materials
Pregabalin and HSA were purchased from Sigma and ethanol (99.9%), Na2HPO4, NaH2PO4,
warfarin and ibuprofen were purchased from Merck.
2.1.1. Preparation of stock solutions. Pregabalin solution was prepared in the buffer solution
adjusted to pH 7.4 with 0.1 M Na2HPO4 and NaH2PO4. HSA stock solution (10-4 M, based on its
molecular weight of 66,000) was prepared in 0.01 M phosphate buffer of pH 7.4 and was kept in
the dark at 4 C. Doubly-distilled water was used throughout the experiment.
2.2. Synthesis of dichloride dihydroxy(ethylenediamine)platinum(IV) complex
A solution containing 0.1 g of K2PtCl4 dissolved in water was treated with ethylene diamine and
stirred for 30 min at room temperature. The suspension was cooled and the product was filtered.
0.04 g of the suspension was cooled down to room temperature and filtered dissolved in 8 mL of
oxygenated water and after stirring at room temperature for 15 min, a powdery yellow product
was filtered and dried at room temperature.
The compound was characterized by elemental analysis and IR. The result of the
elemental analysis is as follows: Analytically found composition of [C2H10Cl2N2O2 Pt]: C, 6.70;
H, 2.74; N, 7.68 %. Corresponding calculated composition: C, 6.66; H, 2.77; N, 7.77%. The IR
spectrum for [C2H10Cl2N2O2 Pt] is shown in figure 3.
2.3. Synthesis of [Pt(en)(Cl)2(Pregabalin)2] complex
A mixture containing [Pt(en)(Cl)2(OH)2] (0.015 g) and Pregabalin (0.03 g) in DMF (5 mL) was
heated in an oil bath at 65 °C for 30 min and afterwards at 35 °C for 1 h (the temperature was
stabilized at desired temperature using a thermometer and heater). The platinum complex was
dried at room temperature and washed with acetone and diethyl ether (figure 2b). Analytically
found composition of [C18H40PtCl2N4O4]: C, 33.84; H, 5.90; N, 9.04 %. Corresponding
calculated composition: C, 33.64; H, 6.23; N, 8.72 %.
4

2.4. Methods
2.4.1. Fluorescence spectra. Fluorescence measurements were carried out with a JASCO
spectrofluorimeter (FP 6200) equipped with a thermostated bath using a 1.0-cm quartz cell and a
Xenon ampuxl-159. λex = 280 nm was used for fluorescence quenching study. The maximal
fluorescence emission of HSA was located at 330 nm. Fluorescence spectra were recorded at
286, 298 and 310 K in the range of 300-500 nm. The interaction between the HSA and Pt(IV)
complex was carried out as follows: fixed amounts of the HSA were titrated with increasing
amounts of Pt(IV) complex. These samples were excited at 630 nm and emission was observed
between 640 and 730 nm.
2.4.2. Displacement experiments. The displacement experiments were carried out at 298 K
using the site probes warfarin and ibuprofen by keeping the concentration of the protein and also
probe constant in 10-5 M and 10-4 M, respectively. The fluorescence quenching titration was
applied to define the binding constants of complex-HSA structure in the presence of above site
probes for it’s I and II.
2.4.3. UV-vis spectrophotometry. UV-vis absorption spectra were measured on an Agilent UV-
vis spectrophotometer Model 8453 using a 1.0-cm cell. Absorption titration experiments were
carried out by keeping the concentration of HSA constant (1.0×10-5 M) while varying the Pt(IV)
complex concentration from 0.0 to 1.3×10-4 M (ri = [complex]/[HSA] = 0.0-2.6). Absorbance
values were recorded after each successive addition of HSA solution and equilibration (ca. 10
min).
2.4.4. Circular dichroism studies. Circular dichroism (CD) measurements were recorded on a
JASCO (J-810) spectropolarimeter using a 1.0-cm quartz cell by keeping the concentration of
HSA constant (3.0×10-6 M) while varying the Pt(IV) complex concentration from 0 - 3.3×10-6 M
(ri = [complex] / [HSA]) = 0.0, 0.2, 0.3).
1
2.4.5. Characterization methods. H NMR spectra were recorded using a Bruker Avance
DPX200 MHz (4.7 T) spectrometer with DMSO as solvent. The elemental analysis was
performed using a Heraeus CHN elemental analyzer. IR spectra were obtained with an ABB
5

