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Journal of Biomolecular Structure and Dynamics
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Formation of the inclusion complex of water
soluble fluorescent calix[4]arene and naringenin:
solubility, cytotoxic effect and molecular modeling
studies
Mehmet Oguz, Asif Ali Bhatti, Berna Dogan, Serdar Karakurt, Serdar Durdagi
& Mustafa Yilmaz
To cite this article: Mehmet Oguz, Asif Ali Bhatti, Berna Dogan, Serdar Karakurt, Serdar
Durdagi & Mustafa Yilmaz (2020) Formation of the inclusion complex of water soluble fluorescent calix[4]arene and naringenin: solubility, cytotoxic effect and molecular modeling studies, Journal of Biomolecular Structure and Dynamics, 38:13, 3801-3813, DOI: 10.1080/07391102.2019.1668301 To link to this article: https://doi.org/10.1080/07391102.2019.1668301
View supplementary material Published online: 11 Oct 2019.
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Formation of the inclusion complex of water soluble fluorescent calix[4]arene
and naringenin: solubility, cytotoxic effect and molecular modeling studies
Mehmet Oguza,b , Asif Ali Bhattia,c , Berna Dogand , Serdar Karakurte , Serdar Durdagid and Mustafa Yilmaza
a
Department of Chemistry, Selcuk University, Konya, Turkey;bDepartment of Advanced Material and Nanotechnology, Selcuk University, Konya, Turkey;cDepartment of Chemistry, Government College University Hyderabad, Hyderabad, Pakistan;dComputational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey;eDepartment of Biochemistry, Selcuk University, Konya, Turkey
Communicated by Ramaswamy H. Sarma
ABSTRACT
Naringenin is considered as an important flavonoid in phytochemistry because of its important effect on cancer chemoprevention. Unfortunately its poor solubility has restricted its therapeutic applications. In this study, an efficient water-soluble fluorescent calix[4]arene (compound 5) was synthesized as host macromolecule to increase solubility and cytotoxicity in cancer cells of water-insoluble naringenin as well as to clarify localization of naringenin into the cells. Complex formed by host–guest interaction between compound 5 and naringenin was analyzed with UV–visible, fluorescence, FTIR spectroscopic techniques and molecular modeling studies. Stern–Volmer analysis showed binding constant value of Ksv3.5 107M1suggesting strong interaction between host and guest. Binding capacity shows 77%
of naringenin was loaded on compound 5. Anticarcinogenic effects of naringenin complex were eval-uated on human colorectal carcinoma cells (DLD-1) and it was found that 5-naringenin complex inhib-its proliferation of DLD-1 cells 3.4-fold more compared to free naringenin. Fluorescence imaging studies show 5-naringenin complex was accumulated into the cytoplasm instead of the nucleus. Increased solubility and cytotoxicity of naringenin with fluorescent calix[4]arene makes it one of the potential candidates as a therapeutic enhancer. For deep understanding of host–guest interaction mechanisms, complementary multiscale molecular modeling studies were also carried out.
ARTICLE HISTORY Received 10 July 2019 Accepted 28 August 2019 KEYWORDS Water soluble; calix[4]arenes; naringenin; inclusion complexes; fluorescence; solubilization; cytotoxicity; molecu-lar docking 1. Introduction
Flavonoids are secondary metabolites of plants, synthe-sized via Shikimic acid pathway following pentose
phos-phate pathway and glycolysis (Cao et al., 2019;
Crozier, Clifford, & Ashihara, 2008). They are known as
micronutrients that are widely identified in foods of plant origin. These are recognized as potent antioxidants, pos-sessing the bioactive potential to reduce cancer risk,
pre-vent cardiovascular disease and neurodegenerative
disorders (Lee et al., 2008; Tu, Liu, et al., 2015;
Williams, Spencer, & Rice-Evans, 2004; Yadav et al., 2016). One of the important subgroups of flavonoids is flavonone that possesses anticancer activity beyond antioxidation effect that arise due to its capability to scavenge free radi-cals. It can have effects on cell proliferation, inhibition of angiogenesis and subcellular signaling with stimulation of
DNA repair enzymes (Erlund,2004; Gao et al.,2006; Kumar,
Birundha, Kaveri, & Devi, 2015; Meshram et al., 2019;
Sakalli, Burkina, Pilipenko, Zlabek, & Zamaratskaia, 2018; Selvaraj, Krishnaswamy, Devashya, Sethuraman, & Krishnan,
2014). Among flavanone, naringenin is considered as an
phytoestrogen with weak estrogenic and antiestrogenic activities that inhibits proliferation of colon cancer cells and melanoma cells (Frydoonfar, McGrath, & Spigelman,
2003; Isobe et al., 2018; T€urkkan, €Ozy€urek, Bener, G€uc¸l€u, &
Apak, 2012). A number of properties has been evaluated
for this flavanone such as it assists in reducing rate of migration of hepatocarcinoma and pancreatic cancer cells and decreases metastasis of melanoma cells up to 63%
(Lentini, Forni, Provenzano, & Beninati, 2007; Tu, Wang,
et al., 2015). Furthermore, reports suggest that naringenin
inhibits cytochrome P450 activity which can change pharmacokinetics in human liver and decrease estrogen synthesis and also has chemoprevention effects on cancer and suppresses toxic effects of aflatoxin B1 in human liver
(Buening et al., 1981; Moon, Wang, & Morris, 2006).
