Journal of Biomolecular Structure and Dynamics

ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20

Exploration of interaction mechanism of tyrosol as

a potent anti-inflammatory agent

Tara Chand Yadav, Naresh Kumar, Utkarsh Raj, Nidhi Goel, Pritish Kumar
Vardawaj, Ramasare Prasad & Vikas Pruthi

To cite this article: Tara Chand Yadav, Naresh Kumar, Utkarsh Raj, Nidhi Goel, Pritish Kumar
Vardawaj, Ramasare Prasad & Vikas Pruthi (2019): Exploration of interaction mechanism of tyrosol
as a potent anti-inflammatory agent, Journal of Biomolecular Structure and Dynamics

To link to this article: https://doi.org/10.1080/07391102.2019.1575283

Published online: 19 Mar 2019.

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2019.1575283

Exploration of interaction mechanism of tyrosol as a potent anti-

inflammatory agent
Tara Chand Yadava
, Naresh Kumarb
, Utkarsh Rajc, Nidhi Goeld, Pritish Kumar Vardawajc, Ramasare
Prasada and Vikas Pruthia
a
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India; bDiscipline of Biosciences and Biomedical
Engineering, Indian Institute of Technology Indore, Indore, India; cDepartment of Bioinformatics, Indian Institute of Information Technology
Allahabad, Allahabad, India; dDepartment of Chemistry, Institute of Science, Banaras Hindu University, Varanasi, India

Communicated by Ramaswamy H. Sarma

ABSTRACT ARTICLE HISTORY

Drug discovery for a vigorous and feasible lead candidate is a challenging scientific mission as it requires expert- Received 14 November 2018
ise, experience, and huge investment. Natural products and their derivatives having structural diversity are Accepted 22 January 2019
renowned source of therapeutic agents since many years. Tyrosol (a natural phenylethanoid) has been extracted
KEYWORDS
from olive oil, and its structure was confirmed by elemental analysis, FT-IR, FT-NMR, and single crystal X-ray crys-
Tyrosol; ab initio; COX-2;
tallography. The conformational analysis for tyrosol geometry was performed by Gaussian 09 in terms of density ADMET; molecular docking;
functional theory. Validation of bond lengths and bond angles obtained experimentally as well as theoretically molecular dynam-
were performed with the help of curve fitting analysis, and values of correlation coefficient (R) obtained as 0.988 ics simulation
and 0.984, respectively. The charge transfer within the tyrosol molecule was confirmed by analysis of
HOMO!LUMO molecular orbitals. In molecular docking with COX-2 (PDB ID: 5F1A), tyrosol was found to pos-
sess satisfactory binding affinity as compared to other NSAIDs (Aspirin, Ibuprofen, and Naproxen) and a COX-2
selective drug (Celecoxib). ADMET prediction, drug-likeness and bioactivity score altogether confirm the lead/
drug like potential of tyrosol. Further investigation of simulation quality plot, RMSD and RMSF plots, ligands
behavior plot as well as post simulation analysis manifest the consistency of 5F1A-tyrosol complex throughout
the 20 ns molecular simulation process that signifies its compactness and stability within the receptor pocket.

Abbreviations: ADMET: Absorption, Distribution, Metabolism, Excretion and Toxicity; Å: Angstrom;

COX-2: Cyclooxygenase-2; DFT: Density Functional Theory; DMF: Dimethylformamide; FMO: Frontier
Molecular Orbital; FT-IR: Fourier-transform Infrared Spectroscopy; FT-NMR: Nuclear Magnetic Resonance
Spectroscopy; HOMO: Highest Occupied Molecular Orbital; LUMO: Lowest Unoccupied Molecular
Orbital; MD: Molecular Dynamics; NS: Nanosecond; NSAIDs: Non-steroidal anti-inflammatory drugs;
OPE: Osiris Property Explorer; RMSD: Root-Mean-Square Deviation; RMSF: Root Sean Square Fluctuation

CONTACT Vikas Pruthi [email protected]; [email protected] Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee,
Uttarakhand 247667, India

Tara Chand Yadav and Naresh Kumar contributed equally as first author.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2019.1575283.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 T. C. YADAV ET AL.