BOMEM MB 104 FT-IR spectrometer using KBr disks in the range 500-4000 cm−1. The
formation of the platinum(IV) complex as a mononuclear compound was also confirmed by mass
spectrum. A Q600 TGA (TA Instruments, USA) was used to perform isothermal experiment
where the heating rates were suitably controlled at 10 °C min-1 under argon atmosphere.
Computational methods consist of RHF/PM6 method that applies computational chemistry
methods (theoretical models) to many standard tasks that provide calculated data applicable to
the determination of molecular shape conformation, structure (equilibrium and transition state
geometry), NMR, IR, Raman, and UV-visible spectra, molecular (and atomic) properties,
reactivity, and selectivity.
2.4.6. Molecular docking. MGL tools 1.5.4 with Auto Grid 4 and Auto Dock 4 [13, 14] were
used to set up and perform blind docking calculations between the complex and HSA. The
crystal structure of HSA (PDB ID: 1BMO) was downloaded from the Protein Data Bank.
Receptor (HSA) and ligand (complex) files were prepared using AutoDock Tools. The HSA was
enclosed in a box with number of grid points in x×y×z directions, 78×64×84 and a grid spacing
of 1 Å. The center of the grid was set to 29.607, 31.782, and 23.488 Å. The amounts of
independent docking runs performed for each docking simulation were set to 100. Cluster
analysis was performed on the docked results using a root mean square (RMS) tolerance of 0.5
Å, and the initial coordinates of the ligand were used as the reference structure. The structures of
molecules were drawn and optimized using HyperChem (HyperCube Inc., Gainesville, FL). The
output from AutoDock was rendered with PyMol [15].
3. Results and discussion
3.1. Characterization of Pt(IV) complex
The complex [Pt(en)(Cl)2(pregabalin)2] was prepared by the reaction of [Pt(en)(Cl)2(OH)2] and
pregabalin in DMF (scheme 1). The structural characteristics of Pt(IV) complex were elucidated
1
by employing elemental analysis, IR, H NMR spectral studies, mass spectrum, TGA analysis
and computational method. The complex is air stable.
6

K2PtCl4 + en
Pt(en)Cl2 + 2KCl (1)
Pt(en)Cl2 + H2O2Pt(en)Cl2(OH)2 (2)
Scheme 1
Elemental analysis of C, H and N with theoretical calculation and computational method
is one of the most essential way to characterize or prove the elemental composition of an
inorganic sample. The obtained percentages from the theoretical and computational methods of
the corresponding elements are completely matched.
FT-IR spectra of the platinum complex and free drug are shown in figure 4. The band of
free pregabalin at 1213.65 cm-1 is assigned to v(C-O) stretching vibration. In the IR spectrum of
Pt(IV) complex, this band shifted 10.86 cm-1 to lower energy because coordination occurred with
acetate form. In the acetate form, resonance between the C-O bond and the electro-positive
effects of platinum reduce the peak frequency [16]. In addition, the spectrum of platinum(IV)
complex has exhibited one new absorption band at 478.28 cm-1 which is assigned to Pt-O
stretching vibration coupled with the stretching of the dicarboxylate group [17]. The hydroxyl
group disappeared and a sharp absorption band for platinum complex at 3487.69 cm-1 was
observed which indicates v(N-H).
1H-NMR spectra of pregabalin in D2O and its Pt(IV) complex in DMSO are shown in
figure 5. In the 1H NMR spectrum of pregabalin, signals attributed to methyl groups appeared at
0.81 ppm. A septet at 1.58 ppm assigned to H1 and H2 appeared as doublet of doublet at 1.15
ppm. H3 was assigned as a quintet at 2.06 ppm and H4 signal was observed at 2.9 ppm. Finally,
7