Furthermore, naringenin significantly inhibits migration of bladder cancer cells through inhibition of AKT and NF-kB (Liao et al.,2014).
However, the poor water solubility of this flavanone makes it less bioactive. To tackle the problem of solubility of this
CONTACT Mustafa Yilmaz myilmaz42@yahoo.com Department of Chemistry, Selcuk University, Konya 42075, Turkey; Serdar Durdagi
serdar.durdagi@med.bau.edu.tr Computational Biology and Molecular Simulations Laboratory, Department of Biophysics, School of Medicine, Bahcesehir University, Istanbul, Turkey.
Supplemental data for this article is available online athttps://doi.org/10.1080/07391102.2019.1668301.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2020, VOL. 38, NO. 13, 3801–3813
compound, different methods are being used in pharmaceutical applications, such as reduction in particle size, modification of crystal structure by the formation of various polymorphic forms, ionization of the molecule, addition of solubilizing agents or improvement of the wettability of the powder, surfactant-stabi-lized nanosuspension formation are formed (Gholami &
Bordbar, 2014; Kanaze et al., 2010; Sahoo et al., 2011;
Semalty, Semalty, Singh, & Rawat,2010; Qian et al.,2019; Zhou, Yang, et al.,2018; Zhao et al.,2018). Moreover, in recent years, supramolecular chemistry has gained large attention in drug delivery system and enhancement of solubility of the water-insoluble drugs (Bono et al.,2018; Drakalska et al., 2014). Thus, different macromolecules have been used as drug carriers (Guo, Wang, Wang, & Liu, 2012; Semalty, Tanwar, & Semalty, 2014;
Tommasini, Calabro, Raneri, Ficarra, & Ficarra, 2004;
Tozuka, Kishi, & Takeuchi, 2010; Yang, 2011; Yilmaz & Sayin,
2016). Among them, calixarenes are well-established
macromo-lecules with excellent host properties, particularly water-soluble versions with sulfo functionality at upper rim facilitates the
water solubility of guest molecules (Guo & Liu, 2014;
Mareeswaran et al., 2014; Ran et al., 2018; Ukhatskaya et al.,
2010; Yang , Zhao, et al.,2016; Zhou, Li, & Yang,2015). Besides, enhancement of solubility of the drug, drug carrier with fluores-cence imaging strategies are expanding rapidly for tracking drug in targeted cells. Initially, fluorescence was mostly used from small organic dyes attached by means of antibodies to the protein of interest (Giepmans, Adams, Ellisman, & Tsien,
2006; Yang, Ran, et al.,2016; Yang, Xie, et al.,2016; Yang et al.,
2015). But in changing tactics, fluorescence technique can open new possibilities in safe and effective drug delivery systems along with synergistic advancements in fluorescent probes, tar-geting strategies, instrumentation and data analysis that enable high throughput screening, single-molecule (Das, Ghosh, Koley,
& Singha Roy,2018; Parveen, Misra, & Sahoo,2012; Zhou, Zhao, et al.,2018).
In this study, we have synthesized a water-soluble cal-ix[4]arene containing fluorescent moiety at the lower rim (compound 5) and formed the water-soluble inclusion complex with naringenin. The complex was investigated for proliferation of human colon cancer cells and clarifica-tion of localizaclarifica-tion in living cells. Molecular modeling stud-ies were also applied to better understand the interaction
between host–guest complex formed by naringenin and
the calix[4]arene derivative.