1. Introduction antioxidants with chemo-protective effects are reported to

suppress the expression of COX-2 by disturbing the signaling
Natural products are admirable source in drug discovery due
pathway that regulates the COX-2 gene (Chinery et al., 1998).
to their structural diversity as well as wide range of bio-
Therefore, COX-2 gene is also used as a biomarker against
logical activities (Beutler, 2009; Harvey, 2008; Koehn & Carter,
cancer to assess the chemo-protective effects of natural phy-
2005; Kumar et al., 2016; Shen, 2015). In past, there are large
tochemicals, and the discovery of novel COX-2 inhibitor is
numbers of articles have been published on the extraction of
considered to be a ray of hope for the prevention of tumori-
natural bioactive molecules from plants, including polyphe-
genesis and inflammation (Cheki et al., 2018; Dhanjal et al.,
nols and their beneficial effects on human being (Ferguson,
2015; Kumar & Pruthi, 2015; Kumar et al., 2018; Nile, Ko, Kim,
Zhu, & Harris, 2005; Fuggetta, Cottarelli, Lanzilli, Tricarico, &
& Keum, 2016; Pang, Hurst, & Argyle, 2016).
Bernini, 2012; Kumar & Pruthi, 2014; Kumar, Pruthi, & Goel,
Herein, extraction and structural elucidation of tyrosol
2015; Manach, Williamson, Morand, Scalbert, & R
em
esy, 2005;
from olive oil followed by ab initio calculations, molecular
Montedoro, Servili, Baldioli, & Miniati, 1992; Ou, Li, & Gao, docking, and molecular dynamics simulation studies with
1999; Toshihiro et al., 2000). In the Mediterranean area, the COX-2 have been carried out to investigate its role as well as
daily consumption of wine and virgin olive oil is related to mechanism in anti-inflammatory reactions and comparison
the protection against several types of cancer, and the bene- with clinically approved anti-inflammatory drugs (aspirin, ibu-
fits are mainly contributed by the phenolic compounds such profen, naproxen, and celecoxib). This is the first report of
as tyrosol (Kontou, Psaltopoulou, Panagiotakos, Dimopoulos, structure determination and comparison of experimental and
& Linos, 2011). Tyrosol, 2-(4-hydroxyphenyl)-ethanol, a deriva- theoretical data on tyrosol to the best of our knowledge.
tive of phenylethyl alcohol, is one of the key bioactive phen-
olic compound, abundantly found in olive oil, white wine,
and plant extracts (Thibault, Arseneault, Longpre, & 2. Experimental
Ramassamy, 2011). It possesses scavenging properties on
2.1. Chemicals and extraction
peroxynitrite and superoxide anion (Bertelli et al., 2002;
Puerta, Mart
ınez Dom
ınguez, Ru
ız-Gut
ıerrez, Flavill, & Hoult, The sample used for the extraction of tyrosol was oil of olive.
2001). Tyrosol also exhibits anti-genotoxic activity, averts The reference compounds (tyrosol) and chromatographic sol-
apoptosis in keratinocyte, reduces the expression of cell vents like methyl alcohol, ethyl acetate, acetonitrile, heptanes,
adhesion molecule resulting in prevention of endothelial syringic acid, gallic acid, DMF etc. others were analytical grade
dysfunction, inhibition of platelet-induced aggregation, and purchased from TCI Chemicals and Himedia, India. Tyrosol
antioxidant, anticancer, anti-depressant, anti-stress, cardio- was extracted in modifications of earlier reported methods
protective, anti-osteoporosis, anti-inflammatory, anti-hyper- (Adhami et al., 2015; Brenes, Garc
ıa, Garc
ıa, & Garrido, 2000;
glycemic, and neural protective effects (Ahn et al., 2008; Romero & Brenes, 2012). Briefly, 2.0 g olive oil sample was
Bertelli et al., 2002; Bu et al., 2007; Carluccio et al., 2003; treated with 2.0 mL (3 times) of DMF followed by washing with
Chandramohan, Pari, Rathinam, & Sheikh, 2015; Dinnella, hexane. N2 gas was fizzed into extract for the removal of
Minichino, D’Andrea, & Monteleone, 2007; Giovannini et al., remaining hexane. Extract was filtered through 0.22 lm pore
1999; Loru et al., 2009; Panossian et al., 2008; Panossian, size filter and introduced into the chromatograph. Separation
Wikman, Kaur, & Asea, 2009; Petroni et al., 1995; Puerta et al., of tyrosol was attained by using an elution gradient technique
2001; Salucci et al, 2015; Thibault et al., 2011). Keeping the (initial composition of 90% water and phosphoric acid at pH
broad spectrum of biological functions of tyrosol in mind, 3.0) and 10% MeOH. Concentration of MeOH was raised to
there is a necessity to gain more insight toward its 3D-geom- 30% over 10 min and it was continued for 20 min.
etry, structural characteristics, and bioactive mechanism. Subsequently, MeOH was lifted up to 40, 50, 60, 70, and 100%
Cyclooxygenase (COX), known as prostaglandin-endoperox- in 5 min internals at 1 mL/min flow rate and 35  C temperature.
ide synthase (PTGS), is a rate determining enzyme in the syn-
thesis of eicosanoids. It has two isoforms; COX-1 and COX-2,
2.2. Instrumentation
which differs mainly in their expression pattern (Funk, Funk,
Kennedy, Pong, & Fitzgerald, 1991; Hempel, Monick, & Crystallized tyrosol was dried completely preceding to elem-
Hunninghake, 1994). COX-1 is constitutively expressed, ental analysis, and checked by Elementar Vario EL III analyzer.
mainly obtained from cell lines of mammalian tissues, and The FT-IR/NMR spectra were recorded on Tensor 27, BRUKER
often regarded as a housekeeping enzyme. It is primarily FT-IR spectrometer, and Avance 500 Bruker Biospin Intl
involved in cell-to-cell signaling, tissue homeostasis, cytopro- 500 MHz, respectively. Melting point recorded on a JSGW
tection, kidney and platelet function, (Kelley et al., 1997) and apparatus and was uncorrected. X-ray data was collected by
also helps to maintain normal mucus lining of the stomach Bruker Kappa Apex four circle-CCD diffractometer having
(Dubois et al., 1998), whereas COX-2 is responsible for the graphite monochromated Mo Ka radiation (k ¼ 0.71070 Å).
formation of key biological mediators such as prostanoids. SAINT v7.34 and SADABS programs were used for Lorentz-
Earlier experimental studies confirm the role of COX-2 Polarization and empirical absorption corrections, respect-
enzyme in numerous inflammatory processes and different ively (SAINT, 2004; Sheldrick, 1996). Crystal structure of tyro-
carcinomas (Mestre et al., 1999). Fascinatingly, many sol was solved by using direct methods, while structure
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 3

