Synthesis, anticancer activity, and β-lactoglobulin binding interactions of multitargeted kinase inhibitor sorafenib tosylate (SORt) using spectroscopic and molecular modelling approaches

Article abstract of DOI:10.1002/bio.3929

Sorafenib tosylate (SORt) is an oral multikinase inhibitor used for treatment of advanced renal cell, liver, and thyroid cancers. In this study, this drug was synthesized and its antiproliferative activities against HCT116 and CT26 cells were assessed. The interaction of SORt with β-lactoglobulin (BLG) was studied using different fluorescence techniques, circular dichroism (CD), zeta potential measurements, and docking simulation. The results of infrared (IR), mass, ^HNMR, and ^CNMR spectra demonstrated that the drug was produced with high quality, purity, and efficiency. SORt showed potent cytotoxicity against HCT116 and CT26 cells with IC₅₀ of 8.12 and 5.42 μM, respectively. For BLG binding of SORt, the results showed that static quenching was the cause of the high affinity drug–protein interaction. Three-dimensional fluorescence and synchronous spectra indicated that SORt conformation was changed at different levels. CD suggested that the α-helix content remained almost constant in the BLG–SORt complex, whereas random coil content decreased. Zeta potential values of BLG were more positive after binding with SORt, due to electrostatic interactions between BLG and SORt. Thermodynamic parameters confirmed van der Waals and hydrogen bond interactions in the complex formation. Molecular modelling predicted the presence of hydrogen bonds and electrostatic forces in the BLG–SORt system, which was consistent with the experimental results.

Full text of DOI:10.1002/bio.3929

Saidijam Massoud (Orcid ID: 0000-0002-1711-5619)
Synthesis, anticancer activity and β-lactoglobulin binding interactions of
multi-targeted kinase inhibitor sorafenib tosylate (SORt) by spectroscopic
and molecular modeling approaches
1
1
1
Hamid Tanzadehpanah , Asrin Bahmani , Neda Hosseinpour Moghadam , Hamid
2
Gholami ,
1
3
1,
Hanie Mahaki , Abbas Farmany and Massoud Saidijam *
1
Research Center for Molecular Medicine, Hamadan University of Medical Sciences,
Hamadan, Iran
2
Department of Biochemistry, School of Medicine, Hamadan University of Medical
Sciences, Hamadan, Iran
3
Dental Research Center, School of Dentistry, Hamadan University of Medical Sciences,
Hamadan, Iran.
*
Corresponding author Email: Sjam110@yahoo.com
First author Email: H.Tanzadehpanah@gmail.com
Tel: +98 8138276299
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/bio.3929
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Abstract
Sorafenib tosylate (SORt) as an oral multi-kinase inhibitor is used for the treatment of
advanced renal cell, liver and thyroid cancers. This drug was synthesized in this work, and its
antiproliferative activities against HCT116 and CT26 cells were assessed. Afterward, the
interaction of SORt with β-lactoglobulin (BLG) was studied using different fluorescence
techniques, circular-dichroism (CD), zeta potential measurements and docking simulation.
H
C
The results of IR, Mass, NMR, and NMR spectra exhibited that the drug was produced
with a high quality, purity and efficiency. SORt showed potent cytotoxicity against HCT116
and CT26 cells with IC50 = 8.12 and 5.42 µM, respectively. Concerning the BLG binding of
SORt; the results showed that a static quenching mechanism is the cause of high affinity for
drug-protein interaction. Three-dimensional fluorescence and synchronous spectra proposed
that SORt conformation has been changed at different levels, as well as circular dichroism
suggested the amount of α-helix remains almost constant in the BLG–SORt complex,
whereas those of random coil were decreased. The Zeta-potential values of BLG were more
positive after binding with SORt, which is due to the existence of electrostatic interactions
between BLG and SORt. Furthermore, thermodynamic parameters confirmed the van der
Waals and hydrogen bond interactions in the complex formation. Finally, Molecular
modeling showed the presence of hydrogen bond and electrostatic force in the BLG–SORt
system, which was consistent with the experimental results.
Keywords: Sorafenib; β-lactoglobulin; MTT assay; Multi-spectroscopy; Docking.
Abbreviations
BLG: Beta-lactoglobulin
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CD: Circular Dichroism
CDI: 4-chloro-3-(trifluoromethyl)aniline, N,N-carbonyldiimidazole
c-Kit: c-Kit protein
DMF: 4-Aminophenol, potassium tert-butylate, potassium carbonate, N,N-
Dimethylformamide
FBS: fetal bovine serum
Flt-3: FMS-like tyrosine kinase 3
FT-IR: Fourier Transform Infrared
MOE: Molecular Operating Environment
NMR: Nuclear magnetic resonance
PDGFRβ: platelet-derived growth factor receptor β
SORt: sorafenib tosylate
VEGFR: vascular endothelial growth factor receptor
ΔG: standard Gibbs free energy change
ΔH: standard enthalpy change
ΔS: standard entropy change
Highlights
Sorafenib tosylate (SORt) was synthesized with high quality, purity and efficiency.