2. Materials and methods 2.1. Materials
All the chemicals used in this work were of reagent grade or analytical grade and obtained from various commercial sources. All aqueous solutions were prepared with deionized water that was obtained via a Millipore milli-Q Plus water purification sys-tem and 100/ATR Sampling Accessory. pH was measured
through Orion 410Aþ pH meter. CEM-MDS 2000 closed vessel
microwave system was used in this study to prepare real and certified samples. IR spectra were performed on a Perkin Elmer spectrum 100 FT-IR spectrometer (ATR). UV/Vis spectra were
measured with Shimadzu 1700 UV–Vis spectrophotometer.
Bio-imaging studies were performed by using ZOE Fluorescent Cell Imager (Bio-Rad, CA, USA).
2.2. Synthesis
Figure 1 represents the schematic rout for the synthesis of
compound 5 according to the previously reported method.
Figure 1. The schematic route for the synthesis of dansyl p-sulfonatocalix[4]arene (compound 5). (i) BrCH2COOCH3/K2CO3/Acetone. (ii) Tris(2-aminoethyl)amine/
Synthesis of compound 5 was confirmed by1HNMR. % Yield: 71,1HNMR (D2O, 25 C) dH:2.16 (m, 2 H, CH2N), 2.39 (m, 4 H,
CH2N) 2.65 (s, 6 H, N(CH3)2), 3.01 (m, 8 H, NHCH2), 3.61 (m,
4 H, ArCH2Ar), 4.15 (m, 2 H, NHSO2),4.31 (m, 4 H, ArCH2Ar),
4.69 (s, 4 H, OCH2þH2O(solvent)) 7.21 (s, 4 H, Ar-H/Calix), 7.50
(s, 4 H, Ar-H/Calix), 7.67 (m, 2 H, ArHdans). 8.03 (m, 2 H,
ArHdans), 8.13 (m, 1 H, ArHdans), 8.28 (m, 1 H, ArHdans) (Collins
et al., 1991; Liu, He, Qing, Xu, & Qin, 2005; Oguz, Bhatti, Karakurt, Aktas, & Yilmaz,2017).
2.3. Preparation of inclusion complex
100 mg of compound 5 was taken in 50 mL Erlenmeyer flask and dissolved in 10 mL water with adjusting pH up to 8.0. In a separate Erlenmeyer flask, the equimolar amount of narin-genin was dissolved in 3 mL methanol and mixed with the solution containing compound 5 and stirred at room tem-perature for 4 hours at room temtem-perature. Methanol was removed using vacuum distillation and the water phase was filtered by using 0.45lm PTFE filter to get the water-soluble material. The filter was washed with methanol and remaining amount of naringenin was measured by the spectro-scopic method.
Binding capacity % (BC %) was calculated by applying the following formula:
BCð%Þ ¼Total mass of drugFree drug in supernatant Total mass of drug 100
(1)
2.4. Determination of binding constant and solubility constant
Stern–Volmer analysis was utilized to probe the nature of
the quenching or enhancement process in the complexation of the drug (Lakowicz & Weber,1973; Zhu, Sun, Wang, Xu, & Wang,2017).
Io
I ¼ 1 þ Ksv½ Q (2)
where Iois the fluorescence intensity for compound 5 in the
absence of naringenin, Io is the fluorescence intensity of
compound 5 in the presence of naringenin. Ksv static
quenching constant and Q is the quencher concentration.
Benesi–Hildebrand equation was used to determine the
binding constant for compound 5-naringenin
complex complex. 1 I Io¼ b a b 1 K M½ þ1 (3)
where I0 represents the fluorescence intensity of compound
5 and I is the fluorescence intensity of for 5-naringenin com-plex, [M] is the concentration of the naringenin and K is the binding constant for complex. From the plot of 1/I– I0vs. 1/
[M], value of b/a– b can be found.
The aqueous solubility of naringenin was determined with increasing concentrations of the compound 5 at pH 7.0, by using Higuchi and Conners method (Higuchi & Connors,
1965). 10 mg of naringenin was taken in each test tube
containing 10 mL aqueous solution (1 105 to 1 104).
The mixtures were equilibrated for 4 h and filtered through 0.45lM nylon filter. The concentrations of dissolved substan-ces in water were determined by absorption spectroscopy.
Ks¼
Slope So ð1 SlopeÞ
(4)
where So is the intrinsic solubility of naringenin and slope
value was obtained from the plot using Higuchi and Conners method.