1
H5 signal was at 2.2 ppm. Comparison of the H NMR spectrum of pregabalin and Pt complex
shows that this drug is coordinated to platinum center.
The formation of the platinum(IV) complex as a mononuclear compound was also
confirmed by mass spectrum which is shown in figure 6. The molecular ion of the Pt(IV)
complex shows a peak at m/z = 642.4 (calculated: 642). The initial fragmentation (m/z = 582.3)
(calculated: 581.9) is due to the elimination of ethylenediamine ion peak. The next ion peak at
m/z = 424.07 (calculated: 423.6) confirms elimination of the first pregabalin molecule. The ion
peak at m/z = 366.07 (calculated: 365.6) is due to the loss of C4H10 (a part of the second drug
molecule). Next ion peak at m/z = 265.84 (calculated: 265.44) is due to the loss of C4H6NO2
(part of the second drug molecule). Finally, the combination by the loss of the two chlorine
groups has inferred from the peak at m/z =194.84 (calculated: 194.44).
TGA profile of Pt(IV) complex (figure 7) shows weight loss in three steps with
temperature. TG curve shows that the platinum complex is thermally stable up to 154 C and
then it undergoes melting with decomposition. The first stage occurs in the range 162-191 C
and second mass loss occurs between 191-215 C is due to loss of drug molecules, supported by
the weight loss in TG (found: 50.7%; calc: 49.29%). The remaining part of the complex that
decomposes at 215-258 C (found: 18.8%; calc: 20.42%) is due to loss of ethylenediamine and
chlorine molecules. The weight losses above 200 C are attributed to the removal of chlorine
(Cl2) molecule [18]. The overall mass loss is 69.5% (calc. 69.71%) leaving Pt as a metallic
residue {calculated (found) %: 30.29 (30.5)}.
3.1.1. Computational methods and theoretical results. The main structures of pregabalin
ligand (I) and its different complex structures with Cl, en (ethylenediamine) and Pt4+ by 2:2:1:1
ratio were optimized by QM calculations. The calculations on the structures of the complex
[Pt(en)(Cl)2(pregabalin)2] forms have been undertaken by RHF/PM6 method. The Hamiltonian
of Pt was identified by PM6 as a hybrid method of the ab initio and semi-empirical methods. The
most stable optimized structure of complex [Pt(en)(Cl)2(Pregabalin)2] was determined and
reported. The results of the calculations have demonstrated that two pregabalin ligands (I) were
coordinated with Pt4+ from -COO functional groups. The complex was examined and optimized
by –NH2 functional groups of ligands (I), but because of the steric restraint around Pt4+ this form
was by 51 kcal mol-1 unstable than the first discussed complex (the most stable form).
8

The HOMO and LUMO energy levels, ΔEHOMO-LUMO and the structural data of the
reported [Pt(en)(Cl)2(Pregabalin)2] complex (with point group C1) were utilized after
optimization of the equilibrium geometries. All of the calculations were performed by
Spartan‘10 package [19]. In figure 8, we have shown: (A) the HOMO and LUMO energy levels
and ΔEHOMO-LUMO of complex [Pt(en)(Cl)2(pregabalin)2] (2:2:1:1 ratio), (B) the HOMO and
LUMO orbital shapes of complex [Pt(en)(Cl)2(pregabalin)2], (C) ball and spoke model of
complex [Pt(en)(Cl)2(pregabalin)2], (D) tube model of complex [Pt(en)(Cl)2(pregabalin)2], and
(E) the magnified part of complex [Pt(en)(Cl)2(pregabalin)2] around Pt4+ and its selected
structural data of the most stable optimized structure.
The
HOMO
and
LUMO
energy
levels,
ΔEHOMO-LUMO
of
complex
[Pt(en)(Cl)2(Pregabalin)2] were -8.67, -1.49 and 7.20 eV, respectively (figure 8A). As seen in
figure 8B, in HOMO orbital of the complex, the distribution was focused around Pt4+, whereas in
LUMO orbital it was concentrated around the pregabalin ligand (I). In figure 8E, the selected
structural data (bond lengths (in Å) and bond angles (in )) of the most stable optimized structure
were reported. The bond angle of O1-Pt4+-O2 was obtained at 170.9 and it shows a distortion
about 9.1 from 180. This form of the complex includes the H-bonds that were demonstrated in
the magnified part of the complex (figure 8E). The longest and closest H-bond lengths were
1.886 and 2.347 Å, respectively.
3.2. Biological studies
3.2.1. Fluorescence quenching mechanism of HSA by Pt(IV) complex. The quenching of
fluorescence of proteins may be applied as a useful tool to derive information about protein-drug
interaction. The degree of fluorescence quenching offers a change of the protein structure and
micro-environment around its residues [20]. The fluorescence of HSA is due to the tryptophan,
tyrosine and phenylalanine residues. However, the intrinsic fluorescence of HSA is essentially
due to tryptophan alone, because phenylalanine has a very low quantum yield and the
fluorescence of tyrosine is totally quenched if it is ionized or is near an amino group, a carboxyl
group, or a tryptophan residue [21, 22].
Some small molecules can alter the microenvironment of tryptophan residues, which can
produce changes of intrinsic fluorescence intensity of HSA. The fluorescence spectra of HSA
with various concentrations of Pt(IV) complex are shown in figure 9. The fluorescence of HSA
9