SORt showed potent cytotoxicity against HCT116 and CT26 colon cancer cells.
Circular dichroism suggested the amount of α-helix remains almost constant in the
BLG–SORt complex.

Molecular modeling also showed the presence of hydrogen bond and electrostatic
force in the BLG–SORt system.
1
. Introduction
Colon cancer is a significant health problem, and by 2019, in the United States, it is
anticipated that 101,420 people to contract the disease, which 51,020 will die [1]. A
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convenient option for colon cancer treatment is oral consumption of Sorafenib tosylate
SORt). SORt an active multikinase inhibitor by affecting all three variants of vascular
(
endothelial growth factor receptors (VEGFR 1, 2 and 3), FMS-like tyrosine kinase 3 (Flt-3),
platelet-derived growth factor receptor β (PDGFRβ), c-Kit protein (c-Kit), and RET receptor
tyrosine kinases induces its anti-cancer activity [2, 3]. Biological activities of SORt depend
on its metabolism and bioavailability. Poor solubility in aqueous media limits the use of
SORt as an efficient medicine. Therefore, it is necessary to use a suitable carrier to promote
its activity [4].
Beta-lactoglobulin (BLG) is a typical globular protein with a molecular weight of 18.3 kDa
and a size of nearly 2 nm [5]. In the sequence of this protein, there are two tryptophan
residues (Trp-19 and Trp-61) that play a role in fluorescence spectroscopy studies as the main
fluorophores [6]. The structure of BLG constitutes of nine antiparallel beta-strands that form
two beta-sheets, and three turn’s α-helix. The β-sheets form a flattened conical barrel named
calyx, which is the center of interaction with small drugs [7]. BLG has six potential sites for
ligand binding. The first site is the Calix position; the second site is between the beta strings
C and D in the CD loop. The third site is formed by the alpha-helix c-terminal and the beta
strands F, G, H and A. The fourth site is between Alpha-Helix and the beta-strand I. The fifth
site is near Loop H. The sixth site is between the CD and DE loops [8]. Some studies have
shown that β-lactoglobulin protein does not bind to any of the ligands through covalent
bonding, and most bonds are hydrophobic and hydrogen interactions [9]. The result showed
controlled release over a long period of time. Generally, when medications are taken orally,
problems such as a broken stomach or irritation in the stomach appear. Therefore, there has
always been an attempt to provide a molecule that can, while carrying the drug, allow it to
pass intact through the stomach, eventually and release it into the target tissue (intestine) [10].
The structure of BLG is strongly dependent on the pH and ionic strength of the environment
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due to the presence of many charged groups in its sequence. Resistant to low pH in the
presence of pepsin degradation is another characteristic of BLG [11]. It remains wholesome
after gastric digestion and can be detected in the small intestine and colon. This property of
protein encouraged its use as a carrier for delivering drugs into the colon and developing a
new drug delivery system in colon cancer treatment [12].
In the present study, we first synthesized SORt with high purity, and then its anti-cancer
activity was investigated on two colon cancer cell lines of HCT116 and CT26. Furthermore,
due to the unique properties of BLG, it can be a suitable carrier for SORt. So, we have
focused on the interactions of SORt with BLG using fluorescence spectroscopy, circular
dichroism, zeta potential measurement and molecular modeling, to use it in some drug
delivery systems.
2
2
4
. Martials, devices and methods
.1. Martials
-Aminophenol, potassium tert-butylate, potassium carbonate, N,N-Dimethylformamide
(
DMF), and p-toluenesulfonic acid were purchased from Merck (Darmstadt, Germany). 4-
chloro-3-(trifluoromethyl)aniline, N,N-carbonyldiimidazole (CDI) and β-Lactoglobulin were
obtained from Sigma (St. Louis, Mo, USA). 4-chloro-N-methyl-2-pyridinecarboxamide was
bought from TCI (TCI America, Inc., Portland, OR, USA). HCT116 and CT26 cell lines were
purchased from National Cell Bank, Pasteur Institute, Iran. The anticancer drug Regorafenib,
used as a positive control in MTT assay, was purchased from Shaanxi Yuan Tai Biological
Technology Co. Ltd, Xi’an City, PR China. All materials for cell culture were obtained from
Gibco (Gibco, USA). Other materials were obtained from high-quality commercial suppliers
and no additional purification was required.