2.5. Cell culture studies
The human colon cancer cell line, DLD1 was provided from ATCC (American Type Culture Collection, ATCC, Rockville,
MD, USA) and cultured in RPMI-1640 (ATTC-30-2001)
medium. All media were supplemented with 10% fetal
bovine serum, 1% L-glutamine and 1%
penicillin/strepto-mycin. This growth medium was also used for dilution of other chemicals. The cell lines were maintained at 37C in a
5% CO2atmosphere and 95% humidity.
2.6. Cell proliferation assays
DLD-1 cells were seeded into 96-well plates (1 104 cell/
well) as three replicates in order to determine the IC50value
of naringenin, compound 5 and 5-naringenin complex were incubated for 48 h at 37C in 5% CO2. The effect of
naringe-nin, compound 5 and 5-naringenin complex on the
prolifer-ation of DLD-1 cells and IC50 values were determined by
using various concentrations ranging from 0.1 to 200mM.
The differences in cell growth were monitored by Alamar blue method in which conversion of resazurin (blue color) to resorufin (pink color) measured by spectrophotometrically.
2.7. Cell imaging studies
Following the calculation of IC50 values, DLD-1 cells were
seeded on 18 mm glass coverslips and allowed to adhere for 24 h. Following to incubation, the medium was replaced with fresh one containing 5-naringenin complex and incubated
1 h at 37C in a 5% CO2atmosphere and 95% humidity. The
images were obtained by ZOE Fluorescent Cell Imager (Bio-Rad, California, USA).
2.8. Computational methods 2.8.1. Geometry optimizations
The crystal structure of tetrahydroxycalix[4]arene was downloaded from Cambridge Structural Database (CSD identifier WUVKON, Deposition number 184678) (Atwood,
Barbour, & Jerga, 2002) and it was modified to compound
5 using Maestro molecular modeling package (2015). The structure was first minimized using OPLS3 force field
(Harder et al., 2016) to remove steric clashes. The pKa of
ionizable groups such as amines, amides, hydroxy groups etc., were predicted by MarvinSketch program (v., 16.1.4.0,
compound found as –4. MacroModel module of Maestro was used to generate different conformers of compound 5 using OPLS3 force field force field and 115 conformers were generated. The structure of the conformer with the lowest energy was then optimized by quantum mechanical (QM) calculations at density functional theory (DFT) level.
The hybrid functional B3LYP (Becke, 1988; Lee, Yang, &
Parr, 1988) with basis set of 6-31G was used for first
optimization at gas phase with convergence criteria of
5.0 106 Hartree. The final structure obtained at this
basis set was then subjected to further optimization again
with B3LYP functional but using 6-31Gþþ basis set. The
atomic charges from electrostatic potential (ESP) fitting was also obtained by DFT calculations (at
B3LYP/6-31Gþþ level) to be used for further docking and
molecular dynamics (MD) simulations.
The 3D structure of naringenin was downloaded from PubChem database and LigPrep module of Maestro with OPLS3 force field was used to optimize the geometry. In
addition, Epik (Shelley et al., 2007) was used to generate
ionization and tautomeric states for molecules in aqueous solution at pH 7.4. As there was one chiral center, both R
and S isomers were generated to be used in host–guest
complex formation. MacroModel module was used to gen-erate different conformers for both isomers (14 different conformers for each isomer). The structures of all conform-ers were then optimized by DFT calculations at
B3LY3/6-31Gþþ level and ESP charges were also calculated for
each conformer.
2.8.2. Molecular docking
The initial host–guest interaction between compound 5 and
R/S naringenin was predicted by molecular docking methods. The Glide docking with extra precision (XP) option (Friesner et al.,2004) was used to predict the initial complex structure. In docking simulations, compound 5 was considered as receptor (host) while naringenin R/S were treated as ligand (guest). The center of the binding cavity of compound 5 (eg grid center) was determined according to following Cartesian
coordinates x ¼ –3.40 Å, y ¼ –2.50 Å and z ¼ 1.43 Å. The
inner box was set as 10 Å 10 Å 10 Å while the outer box
was set as 20 Å 20 Å 20 Å. To soften the potential for
the nonpolar parts of the host compound 5, the van der Waals radii of atoms with partial atomic charges less than 0.25 were scaled by a factor of 0.8. The conformers of R/S naringenin generated by MacroModel and further optimized by DFT calculations were used for ligand docking with Glide/ XP. For each isomer of naringenin, at most 100 docking poses were requested. The intramolecular hydrogen bonds
were asked to be rewarded and planarity of conjugated p
groups were enhanced. The potential of nonpolar part of ligands were also softened by scaling of van der Waals radii of atoms with partial atomic charges less than 0.15 by a fac-tor of 0.6. The previously calculated ESP charges of both host and guest molecules were used. Docking poses obtained were clustered based on the root mean square deviations (RMSD) of ligands, R/S naringenin, with a cutoff value of 2.0 Å.