solution, refinement and data output were accomplished 2.5. Molecular docking
with the SHELXTL program (Sheldrick, 1990, 2000). Hydrogen
Molecular docking of tyrosol, aspirin, naproxen, ibuprofen,
atoms were located in geometrically calculated positions
and celecoxib with aspirin-acetylated human COX-2 (PDB ID:
using riding model, while non-hydrogen atoms were enlight-
5F1A) receptor has been carried out using GLIDE v6.7
ened anisotropically. Images were produced in the crystal lat-
(Schrodinger, LLC, New York, 2015). Extra precision (XP)
tice with DIAMOND software (Brandenburg, 1999;
method was applied for docking purpose of all five com-
Klaus, 1999).
pounds into the defined binding cavity of receptor devoid of
constraint and positioned on the basis of docking score.
Glide internally generates different conformations of com-
2.3. DFT and statistical studies
pounds, which are then passed-on through a series of filters.
Geometry of tyrosol, aspirin, naproxen, ibuprofen, and cele- In XP docking, compounds exhibiting good scores for a
coxib were optimized at 6-311G (d,p) basis set with a hybrid range of interactions achieved by extensive sampling with
function B3LYP by Gaussian 09 (Becke, 1993; Frisch, 2009). advanced scoring and avoiding penalties are selected to
Conformational analysis was performed to find most stable eliminate the false positives. Finally, docking results are
conformation of tyrosol (Hockridge et al., 1999). Inputs for assessed on the basis of scoring function given by Glide
geometry optimization were given in the form of z-matrix score or Glide G-score, which can be represented as:
constructed by GaussView 5.0 (Goel & Kumar, 2018). GScore ¼ 0:065  VanderWaalsenergy þ 0:130
Structural parameters for optimized geometry like total
 Coulombenergy þ Lipo þ Hbond þ Metal þ BuryP
energy, dipole moment, bond lengths, bond angles, and
þ RotB þ Site
HOMO–LUMO energy have been computed at the same
basis set (Koopmans, 1934; Tsuneda & Hirao, 2014; Zhang & where, hydrophobic interactions are indicated as Lipo, metal
Musgrave, 2007). The values of correlation coefficient (R) binding by Metal, buried polar group penalty was repre-
were calculated for statistical validation between crystal sented as BuryP, penalty for freezing rotatable bonds was
structure and DFT optimizations data for bond lengths and shown by RotB and polar interactions existing in the active
angles (Kumar et al., 2015). site represented by Site. A comparative analysis of tyrosol
with well-known clinically marketed drugs (aspirin, naproxen,
ibuprofen, and celecoxib) was carried out on the basis of
2.4. Calculation of ADME and toxicity docking score and involved interacting residues of
active site.
In the course of development of a new pharmacologically
active molecule, in vitro assessment at an early stage using
ADMET (Absorption, Distribution, Metabolism, Excretion, and 2.5.1. Ligand preparation
Toxicity) and in vivo evaluation of pharmacodynamics param- Optimized 3D geometries of currently prescribed drugs for
eters offers a basis for identification of new molecular enti- clinical purpose along with tyrosol were converted into .pdb
ties which may serves as probable lead drug entrant with file format prior to molecular docking (Kumar & Garg, 2014).
suitable efficacy, metabolism, and pharmacokinetics. The Ligand processing followed by expansion of protonation and
ADME/TOX descriptors of a chemical entity allow the tautomeric states (7.0 þ 2.0 pH units) was done by LigPrep
researchers to comprehend the necessary safety and efficacy along with Epik module (Shaw, Yibing, John, Michael, & Ron,
information for regulatory approval. QikProp v4.4, 2005). For each ligand, a total of five stereoisomers with min-
(Schro€dinger, LLC, New York, 2015) was used to compute the imum energy conformations were produced, and the lowest
ADME descriptors like molecular weight (MW), total polar energy conformer with accurate chirality was selected for fur-
surface area, dipole moment, solvent accessible surface area, ther study.
hydrophobic and hydrophilic component of SASA, MDCK
(Madin-Darby Canine Kidney), Caco2-cell (heterogeneous 2.5.2. Preparation of target receptor and active site
human epithelial colorectal adenocarcinoma cell lines), pre- identification
dicted polarizability, predicted hexadecane/gas partition The 3D X-ray crystal structure of aspirin-acetylated human
coefficient, predicted octanol/gas partition coefficient, pre- COX-2 at a resolution of 2.38 Å was obtained from the RCSB-
dicted water/gas partition coefficient, predicted octanol/ PDB (Protein Data Bank; http://www.rcsb.org/) with PDB ID:
water partition coefficient, predicted aqueous solubility, con- 5F1A (Lucido, Orlando, Vecchio, & Malkowski, 2016). The pro-
formation-independent predicted aqueous solubility, etc. for tein structure of salicylate bound human COX-2 complex was
tyrosol, aspirin, naproxen, ibuprofen, and celecoxib. A dis- further processed and analyzed to assess conformational sta-
tinctive stress has been laid on the prediction of IC50 value bility. Steric clashes, hetero-atoms and non-essential water
for blockage of HERG Kþ channels for all the screened dual molecules were scraped off, as well as hydrogen bond and
inhibitors to check their potency. Toxicity prediction was car- proper bond orders were assigned to crystal structure with
ried out by using Osiris Property Explorer (OPE; a web-based the help of Maestro Protein Preparation Wizard Workflow
tool) of five docked compounds (http://www.organic-chemis- program suite v10.2, Schro €dinger, LLC, New York, NY, 2015,
try.org/prog/peo/). and the default parameters were used (Sastry, Adzhigirey,
4 T. C. YADAV ET AL.