2
.2. Devices
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All melting points were measured using Barnested/Electrothermal IA-9100 and values were
uncorrected. Bruker FT-IR Alpha was used to identify the functional groups. NMR-Bruker-
5
00-UltraSheild was used to analyze the number and types of C and H atoms. To obtain the
mass spectra of the derivatives, Quadrupole technique and Agilent Technologies- 5975C- EI-
0eV apparatus were used. All fluorescence spectra were achieved by employing a FP-6200
7
spectrofluorometer, with 1.0 cm quartz cells. An Aviv Model 215 Circular Dichroism
spectrometer with a cell path length of 1cm, at room temperature, was used to the record of
CD spectra. The zeta potential measurements were examined using a Zeta-sizer, Nano-series–
ZS (Malvern Instruments Ltd).
2
.3. Synthesis of sorafenib tosylate (SORt)
Synthesis of SORt was performed in 3 steps (Scheme 1).
2
.3.1. Synthesis of 1-[4-Chloro-3-(trifluoromethyl) phenyl]-3-(4-hydroxyphenyl) urea
4
-Chloro-3-(trifluoromethyl) aniline (1mmol) was added to the dichloromethane and was
stirred until a clear solution was obtained. After 10 min, N,N-carbonyldiimidazole (1mmol)
was added to the reaction mixture and the reaction was continued at reflux at 50°C. After 16
hours, the reaction was complete and 4-chloro-3-(trifluoromethyl) phenyl isocyanate was
obtained (Scheme 1, route a). Then 4-aminophenol (1mmol) was added to the precursor
product in dichloromethane. The reaction was continued at 25°C for 16h and finally,
intermediate was obtained (Scheme 1, route b). Finally, the dichloromethane was distillated
under reduced pressure and the intermediate was obtained as a white crystal without further
purification.
2
.3.2. Synthesis of 4-{4-[({[4-Chloro-3-(trifluoromethyl-phenyl]amino}carbonyl)amino]
phenoxy}-N-methylpyridine-2-carboxamide (sorafenib)
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The intermediate (1mmol) was dissolved in N,N-dimethylformamide. After the clear solution
was obtained, potassium tert-butylate (1.5 mmol) was added to the reaction mixture and the
reaction continued for 2h at 25°C. Then, 4-chloro-N-methyl-2-pyridinecarboxamide (1mmol)
and potassium carbonate (0.5 mmol) were added to the reaction mixture. The reaction
temperature was adjusted to about 80°C and the reaction continued for 8h. After 8 hours, the
reaction was completed (Scheme 1, route c). To separate and purify the product, the reaction
mixture was cooled to 25°C. The extraction process was then carried out by mixing water and
ethyl acetate. The formed salts and polar components were dissolved in water and the non-
polar components were dissolved in the nonpolar phase, namely ethyl acetate. Ethyl acetate
was distillated under reduced pressure and sorafenib remained solid white.
2
.3.3. Synthesis of 4-{4-[({[4-Chloro-3-(trifluoromethyl-phenyl]amino}carbonyl)amino]
phenoxy}-N-methylpyridine-2-carboxamide tosylate (sorafenib tosylate)
Sorafenib (1mmol) was added to the ethanol and was stirred for 10 minutes until a clear
solution was obtained. Subsequently, p-toluenesulfonic acid (1.1 mmol) was added to the
reaction and stirred at 25°C for 15h (Scheme 1, route d). The solvent was distillated under
reduced pressure and the final precipitate was washed several times with cold ethanol.
2
.4. MTT assay
HCT116 and CT26 were grown in DMEM and RPMI 1640, respectively. The mediums were
containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin. A humidified
atmosphere incubator provides conditions of 95% air, 5% CO and 37ºC for the cancer cells.
2
To determine of cytotoxicity activity, about 4,000 cells were seeded into 96 well plates and
incubated for 24 h. After incubation, the cells were treated with various concentrations of
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SORt (0.5, 1, 2, 4, 8 and 16 µM) for three days. Then, MTT reagent was added to each well
fifteen microliters, 5 mg/ml) and the plates were incubated for 4 hours. The medium was
(
removed and 150 µl DMSO was added to each well. After 20 minutes, the absorbance at 630
nm was measured [13, 14]. In MTT assay, regorafenib was employed as the positive control.
Regorafenib, as a small molecule, has a chemical structure and biochemical profile similar to
that of SORt. The only difference is the addition of a fluorine atom to the center of
regorafenib, which increases its efficiency in anti-cancer activity. The cytotoxicity data were
analyzed using one-way ANOVA followed by the post-hoc Tukey’s test [15].
