Kinetic and Thermodynamic Investigation of Human Serum Albumin Interaction with Anticancer Glycine Derivative of Platinum Complex by Using Spectroscopic Methods and Molecular Docking

Article abstract of DOI:10.1007/s12010-019-03078-y

In this paper, a new anticancer Pt (II) complex, cis-[Pt (NH₃)₂(tertpentylgly)]NO₃, was synthesized with glycine-derivative ligand and characterized. Cytotoxicity of this water-soluble Pt complex was studied against human cancer breast cell line of MCF-7. The interaction of human serum albumin (HSA) with Pt complex was studied by using UV-Vis, fluorescence spectroscopy methods, and molecular docking at 27 and 37?°C in the physiological situation (I = 10?mM, pH = 7.4). The negative ΔHb0 and positive ΔSb0 indicated that electrostatic force may be a major mode in the binding between Pt complex and HSA. Binding constant values were obtained through UV-Vis and fluorescence spectroscopy that reveal strong interaction. The negative Gibbs free energy that was obtained by using the UV-Vis method offers spontaneous interaction. Fluorescence quenching the intensity of HSA by adding Pt complex confirms the static mode of interaction is effective for this binding process. Hill coefficients, n_H, Hill constant, k_H, complex aggregation number around HSA, , number of binding sites, g, HSA melting temperature, T_m, and Stern-Volmer constant, k_SV, were also obtained. The kinetics of the interaction was studied, which showed a second-order kinetic. The results of molecular docking demonstrate the position of binding of Pt complex on HSA is the site I in the subdomain IIA.

Full text of DOI:10.1007/s12010-019-03078-y

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https://doi.org/10.1007/s12010-019-03078-y
Kinetic and Thermodynamic Investigation of Human
Serum Albumin Interaction with Anticancer Glycine
Derivative of Platinum Complex by Using Spectroscopic
Methods and Molecular Docking
Afrooz Shiekhzadeh1 & Nasrin Sohrabi1 & Mahboube Eslami Moghadam2
&
Mohsen Oftadeh1
Received: 8 February 2019 /Accepted: 5 July 2019/
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
In this paper, a new anticancer Pt (II) complex, cis-[Pt (NH3)2(tertpentylgly)]NO3, was
synthesized with glycine-derivative ligand and characterized. Cytotoxicity of this
water-soluble Pt complex was studied against human cancer breast cell line of
MCF-7. The interaction of human serum albumin (HSA) with Pt complex was studied
by using UV-Vis, fluorescence spectroscopy methods, and molecular docking at 27
and 37 °C in the physiological situation (I = 10 mM, pH = 7.4). The negative ΔHb0
and positive ΔS0b indicated that electrostatic force may be a major mode in the
binding between Pt complex and HSA. Binding constant values were obtained through
UV-Vis and fluorescence spectroscopy that reveal strong interaction. The negative
Gibbs free energy that was obtained by using the UV-Vis method offers spontaneous
interaction. Fluorescence quenching the intensity of HSA by adding Pt complex
confirms the static mode of interaction is effective for this binding process. Hill
coefficients, nH, Hill constant, kH, complex aggregation number around HSA, <J>,
number of binding sites, g, HSA melting temperature, Tm, and Stern-Volmer constant,
kSV, were also obtained. The kinetics of the interaction was studied, which showed a
second-order kinetic. The results of molecular docking demonstrate the position of
binding of Pt complex on HSA is the site I in the subdomain IIA.
.
.
.
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Keywords Human serum albumin Fluorescence quenching Pt complex Tertpentyl glycine
Molecular docking
* Nasrin Sohrabi
sohrabnas@pnu.ac.ir; nsohrabi48@jmail.com
* Mahboube Eslami Moghadam
eslami_moghadam@ccerci.ac.ir; eslami_moghadam@yahoo.com
Extended author information available on the last page of the article