decreased with the increasing concentration of Pt(IV) complex, showing that Pt(IV) complex
interacted with HSA and quenched its intrinsic fluorescence. The quenching mechanism can be
classified as static quenching and dynamic quenching, and they represent two very different
quenching processes [23]. To analyze the mechanism (static or dynamic) of fluorescence
quenching the Stern-Volmer equation can be used, Eq. (1) [24]:
F0
(1)
 1  K q 0

Q

 1  K
Q
 
sv
F
where F0 and F are the fluorescence intensities in the absence and presence of quencher,
respectively. KSV is the Stern-Volmer quenching constant, which was determined by linear
regression of a plot of F0/F versus [Q]. Kq is the quenching rate constant of biomolecule, τ0 is the
average lifetime of the fluorophore without quencher, the value of τ0 of the biopolymer is 10-8 s
[23], and [Q] is the concentration of quencher.
For dynamic quenching, the maximum scattering collisional quenching constant of
various quenchers is 2.0×1010 M-1s-1 [25-27]. However, Kq is much higher than 2.0×1010 M-1s1,
suggesting that there is a non-fluorescent complex is formed between the drug and protein, i.e.,
static quenching.
The dynamic quenching only results from the excited state of quenching molecule with
no function on the absorption spectrum of quenching substances [28], while a complex of HSA
and other compounds forms in static quenching, so there will be some changes in the UV-visible
spectra of the HSA [29].
According to the UV-vis spectra, the fluorescence quenching of HSA in present study
seems to be primarily caused by complex formation between HSA and Pt(IV) complex (i.e.,
static quenching).
3.2.1.1. Binding constant and binding sites. For static quenching, the relationship between
fluorescence quenching intensity and the concentration of quenchers can be demonstrated by the
double logarithm regression curve, Eq. (2) [30]:
Log{(F0-F)/F} = log Kb + n log[Q]
(2)
10

where Kb is the binding constant, n is the number of binding sites per HSA and [Q] is the
concentration of the free quencher. The values of Kb and n were calculated from the values of
intercept and slope of the plots, respectively, and the result is shown in table 1. According to
previous studies, pregabalin does not have any affinity to protein, therefore, pregabalin does not
interact with HSA [3], but platinum complex interacts with HSA. The results illustrated that the
binding constant between the complex and HSA decreases with respect to increasing the
temperature. The values of n equal to 1 showed that there is just a single binding site in HSA for
Pt(IV) complex.
3.2.1.2. Thermodynamic parameters and nature of the binding forces. Known interaction forces
between drug and biomacromolecule may consist of hydrophobic forces, electrostatic
interactions, van der Waals interactions and hydrogen bonds. According to the data obtained
from enthalpy changes (ΔH°) and entropy changes (ΔS°), the model of interaction between a
drug and biomolecule can be concluded [31].
According to the data of enthalpy change (ΔH) and entropy change (ΔS), the model of
interaction between the drug and biomolecule can be concluded: (1) ΔH > 0 and ΔS > 0,
hydrophobic forces; (2) ΔH < 0 and ΔS < 0, van der Waals interactions and hydrogen bonds; (3)
ΔH < 0 and ΔS > 0, electrostatic interactions [32]. The thermodynamic parameters can be
calculated from van’t Hoff equation (Eqs. 3 and 4):
Ln(Kb) = -H/RT+S/R
G = H-TS
(3)
(4)
where Kb is the binding constant at the corresponding temperature and R is the gas constant. ∆H,
∆G and ∆S are standard enthalpy change, standard free energy change and standard entropy
change, respectively. Parameters demonstrating a complete thermodynamic profile of molecular
interactions are shown in table 2. Hydrogen bonding and van der Waals interactions are usually
characterized by negative standard enthalpies and entropies of interaction [33].
3.2.1.3. Site-selective binding of Pt(IV) complex on HAS. HSA is composed of three structurally
similar spherical domains, each of which contains two subdomains, denominated subdomain IA,
IB, IIA, IIB, IIIA, and IIIB. Site I of HSA demonstrated dependency for warfarin,
11