Day, Annabhimoju, & Sherman, 2013). SiteMap, v3.4, was created to confirm the complete solvent coverage of
Schro€dinger, LLC, New York, NY, 2015 was utilized to ascer- entire surface 5F1A-tyrosol system creating a box volume of
tain the active site of 5F1A protein structure (Halgren, 2009), 613,915 Å. Net charge on the system was neutralized and
residues lying within the range of 5 Å in vicinity of Ser530 balanced by adding two Naþ ions. To provide the real time
were defined as active site residues and the same has also environment for simulation purpose, 0.15 M NaCl was also
been substantiated from the literature as well around which added to stabilize the system (Bowers et al., 2006).
the docking receptor grid was generated. The preparation
and processing of receptor provides all the necessary envir-
onmental settings and validates the protein structure in-silico 2.6.2. Molecular dynamics
which brings the structure to mimic the in vitro surrounding System building of 5F1A-tyrosol complex was followed by
condition. The ingrained default constraints were utilized for molecular dynamics simulation of the complex. Periodic
cutoff, neutralization, scaling, and dimension of the binding boundary conditions were applied and all the bad contacts
pocket. The electrostatic grid box was generated around the of residues were removed by energy minimization with the
target receptor via Glide v6.6, Schro €dinger, LLC, New York, help of hybrid method steepest decent and the limited-
NY, 2015 module (Halgren et al., 2004). The dimensions of memory Broyden  Fletcher  Goldfarb  Shanno (LBFGS)
the receptor grid were kept as: Inner box: X ¼ 10, Y ¼ 10, algorithms (Guo et al., 2010). Prior to equilibration and final
Z ¼ 10, Outer box: X ¼ 30, Y ¼ 30, Z ¼ 30 and Grid center MD production run, the system was minimized by applying
X ¼ 42.73463, Y ¼ 26.3990, Z ¼ 240.0050. 1 kcal/mol/Å of convergence threshold followed by 2000 iter-
ations and pre-equilibration was carried out with the help of
ingrained relaxation protocol employed in Desmond. The
2.6. Molecular dynamics simulations complete system was exposed to 300 K and 1 bar pressure
Structural and functional veracity of proteins owes to its for 20 ns at NPT ensemble of MD simulation with recording
molecular function which is ultimately influenced by its con- interval maintained at 10 ps for total energy (kcal mol). The
formational stability and integrity, can be studied by using structural variations along with dynamic behavior were
atomic level perturbation in simulated real time environment examined by determining the stability of protein via calcula-
via MD (Molecular dynamics) simulation. In our present tion of root mean square deviation (RMSD) – defines the sta-
investigation, MD simulation has been carried out to assess bility of protein, potential energy and RMSF determining the
the conformational stability of protein-ligand complex by thermal movement of an individual residue and their fluctu-
using Desmond tool (Bowers et al., 2006, Maestro-Desmond ation, its scoring guarantees the overall alignment of the sys-
Interoperability Tools, version 4.1, Schro€dinger, New York, tem in surrounding and observance of large structural
2015). MD simulations analysis for tyrosol in complex with changes and stable nature of the receptor–ligand complex
aspirin-acetylated human COX-2 receptor was evaluated on can be analyzed (Raj, Kumar, Gupta, & Varadwaj, 2015).
the basis of binding affinity, number of H-bonds, ADME and
toxicity parameters. Stability and conformational behavior 3. Results and discussions
was assessed by analyzing the simulation quality plots, pro-
tein–ligand RMSD, protein–ligand contact graph and root 3.1. Elemental analysis, FT-IR, and FT-NMR studies
mean square fluctuation (RMSF) plot throughout the
Purified tyrosol was further processed for its structural char-
20 ns simulation.
acterization. Melting point of purified tyrosol was recorded
as 91–93  C. Elemental analysis calculated for tyrosol
2.6.1. System building (C8H10O2, 138.16) was C: 69.54% and H: 7.30%, while experi-
The receptor–ligand complex system was processed and side mentally found C: 68.21% and H: 7.23% for the same.
chains of different residues are refined, followed by the strain According to the FT-IR spectrum (KBr, Figure S1), tyrosol
minimization using protein preparation interface of Maestro exhibits the transmittance peaks at 3426 cm1, which is due
(Schro€dinger, LLC, New York, NY, 2015) prior to MD simula- to asymmetric stretching vibrations of phenolic as well as
tions. In the course of complex processing, all the missing alcoholic O–H groups. In addition, stretching band at
atoms of 5F1A-tyrosol system were added and processed 1262 cm1, this reflects the presence of C–O. The weak
complex was then introduced to the system builder module absorption of 2930 cm1 has been designated to mas(C–H)
of Desmond, encompassing ions and solvation tabs. stretching vibration, C ¼ C stretching vibrations occurs at
Undesirable water molecules were scraped off from the crys- 1632 and 1563 cm1. The bands in the region of
tal structure of complex and TIP3P water model system was 950–550 cm1, represent strong C–H bending transmittance.
1
chosen for the solvation of 5F1A-tyrosol complex by means H-NMR (500 MHz, DMSO-d6, ppm, Figure S2) d: 2.49-2.61 (dd,
of solvation panel (Jorgensen, Chandrasekhar, Madura, 2H, –CH2, J ¼ 6 Hz), 3.49–3.54 (dd, 2H, –CH2, J ¼ 2.5 Hz),
Impey, & Klein, 1983). Optimized potentials for liquid simula- 4.56–4.59 (dd, 1H, Alcohol, J ¼ 1.5 Hz), 6.64–6.66 (d, 2H,
tions (OPLS_2005) force-field was utilized for amino acid Aromatic, J ¼ 1 Hz), 6.97–6.99 (d, 2H, Aromatic, J ¼ 6.1 Hz),
interaction (Shivakumar et al., 2010), encompassing SPC 9.168 (s, 1H, –OH exch). The presence of exchangeable pro-
(Simple point charge) solvent archetype for docked complex tons (phenolic and carboxylic) in tyrosol molecule has been
and orthorhombic water box of dimension (10  10  10 Å) confirmed by adding a few drops of D2O in NMR sample
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 5

Figure 1. Ball and stick models of tyrosol’s geometry (a) solid state single crystal, (b) DFT optimized, and (c) 3D packing showing various O–HO non-covalent
interactions. Color code: C, gray/black; H, orange/white; O, red.

tube. 13C-NMR (125 MHz, DMSO-d6, ppm, Figure S3) d: 38.69, Table 1. Crystallographic data collection and refinement parameters for iso-
lated tyrosol.
63.06, 115.39, 130.14 and 155.88.
Empirical formula C8H10O2
Color White
3.2. Crystal structure Formula weight (g mol1) 138.16
Crystal system Monoclinic
Tyrosol crystallizes in monoclinic system with space group P Space group P 21
a (Å) 5.3179(3)
21 (Figure 1(a)). Refinement parameters, crystallographic b (Å) 8.3579(4)
data, bond lengths, bond angles, isotropic and anisotropic c (Å) 8.1495(4)
displacement parameters are provided in Table 1 and Tables a ( ) 90.0
b ( ) 94.705(4)
S1–S3. The single crystal X-ray structure analysis reveals that c ( ) 90.0
one tyrosol molecule is coordinated to four other tyrosol V (Å3) 361.00(3)
molecules through O–HO non-covalent interactions Z 2
DCalc (g/cm3) 1.271
(O1–H1O2, 1.889(3) Å; O2–H2AO1, 1.992(3) Å), and associ- m (Mo Ka) mm1) 0.090
ated to create a two dimensional ladder like perspective Crystal size (mm) 0.32  0.26  0.19
hmax ( ) 2.508–28.306
view (Figure 1(c)).
Reflections measured 1727
Observed reflections 1369
Data/restraints/parameters 1727/1/93
3.3. Geometry optimization, FMOs and Final R indices[I > 2(I)}]
statistical analysis R1a 0.0406
wR2b 0.0978
Conformational analysis for tyrosol (Figure S4) has been car- CCDC number 1584517
P P P P
ried out, and the lowest energy structure (Figure S4e) was
a
R1 ¼ kFojjFck = jFoj, bwR2 ¼ { [w (Fo2  Fc2) 2] / w (Fo2)2.
processed for further theoretical calculation and comparison
with X-ray crystal structure (Hockridge et al., 1999). It showed diffraction (Figure 1(a)). Computation of frontier molecular
the positive harmonic vibrational frequencies, indicated glo- orbitals (FMOs), which plays a major role in the prediction of
bal minimum on the potential energy surface. It also showed electronic and optical properties of a molecule, has been
a resemblance with the structure obtained by single crystal achieved by HOMO (highest occupied molecular orbital) and
6 T. C. YADAV ET AL.

