BIOMAC-17258; No of Pages 10

International Journal of Biological Macromolecules xxx (xxxx) xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Cholesterol-lowering drugs the simvastatin and atorvastatin change the

protease activity of pepsin: An experimental and computational study
Mohsen Shahlaei a, Paria Zamani b, Negin Farhadian c, Fatemeh Balaei d, Mohabbat Ansari a, Sajad Moradi a,⁎
a
Nano Drug Delivery Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
b
Pharmaceutical Sciences Research Center, Health Institute, School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran
c
Substance Abuse Prevention Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
d
Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the effect of long-term use drugs of cholesterol-lowering atorvastatin and simvastatin on the activ-
Received 6 May 2020 ity and molecular structure of pepsin as important gastric enzyme was investigated by various experimental and
Received in revised form 11 November 2020 computational methods. Based on the results obtained from fluorescence experiments, both drugs can bond to
Accepted 12 November 2020
pepsin and quench the fluorescence intensity of protein through the static quenching mechanism. Also analysis
Available online xxxx
of the thermodynamic parameters of binding the drugs to pepsin showed that the main forces in the complex
Keywords:
formation for both are hydrophobic interactions and van der Waals forces. The effects of the drugs on the enzy-
Atorvastatin matic activity of pepsin were then investigated and results showed that in the presence of both drugs the catalytic
Simvastatin activity of the enzyme was significantly increased in lower (0.3–0.6 mM) concentrations however about the ator-
Pepsin vastatin, increasing the concentration (0.9 mM) decreased the protease activity of pepsin. Also as a result of the
Enzyme activity FTIR studies, it was found that binding of the drugs to protein did not significant alteration in the structure of the
Spectroscopy study protein. In order to obtain the atomic details of drug-protein interactions, the computational calculations were
Molecular dynamics simulation performed. The results in good agreement with those obtained from the experimental for interaction; confirm
that the drugs both are bind to a cleft near the active site of the protein without any change in the structure of
pepsin. Overall from the results obtained in this study, it can be concluded that both simvastatin and atorvastatin
can strongly bond to a location close to the active site of pepsin and the binding change the enzymatic activity of
protein.
© 2020 Published by Elsevier B.V.

1. Introduction located between the main domains of N terminal (Residues 1–172)

The statin drugs which mainly act as 3-hydroxy-3-methylglutaryl-
coenzyme A (HMG-CoA) reductase inhibitors are widely used for lower-
ing the cholesterol especially in patients who are in risk with cardiovas-
cular disorders [1]. Atorvastatin (ATC) [1,2] and Simvastatin (SIM) [3,4]
both are used for long duration times even by the rest of life and in the
case of such medications, their side effect is more challenging because
they are longer in interaction with macromolecules in the body.
Pepsin is a globular shape enzyme which is secreted from the main
cells in the stomach as a digestive protease and its main role is to con-
vert proteins in foods into smaller digestible peptides [5]. In fact, the
main function of this enzyme is to cleave amide bonds in proteins
next to the aromatic amino acids including tryptophan, tyrosine and
phenylalanine. This protein contains 326 amino acids and has a molec-
ular weight of about 35 kDa. The basic structure of this protein is com-
posed of the β-sheets and the catalytic principle part of the enzyme is
⁎ Corresponding author.
E-mail address: sajad.moradi@kums.ac.ir (S. Moradi).
and C terminal (Residues 173–326). The active site contains the amino
acids Asp-32 and Asp-215, which one of them become protonated and
another is deprotonated [6].
Previous studies have demonstrated that not only the acid but also
the pepsin containing gastric acid is the main influencing factor for pep-
tic ulcers [7–9]. In the healthy and hemostasis state, the amount of pep-
sin and mucus formation is in equilibrium and the rare of mucus
digestion by pepsin is equal to those of new mucus production by the
gastric cells. Both increasing in pepsin secretion and its activity can dis-
rupt this balance and causes the stomach to undergo peptic ulcer [9] and
more the long time imbalance more the ulcer formation and its severity.
This especially is important about the edible molecules which are in di-
rect interaction with the pepsin. Among them, the orally administrated
drugs in particular those which are used for long term periods are of the
important candidates to be causative agents for gastric ulcers.
Too many studies have conducted to investigate the interaction and
effects of several food and medicinal molecules on the structure and ac-
tivity of pepsin [10–16]. Zeng et al. have shown that the nobiletin sup-
press protease activity of the pepsin by binding to its binding site

https://doi.org/10.1016/j.ijbiomac.2020.11.095
0141-8130/© 2020 Published by Elsevier B.V.