phenylbutazone, etc., while site II for ibuprofen, diazepam, flufenamic acid, etc. To recognize
binding site of the Pt(IV) complex with HSA, the site marker competitive experiment was
performed. Warfarin and ibuprofen which specifically bound to a known site on HSA were used
as site marker fluorescence probes to monitor sites I and II of HSA, respectively [34]. Different
amounts of the Pt(IV) complex were added to a solution containing fixed amounts of HSA and
site probe and fluorescence intensities were noted down upon excitation at 290 nm (figure 10).
The values of binding constant (Kb) at room temperature were obtained to be 2.7×103M-1 for
Pt(IV) complex-HSA-warfarin system and 3.7×103 M-1 for Pt(IV) complex-HSA-ibuprofen
system while the binding constant (Kb) for Pt(IV) complex-HSA system were obtained to be
3.8×103 M-1. The results showed that the binding site for Pt(IV) complex and warfarin was the
same in HSA. So, the Pt(IV) complex was most likely bound to the hydrophobic pocket located
in subdomain IIA (site I) [35].
3.2.2. UV-vis absorption spectroscopy. Spectroscopy techniques can be applied to understand
the structural changes of protein and to check protein-ligand complex formation. UV-vis
absorption measurement is an easy but effective method in detecting complex formation [36].
The interaction between Pt(IV) complex and HSA was defined by using the data of the UV-vis
absorption spectra (figure 11). The UV absorption intensity of HSA increased with the variation
of complex concentration. This also indicated the change in polarity around the tryptophan
residue. As a result, the binding of Pt(IV) complex to HSA molecule might lead to altering in
protein conformation [37].
3.2.3. Changes of the HSA secondary structure induced by Pt(IV) complex. Circular
dichroism (CD) spectroscopy is a perfect method that plays an important role in the
characterization and examination of proteins, polypeptides and peptide structures especially for
secondary structure. The CD spectra of HSA showed two negative bands at 208 and 222 nm,
which are characteristic features which correspond to the nπ* and ππ* transitions of
peptides in α-helical protein structure [38]. To investigate the effect of complex on HSA
secondary structure, CD measurement was carried out.
The CD results were expressed in terms of mean residue ellipticity (MRE) in
deg∙cm2∙dmol-1 according to the following equation (Eq. 5):
12

observedCD
(m deg ree )
MRE

(5)
C
p nl  10
where Cp is the molar concentration of the protein (HSA), n is the number of amino acid residues
(585 for HSA) of the protein and l is the path length (1 cm). The α-helical contents of free and
combined HSA were calculated from MRE values at 208 nm using Eq. (6):
MRE
 4000
208
(6)
  Helix (%)

 100
33000  4000
The CD measurement of HSA in the absence as well as in the presence of Pt(IV) complex are
shown in figure 12. Upon interaction with Pt(IV) complex, the CD spectra of HSA got alteration
and shows a decrease in ellipticity without affecting the shape of spectrum. This showed that
Pt(IV) complex increase the alpha-helical structural content of HSA (42%- 47%- 50%) [39].
3.2.4. Molecular docking analysis. The docked structure (figure 13) shows that complex is
located within the binding pocket of site 1 (subdomain IIA) of HSA. On the other hand, there
was hydrogen bond between hydrogen atom of the complex with the OD1 group of ASP 259
(1.53 Å) (figure 14). This indicated that hydrogen bond played an important role in the binding
of complex to HSA. From the docking simulation the observed free energy change of binding
(ΔG) for the HSA+complex is calculated to be -4.24 kcal∙mol-1, which is slightly lower than the
experimental free energy of binding (-4.29 kcal∙mol-1) obtained from the fluorescence data. This
apparent mismatch in the free energy changes could be due to the exclusion of the solvent and/or
rigidity of some other receptors in the molecular docking studies. Furthermore, the binding
constant obtained from the fluorescence data was correlated with the free binding energy of
docked model. Basic formula of binding constant and Gibbs free energy is shown in Eq. (7):
(7)
∆G = - RTLnK
The binding constant obtained from the fluorescence data (1.45×103 M-1) matches roughly to the
binding constant calculated by docked complex-HSA model (1.32×103 M-1). Hence, it can be
concluded that complex-HSA docked model is in approximate correlation with our experimental
results.
13