2
.5. Fluorescence measurements
The measurements were done at a excitation wavelength of 280 nm, and the emission spectra
were recorded from 290 to 450 nm [16]. Briefly, two milliliters of BLG (final concentration,
-6
-6
2
1 × 10 M) were titrated by serial addition of SORt (0 - 10 × 10 M), at 298, 305 and 310
temperatures and pH 7.4. In this study, the absorption of exciting light and reabsorption of
emitted light were used to decrease the inner filter effect (IFE) using the following equation:
퐹
푐표푟
= 퐹표푏푠 × 푒푥푝(퐴ꢀꢁ+퐴ꢀ푚)/2
where Fcor and Fobs are the corrected and observed fluorescence intensities, respectively. Aex
and Aem are the absorption of SORt at the excitation and the emission wavelength,
respectively.
Synchronous fluorescence spectra were assessed at two scanning intervals of Δλ (Δλ = λem
ex) [17]. Δλ values were set at 15 and 60 nm. The concentrations of SORt and BLG were like
the description above.
–
λ
Excitation and emission wavelength ranges to estimate three-dimensional fluorescence spectra
were 220 to 350 and 220 to 500 nm, respectively [18]. The final concentrations of SORt and
-6
-6
BLG were 10 × 10 M and 21 × 10 M, respectively.
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2
.6. Circular dichroism (CD) spectroscopy
The CD spectra were obtained over a wavelength range of 200–260 nm, with a scanning rate
-1
at 50 nm min as an average of two scans for each CD spectrum [19]. The concentration of
-6
BLG solution was 21 × 10 M, whereas the final concentrations of SORt in complex with
-6
-6
BLG were 5 × 10 and 10 × 10 M.
2
.7. Zeta potential measurements
The formation of the BLG-SORt complex can be further confirmed by the zeta potential
measurements. One milliliter of BLG was prepared in 0.05 M phosphate buffer and room
−6
temperature (pH 7.4), that way, the final concentrations of BLG and SORt were 21 × 10 and
-6
0
.0 – 5 ×10 M, respectively.
2
.8. Molecular modeling
Molecular simulation based on blind docking was accomplished using Molecular Operating
Environment (MOE) software [20]. ChemDraw Ultra software version 8.0 was applied for the
SORt preparation. The geometry of SORt was optimized by applying HyperChem program
version 7 (http://www.hyper.com). The crystal structure for BLG was obtained from the
Protein Data Bank (PDB code: 2GJ5). First, all the water molecules were removed and
hydrogen atoms were added to the protein file, as required in the Lamarckian Genetic
Algorithm. The docking parameter file was set to 100 runs. Finally, the best docking energy
result (the lowest energy) was assumed as the possible candidate for ligand-protein interaction
and was used for the final analysis with the Discovery Studio Visualizer 2.5.5 and MOE
softwares [21].
3
3
. Results and discussions
.1. Synthesis of sorafenib tosylate (SORt)
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According to the general method and the synthetic details described in the preceding steps,
the products were separated and purified at each step. Their physical properties and spectra
H
information are reported below sections. It should be noted that the IR, Mass, NMR, and
CNMR spectra for SORt are presented in Figures 1 and 2.
3
.1.1. Synthesis of 1-[4-Chloro-3-(trifluoromethyl) phenyl]-3-(4-hydroxyphenyl) urea
The Characterization of the target compound is described below.
White solid, Mp: 200°C, IR (KBr): υ (cm-1) = 3325-2500 (NH, OH), 1710 (C=O).
1
H NMR (DMSO-d6): δ (ppm) = 7.17 (d, 3J= 8Hz, 2H-Ar), 7.59 (d, 3J=8Hz, 2H-Ar), 7.60
(
d, 3J=8Hz, 1H-Ar), 7.70 (dd, 3J=8Hz, 4J=2.5Hz, 1H-Ar), 8.1 (d, 4J=2.5Hz, 1H-Ar), 9.39 (s,
1
1
1
H, NH), 9.56 (s, 1H, NH).
3C NMR (DMSO-d6): δ (ppm) = 115.4, 116.4, 119.8, 119.9, 120.8, 123.1, 125.1, 130.7,
31.7, 140.8, 152.8, 154.1.
EI-MS (70 eV): m/z (%) = 330.6 (M+•).
3
.1.2. Synthesis of sorafenib (SOR)
The characterization of SOR is described as below.