Please cite this article as: M. Shahlaei, P. Zamani, N. Farhadian, et al., Cholesterol-lowering drugs the simvastatin and atorvastatin change the
protease activity of pepsin: ..., , https://doi.org/10.1016/j.ijbiomac.2020.11.095
M. Shahlaei, P. Zamani, N. Farhadian et al.
cavity electrostatically [17]. Moradi et al. comparatively investigated the
drug interaction of aspirin and warfarin with pepsin and have reported
that the drugs are bonded to protein by h-bonding/van der Waals and
hydrophobic forces respectively. Also the results indicated that the en-
zymatic activity of protein increased by aspirin and decreased by warfa-
rin [18]. In another study Ying et al. have demonstrated that the
curcumin binds to pepsin mainly by hydrophobic interactions and pro-
ceed some changes in its secondary structures [19]. On the other hand
because of the importance of interactions between astatines and biolog-
ical macromolecules several studies have conducted in order to investi-
gating these intermolecular phenomena. However they are all in
pharmaceutically level and there is no evidence for binding details of
the interaction [20,21]. For example Hockman et al. were studied the
bio interaction of ATC and SIM with P-glycoprotein (P-gp) in order to in-
vestigate the effect of drugs on the pump function of P-gp [22]. By the
now there is no study to investigate the molecular interactions and ef-
fect of stating drugs on pepsin.
Molecular modeling approaches by the help of mathematics enable
scientists to perform the study of phenomena in atomic and molecular
levels which some of them are not accessible in experimental situations
[23]. They also make the researches to be done as a time and cost-
effective manner [24]. In biology and medicine, these methods are
used for both predicting the experiments and screening or attain the
molecular details of an experiment for improvement of new more suc-
cessful ones [25–27].
The statin drugs are used for long terms and can interact with the
main stomach enzyme of pepsin. The long duration of drug-protein in-
teraction can severely affect the function of such biological macromole-
cules. As the enzymatic activity of pepsin is important in the matter of
gastric ulcers, it is necessary to investigating the interactions between
the long term administrated drugs with this protein. By the now there
is no report on the study of atorvastatin and simvastatin drugs on struc-
ture and function of the main stomach protease of pepsin. Considering
the aforementioned remarks, in this study, the interactions and effects
of tow long term used drugs of atorvastatin and simvastatin were inves-
tigated on the pepsin.
2. Materials and methods
2.1. Chemical materials and reagents
Pepsin from the porcine gastric mucosa (CAS Number 9001-75-6),
atorvastatin (CAS Number: 344423-98-9) and simvastatin (CAS Num-
ber: 79902-63-9), casein (CAS Number: 9000-71-9), and Folin's reagent
(CAS Number 521-24-4) were purchased from MERCK. To prepare an
enzyme solution with the required concentrations, 0.2 M sodium
citrate–citric acid buffer, pH 2.5 (including 0.1 M NaCl) was prepared.
The enzyme solution was freshly prepared immediately before use.
2.2. UV/visible absorption measurements
The UV/visible spectra of protein (100 μM) in the absence and pres-
ence of various concentrations of the drugs were taken and recorded by
using an Agilent 8453 UV spectrophotometer with quartz cuvettes of
1 cm width. The concentrations of each drug were 30, 70, 100, 130
and 150 μM and 3 minute time intervals were selected for each sample
recording. The titrations were done in such a way that the total addition
of protein solution was less than 0.3% of total volumes in order to
change in concentrations do not have significant effect on protein
absorption.
2.3. Recording and study of fluorescence spectra
For all the fluorescence studies, the spectra were recorded by using
PerkinElmer (LS55) fluorescence spectrofluorometer. The excitation
wavelength was set to 290 nm and the emissions from 300 to 500 nm
2
International Journal of Biological Macromolecules xxx (xxxx) xxx
were collected at three different temperatures of 290, 300, and 310 K.
A concentration of 1 μM for pepsin was titrated by different concentra-
tions of 100, 300, 500, 700 and 900 μM for each drug [28]. The Stern–
Volmer equation (Eq. (1)) and its modified version (Eq. (2)) were
then used to further investigating the quenching mechanism and ther-
modynamic parameters of interaction.
F0
¼ 1 þ K q τ0 ½Q  ¼ 1 þ K SV ½Q  ð2Þ
F
log fðF 0 −F Þ=F g ¼ log K b þ nlog½Q  ð3Þ
Here the value of F0 and F are respectively the fluorescence intensity
of macromolecule in the absence and presence of ligand, τ0 is the fluo-
rescence life-time of fluorophore [4] and [Q] is the concentration of
quencher. The terms KSV and Kq and Kb represent the Stern Volmer,
quenching and binding constants respectively [29].
2.4. Measuring protease activity
The effect of the drug interaction with pepsin on the enzymatic ac-
tivity of protein at pH 2.5 was investigated using the modified Anson's
method. In brief, 500 μM of pepsin in sodium citrate/citric acid buffer
followed by the addition of the various concentrations of the drugs.
The solutions were then incubated for 5 min. The freshly prepared solu-
tion of casein (100 μl 3% w=v) was added to the protein/drug solution
and incubated at 37 °C. After 15 min, the reaction was stopped by
adding 1 ml of Trichloroacetic acid (5% w=v TCA) and the aggregated pro-
tein was separated from the solution by centrifugation at 13,000 rpm for
10 min. It should be noted that the casein solution in the presence of
pepsin and TCA or divalent ions such as Ca++ is converted to two
main products of digested peptides and insoluble paracasein [30]. The
paracasein is then removed from the remnant supernatant which is
containing the solved digested peptides. Then, 100 μl of folin reagent
and 200 μl of sodium hydroxide solution (10 M) were added to 1 ml
of the collected supernatant. Finally, the absorbance of the solution at
625 nm was taken and the activities were calculated using the following
equation (Eq. (3)) [31].
PAU=ml ¼ A:V=A1 :t ð1Þ
where A is represents the absorbance of a sample, A1 is indicates the ab-
sorbance of 1 mEq of tyrosine (1620), V and t refer the sample volume
and reaction time, respectively.
2.5. FTIR spectroscopy studies