4. Conclusion
According to previous studies, pregabalin does not interact with plasma proteins [3]. The aim of
this study was to synthesize a new platinum complex, [Pt(en)(Cl)2(Pregabalin)2], to study
binding of the platinum complex to plasma protein and to investigate the new biological effects
of this complex. According to experimental and theoretical methods, this new platinum complex
shows the ability to interact with the protein.
(I) Fluorescence study showed that the quenching of HSA by Pt(IV) complex is a result of
the formation of HAS-Pt(IV) complex structure; according to thermodynamic parameters
(negative ΔH and ΔS value), hydrogen bonding and van der Waals forces played major
roles in the binding process between Pt(IV) complex and HSA [40].
(II) UV-vis and CD spectra of HSA in the presence of Pt(IV) complex showed that the
binding of Pt(IV) complex causes the conformational change in protein.
(III) Docking study suggested that the binding of Pt(IV) complex to HSA is located within site
1 (subdomain IIA) by hydrogen forces.
(IV) The calculations on the structures of the Pt(IV) complex forms undertaken by RHF/PM6
method have shown that theoretical and experimental calculations are consistent with
each other.
Acknowledgements
Financial support from the Razi University Research Center is gratefully acknowledged.
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16

Table 1. Binding constants (Kb) and the number of binding sites (n) of the
HSA-complex system.
T (K)
286
R2
n
Kb ± 0.03 (M-1)
4482.9
0.98
0.99
0.99
1.18
298
0.9535
0.8554
3839.7
310
1456.8
Table 2. Thermodynamic parameters for the binding of the complex to
HSA.
ΔG ± 0.02
ΔH ± 0.02
ΔS ±2
T (K)
(kJ mol-1)
(kJ mol-1)
(J mol-1 K-1)
286
298
310
-96.1461
-95.7405
-95.3349
-
-
-105.812
-
-33.798
-
17

Graphical abstract
18

Figure 1. The structure of pregabalin drug.
a
b
Figure 2. The structure of (a) satraplatin and (b) Pt(IV) complex.
19

Figure 3. IR spectrum of dichloride dihydroxy(ethylenediamine) platinum(IV) complex.
20

a
b
Figure 4. IR spectrum of (a) pregabalin and (b) Pt(IV) complex.
21

a
b
Figure 5. 1H NMR spectrum of (a) pregabalin in D2O and (b) Pt(IV) complex in DMSO.
22

Figure 6. Mass spectrum of the platinum complex.
Figure 7. TGA plot of Pt(IV) complex.
23

Figure 8. (A) The HOMO and LUMO energy levels and ΔEHOMO-LUMO of
[Pt(en)(Cl)2(pregabalin)2]. (B) The HOMO and LUMO orbital shapes of
[Pt(en)(Cl)2(pregabalin)2]. (C) Ball and spoke model of [Pt(en)(Cl)2(pregabalin)2]. (D) Tube
model of [Pt(en)(Cl)2(pregabalin)2]. (E) The magnified part of [Pt(en)(Cl)2(pregabalin)2] around
Pt4+ and its selected structural data.
450
400
350
300
250
200
150
100
50
0
305
355
405
455
505
Wavelength
Figure 9. Fluorescence spectra of the interaction of Pt(IV) complex with HAS.
24

250
200
150
100
50
HSA
HSA+war
increasing complex
0
314
364
414
464
514
564
Wavelength
a
HSA
200
150
100
50
HSA+Ibu
increasing complex
0
310
360
410
460
510
WavWavelengthe
b
Figure 10. Effect of site marker: (a) warfarin and (b) ibuprofen to Pt(IV) complex-HSA system.
25

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
230 250 270 290 310 330 350 370 390 410 430 450 470 490
Wavelength
Figure 11. UV absorption spectra of HSA in the presence of different concentrations of Pt(IV)
complex. C[HSA] = 1.0×10-5 M, and C[complex] = 0.0 to 1.3×10-4 M (ri = [complex] / [HSA] =
0.0-2.6).
10
5
0
199
209
219
229
239
249
259
269
-5
-10
-15
-20
-25
-30
-35
HSA
ri=0.2
ri=0.3
Wavelength
Figure 12. CD spectra of HSA in the absence and presence of Pt(IV) complex (ri = [complex] /
[HSA] = 0.0-0.2-0.3). C[HSA] = 3×10-6 M, and C[complex] = 0.0 – 3.3×10-6 M.
26

Figure 13. Modeling structure of HSA and complex; the subdomains are displayed.
Figure 14. The hydrogen bond interaction (yellow dashed line) between complex (lines) and
ASP-259 (publication).
27