2.8.3 Molecular Dynamics (MD) Simulations
A representative host–guest complex structure from each
cluster of docking poses as well as top docking poses for both isomers were subjected to MD simulations. For R iso-mer, six different complex structures were considered for MD simulations, whereas for S isomer, eight different
host–guest complexes were used. While ESP charges
obtained by QM calculations were used in host-guest com-plexes, OPLS3 force field was utilized to obtain other force
field parameters in Desmond. (Bowers et al.,2006), . Each
system was then solvated using TIP3P water model
(Jorgensen, Chandrasekhar, Madura, Impey, & Klein, 1983)
in orthorhombic boxes with the sizes of boxes calculated by buffer method and distances along all three directions were chosen as 25 Å. For each system, first the default relaxation protocol of Desmond was applied in which a series of minimizations and short MD simulations were performed to relax the model system before production MD simulations. The production MD simulations were per-formed under NPT (constant pressure and temperature) ensemble for 100 ns. During production simulations
Nose-Hoover thermostat (Nose-Hoover, 1985; Nose,1984) was utilized
while the initial temperature was set as 310 K. The pres-sure was set at 1.01325 bar and it was initially controlled by the Martyna-Tobias-Klein method (Martyna, Tobias, &
Klein, 1994) with isotopic coupling style and relaxation
time of 2 ps. The equation of motions in dynamics was cal-culated using the multi-step RESPA integrator with time steps of 2.0, 2.0 and 6.0 fs for bonded, near non-bonded and far non-bonded interactions, respectively. For the
short-range electrostatic and Lennard–Jones interactions
the cutoff value was set as 9.0 Å. Long-range interactions were estimated using Particle mesh Ewald (Ananchenko
et al., 2007) method (Darden, York, & Pedersen, 1993;
Ewald, 1921) along with periodic boundary conditions. The
trajectories obtained by MD simulations were also clus-tered based on the RMSD fluctuations of the ligand R/S naringenin. The MM/GBSA approach (Lyne, Lamb, & Saeh,
2006; Sirin et al., 2014) implemented in Prime module of
Maestro with solvent model of VSGB 2.0 (Li et al., 2011)
was used to calculate the binding free energies.
3. Result and discussion
Controlled drug delivery requires the understanding of drug design, stability and metabolism together with the complexities imposed by the biological systems such as drug-target interaction. Fluorescence technique provides a comprehensive tool for investigating many aspects of drug
delivery in single cells and whole tissue (White &
Errington, 2005). Many drugs are inherently
auto-fluores-cent and therefore can be tracked using microscopy tech-niques, while others need ancillary methodologies. In this scenario, by maintaining the drug stability, efficient carrier along with fluorescence probe is required (Johnson &
Spence, 2010). The purpose of this work is to utilize
water-soluble fluorescence calixarene as solubility enhancer for naringenin, as well as to increase the cytotoxicity of
naringenin in cancer cells.. Compound 5 was synthesized and characterized according to the previously published
method (Oguz et al.,2017). The compound 5 was selected
due to presence of different functionalities such as -SO3H
at upper rim that provide good environment to calixarene to accommodate guest species, while fluorescent dansyl moiety at lower rim help to identity the presence of com-plex in living cell. Therefore, compound 5 may act as an ideal derivative for this study (Figure 3).
3.1. FTIR analysis
Interaction of naringenin with compound 5 was confirmed with FTIR spectroscopy by comparing individual component with
complex. Figure 2(a) shows spectrum of compound 5 with
broad band at 3359 cm1 for –OH group, characteristic band
for –SO3 appeared at 1189 and 1041 cm1, respectively.