White solid, Mp: 218°C, IR (KBr): υ (cm-1) = 3300-3100 (3NH), 1707 (C=O), 1615 (C=O)
1
H NMR (DMSO-d6): δ (ppm) = 2.84 (d, 3J=4.5Hz, 3H, NHCH3), 7.17 (d, 3J= 8Hz, 2H-
Ar), 7.33 (dd, 3J=6.5Hz, 4J=2.5Hz, 1H-Ar), 7.59 (d, 3J=8Hz, 2H-Ar), 7.60 (d, 3J=8Hz, 1H-
Ar), 7.70 (dd, 3J=8Hz, 4J=2.5Hz, 1H-Ar), 7.77 (d, 4J=2.5Hz, 1H-Ar), 8.1 (d, 4J=2.5Hz, 1H-
Ar), 8.61 (d, 3J=6.5Hz, 1H-Ar), 9.17 (brd, 3J=4.5Hz, NHCH3), 9.39 (s, 1H, NH), 9.56 (s,
1
1
1
H, NH).
3C NMR (DMSO-d6): δ (ppm) = 25.7, 109.9, 114.05, 116.08, 119.7, 120.7, 121.1, 121.7,
22.3, 123.3, 131.4, 137.3, 138.07, 138.9, 146.5, 146.8, 151.8, 160.3, 168.3.
EI-MS (70 eV): m/z (%) = 4.64.2 (M+•).
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3
.1.3. Synthesis of sorafenib tosylate (SORt) as the final product
The characteristic of SORt, which is the final product, is described as below (Figures 1 and
).
White solid, Mp: 223 °C, IR (KBr): υ (cm-1) = 3500-2500 (NH and OH), 1723 (C=O), 1689
C=O), 1598 (S=O), 1556 (S=O).
H NMR (DMSO-d6): δ (ppm) = 2.29 (s, 3H, CH3), 2.84 (d, 3J=4.5Hz, 3H, NHCH3), 7.17
2
(
1
(
(
d, 3J= 8Hz, 2H-Ar), 7.22 (d, 3J=9Hz, 2H-Ar), 7.33 (dd, 3J=6.5Hz, 4J=2.5Hz, 1H-Ar), 7.59
d, 3J=8Hz, 2H-Ar), 7.60 (d, 3J=8Hz, 1H-Ar), 7.67 (d, 3J=9Hz, 2H-Ar), 7.70 (dd, 3J=8Hz,
4
1
J=2.5Hz, 1H-Ar), 7.77 (d, 4J=2.5Hz, 1H-Ar), 8.1 (d, 4J=2.5Hz, 1H-Ar), 8.61 (d, 3J=6.5Hz,
H-Ar), 9.17 (brd, 3J=4.5Hz, NHCH3), 9.39 (s, 1H, NH), 9.56 (s, 1H, NH), 13.13 (brs, 1H,
OH).
1
1
1
3C NMR (DMSO-d6): δ (ppm) = 20.2, 25.7, 109.9, 114.05, 116.08, 119.7, 120.7, 121.1,
21.7, 122.3, 123.3, 124.9, 127.8, 131.4, 137.3, 138.07, 138.9, 143.7, 146.5, 146.8, 147.7,
51.8, 160.3, 168.3.
EI-MS (70 eV): m/z (%) = 4.64.2 (M+•).
3
.2. Bioassays for anticancer activity of SORt
After the synthesis and characterization of SORt, its anticancer activity was investigated. For
this subject, two cell lines of HCT116 and CT26 were selected. These cell lines have
epithelial cell morphology and are rapid-growing [22, 23]. The results of the cytotoxicity
assay show that CT26 cells are more sensitive to SORt than HCT116 cells, the IC50 values
were also 5.42 and 8.12 μM, respectively. The results of IC50 calculation for anticancer the
drug of regorafenib as positive control exhibited 17.40 and 28.62 μM for CT26 and HCT116
cells, respectively (Fig. 3). Considering the anticancer effects of SORt, a summarization of
another study has been represented; Yao, et al. after design, synthesis and antiproliferative
activity of a collection of SOR derivatives, demonstrated that one of the derivatives shown
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more potent effect than that of SOR. Furthermore, two compounds have almost the same
impact as SOR and the rest have less effect than SOR. In their study, the IC50 value for SORt
for the HCT116 cell line was 7.8 ± 1.1, which is closer to our results [2].
3
3
.3. Study of the interaction between SORt and BLG
.3.1. Fluorescence measurement study
The interaction of SORt with BLG was investigated using fluorescence spectroscopy. This
technique can provide information on the binding of a small molecule to the protein, such as
intermolecular distances, binding sites, binding modes, binding constants, and binding
mechanisms [24]. BLG has two tryptophan residues (Trp) and four tyrosine (Tyr) residues
per monomer [25]. They are as aromatic amino acids and contribute to the fluorescence
emission of BLG. Fig. 4A depicts the variation of the emission intensity of BLG solution
with addition to different concentrations of SORt. The emission spectra of BLG displayed a
peak at 317 nm. The addition of different concentrations of SORt to the fixed concentration of
BLG resulted in a significant quenching in the fluorescence intensity. The quenching takes
place when the SORt is sufficiently close to the fluorescence residues of the BLG. So, it can
be concluded that several parameters such as ground state complex formation, collisional
quenching, reactions in the excited-state, rearrangement of molecules and energy transfer
between the ligand and the macromolecule have been occurred in the system.