FTIR spectra of free pepsin (APO state) and in the presence of drugs
in the ratio of 1:5 were recorded using a Shimadzu IR2000 spectropho-
tometer in the range of 1200–1800 CM−1 and resolution of 8 CM−1 by
100 scans [32].
2.6. Molecular modeling studies
Computational studies are used to investigate the biological phe-
nomenon in molecular levels as in silico manner [33,34]. At the first
step in order to predict the resulting protein-ligand complex, the molec-
ular docking studies were performed using the autodock package [35].
Initially, the 3D structure of pepsin was obtained from the protein
data bank (PDB ID; 1PSO) [36]. The molecular structures of atorvastatin
and simvastatin were prepared and optimized in Avogadro software
(https://avogadro.cc). The structures containing atomic partial changes
and atom typing for the autodock force field were obtained by MGLtools
molecular package. Also, all bonds for the drugs were considered as ac-
tive [37]. Using the Autogrid4, energetic maps for atom types related to
each ligand were calculated in a search space consist of 126 point by the
grade spacing of 0.47 nm in all directions for which all parts of the
M. Shahlaei, P. Zamani, N. Farhadian et al.
protein are accessible to interaction sites. Drug binding to pepsin was
performed using Autodock4 under the Lamarckian genetic algorithm
for 250 runs [38]. In the next step, the interaction between pepsin and
each drug was dynamically investigated using molecular dynamics
(MD) simulations. Low-energy drug-protein complexes in the biggest
cluster were selected as initial structures for further MD analysis. At
first, the topology information for the drugs was prepared in the Auto-
mated Topology Builder (ATB) server [39]. The simulations were per-
formed by using the gromos 53a6 force field of the GROMACS package
(version 2018). A SPC/E model of water was applied to filling the simu-
lation box fallowing by neutralization of the systems by the appropriate
amount of Na or Cl ions [40]. The energy of the system was minimized in
the steepest descend method. By applying NVT and NPT ensembles, the
temperature and pressure were coupled in 310 K and 1 bar respectively
using a v-rescale thermostat and Parinello-Rahman barostat [41]. Fi-
nally, a 50 ns of MD simulations were done on all systems using the
leap-frog algorithm [42].
3. Results and discussion
3.1. UV–vis absorption spectra of pepsin-ligand complexes
The most accessible experimental technique to check the probability
of interactions between the macromolecules and the ligands is the UV–
vis spectroscopy [31]. In this regards the changes in the absorption spec-
trum of pepsin in the presence of atorvastatin and simvastatin are
shown in Fig. 1. As can be seen, the absorption intensity in the presence
of both drugs has decreased, which could indicate the interaction be-
tween pepsin and ligands. The figure shows that in the presence of sim-
vastatin (Fig. 1A), the decrease in the intensity of protein absorption is
more intense which can imply either more potent interaction or differ-
ences in interaction location of simvastatin (Fig. 1B). As it is mentioned
in methods, the maximum values of titrated drug solutions were less
than 0.3% of total volume in order to eliminate the effect of change in
protein concentration. Also the time intervals were set to the time
3 min in which there were no longer change in UV absorption. Hence
it can be concluded that the decrement in absorption curves is related
to binding of the drugs to the protein.
Fig. 1. UV spectroscopy of pepsin (blue line) in the presence of different concentrations 100 (red line), 300 (green line), 500 (violet line), 700 (light blue line) and 900 (orange line) μM of
A) simvastatin and B) atorvastatin.
3
International Journal of Biological Macromolecules xxx (xxxx) xxx
3.2. Fluorescence spectroscopy
In order to obtain more comprehensive information on the nature
and mechanism of interactions between pepsin and drugs along with
determining the thermodynamic parameters of interactions the fluores-
cence spectroscopy methods were used [43]. To perform the experi-
ments, the protein solution was titrated with different concentrations
of each drug at different temperatures. The spectra were then recorded
and shown in Fig. 2. These results illustrated that the intensity of pepsin
fluorescence is decreased and this can be a sign for protein interaction
with drugs. This interaction can be either stable complex formation or
momentary collision. To examine this, it is necessary to evaluate the
mechanism of fluorescence quenching of protein by using Stern-
Volmer equation (Eq. (2)).
Further calculating of the binding constant and the number of bind-
ing sites of ligand on the protein are also possible by using another well-
known modified Stern Volmer equation (Eq. (3)) [44].
The final results of the calculations are provided in Fig. 3 and Table 1.
As can be seen in Fig. 3, the slope of the linear plot of florescence
quenching against concentration of quencher is decreased by increasing
in the temperature. In the other words by increasing in temperature the
quenching effect of drugs is decreased which can be a sign for
destabilizing of protein-drug complexes. This is clearly a sign for static
quenching which is a result for the formation of a stable complex be-
tween protein and drugs. However, the slope of the curves related to
the protein in complex with simvastatin is much higher than those of
atorvastatin which can indicate more potent binding of this drug to pep-
sin, the slope depression about atorvastatin is less than those about SIM.
This can be because of more hydrophobicity of ATC than SIM that causes
some hydrophobic interactions with protein and makes some stability
in the complex in higher temperatures. Also from the results reported
in Table 1, it is observed that in the lowest temperatures the number
of binding sites and as a result binding constant for atorvastatin is higher
than those for simvastatin. Another method for determining the
quenching mechanism is using the value of quenching constant in
which according to previous reports, if it is greater than the value of
scattering collision quenching constant (2.0 × 1010 mol−1 s−1) [3
HCTZ], then it can be considered that the mechanism of quenching is
as static type [32].
M. Shahlaei, P. Zamani, N. Farhadian et al. International Journal of Biological Macromolecules xxx (xxxx) xxx