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Abbreviations
HSA
PDB
Human serum albumin
Protein Data Bank
Tertpentylgly 1,1-Dimethyl propyl glycine
Introduction
Human serum albumin (HSA), as the most plentiful carrier protein, has many significant
physiological activities. Among its functions, metallodrugs get free in targeting organs via
binding with albumin protein. Hence, HSA cannot protect metallodrugs after binding against
oxidation, aquation, distribution, and the stability of drug changes [1].
Metal drugs are bound and unbound to HSA in plasma circulation. The unbound drugs spread
in the blood, and they are metabolized distributed intracellularly via particular forwarding systems
[2]. Also, free drug molecules can interact effective with hurt targets [3]. In most case, the
unbound drug concentration in plasma should be determined and controlled [3].
Therefore, the investigation of reversible HSA-complex interactions is necessary for
following the pharmacokinetics and dynamics effects of drugs. Also, in more cases, the
solubility of hydrophobic drugs can be improved by interaction with serum albumin [4].
Hence, protein-drug binding study provides valuable data for understanding the effect of drug
in vivo processes. Platinum (II) complexes as anticancer drugs, for example, cisplatin, are
known and used with high chemotherapeutic affect for treatment of several types of cancer,
some tumors, and ovarian and head and neck carcinomas [5].
Binding of Pt complexes on serum albumin is necessary for their biotransformation in
biological fluids, but because of binding of platinum center with protein contains sulfur atoms,
serious side effects cause. Hence, more Pt complexes are designing, and new agents are being
synthesized with lower toxicity [6]. In this case, redox activity of metal center and selectivity bind
to bimolecular target should be attended. Some methods and several spectroscopic techniques
such as fluorescence and absorption employ conformational properties of the protein and drug-
protein [7, 8]. In recent years, various investigations have been done on the interaction of
anticancer platinum and palladium derivatives with human serum albumin [9–20].
In the present research, a platinum (II) complex, cis-[Pt (NH3)2(tertpentylgly)]NO3, was
synthesized and characterized (Scheme 1). The HSA interaction with this water-soluble Pt
complex was studied by using electronic absorption and fluorescence spectroscopies and also
Scheme 1 The proposed structure
of cis-[Pt
(NH3)2(tertpentylgly)]NO3
complex
O
H3N
H3N
O
Pt
NH
NO3

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modeled by molecular docking. Based on these studies, the important information about
thermodynamic parameters and the modes and mechanism and also the location of the
connection were determined. Then, the enthalpy (ΔΗ°b), entropy (ΔSb° ), and Gibbs free energy
(ΔG°b) changes, melting point of HSA in the absence and presence of complex, Tm, the
aggregation number complex around HSA, 〈J〉, computed and estimated the number of binding
sites, g, binding constants, Kb, Hill coefficient, nH, Stern-Volmer coefficient, Kb, quenching
constant, kq, and the mechanism of quenching. In addition, cytotoxicity of this new complex
has been tested against human breast cancer cell line of MCF-7. These results would be
expected to help realize the binding mode of this Pt (II) complex to protein.
Experimental
Materials and Instrumentation
Silver nitrate, potassium iodide, tertpentyl amine, ammonia, sodium bicarbonate, and potassium
tetrachloroplatinate were purchased from Merck (Germany). Solvents such as acetone, ethanol,
and anhydrous diethyl ether were obtained from Merck Co. (Germany). Human serum albumin
(HSA, purity > 99.0%) was bought from Sigma-Aldrich Co. (USA) and stored at 4.0 °C. The
stock solution of the Pt (II) complex (2 mM) was prepared with Tris-HCl buffer containing
NaCl (10 mM, pH 7.4). The HSA stock solution was diluted to 0.03 mM with the same buffer.
Infrared spectra were measured on a FTIR 8400 Shimadzu spectrometer from 4000 to 400
1
cm−1using KBr pellets. H NMR (300 MHz) spectrum was collected on Bruker BRX-250
Avance spectrometer with reference as DMSO − d6 solvent. The melting point of complex was
determined on a Buchi (Flawil, Switzerland) melting point B-545. Conductivity measurement
was carried out by a TetraCon 325 electrode connected to a Terminal 740 (WTW, Germany)
with a cell constant of 0.951 using conductivity water as the solvent. Elemental analysis (C, H,
and N) of complex that was performed by a CHN has been analyzed by Elementar
Analysensysteme GmbH-Vario EL III.
All fluorescence spectra were carried out on a Scinco FS-2 fluorescence spectrophotometer
in 200–700 nm. UV spectra of free HSA and HSA in the presence of various concentrations of
Pt (II) complex were measured using the PerkinElmer Precisely Lambda 25 Spectrophotom-
eter in 200–700 nm.
Synthesis of cis-[Pt (NH3)2(tertpentylgly)]NO3
Tertpentyl glycine was synthesized the way it was previously published and used for synthesis of
complex [21]. Also cis − [Pt(NH3)2I2] was prepared according to the method described previously
[22]. 0.183 mmol (0.088 g) cis − [Pt(NH3)2I2] and 0.367 mmol (0.062 g) AgNO3 were suspended in
18 mL double-distilled water under continuous stirring condition for 24 h at room temperature and
then refluxed at 50 °C for 30 min under darkness. Then, gray AgI precipitate was removed by
centrifugation. After that, the solution of 0.183 mmol (0.033 g) tertpentyl glycine hydrochloride and
10 mL solution of 0.366 mmol (0.03 g) NaHCO3 in water were added to above centrifuged solution
and stirred at 40 °C for 4 h. The solution was evaporated to 1 mL until the brown precipitate
appeared. The obtained product was decanted and washed by chilly acetone and dried in a
desiccator. Scheme 2 shows the formation of complex.