Table 2. Molecular properties calculated for tyrosol at DFT/B3LYP/6-311G

Lipinski, Lombardo, Dominy, & Feeney, 1997; Lombardo,
basis set.
Gifford, & Shalaeva, 2003; Navia & Chaturvedi, 1996; Yadav
Parameters (unit) Computed values et al., 2018). ADMET calculation using QikProp program of
Total energy (eV) 12556.2849 mentioned drugs along with tyrosol has been given in Table
Dipole moment (Debye) 2.4798
EHOMO (eV) 6.0851 3. The pharmacokinetic screening of tyrosol molecule was
ELUMO (eV) 0.3505 found in the satisfactory parameters of Lipinski’s rule of five.
EHOMO – ELUMO 5.7343
Chemical potential (eV) 3.2175
Octanol–water partition coefficient (QP logP (o/w)), conform-
Chemical hardness (eV) 3.2175 ation-independent aqueous solubility (CIQP log S), and the
Chemical softness (eV)1 0.1554 predicted aqueous solubility (QP log S), reported as critical
Electronegativity (eV) 2.7898
Electrophilic index (eV) 1.2095
factors in determining absorption and distribution properties
of drug inside the body was found in the suitable range,
whereas cell permeability (QPPCaco), a vital feature influenc-
LUMO (lowest unoccupied molecular orbital) analysis. HOMO ing the access (to biological membranes) along with metab-
is known as the ability of a molecule to donate an electron, olism of drugs and the percentage human oral absorptions
while LUMO corresponds to the ability to obtain an electron. values were observed in acceptable range. General rule of
The energy gap between HOMO and LUMO is used to calcu- thumb says, greater the bioactivity score higher will be the
late the optical and electronic properties viz. chemical hard- possibility of the molecule to be active. Compounds possess-
ness, chemical softness, kinetic stability, and optical ing bioactivity score >0.00 manifest substantial biological
polarizability of a molecule (Goel & Kumar, 2017; Pearson, actions, while score ranging from -0.50 to 0.00 were of mod-
1988; Vasilescu & Adrian-Scotto, 2010). The values of these erately active compounds, and score found below -0.50 were
parameters are provided in Table 2. As shown in Figure 2(a), assumed to be inactive (Verma, 2012). The predicted bioac-
the pictorial representations of isodensity plots energy transi- tivities viz. as GPCR (G-protein coupled receptor), LI (ligand
tion for tyrosol clearly confirm that in ground state (HOMO), gated ion channel modulator), KI (kinase inhibitor), NRL
the density is distributed throughout the molecule mainly at (nuclear receptor ligand), PI (protease inhibitor), and EI
benzene ring, while in case of LUMO the density is con- (enzyme inhibitor) computed by using MOLINSPIRATION
densed around the upper and lower four carbon atoms of server and have been shown in Table 3.
benzene ring. The phenol group also lost the density from
HOMO!LUMO indicating its role in biological properties of
tyrosol. Most stable geometry of tyrosol (Figure 1(b)) con- 3.5. Interaction study of the protein–ligand complex
tains 37 occupied and 73 unoccupied virtual molecular orbi- All the reference compounds along with tyrosol are listed in
tals, respectively out of 110 alpha molecular orbitals. Table 4 with their docking score, Glide g-score, and Glide e-
Comparisons between simulated and experimentally model. These ligands are ranked based on their docking
obtained bond lengths and bond angles data have been score. The 2D receptor–ligand interactions of the mentioned
summarized in Table S1. These data were statistically ana- compounds are shown below in Figure 3, as protein–ligand
lyzed for its mathematical validation, and the values of cor- interaction diagram. Tyrosol shows docking score of
relation coefficient (R) for bond lengths and bond angles 5.528 kcal/mole and forms hydrogen bond with backbone
were found to be 0.988 and 0.984, respectively, in curve fit- of Met522 amino acid residue, polar interaction with Ser530,
ting analysis (Figure 2(b), (c)). Commenting on these findings, hydrophobic interaction with Val349, Leu352, Ala527, Tyr348,
we inferred that simulated values for structural parameters Tyr385, Phe381, Leu384, Gly526, Trp387, Phe518, and Val523
lie statistically closed to the experimentally obtained results amino acid residues. Celecoxib has docking score of
and outcome in these cases to be worthy in respect to their 7.145 kcal/mole and forms H-bond with backbone of
statistical significance. Phe518 and Leu352 amino acid residue, polar interaction
with Ser530, Ser353, Gln192 and His90, p–p interaction with
3.4. ADMET analysis positively charged residue Arg120, positive charge interaction
with Arg513 and hydrophobic interaction with Val349,
Most of the lead molecules often turn out to be unsuccessful Val523, Val116, Leu359, Leu384, Leu531, Ala527, Ala516,
before clinical solicitation due to their poor pharmacokinetic Tyr348, Tyr355, Tyr385, Phe381, Gly526, Trp387, and Met522.
properties (Darvas et al., 2002). Therefore, it is extremely sig- Aspirin, the top scoring compound exhibits docking score
nificant to design and develop such kind of lead molecules 7.499 kcal/mol and form two hydrogen bonds with side
which can offer better gut absorption, safely reach the target chains of Ser530 and Tyr385 amino acid residues, polar inter-
site and should not transform into toxic metabolite before it action with positively charged Ser530 residue, hydrophobic inter-
reaches to the desired site of action followed by its safe actions with residues Val349, Tyr348, Phe205, Leu384, Phe381,
elimination prior to its accumulation inside the body result- Met522, Leu352, Ala527, Trp387, Phe518, Val523 and Gly526.
ing in harmful side effects. Evaluation of pharmacokinetic Ibuprofen shows the value of docking score as 6.636 kcal/mole
factors in initial stages of designing and development of and it forms two H-bonds; one with side chain of Tyr355, while
novel lead candidate using computational methods is exten- other with side chain of Arg120. Polar interactions were also
sively prevalent nowadays (Gleeson, Hersey, & Hannongbua, noticed with Ser530 as well as Ser353, and hydrophobic interac-
2011; Hodgson, 2001; Kumar, Goel, Yadav, & Pruthi, 2017; tions with Leu359, Ala527, Val349, Leu384, Trp381, Gly526,
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 7

Figure 2. FMOs diagram (a) correlation graph between calculated and experimental bond lengths, (b) and bond angles, (c) for tyrosol.