Fig. 2. Fluorescence spectra of pepsin (blue line) in the presence of different concentrations 100 (red line), 300 (green line), 500 (violet line), 700 (light blue line) and 900 (orange line) μM
of A) simvastatin and B) atorvastatin.

Therefore base on the value of calculated Kq represented in Table 1 it 3.3. Calculation of thermodynamic parameters
can be concluded that the quenching mechanism of pepsin by atorva-
statin and simvastatin is a static manner that confirms the formation By using the sign and magnitude of thermodynamic parameters the
of stable interaction between the molecules. ΔH and ΔS, it is possible to determine the nature of major forces in the

Fig. 3. Stern–Volmer plots and the modified Stern–Volmer for A) atorvastatin and B) simvastatin binding to pepsin.

4
M. Shahlaei, P. Zamani, N. Farhadian et al. International Journal of Biological Macromolecules xxx (xxxx) xxx

Table 1 Then, the Gibbs free energy (ΔG) of interaction was determined by
The binding constant (Kb), the Stern–Volmer quenching constant (KSV), quenching rate applying the given values of ΔH and ΔS in the Eq. (5).
constant (kq) and the number of binding site (n) for binding of atorvastatin and simva-
statin to pepsin at different temperatures.
ΔG0 ¼ ΔH 0 −TΔS0 ð5Þ
Drug Temperature Kb KSV Kq n
(K)
The values of thermodynamic parameters obtained from the interac-
Atorvastatin 290 5727.47 7874 7.874 E+11 1.16
tion of pepsin and both drugs are listed in Table 2. As can be seen about
300 2213.60 6923 6.923 E+11 0.87
310 1075.43 5905 5.905 E+11 0.76
both of the drugs, the sign for thermodynamic values of ΔH and ΔS are
Simvastatin 290 3311.11 49,160 4.916 E+12 0.61 negative which indicating that the main forces in these interactions are
300 1949.45 36,832 3.683 E+12 0.51 as hydrogen bond and van der Waals force [46]. Also from the negative
310 870.63 27,974 2.797 E+12 0.47 value of ΔG in Table 2 it can be said that protein-drug bindings is a spon-
taneous process in all temperatures and by increasing temperature, the
binding energies are decreased.
Table 2
Thermodynamic parameters for binding of atorvastatin and simvastatin to the pepsin. 3.4. Study of pepsin enzyme activity

Complex T (K) ΔS° (J mol−1 K−1) ΔH° (kJ mol−1) ΔG°(kJ mol−1)
The main effect of each ligand on any macromolecule is affecting its
Atorvastatin-pepsin 290 −248.17 −90.12 −18.15 function; hence the enzymatic activity of pepsin in the presence of
300 −15.66
drugs is examined. For this purpose, the enzyme activity was calculated
310 −13.18
Simvastatin-pepsin 290 −288.147 −29.41 −22.12 using equation (Eq. (1)) and the results are listed in Table 3. As shown in
300 −19.24 this table, the enzymatic activity of pepsin in the presence of both drugs
310 −16.36 at lower concentrations has increased, however in the case of atorva-
statin at higher drug concentrations, enzymatic activity is reduced. Con-
sidering other results obtained in this study this may be due to the fact
that there is more than one site for binding the ATC to the protein, and
Table 3 the binding of the drug in the second location has a reduction effect on
Activity of pepsin in the absent and present different concentrations of atorvastatin and
the protease activity of the enzyme.
simvastatin.