3
.3.2. Calculation of the binding constants between BLG and SORt
Generally, fluorescence quenching is not out of two states; they are static or dynamic.
Dynamic quenching relies on intermolecular collision in a fluid environment, while static
quenching depends on forming a stable ground state complex [26]. The type of quenching
caused by SORt on BLG can be estimated from the variation of the quenching signal with
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temperature. For this reason, the fluorescence quenching data were analyzed by the well-
known Stern–Volmer equation:
퐹0
=
1 ꢂ 퐾 휏 [푄] = 1 ꢂ 퐾 [푄]
푞 0 푆푉
퐹
The fluorescence intensities before and after the addition of SORt, are F
kq, τ , [Q] and Ksv respectively are the Stern–Volmer dynamic quenching constant, the
average life-time of the biomolecule without quencher (τ
0
and F ,respectively.
0
-8
0
=10 s), the concentration of the
SOR, and the quenching rate constant of the biomolecule. To study the fluorescence
quenching mechanism, we investigated the fluorescence quenching effect of The Stern–
Volmer curves of F /F versus [Q] at temperatures of 298, 305 and 310 (Fig. 4B) [27]. The
0
calculated of the Ksv and kq are summarized in table 1. The results represented at table 1
indicate that the quenching procedure is initiated by astatic quenching because rate constants
decrease with increasing temperature. In addition, the quenching constant for BLG-SORt
1
0
-1
12
-1
complex is greater than 2.0 × 10 M s (24.45 × 10 M s ). This shown that fluorescence
quenching does not follow the dynamic collision quenching, and most probably, a static
mechanism is responsible for the formation of the complex [28, 29]. Furthermore, the Ksv
values for the interactions of the SORt with BLG at different temperatures decreased with
increasing temperature; confirming the static interaction between the drug and the protein.
The binding constant and the number of binding sites of SORt on BLG were calculated by the
Hill equation [30]:
퐿ꢃ푔(퐹 − 퐹0)
=
퐿ꢃ푔퐾 ꢂ 푛퐿ꢃ푔[푄]
푏
퐹
where F
the binding constant and the number of binding sites. The binding constant K
different temperatures (298, 305 and 310) were calculated from the intercept and slope of the
plot of log (F-F /F) versus log [Q], respectively [31] (Fig. 4C) (Table 1).
0
, F, K
b
and n respectively are the fluorescence intensities without and with enhancer,
b
and n at three
0
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3
.3.3. Determination of the thermodynamic parameters
To further characterization of the interaction forces between SOR and BLG, the
thermodynamic parameters were analyzed. The thermodynamic parameters of standard Gibbs
free energy change (ΔG), standard enthalpy change (ΔH) and standard entropy change (ΔS)
can donate valuable information including hydrogen bonds, van der Waals force, electrostatic
force, hydrophobic interaction force, which are mainly interaction forces between a drug
molecule and bio-macromolecule. The binding of proteins with drug molecules can be
concluded from the thermodynamic parameters: if ΔH > 0 and ΔS > 0 the main binding
forces are hydrophobic interaction, ΔH < 0 and ΔS < 0 the main binding forces are van der
Waals or hydrogen bond interactions and ΔH < 0 and ΔS > 0 hydrogen bonds and
hydrophobic interactions are important forces for the formation of the complex. Moreover,
the negative symbol in ΔG values reveals the spontaneous nature of the reaction process
[
32]. In this study, the thermodynamic parameters were calculated via the van’t Hoff
equation:
ΔH ΔS
퐿푛 퐾 = −
ꢂ
푅푇
푅
where K is the binding constant, T is the absolute temperature, and R is the universal gas
−
1
−1
constant (8.314 J mol K ). ΔH and ΔS were obtained from the slope and intercept of the
linear plot based on lnK versus 1/T (Fig 4D). The free energy change (ΔG) is estimated from
the following equation [33, 34]:
훥퐺 = 훥퐻 − 푇훥ꢄ
The values of ΔH, ΔS, and ΔG for SORt binding to BLG are listed in Table 1. The negative
values of ΔH, ΔS and ΔG suggested that van der Waals and hydrogen bond interactions were
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the main binding forces in the interaction process between SORt and BLG, and has been
occurred a spontaneous nature of the reaction process.