Tyr385, Met522, Phe518, Leu352, Val523, Leu531, and Val116 depth analysis of results observed during MD simulation to
amino acid residues. Naproxen shows 7.352 kcal/mol docking evaluate the stability of tyrosol with 5F1A receptor (Aspirin-
score and form H-bond with side chain of Ser530, polar inter- Acetylated Human COX-2, Docking score = 5.528, GLIDE
action with Ser353, hydrophobic interactions with Tyr355, g-score = 5.528 and GLIDE e-model = 6.940) complex
Leu359, Leu531 Val349, Val523, Ala527, Trp583, Tyr385, Trp548, obtained from docking. Desmond is an extremely efficient
Phe381, Met522, Leu384, Phe518, Leu352, Val116, Val525, Gly526 high performance MD simulation package available to carry
amino acid residues and positive charge interaction with residue out simulations for bio-molecular systems employing parallel
Arg120. We have also calculated the change in the torsion angles algorithms and numerical techniques to accomplish accuracy.
for tyrosol in free state as well as complex with COX-2 (Table S4)
and noticed almost same values, indicating its structure stability
during the interaction process. 3.6.1. Simulation quality plot analysis
During the course of 20 ns simulation process, system behav-
ior of protein–ligand complex was mainly observed via tem-
3.6. Dynamics simulations study perature (T), pressure (P), volume (V), potential energy (E_p),
A variety of fundamental biological features including con- and total energy (E) plots as shown in Figure 5. Volume of
formational behavior of proteins (Balasco, Barone, & system ranges from 590,000 to 575,000 nm3 and average
Vitagliano, 2015), unraveling of key structural insights as well volume of the system was maintained 576239.398 nm3
as designing and development of novel molecules (Arfeen, throughout the simulation. P (1000 to þ300 bar) and T
Patel, Khan, & Bharatam, 2015) can be studied by using (300 K) of the system were also nearly constant during the
molecular dynamic simulations via atomic-level perturbation. simulation (Figure 4). The behavior of potential energy (E_p)
A molecular dynamics time step consists of a computation- is consistent (190,500 to 192,500 kcal/mol), while total
ally intensive force calculation for each atom of a chemical energy (E) increases suddenly from -170,000 to -155,000 kcal/
system, following less intensive integration step which aids mol and then gradually becomes constant as the time of the
in movement and positioning of atoms according to classical simulation increases till it reaches -150,000 kcal/mol. Plot ana-
Newtonian laws of motion. Herein, we are displaying in- lysis infers that the protein ligand complex shows consistent
8 T. C. YADAV ET AL.

Table 3. ADMET profiling calculated for tyrosol, aspirin, naproxen, ibuprofen, and celecoxib.
ADME descriptors Aspirin Ibuprofen Naproxen Tyrosol Celecoxib Range
MW 190.239 214.347 242.358 144.213 381.372 500
SASA 400.502 488.846 489.334 370.974 621.969 300–1000
FOSA 287.161 399.128 405.817 265.809 89.007 0–750
FISA 113.341 89.718 83.517 105.165 151.466 7–330
Volume 667.639 840.079 857.861 589.253 1079.981 500–2000
Donor HB 3 2 2 2 2 0–6
Acceptor HB 6.8 3.4 5.1 3.4 5.5 2–20
Glob 0.922405 0.880792 0.892286 0.916268 0.818478 0.75–0.95
QP polrz 16.712 23.613 24.967 14.86 38.233 13–70
QPlogPC16 6.159 7.174 7.374 5.001 10.758 4–18
QPlogPoct 12.584 11.784 13.134 9.063 19.79 8–35
QPlogPw 11.222 6.46 8.315 6.723 11.941 4–45
QPlogPo/w 0.04 2.336 1.884 0.635 3.346 2–6.5
CIQPlogS 0.831 2.157 2.124 0.935 5.728 6.5–0.5
QPlogKHSA 0.796 0.051 0.198 0.558 0.37 1.5–1.5
QPlogHERG 2.788 3.461 3.275 2.887 5.693 10–(5)
QPPCaco 833.853 1396.712 1599.228 496.823 362.702 25–500
QPlogBB 0.686 0.569 0.437 0.513 0.758 3.0–1.2
QPPMDCK 406.497 709.894 821.775 493.011 799.513 <25 poor
>500 great
QPlogKp 3.032 2.597 2.579 3.074 3.215 8.0–0.1
% Human Oral Absorption 78.991 96.912 95.321 84.333 92.349 >80 high
<25 low
Tumorigenic Yes No No No No NA
Mutagenic Yes Yes Yes No No NA
Irritant No No No No No NA
Reproductive effect Yes Yes Yes No No NA
Drug-likeness 1.63 0.08 0.36 4.13 8.10 NA
Drug score 0.48 0.31 0.26 0.32 0.36 NA
GPCR ligand 0.76 0.17 0.11 0.80 0.06 NA
Ion channel modulator 0.32 0.01 0.06 0.13 0.27 NA
Kinase inhibitor 1.06 0.72 0.38 0.87 0.01 NA
Nuclear receptor ligand 0.44 0.05 0.14 0.65 0.28 NA
Protease inhibitor 0.82 0.21 0.26 0.97 0.06 NA
Enzyme inhibitor 0.28 0.12 0.15 0.21 0.17 NA