Concentration of Activity of pepsin at present Activity of pepsin at present 3.5. FTIR spectroscopy
drugs SIM ATC

Blank 100 100 FTIR spectroscopy is of the best techniques for probing conforma-
0.3 mM 129.49 131.65
tional change in the protein secondary structure upon binding a ligand.
0.6 mM 133.81 128.77
0.9 mM 138.13 124.46
The amide band I in 1600–1700 CM−1 related to C_O stretching and
amide band II in 1500–1600 for C\\N stretching coupled with N\\H
bending are more sensitive to change in protein structure so, can be
used for such studies [47,48].
interaction between the protein and the ligand. In this regards the van't The FTIR spectra of protein in the absence and presence of both
Hoff equation (Eq. (4)) was used and by plotting the values of lnKb drugs (ATC and SIM) was recorded and shown in Fig. 4. The amide I
against 1/T, the ΔH and ΔS were obtained [45]. band at the position 1500–1550 and a peak in 1650 related to beta-
sheets of protein presented in all systems without significant right or
lnK b ¼ −ðΔH 0 =RT Þ þ ðΔS0 =RÞ ð4Þ left shifts. Hence it can be concluded that no sever structural change

Fig. 4. FTIR results of pepsin in the absent and present of atorvastatin and simvastatin.

5
M. Shahlaei, P. Zamani, N. Farhadian et al. International Journal of Biological Macromolecules xxx (xxxx) xxx

Table 4 with the results obtained in the experimental methods. About the sim-
Results of molecular docking studies on the interaction of atenolol and diltiazem with pep- vastatin, the interaction energy was as −10.61 kcal/mol. Hydrogen
sin protein.
bonding and van der Waals interactions are also involved in the binding
Pepsin-simvastatin Pepsin-atorvastatin of SIM to pepsin. Fig. 5 has further indicated a greater penetration of
Number of runs in cluster rank 146 162 simvastatin into the active site cavity.
Intermolecular interaction energy −10.61 −8.78
(kcal/mol)
3.6.2. Molecular dynamic simulation studies
Molecular dynamics simulations can provide more time-dependent
information on the dynamics of understudy systems. At first, from the
has happened in the structure of the protein. These results indicating
MD simulations it is observed that the drugs are attached to the active
that the drug interaction of ATC and SIM with pepsin did not have an ad-
site of protein during the simulation time and never separated from
verse effect on protein structure.
protein because of stable complexes have formed with protein
(Fig. 6). As can be seen from Fig. 6 in the case of ATC the location if bind-
3.6. Molecular modeling ing is not significantly changed on the protein but the drug SIM have
mover from its location in the first frame at the end of simulations.
3.6.1. Docking The structural changes of pepsin in interaction with simvastatin and
The interaction energies and more molecular details of the drug- atorvastatin were then investigated. The overall changes in the position
pepsin interaction were studied by docking analysis and the results of the protein backbone by an analysis called Root Mean Square Devia-
are shown in Table 4 and Fig. 5. As can be seen from this figure the inter- tion (RMSD) was performed comparatively in the presence of simva-
action location for both drugs is in the cleft closed to the active site and statin and atorvastatin and the results are shown in Fig. 7. The results
the amino acids involved in interaction have many similarities for both illustrated that the values and fluctuations of RMSD for pepsin in the
drugs. The Asp 32 and Asp 215, the main catalytic residues are in direct presence of simvastatin were higher than those related for atorvastatin
interaction with the drugs. In the case of atorvastatin, the intermolecu- and both are lower than that of free pepsin. In the case of free protein
lar energy of interaction is −8.78 kcal/mol, and the nature of energies is (APO state) after a primary jump in RMSD value to the 0.4 nm the
as hydrogen bonds and van der Waals forces, which is in agreement relative equilibration in the system has archived by some fluctuations.

Fig. 5. Docking analysis for drug interactions with pepsin for A) simvastatin and B) atorvastatin.

6
M. Shahlaei, P. Zamani, N. Farhadian et al. International Journal of Biological Macromolecules xxx (xxxx) xxx

Fig. 6. Drug-protein complexes before and after 50 ns of MD simulations for A) simvastatin and B) atorvastatin.

Other systems contain the drugs have an RMSD value of 0.3 nm formation between ATC and SIM with pepsin has led to less freedom
with higher fluctuations in the complex system of SIM-pepsin. The in protein mobility.
lower fluctuation in RMSD is happened in the pepsin in interacted Another parameter evaluated in the computational studies is the
with ATC. These results altogether indicating that the complex measurement of the flexibility of each residual during the simulation

Fig. 7. RMSD analysis for interaction of simvastatin and atorvastatin with pepsin.

7
M. Shahlaei, P. Zamani, N. Farhadian et al. International Journal of Biological Macromolecules xxx (xxxx) xxx

Fig. 8. Results of RMSF in pepsin residues in complex with simvastatin and atorvastatin.