Characteristic band appeared at 1630 cm1 assigned to C-N
band for amide and at 792 cm1 for aromatic ring (Valand,
Patel, & Menon, 2015). Naringenin shows broad peak at
3287 cm1for -OH vibration, 1628 and 1588 cm1are assigned to the stretching vibration for C¼ O and C ¼ C aromatic group,
1309 and 1150 cm1 are correspond to the C–O–C vibration
and 1065 cm1correspond to C–H of the aromatic group
bend-ing vibration (Semalty et al., 2010). The complex shows quite different characteristics bands with broadening of OH stretching band at 3367 cm1due to interaction of phenolic–OH of
narin-genin with SO3 through hydrogen bonding. Presence of broad
band at 1600 cm1is due to merging of different bands (C¼ O and C¼ C), this can also be attributed to p-p stacking interac-tions of naringenin and calixarene cavity, that confirms forma-tion of complex (Figure 3).
3.2. UV–visible and fluorescence study
Confirmation of inclusion complex was carried out by
analyz-ing it through UV–Vis and fluorescence spectroscopy. As
evi-denced from the absorbance and emission spectra (Figure
4(a,b)), compound 5 and 5-naringenin complex have
differ-ent profiles. The blue shift in 5-naringenin complex from 289
Figure 2. FTIR spectra of compound 5 (a), naringenin (b) and 5-naringenin complex (c).
to 324 nm (Figure 4(a)) can be attributed to the interaction of phenolic moieties of naringenin with sulphate present at upper rim of calixarene that enforce hydrophilic interactions
(Valand et al.,2015). Compound 5 is highly fluorescent with
dansyl moiety at its lower rim. We can use fluorescent spec-troscopy to analyze the effect on intensity of compound 5 by titrating it with naringenin that is regarded as
non-fluor-escent.Figure 4(b)shows the increase in intensity at 505 nm
in fluorescence spectra attributed structural changes due to holding the naringenin in calix[4]arene cavity (Mareeswaran et al.,2014). This result clearly demonstrated that the entrap-ment of hydrophobic naringenin in the hydrophilic environ-ment created by the compound 5 is crucial for the solubilizing process.
3.3. Determination of Stoichiometric ratio, binding and solubility constant
Effect on fluorescence intensity with changing concentration of naringenin was observed. Data obtained was fitted in
modified Benesi–Hildebrand equation (Eq. 3). From the plot
of 1/I– I0 and 1/[M] (Figure 5(a)) binding constant K was
cal-culated for 5-naringenin complex as 1.2 107 M1. From
Stern–Volmer plot (Figure 5(b)), Ksv was determined as
3.5 107 M1. The Ksvvalue suggests the high binding
affin-ity of compound 5 for the naringenin in aqueous media. Binding of naringenin with compound 5 was also evident from binding constant
Figure 4. UV–visible spectra (a) and fluorescence response (b) of 5, naringenin and 5-naringenin complex.
Figure 5. Benesi–Hildebrand plot compound 5 (1.5 106M) and naringenin (1 108M to 3.3 108M) (a). Stern–Volmer plot (b).
Figure 6. Phase solubility diagram for 5-naringenin as per Higuchi Connors method.
Figure 7. Effects of naringenin and 5-naringenin complex on viability and pro-liferation of DLD-1 cells, the viability of DLD-1 cells after treatment with differ-ent concdiffer-entrations of naringenin and 5-naringenin measured by Alamar blue assay, IC50values were determined from the sigmoidal plot of compound
Solubility studies were carried out using phase solubility technique devised by Higuchi and Connors (1965) (Figure 6).
Many phenolic and flavonoid compounds have
anticarcinogenic and antiproliferative properties, however, the main problem is inadequate solubility which decreases the bioavailability of the compound. One of the main active
Figure 8. Fluorescence images and their corresponding bright-field transmission images: Bright-field (right panels, (aand dw), DLD-1 cells incubated with
5-narin-genin complex (10mM) (middle, (b and ew)) and overlap of images of bright field and fluorescence (left panels, (c and fw)), respectively. , 20 magnification andw, 40 magnification.
Figure 9. The optimized structure of compound 5 by B3LYP hybrid functional with 6-31Gþþ basis set. Gray, red, blue, yellow and white colored spheres repre-sent C, O, N, S and polar H atoms, respectively. Yellow dashed lines show the hydrogen bonds. (a and b) Side views and (c and d) bottom and top views, respectively.
compounds in grapefruit is naringenin which affects the metabolism of many xenobiotics including drugs and proto-xicants, metabolized by cytochrome P450 enzymes especially,
CYP3A4 (Edwards & Bernier, 1996). Hence, inhibition of
CYP3A4 has crucial importance to prevent cytotoxicity of those hazardous compounds. Naringenin was proved to inhibit the activity of this enzyme (Henderson, Miranda, Stevens, Deinzer, & Buhler,2000).