behavior into the system, and can carry out the inhibition of Table 4. Docking and GLIDE score of tested ligands with COX-2 (PDB
human COX-2 by tyrosol. ID: 5F1A).
Ligand name Docking score GLIDE g-score GLIDE e-model
Tyrosol 5.528 5.528 6.940
3.6.2. RMSD and RMSF analysis Asprin 7.499 7.499 0.863
Functional veracity of protein–ligand complex is governed by Naproxen 7.352 7.352 32.263
Celecoxib 7.145 7.146 47.630
their folding and movement of backbone within the system. Ibuprofen 6.636 6.366 14.466
RMSD analysis of receptor offers insight of the structural
deviations ensuing in Ca, backbone, and side chain residues
2.238; median - 2.252), Celecoxib (mean - 2.072; median -
of the target receptor throughout the 20 ns simulation
2.159), and Tyrosol (mean -1.878; median - 1.902) were
(Shivakumar et al., 2010). Calculation of protein_C-alpha,
calculated. The RMSD of side chain atoms with Aspirin (mean
backbone, side-chain, and ligand_RMSD was carried out dur-
- 2.889; median - 3.040), Ibuprofen (mean - 2.536; median -
ing the progression of simulation trajectory of 20 ns in order
2.601), Naproxen (mean - 3.145; median - 3.180), Celecoxib
to compute the mean variation in shift of a group of atoms
for a specific frame with respect to a reference frame. The (mean - 3.014; median - 3.102), and Tyrosol (mean - 2.758;
MD analysis of 5F1A (COX-2) receptor with ligand (Aspirin, median - 2.798) were also calculated. The ligand RMSD of
Ibuprofen, Naproxen, Celecoxib, and Tyrosol) complexes dur- Aspirin (mean - 0.293; median - 0.295), Ibuprofen (mean -
ing simulation process was monitored via RMSD profiles as 0.383; median - 0.388), Naproxen (mean - 0.160; median -
calculated from the atomic fluctuations from MD trajectories. 0.154), Celecoxib (mean - 0.755; median - 0.796), and Tyrosol
All receptor frames were first superimposed on the reference (mean - 0.141; median - 0.138) showed a significant observa-
frames of 5F1A receptor’s C-alpha atoms and side chain tions. Fluctuation in RMSD of the ligand signifies the stability
atoms followed by RMSD calculation on the basis of selected of ligand with respect to the receptor between its active site
atoms as depicted in Figure 5. The RMSD of C-alpha atoms cleft. The RMSD plot of the 5F1A-tyrosol complex depicted in
with Aspirin (mean - 1.920; median - 2.030), Ibuprofen (mean Figure 5 manifests early conformational oscillations occurring
- 1.583; median - 1.611), Naproxen (mean - 2.211; median - within the receptor complex system till 15 ns, which later on
2.226), Celecoxib (mean - 2.207; median - 2.107), and Tyrosol became stabilized and converged afterwards in production
(mean - 1.678; median - 1.878) were computed. The RMSD of phase. RMSD backbone and RMSD_Ca plot indicate steady
backbone atoms with Aspirin (mean - 1.948; median - 2.050), behavior, and also shows deviations initially from 1 to 10 Å
Ibuprofen (mean - 1.619; median - 1.647), Naproxen (mean - and further converged in the succeeding 10 ns signifying
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 9

Figure 3. Molecular docking interactions of (a) Aspirin (b) Ibuprofen (c) Naproxen (d) Celecoxib, and (e) Tyrosol with COX-2 (PDB ID 5F1A).

that the binding affinity and ligands position were main- ligand from the receptor pocket during simulation, but
tained within receptor pocket (Figure 5). Similarly, big fluctu- herein, the ligand illustrates a consistent and steady perform-
ation in RMSD of ligand plot implies away movement of ance throughout entire 20 ns simulation process (range
10 T. C. YADAV ET AL.

Figure 4. Simulation quality analysis plot of volume, temperature, pressure, potential energy, and total energy during the 20 ns simulations.

Figure 5. RMSD plots of ligand, C-alpha, backbone, and side chain for 20 ns.

0.00–0.274 Å; mean 0.141; median 0.138), signifying the protein with respect to the reference during the entire simu-
5F1A-Tyrosol complex stability within the system. While lation. As shown in Figure 6, The RMSF calculation of 5F1A
higher variation in RMSD plot of side chain has been (COX-2) with Aspirin, Ibuprofen, Naproxen, Celecoxib com-
observed initially from 1 to 15 Å and after 15 ns got con- plexes depicts higher fluctuation in the side chain amino
verged became stable showing consistent behavior steadily acid residues as compared to Tyrosol. Consequently, the fluc-
till the termination of simulation process. tuation of core binding site amino acid residue of receptor
RMSF parameter determines the structural and conform- with tyrosol belonging to active site cavity viz., Ser530,
ational variations occurring locally along the receptor. The Tyr385, Leu359, Ala527, Val349, Leu384, Trp381, Gly526,
RMSF is used to analyze the deviation of residues of the Tyr385, Met522, Phe518, Leu352, Val523, Leu531, Val116 and
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 11

Figure 6. RMSF plot of amino acid residues C-alpha (red color) and backbone (blue color) of COX-2 protein with respect to fluctuation (Å) during 20 ns simulation.