by evaluating the root mean square fluctuation (RMSF) of protein. The
results of this study for pepsin in APO state were compared to those in
the presence of drugs simvastatin and atorvastatin (Fig. 8). As can be
seen from the figure, the RMSF values in most residues of interacted pro-
tein are less than those of free pepsin. This indicating the rigidity of pro-
tein when is in complex with drugs. Also, the results show that
atorvastatin is most effective in preventing mobility of pepsin than sim-
vastatin especially about the amino acids in the location of the active
site. In the residue locations of 30–40, 70–80 and 200–230 which their
amino acids are in direct interaction with the drugs, the RMSF value is
clearly decreased. In contrast in some parts of protein such as residues
90–100 that are not in direct contact with the drugs the fluctuation of
amino acids. This result confirms the in vitro results that pepsin enzy-
matic activity was more increased in the presence of atorvastatin
compared to simvastatin which is likely due to the lower flexibility of
active site residues. The lower fluctuation in the active site practically
can help the facility of the substrate to bind to pepsin.
The Radius of gyration (Rg) as a function of time is actually an
index of the compression in the structure of the protein. Rg calcula-
tions for pepsin in different states were performed and results are re-
ported in Fig. 8. For free protein, the value of Rg is increased by the
0.15 nm and maintains constant in the rest of the simulation. This in-
dicating some inflate is occurred in the protein which the addition of
drugs in the system as is presented in Fig. 9 prevents this inflation.
Also, it can be seen that the drug atorvastatin causes the lowest
value of Rg. These are all in agreement with other results in which
the interaction of drugs with protein caused less flexibility and mo-
bility in the system.

Fig. 9. Radius of gyration for interaction of simvastatin and atorvastatin with pepsin.