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H3N
OH2
OH2
2AgNO3
Pt
2AgI
(NO3)2
+
[Pt(NH3)2I2]
H3N
O
H3N
H3N
O
Pt
NH
HN
NO3
O
O
Scheme 2 General procedure of complex formation
cis-[Pt (NH3)2(tertpentylgly)]NO3 (435.08 g/mol): the yield is 69%; mp (160–165 °C);
analytical calculated for C7H20N3O2Pt compound is C, 19.30, H, 4.59, N, 9.65%, analytical
found: C, 18.93, H, 4.20, N, 9.37%; UV: λmax nm (ƐM): 201 (28.8); molar conductance, ɅM
(10−4 M, H2O) = 120 Ω −1 cm2 mol−1; FTIR (cm−1, KBr disk): 3432 (s, N-H), 3283 (s, C-H),
1627 (s, C=O), 1361 (s, (NO3)−); 1H NMR (300 MHz, DMSO-d6, δ in ppm, J in Hz): 1.12 (t,
J = 9 Hz 3H), 1.21 (m, 2H), 1.62 (s, 6H), 2.07 (d, 2H), 4.4 (m, 7H NH).
Cell Culture and Cell Proliferation Assay
Human breast cancer cell line of MCF-7 was purchased from the National Cell Bank of Iran
(NCBI), Pasteur Institute of Iran. The cells were grown on the DMEM medium (Sigma)
μg
mL
supplemented with L-glutamine (2 mM), streptomycin, penicillin (5 ), and 10% heat-
inactivated fetal calf serum at 37 °C under 50%: 95% CO2; air atmosphere. The cytotoxicity
activity of new synthesized complex was studied by MTT assay. The harvested cancer cells of
MCF-7 were seeded into a 96-well plate (1 Â 104 ) and incubated with various concentra-
cell
mL
tions of sterilized cis-[Pt (NH3)2(tertpentylgly)]NO3 (0–800 μM) for 24 h. IC50 value deter-
mination was done according to the published method in Ref [23].
Evaluation of Thermodynamic Parameters
Absorption recording is an easy and suitable method to investigate the complex-HSA forma-
tion [24, 25]. A working solution of HSA and Pt complex was diluted to 15 μM and 0.8 mM,
respectively. Then, the injection of complex to HSA solution was continued up to the point that
no further changes in the absorption were shown. The spectra were recorded from 200 to
700 nm in a quartz cuvette (total cuvette volume was 1000 μL) at 27 and 37 °C.
Fluorescence Studies
The fluorescence intensity of protein may be quenched by adding of metal complex, when
complex interacts with HSA. In this study, the fluorescence intensity was monitored by
excitation wavelength at 280 nm and emission wavelength limited area of 200–700 nm. In