Arg120 are intacted residues, and showed the consistency 3.6.4. Interaction fraction plot of 5F1A-tyrosol complex
throughout the 20 ns simulation. The stability trend for the during simulation
receptor-ligand complexes were found in decreasing order; 5F1A receptor interactions with the ligand tyrosol were
Tyrosol > Aspirin > Celecoxib > Ibuprofen > Naproxen. observed during the course of 20 ns simulation process and
Active site amino acid residues manifest comparatively low a stacked bar chart is also plotted for the same that displays
side chain and backbone fluctuations as compared to other the interaction fraction of the amino acid residues compris-
residues. The small range RMSFs and RMSDs reflects insignifi- ing active site of 5F1A receptor as shown in Figure 8. Amino
cant structural rearrangements in the docking complex dur- acid residues Leu352, Leu384, Tyr385, Trp387, and Ser530
ing 20 ns simulation. hydrophobic groove of COX channel of 5F1A receptor are
involved in the regulation of the activity of the protein. Out
of them, Leu384, Tyr385, Trp387, and Ser530 amino acid resi-
3.6.3. Energy measurement and variation analysis
dues were found to be more promising residues for the cata-
The observed average total energy for 5F1A (COX-2) complex
lytic activity of this receptor as these have interaction
with compounds aspirin, ibuprofen, naproxen, celecoxib and
fraction values of 0.9, 0.8, 0.95 and 0.8, respectively, and they
tyrosol were -153,463 kcal/mol, -153,578 kcal/mol, also were involved in the formation of H-bonds with ligand
-153,444 kcal/mol, -151,008 kcal/mol, and 152,670 kcal/mol after 20 ns simulation process. Leu352, Phe518, and Val523
along with a standard deviation of 1472.459 kcal/mol, amino acid residues were also observed in the creation of
1437.294 kcal/mol, 1481.000 kcal/mol, 1254.944 kcal/mol, and hydrophobic interaction and salt bridge, respectively, with
1462.00 kcal/mol, respectively, during the whole simulation the ligand after 20 ns simulation process.
process. Correspondingly, the recorded average potential
energy (a vital factor in simulation analysis) was
-192,252 kcal/mol for aspirin, -192,319 kcal/mol for ibuprofen, 3.6.5. Ligand property calculation during 20 ns simulation
-192,296 kcal/mol for naproxen, -188,428 kcal/mol for cele- To elucidate the stability of tyrosol in the 5F1A receptor, five
coxib and 192,276 kcal/mol for tyrosol with a standard properties were examined during the course of 20 ns simula-
deviation of 242.469 kcal/mol, 222.039 kcal/mol, 246.311 kcal/ tion: (1) Ligand RMSD—it is root mean square deviation of a
mol, 232.739 kcal/mol and 258.799 kcal/mol, respectively, dur- ligand with respect to reference conformation (normally, first
ing the whole simulation process. From the graphs, it is evi- frame is considered as reference at time t ¼ 0); (2) rGyr
dent that the system 5F1A-Tyrosol was more stable as (radius of gyration) calculates the ligand extendedness, corre-
compared to other systems and remains stable during the sponding to its principal moment of inertia; (3) MolSA
20 ns simulation process as there were not any significant (molecular surface area)—computation of molecular surface
variations happening in the conformation of the protein–li- with a probe of radius 1.4 Å; (5) SASA (solvent accessible sur-
gand complex as shown in Figure 7. Potential energy E_p face area)—it is considered as surface area accessible to
(kcal/mol) plot of the ligand-complex with respect to water molecules; (6) PSA (polar surface area); defined as the
time (ps). solvent accessible surface area of the molecule attributed
12 T. C. YADAV ET AL.

Figure 7. Potential energy (kcal/mol) plots of ligands-COX-2 complex with respect to time (ps).

Figure 8. Histogram for tyrosol binding with COX-2.

due to O2 and N2 atoms. From the plot, it is a clear evident carried out (Figure 10). Before MD simulation, only Meth522
that RMSD of ligand remains constant during the whole residue was found to be interacting via H-bond formation,
simulation process. Overall ligand RMSD was observed below polar interaction with Ser530 and hydrophobic interactions
0.5 Å. In the initial stages of simulation, it shows slight fluctu- with amino acid residues like Tyr348, Leu352, Tyr385, Trp387,
ation from 0.00 to 0.274 Å with an average of 0.141 Å, but Phe518 and Val523, etc. Post-simulation analysis revealed
later on maintains a constant RMSD throughout the simula- better conformational stability of ligand within active site of
tion process. This investigation manifests that the complex 5F1A receptor as the hydrophobic groove was occupied by
acquires a stable conformation during the entire simulation the hydroxy-ethyl moiety of ligand coordinated (H-bond)
process. Throughout the 20 ns simulation, radius of gyration predominantly by Leu384 and Trp387 via side chain and
of ligand ranges from 2.25 to 2.55 Å. MolSA, SASA, and from
backbone respectively, and a hydrophobic interaction with
the prior PSA plots exhibited the steady behavior of the lig-
Leu352. Phenol moiety of ligand interacts via side chain
and during complete process of simulation (Figure 9).
H-bond with Tyr385 and Ser530. From the plot, it is clear
that the conformational integrity of the complex was greatly
3.6.6. Post-simulation analysis of 5F1A-tyrosol complex improved after MD simulation as the ligand was kept tightly
Following MD simulation, the post simulation analyses of the intact within hydrophobic groove of COX channel of tar-
receptor (5F1A) as well as ligand (Tyrosol) interactions were get receptor.
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 13

Figure 9. Variation in the ligand properties with respect to time during the course of 20 ns simulation.

Figure 10. Post simulation interaction diagram of 5F1A-tyrosol after 20 ns simulation.

14 T. C. YADAV ET AL.

4. Conclusions thankful to MHRD and UGC (award letter no. 201718-PDFSS-2017-18-

UTT-17095) for SRF and Postdoctoral fellowship, respectively. Authors
Stimulation of COX-2 enzyme results in production of prosta- are highly thankful to Dr. Nidhi Goel (Department of Chemistry, BHU
glandins in swollen as well as erythematous tissues, and Varanasi) for solving the crystal structure of tyrosol. Tara Chand Yadav
and Naresh Kumar contributed equally in this research article.
therefore, has been acknowledged as a significant target to
design and develop more selective anti-inflammatory agents.
The use of conventional prescription including NSAIDs and Disclosure statement
selective inhibitor such as celecoxib, accounts for the treat-
No potential conflict of interest was reported by the authors.
ment of inflammation, have been reported to be accompany-
ing several unusual side effects, which chiefly includes
cardiovascular and GI (gastro-intestinal) toxicity. Even though, ORCID
the availability of structural information is amass. The charac-
Tara Chand Yadav http://orcid.org/0000-0003-4941-1946
teristics of the drug-enzyme interaction that are responsible
Naresh Kumar http://orcid.org/0000-0002-0551-3302
for determining the COX-2 selectivity of drugs are not always
clear in several condition. No uninterrupted ligand–protein
interactions have been observed with amino acid residues
that are exclusive to COX-2. Henceforth, despite of remark- References
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