8
M. Shahlaei, P. Zamani, N. Farhadian et al.
4. Conclusion
The effects of two long-term orally used cholesterol-lowering drugs
in the class of statins on the structure and enzymatic activity of pepsin
were investigated. For this propose experimental techniques of UV–
vis, fluorescence and FTIR spectroscopies in combination with computa-
tional docking and molecular dynamics approaches were applied. The
results obtained from UV–visible and Fluorescence study revealed that
both drugs have stable interactions with protein and have quenched
the fluorescence intensity of protein by a static manner. The findings
from the FTIR analysis showed that in the presence of drugs the content
of the secondary structures of the protein have not changed signifi-
cantly. Also, the experiments illustrated that at the presence of both
atorvastatin and simvastatin at the concentrations 0.3–0.6 μM, the enzy-
matic activity of pepsin was increased however about the ATC by in-
creasing the concentration to 0.9 μM, the protease activity has
decreased. This can be because of the ATC have multiple binding sites
on the protein and by increasing the concentration other sites which
have incremental effect are occupied. The results of molecular modeling
confirm that a stable binding of drugs to protein is occur close to the ac-
tive site and this interactions have no adverse effect on the dynamic and
structure of pepsin.
Declaration of competing interest
The authors also declare that there is no any conflict of interest.
Acknowledgment
This work was supported by the Kermanshah University of Medical
Sciences, Kermanshah, Iran [grant number of 980377].
References
[1] C. Stancu, A. Sima, Statins: mechanism of action and effects, J. Cell. Mol. Med. 5 (4)
(2001) 378–387.
[2] P. Ziros, Z. Zagoriti, G. Lagoumintzis, V. Kyriazopoulou, R.P. Iskrenova, E.I. Habeos,
G.P. Sykiotis, D.V. Chartoumpekis, I.G. Habeos, Hepatic Fgf21 expression is repressed
after simvastatin treatment in mice, PLoS One 11 (9) (2016), e0162024, .
[3] Z. Zhang, H. Bu, Z. Gao, Y. Huang, F. Gao, Y. Li, The characteristics and mechanism of
simvastatin loaded lipid nanoparticles to increase oral bioavailability in rats, Int. J.
Pharm. 394 (1–2) (2010) 147–153.
[4] J.-H. Shi, Q. Wang, D.-Q. Pan, T.-T. Liu, M. Jiang, Characterization of interactions of
simvastatin, pravastatin, fluvastatin, and pitavastatin with bovine serum albumin:
multiple spectroscopic and molecular docking, J. Biomol. Struct. Dyn. 35 (7)
(2017) 1529–1546.
[5] J.S. Fruton, A history of pepsin and related enzymes, Q. Rev. Biol. 77 (2) (2002)
127–147.
[6] M. Fujinaga, M.M. Chernaia, S.C. Mosimann, M.N. James, N.I. Tarasova, Crystal struc-
ture of human pepsin and its complex with pepstatin, Protein Sci. 4 (5) (1995)
960–972.
[7] I. Samloff, R. Taggart, Pepsinogens, pepsins, and peptic ulcer, clinical and investiga-
tive medicine, Medecine clinique et experimentale 10 (3) (1987) 215–221.
[8] K.D. Bardhan, V. Strugala, P.W. Dettmar, Reflux revisited: advancing the role of pep-
sin, International Journal of Otolaryngology 2012 (2011).
[9] C. Venables, Mucus, pepsin, and peptic ulcer, Gut 27 (3) (1986) 233.
[10] L. Shen, H. Xu, F. Huang, Y. Li, H. Xiao, Z. Yang, Z. Hu, Z. He, Z. Zeng, Y. Li, Investigation
on interaction between ligupurpuroside A and pepsin by spectroscopic and docking
methods, Spectrochim. Acta A Mol. Biomol. Spectrosc. 135 (2015) 256–263.
[11] H.-j. Zeng, R. Yang, H. Liang, L.-b. Qu, Molecular interactions of flavonoids to pepsin:
insights from spectroscopic and molecular docking studies, Spectrochim. Acta A
Mol. Biomol. Spectrosc. 151 (2015) 576–590.
[12] H.-j. Zeng, J. You, H.-l. Liang, T. Qi, R. Yang, L.-b. Qu, Investigation on the binding in-
teraction between silybin and pepsin by spectral and molecular docking, Int. J. Biol.
Macromol. 67 (2014) 105–111.
[13] X. Ma, L. Guo, Q. Wang, J. He, H. Li, Spectroscopy and molecular modeling study on
the interaction between mycophenolate mofetil and pepsin, J. Fluoresc. 26 (2)
(2016) 599–608.
[14] L. Ma, B. Liu, C. Wang, H. Zhang, X. Cheng, The interaction mechanism of nifedipine
and pepsin, Monatshefte für Chemie-Chemical Monthly 149 (11) (2018)
2123–2130.
[15] G. Nan, J. Sun, M. Ding, X. Yang, G. Yang, Interaction behavior between myricetin and
dihydromyricetin with pepsin by spectroscopic and docking methods, J. Mol. Liq.
223 (2016) 128–135.
9
International Journal of Biological Macromolecules xxx (xxxx) xxx
[16] M. Pathak, D. Sharma, N. Sharma, M. Sharma, Spectroscopic and thermodynamic
studies of the binding mechanism of metformin to pepsin, J. Mol. Struct. 1166
(2018) 183–189.
[17] H.-j. Zeng, T. Qi, R. Yang, J. You, L.-b. Qu, Spectroscopy and molecular docking study
on the interaction behavior between nobiletin and pepsin, J. Fluoresc. 24 (4) (2014)
1031–1040.
[18] S. Moradi, N. Farhadian, F. Balaei, M. Ansari, M. Shahlaei, Multi spectroscopy and
molecular modeling aspects related to drug interaction of aspirin and warfarin
with pepsin; structural change and protease activity, Spectrochim. Acta A Mol.
Biomol. Spectrosc. 228 (2020), 117813, .
[19] M. Ying, F. Huang, H. Ye, H. Xu, L. Shen, T. Huan, S. Huang, J. Xie, S. Tian, Z. Hu, Study
on interaction between curcumin and pepsin by spectroscopic and docking
methods, Int. J. Biol. Macromol. 79 (2015) 201–208.
[20] P.-H. Hsyu, M.D. Schultz-Smith, J.H. Lillibridge, R.H. Lewis, B.M. Kerr, Pharmacoki-
netic interactions between nelfinavir and 3-hydroxy-3-methylglutaryl coenzyme a
reductase inhibitors atorvastatin and simvastatin, Antimicrob. Agents Chemother.
45 (12) (2001) 3445–3450.
[21] T.A. Jacobson, Comparative pharmacokinetic interaction profiles of pravastatin, sim-
vastatin, and atorvastatin when coadministered with cytochrome P450 inhibitors,