3
.3.4. Synchronous fluorescence spectroscopy
Synchronous fluorescence spectroscopy is a useful technique to consider the
microenvironment of amino acid residues through observing the maximum fluorescence
wavelength shift corresponding to the polarity variation nearby the fluorophore (Tyr and Trp)
molecules [35]. Therefore, the conformation changes in protein structure are determined by
this method [36]. When difference values (Δλ) between the excitation and emission
wavelengths are 15 and 60 nm, the synchronous fluorescence spectra display characteristics
of the Tyr and Trp residues of the protein, respectively [37]. The results showed that, when
the concentrations of SORt increase, the maximum emission wavelength in Δλ = 60 becomes
slightly red-shifted (from 338 to 341 nm), while in Δλ = 15 do not appear any significant shift
in that (Figures 5A and 5B). This indicates that the conformation of the Trp micro-
environment is changed, while for Tyr the microenvironment remained unaffected. This
protein contains two residues of tryptophan (Trp-19 and Trp-61), which Trp-19 is placed on the
beta-strand A and inside the beta-barrel; Trp-61 is located on the beta-strand C on the outer
surface of the CD loop and is exposed to solvent [38]. The red-shift implies that the
microenvironment of the Trp residues of the protein in the near the interaction site of the drug
is perturbed, leading to a higher degree of exposure to the more polar aqueous environs [39].
3
.3.5. Three-dimensional (3D) fluorescence spectra
When the excitation wavelength, emission wavelength and fluorescence intensity as three
critical parameters in fluorescence spectroscopy be used in one axes, a new spectrum is being
created, which known as 3D fluorescence spectra [40]. Because the contour map gives us
valuable information such as shift at the excitation or emission wavelength around the
fluorescent peak; the advent of a new peak; vanishing of the existing peak, this spectrum was
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chosen to monitor the conformational changes in the BLG under interaction with SORt [41].
There are two peaks in the 3D fluorescence spectra (peaks 1 and 2). Peak 1, which is seen in
the λex = 280 nm and λem = 340 nm and mostly shows the spectra behavior of the Trp and
Tyr residues. Peak 2, that appeared during λex = 235 nm and λem = 337 are related to the
fluorescence spectra behavior of the polypeptide backbone structure of BLG (Figures 5C
and 5D). From the data obtained from 3D fluorescence, it can be said that the interaction
between BLG and SORt is formed, and this interaction has altered the 3D structure of the
BLG. Although 3D fluorescence data cannot quantify the content of α-helix, β-sheet and
random coil, it can qualitatively confirm the conformational change in the protein structure.
3
.3.6. Circular dichroism (CD) analysis
Generally, the interaction of the small molecules with protein cause changes in the protein
polypeptide backbone conformation (secondary structure). One way to observe these changes
is to use CD spectroscopy techniques. Fig. 6A shows the CD spectra of the BLG free and
BLG-SORt system. After analyzing the data using CD analysis using Neural (CDNN)
Networks, the fractions of α-helix, β-sheet and the random coil of BLG without and with
SORt are listed in Table 2. As can be seen from the data in Table 2, the free BLG comprised
2
0.34% α-helix, 43.85% β-sheet, 35.80% random coil. Our results showed the amount of α-
helix remains almost constant in the BLG–SOR system, whereas those of random coil
decreased. Additionally, the content of the β-sheet was slightly increased by the addition of
two amounts (5 and 10 µM) of SORt. Changes in the percentage of β-sheet and random coil
structures in BLG showed that SOR bind to the amino acid residues of the main polypeptide
chain of BLG.
3
.3.7. Zeta-Potential Measurements
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The surface properties of proteins have essential factors in their ionic interactions with other
molecules. The ionization of surface groups of the proteins mainly causes the surface charge
on the proteins. The surface charge of proteins can be measured using a zeta-potential
technique [43]. A result of the zeta potential, which shows the effect of the addition of SORt
on the BLG, is illustrated in Fig. 6B. As the SORt concentration increases, the zeta-potential
values are also increased, which suggests the formation of BLG–SORt complex. This
tendency is indicative of the existence of electrostatic interactions between BLG and SORt
[
44].
3
.3.8. Molecular Docking
Molecular simulation is a useful technique to identify the binding site, understand and
interpret the mechanism of interaction between the BLG is sorafenib. It is also used as an in
silico tool to validate experimental interpretations [45]. In general, there are three major
binding sites in the BLG structure, an internal cavity of the β-barrel (central calyx domain), a
groove between the α-helix and the β-barrel that forms the hydrophobic surface pocket and
the outer surface near Trp19-Arg124 [39]. The docking result showed that, sorafenib is
unable to enter the cavity of β-barrel, and its interaction is close to the β-sheet and α-helix
(
Fig 7A). The binding of SORt to the protein is via hydrogen bonds and electrostatic forces.