Increased bioavailability of naringenin may protect living systems against toxic compounds. Therefore, interaction of naringenin with water-soluble calix[4]arene increases the solubility in water, which protects toxic effects of organic sol-vents. Cytotoxicity studies showed that compound 5 has no toxic effect on proliferation of DLD-1 cells. When compared with free naringenin, cytotoxic effects of 5-naringenin
com-plex was elevated 3.43-fold (p< 0.001) as IC50 value was
decreased from 66mM (free naringenin) to 19.2 mM
(5-narin-genin complex), (Figure 7).
Following the cytotoxicity studies, the cells were
incu-bated with 10mM of 5-naringenin complex for 45 min to
clar-ify localization in living cells. Fluorescence imaging studies showed 5-naringenin complex was accumulated into the cytoplasm instead of the nucleus. Localization of compound 5-naringenin complex in cytoplasm may explain action of compound since one of the important death mechanisms, extrinsic apoptosis was observed to occur in cytoplasm (Figure 8).
3.4. Computational modeling of compound 5-naringenin (R/S)
The DFT optimized geometry of compound 5 at
B3LYP/6-31Gþþ level can be found in Figure 9. The calix[4]arene
part of compound 5 is found at so-called‘pinched cone’
con-formation (Podoprygorina, Bolte, & B€ohmer, 2009; Scheerder et al., 1996; Zuo, Wiest, & Wu, 2011) most likely due to the bridging of upper rim (narrow rim) and intramolecular hydro-gen bonds. In this conformation, the two aromatic rings that are connected via a dansyl group orientated in parallel, while the other two rings are strongly tilted outwards as shown in the single crystal structure of bridged compounds obtained by Podoprygorina et al. (2009). 1,3-alternate conformation of calix[4]arene of compound 5 is also considered and
opti-mized again at B3LYP/6-31Gþþ level, however ‘pinched
cone’ conformation is found to be the lower energy
con-former between the two structures. Moreover, NMR spectrum
of compound 5 also indicates a cone-like structure (Figure
S1b) hence ‘pinched cone’ conformer shown in Figure 9 is
used for further docking and MD studies.
The initial host–guest complex structures between
com-pound 5 and R/S naringenin has been determined by molecular docking studies. The poses generated by Glide/ XP are clustered based on RMSD fluctuations of ligand orientation and considering RMSD value of less than 2.0 Å resulted in five complex structures for R isomer and seven
Figure 10. The representative structures obtained after the MD simulations of compound 5/R-naringenin complex. The ligand naringenin is represented by green colored ball and sticks while compound 5 is represented in gray color. Atom coloring scheme is same asFigure 9and yellow dashed lines represent hydrogen bonds, whereas blue dashed lines representp-p stacking interactions. Ligand electrostatic potential is also displayed as mesh surfaces with blue to red colored areas representing negative to positive partial charges.
complex structures for S isomer with at least five members in each cluster. All docking poses could be found in
Figures S2 and S3. Both isomers of naringenin are partially
included in host compound 5 based on the docking poses and they could not cross completely the cavity of calix[4]-arene most likely due to the hydrogen bonding
interac-tions with SO3– groups at the lower rim of calix[4]arene
and/or the tightening of upper rim part of cavity due to the bringing of 1,3 aromatic rings by dansyl group. This is actually consistent with our previous study in which quer-cetin, another flavonoid, formed a complex with a calix[4]-arene derivative and in silico studies again indicated that it could only be partially included in the calix[4]arene cavity (Yilmaz et al.,2019).
The top-docking poses as well as representative structures from each cluster of docking poses for both isomers have been used as initial structures for MD simulations and 100-ns simulations were run for each complex structure. The trajec-tories obtained by MD are visualized to analyze the stability
of interactions between host and guest compounds.
Regardless of the initial structure of MD simulations, R-narin-genin, in most of the trajectories, is found as attached to the
host compound 5 especially for starting structure in Figure
S2(b). However, the detachment of guest molecule
R-narin-genin is also observed such that guest molecule was outside of the calix[4]arene cavity but still interacting with the host compound. After the clustering of all six 100 ns MD
simula-tions, representative host–guest complex structures for
R-Figure 11. The representative structures obtained after MD simulations complex of compound 5 and S-naringenin. The ligand naringenin is represented by green colored ball and sticks while compound 5 is represented in gray color. Atom coloring scheme is same asFigure 9and yellow dashed lines represent hydrogen bonds, whereas blue dashed lines representp-p stacking interactions. Ligand electrostatic potential is also displayed as mesh surfaces with blue to red colored areas representing negative to positive partial charges.
naringenin were identified (Figure 10). While complete
separ-ation of host–guest complex has also been witnessed in
some trajectories, such as the MD simulations started from initial pose inFigure S2(e) and (f), the re-attachment and/or re-interactions of host–guest are also observed in some cases
as shown inFigure 10(d). In the case of S-naringenin–
com-pound 5 complex, more detachment events are witnessed by analysis of all eight 100 ns MD simulation trajectories.