Am. J. Cardiol. 94 (9) (2004) 1140–1146.
[22] J.H. Hochman, N. Pudvah, J. Qiu, M. Yamazaki, C. Tang, J.H. Lin, T. Prueksaritanont, In-
teractions of human P-glycoprotein with simvastatin, simvastatin acid, and atorva-
statin, Pharm. Res. 21 (9) (2004) 1686–1691.
[23] H.-D. Holtje, G. Folkers, R. Mannhold, H. Kubinyi, H. Timmerman, Molecular Model-
ing: Basic Principles and Applications, John Wiley & Sons, Inc, 1997.
[24] S. Moradi, S. Khani, M. Ansari, M. Shahlaei, Atomistic details on the mechanism of
organophosphates resistance in insects: insights from homology modeling, docking
and molecular dynamic simulation, J. Mol. Liq. 276 (2019) 59–66.
[25] M. Ansari, S. Moradi, M. Shahlaei, A molecular dynamics simulation study on the
mechanism of loading of gemcitabine and camptothecin in poly lactic-co-glycolic
acid as a nano drug delivery system, J. Mol. Liq. 269 (2018) 110–118.
[26] S. Moradi, E. Hosseini, M. Abdoli, S. Khani, M. Shahlaei, Comparative molecular dy-
namic simulation study on the use of chitosan for temperature stabilization of inter-
feron αII, Carbohydr. Polym. 203 (2019) 52–59.
[27] F. Jafari, S. Moradi, A. Nowroozi, K. Sadrjavadi, L. Hosseinzadeh, M. Shahlaei, Explor-
ing the binding mechanism of paraquat to DNA by a combination of spectroscopic,
cellular uptake, molecular docking and molecular dynamics simulation methods,
New J. Chem. 41 (23) (2017) 14188–14198.
[28] A.R. Bizzarri, I. Moscetti, S. Cannistraro, Interaction of the anticancer p28 peptide
with p53-DBD as studied by fluorescence, FRET, docking and MD simulations,
Biochimica et Biophysica Acta (BBA)-General Subjects 1863 (2) (2019) 342–350.
[29] H. Rimac, C. Dufour, Ž. Debeljak, B. Zorc, M. Bojić, Warfarin and flavonoids do not
share the same binding region in binding to the IIA subdomain of human serum al-
bumin, Molecules 22 (7) (2017) 1153.
[30] J. Cerbulis, J. Custer, C. Zittle, Action of rennin and pepsin on α-casein: paracasein
and soluble products, Arch. Biochem. Biophys. 84 (2) (1959) 417–427.
[31] Z. Krejpcio, R. Wojciak, The influence of Al3+ ions on pepsin and trypsin activity
in vitro, Pol. J. Environ. Stud. 11 (3) (2002) 251–254.
[32] F. Balaei, S. Ghobadi, Hydrochlorothiazide binding to human serum albumin induces
some compactness in the molecular structure of the protein: a multi-spectroscopic
and computational study, J. Pharm. Biomed. Anal. 162 (2019) 1–8.
[33] R.R. Nasab, M. Mansourian, F. Hassanzadeh, Synthesis, antimicrobial evaluation and
docking studies of some novel quinazolinone Schiff base derivatives, Research in
Pharmaceutical Sciences 13 (3) (2018) 213.
[34] K. Weber, J. Pringle, M. Osborn, [1] Measurement of molecular weights by electro-
phoresis on SDS-acrylamide gel, Methods Enzymol. (1972) 3–27 Elsevier.
[35] G.M. Morris, R. Huey, A.J. Olson, Using autodock for ligand-receptor docking, Curr.
Protoc. Bioinformatics 24 (1) (2008) (8.14. 1-8.14. 40).
[36] H.M. Berman, T. Battistuz, T.N. Bhat, W.F. Bluhm, P.E. Bourne, K. Burkhardt, Z. Feng,
G.L. Gilliland, L. Iype, S. Jain, The protein data bank, Acta Crystallogr. D Biol.
Crystallogr. 58 (6) (2002) 899–907.
[37] D.S. Goodsell, G.M. Morris, A.J. Olson, Automated docking of flexible ligands: appli-
cations of AutoDock, J. Mol. Recognit. 9 (1) (1996) 1–5.
[38] M.D. Hanwell, D.E. Curtis, D.C. Lonie, T. Vandermeersch, E. Zurek, G.R. Hutchison,
Avogadro: an advanced semantic chemical editor, visualization, and analysis plat-
form, Journal of Cheminformatics 4 (1) (2012) 17.
[39] A.K. Malde, L. Zuo, M. Breeze, M. Stroet, D. Poger, P.C. Nair, C. Oostenbrink, A.E. Mark,
An automated force field topology builder (ATB) and repository: version 1.0, J.
Chem. Theory Comput. 7 (12) (2011) 4026–4037.
[40] K. Binder, Applications of Monte Carlo methods to statistical physics, Rep. Prog.
Phys. 60 (5) (1997) 487.
[41] B. Hess, C. Kutzner, D. Van Der Spoel, E. Lindahl, GROMACS 4: algorithms for highly
efficient, load-balanced, and scalable molecular simulation, J. Chem. Theory Comput.
4 (3) (2008) 435–447.
[42] D. van Aalten, A. Amadei, R. Bywater, J. Findlay, H. Berendsen, C. Sander, P. Stouten, A
comparison of structural and dynamic properties of different simulation methods
applied to SH3, Biophys. J. 70 (2) (1996) 684–692.
[43] H.-N. Hou, Z.-D. Qi, Y.-W. OuYang, F.-L. Liao, Y. Zhang, Y. Liu, Studies on interaction
between vitamin B12 and human serum albumin, J. Pharm. Biomed. Anal. 47 (1)
(2008) 134–139.
[44] S.K. Chaturvedi, E. Ahmad, J.M. Khan, P. Alam, M. Ishtikhar, R.H. Khan, Elucidating
the interaction of limonene with bovine serum albumin: a multi-technique ap-
proach, Mol. BioSyst. 11 (1) (2015) 307–316.
[45] W. Gao, N. Li, Y. Chen, Y. Xu, Y. Lin, Y. Yin, Z. Hu, Study of interaction between
syringin and human serum albumin by multi-spectroscopic method and atomic
force microscopy, J. Mol. Struct. 983 (1–3) (2010) 133–140.
M. Shahlaei, P. Zamani, N. Farhadian et al.
[46] Z. Nan, C. Hao, X. Ye, Y. Feng, R. Sun, Interaction of graphene oxide with bovine
serum albumin: a fluorescence quenching study, Spectrochim. Acta A Mol. Biomol.
Spectrosc. 210 (2019) 348–354.
[47] Z.-Q. Xu, Q.-Q. Yang, J.-Y. Lan, J.-Q. Zhang, W. Peng, J.-C. Jin, F.-L. Jiang, Y. Liu, Inter-
actions between carbon nanodots with human serum albumin and γ-globulins:
the effects on the transportation function, J. Hazard. Mater. 301 (2016) 242–249.
10
International Journal of Biological Macromolecules xxx (xxxx) xxx
[48] N. Moradi, M.R. Ashrafi-Kooshk, S. Ghobadi, M. Shahlaei, R. Khodarahmi, Spectro-
scopic study of drug-binding characteristics of unmodified and pNPA-based acety-
lated human serum albumin: does esterase activity affect microenvironment of
drug binding sites on the protein? J. Lumin. 160 (2015) 351–361.