Hence, there is hydrogen bonding interaction between SORt and BLG through the residue
Asp53, which is consistent with the results of thermodynamics parameters. Furthermore, the
charged amino acids of Asp53, Glu74, Lys75, Lys83 and Asp85 participate in the formation
of electrostatic interactions (Fig 7B), which confirms the results obtained from zeta potential
measurements.
4
. Conclusion
Since the SORt is one of the most widely used drugs among cancer patients, it was
successfully synthesized in this project, and its anticancer properties was confirmed against
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two colon cancer cell lines of HCT116 and CT26. In general, SORt is a small molecule with
the hydrophobic nature; hence it is a limiting factor for its oral administration. One way to
overcome this limitation is to use a carrier that can form a complex with the drug. So, in this
study dietary protein of β-lactoglobulin (BLG) was used for this purpose, and its interaction
with the drug was investigated. Stern–Volmer dynamic quenching constant (kq), steady-state
and fluorescence resonance energy transfer (FRET) results showed that static quenching is
the primary reason for fluorescence quenching of BLG in complex with SORt. The negative
values of thermodynamic parameters, including ΔH and ΔS suggested that van der Waals and
hydrogen bond interactions were the main binding forces in the system of BLG-SORt.
Circular dichroism exhibited that the amount of α-helix remains almost constant in the BLG–
SORt complex. Thus, it became apparent that SORt high affinity can bind to a suitable site at
the BLG protein surface. Therefore, based on the insights into drug and protein binding, in
the future studies we are looking for the design of nanoparticles of SORt based on BLG
protein.
Disclosure statement
No potential conflict of interest was reported by the authors.
Acknowledgement
This work is a part of a research project and was financially supported by Deputy of Research,
Hamadan University of Medical Sciences [Grant number: 9704192187]. Therefore, the
authors are very thankful for the support of this deputy.
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Graphical Abstract
1
- Synthesis of Sorafenib
2
- Confirmed by FT-IR, Mass and NMR 3- Sorafenib Cytotoxicity on HCT116 &
CT26 and its Binding to β-lactoglobulin
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OH
CF3
CF3
O
a
Cl
Cl
N
N
+
+
NCO
NH2
N
N
NH2
4
1
2
3
b
CF3
O
Cl
OH
Cl
O
N
H
+
N
NH NH
5
Intermediate
O
c
CF3
O
Cl
O
N
H
N
NH NH
Sorafenib
d
CF3
O
Cl
O
O
N
H
+
N
NH NH
H
O S O
-
O
Sorafenib tosylate
Scheme 1. Synthesis of Sorafenib tosylate, (a) CH
2
Cl
2
, reflux, 16h (b) CH
2
Cl , r.t., 16h (c) DMF,
2
potassium tert-butylate, K CO , 80°C, (d) EtOH, r.t., 15h
2
3
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A
B
Figure 1. Sorafenib tosylate spectra IR (A) and Mass (B).
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A
B
H
C
Figure 2. Sorafenib tosylate spectra MNR (A) and NMR (B).
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Figure 3: Cytotoxicity effects of Sorafenib tosylate and Regorafenib on two cancer cell lines of
HCT116 (A) and CT26 (B).
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Figure 4. Fluorescence quenching, Stern–Volmer and Hill curves for the interaction between β-
-6
lactoglobulin (BLG) and Sorafenib tosylate (SORt). Fluorescence quenching of BLG (21 × 10 M)
-6
induced by SORt (0.0 to 10 × 10 M) (A), Stern–Volmer curves of BLG-SORt complex at different
temperatures (B), Double logarithmic plots of log((F0 – F)/F) against log[SORt] derived from the
fluorescence quenching at different temperatures and pH 7.4 (C) and van’t Hoff plot for the binding
of SORt with BLG (D).
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Figure 5. Synchronous and three-dimensional fluorescence spectra for the interaction between
β-lactoglobulin (BLG) and Sorafenib tosylate (SORt). Synchronous fluorescence spectra of BLG
-
6
-6
(
(
(
21 × 10 M) in the presence of increasing concentrations of SORt (0.0 to 10 × 10 M) at ∆λ = 15 nm
A), and ∆λ = 60 nm (B). Contour diagrams of three-dimensional fluorescence spectra of BLG free
-6
-6
21 × 10 M) (C), BLG-SORt (SORt concentration 10 × 10 M) (D). T= 298 k, pH 7.4.
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