Some observed host–guest complex structures for
S-naringe-nin after clustering of all MD trajectories could be found in
Figure 11. Though guest S-naringenin leaves the cavity of
calix[4]arene, in most of the trajectories it again re-attaches to the cavity and/or comes close to interact with compound 5 after a time of complete detachment such as shown in
Figure 11(e). We have also witnessed some conformational
changes of complex as seen in Figures 10(c) and11(c) such
that not only the dansyl group which is quite mobile during MD trajectories but also one of the calix[4]arene aromatic
rings changed its conformation to so called ‘partial cone’
conformation (Zuo et al.,2011).
Additionally, MM/GBSA calculations are performed for all MD simulations of R/S naringenin-compound 5 complexes though this method could only give crude binding energies that could not be necessarily compared to experimental
val-ues (Genheden & Ryde, 2015; Godschalk, Genheden,
S€oderhjelm, & Ryde, 2013; Greenidge, Kramer, Mozziconacci, & Wolf, 2013). Nevertheless MM/GBSA energies could aid to understand the kind of interactions that drive the host–guest complex formation. Here we have also observed that there is a distinct difference between binding energy of complex when the guest compound is in cavity of calix[4]arene and when the guest leaves the cavity but continue to interact with the host molecule (as shown inFigures 10(d)and11(e)). For R-naringenin in complex with compound 5 the average
binding energy found as –21.61 ± 7.61 kcal/mol when the
guest is in cavity during all MD trajectories, whereas the binding energy is found as–12.23 ± 7.24 kcal/mol in case that R-naringenin leaves the cavity but still interacts with com-pound 5 during MD simulations. In both cases the binding energy is mostly driven by electrostatic, van der Waals and lipophilic interactions as can be seen from the binding
energy components list given inTable 1. The same
phenom-enon is true for S-isomer of naringenin where in the case that guest compound staying in the calix[4]arene cavity the
binding energy is found as –24.21 ± 4.30 kcal/mol, whereas
the value of binding energy decreases to –13.71 ± 9.10 kcal/
mol when the S-naringenin leaves the cavity but continue interacting with the host compound 5. The driving force for the complex formation again seems to be electrostatic, van der Waals and lipophilic interactions in both cases for S-iso-mer. As mentioned before, binding energy values for both R/
S-naringenin complex formation with compound 5 may not be directly compared to experimental binding energy value –10.70 kcal/mol (calculated from binding constant value of Ksv3.5 107 M1). In summary, it could be said that complex
formation between naringenin (R/S) and compound 5 are found to be favorable and the molecules are strongly inter-acting based on the low binding energies.
4. Conclusion
In this study, water-soluble fluorescent calix[4]arene (5) used as a host for water-insoluble naringenin to enhance its water
solubility and introduce fluorescent property. Host–guest
complex was formed at pH 8.0. Spectroscopic measurements and molecular modeling studies were conducted to broaden the host–guest interaction, which confirmed the efficiency of compound 5 as drug carrier. Moreover, it has been found that 5-naringenin complex possesses anti-proliferative effects
of human colon cancer cells with IC50 value of 19.2mM.
Besides, fluorescent property of compound 5 can track pres-ence of non-fluorescent naringenin in the cells. 5-Naringenin complex has been demonstrated to localize in cytoplasmic part of cell. Compound 5 could be versatile candidate as drug carrier in pharmaceutical industry.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was financially support by the Research Foundation of Selcuk University (Project No.: 19401060).
ORCID
Mehmet Oguz http://orcid.org/0000-0002-3999-620X
Asif Ali Bhatti http://orcid.org/0000-0001-5921-532X
Berna Dogan http://orcid.org/0000-0002-5650-5177
Serdar Karakurt http://orcid.org/0000-0002-4449-6103
Serdar Durdagi http://orcid.org/0000-0002-0426-0905
Mustafa Yilmaz http://orcid.org/0000-0003-2904-160X
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