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Repurposing existing drugs for new AMPK activators

as a strategy to extend lifespan: a computer-aided drug
discovery study
Sepideh Mofidifar . Farzin Sohraby . Milad Bagheri . Hassan Aryapour

Received: 7 October 2017 / Accepted: 4 January 2018

Ó Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Dietary restriction is one of the several to be more potential activators than C2. However, hit-
ways which could putatively extend organisms’ lifes- compounds in ab binding site, exhibited higher
pan, ranging from Saccharomyces cerevisiae to unfavorable binding affinity. Hence, present findings
rodents, by activating the AMP-activated protein can prove to be valuable for discovering new activa-
kinase (AMPK), an ATP/AMP sensor. Extensive tors for AMPK.
researches have shown that aging reduces sensibility
of AMPK and eventually causes energy imbalance in Keywords AMPK  Drug repurposing  Virtual
cells. Research in mammals’ AMPK depicts that this screening  FDA-approved drugs
signaling molecule could control autophagy, improve
cellular stress resistance and suppress inflammatory
responses. Hence, in this study we performed a drug
repurposing of 1908 FDA-approved drugs in order to Introduction
discover putative safe activators of AMPK and to find
new applications for existing drugs. For this purpose, Aging is a multifactorial process, which causes
FDA-approved drugs were screened by virtual screen- deterioration of the physiological function. Studies
ing and the ligand–protein interactions were carefully have shown that caloric restriction could potentially
inspected. Moreover, through MM/PBSA analysis, the decrease aging processes (Hall et al. 2013), which not
binding affinity of hit compounds in c and ab binding only has profound influence on life span, but also
sites were investigated. As Cangrelor, Nacitentan, delays some age-related pathologies, such as cancer
Levoleucovorin and Glisoxepide had lower binding and neurodegenerative diseases (Maswood et al.
affinities; we predicted that they would probably prove 2004). DR is able to increase AMP/ATP and ADP/
ATP rations. Subsequently, the AMPK is allosterically
activated by high concentration of AMP (Salminen
Electronic supplementary material The online version of and Kaarniranta 2012). AMPK is a conserved serine/
this article (https://doi.org/10.1007/s10522-018-9744-x) con- threonine protein kinase that appears to exert a variety
tains supplementary material, which is available to authorized
users. of biological functions. It is a putative regulator of
cell’s homeostasis (Hardie and Hawley 2001), which
S. Mofidifar  F. Sohraby  M. Bagheri  sustains cellular life span by phosphorylating a large
H. Aryapour (&)
number of downstream proteins. Therefore, in
Department of Biology, Faculty of Science, Golestan
University, Gorgan, Iran response to energy balance, AMPK activates catabolic
e-mail: h.aryapour@gu.ac.ir processes, while inhibiting anabolic pathways (Rana


et al. 2014). Consequently, mTOR proteins that are the simulation, binding free energy calculations and
regulators of metabolism and cellular growth are related analyses were carried out to investigate the
switched off (Dalle Pezze et al. 2016) and sirtuin is stability of resultant complexes. Finally, several drugs
switched on as an anti-aging protein (Cantó et al. were predicted to be the potential activators of AMPK.
2009) (Fig. S1 in supporting information).
Structurally, AMPK is a heterotrimeric kinase
expressed in many cells. It is composed of one Materials and methods
catalytic subunit a and two regulatory subunits b and
c (Fig. S2 in supporting information) (Steinberg and Protein preparation
Kemp 2009). The a subunit consists of N-terminal
kinase domain (KD) and C-terminal subunit interact- The X-ray micrograph of the crystal structure of
ing domain (SID). Phosphorylation of threonine 172 in human AMPK (PDB code 4ZHX) was obtained from
KD domain has crucial function in activating the Protein Data Bank. Due to large number of missing
AMPK that is carried out by the signaling pathways of amino acids in 4ZHX (112 aa in a, 95 aa in b and 36 aa
calcium/calmodulin-dependent protein kinase kinase in c subunit), Swiss-Model server was used to build a
(CaMKK) and liver kinase B1 (LKB1); two major reliable 3D model of the protein using amino acid
upstream activators of AMPK (Crute et al. 1998). sequence (Bordoli et al. 2009). Then the model was
The b subunit of AMPK contains two main used as template in modeler for refining missing parts
domains: carbohydrate binding domain (CBD) and of the experimental structure.
subunit binding sequence at the C-terminal, which
interact with both a and c subunits (Townley and Molecular docking
Shapiro 2007). ‘‘ADaM’’ (allosteric drug and metabo-
lite) site is located in the interface between a and b In this study, a total of 1908 FDA-approved drugs were
subunits, where a large number of AMPK activators retrieved from DrugBank database (on 18/01/2017).
bind to it (Cameron and Kurumbail 2016). The These 2D drugs were converted to their 3D forms by
synthesis of activators in this site prevents the dephos- molconverter from Jchem toolkit (Version,
phorylation of AMPK by phosphatases (Li et al. 2015). ChemAxon). After that, virtual screening was per-
The c subunit is sensitive to AMP: ATP ratio within formed by LeDock, which had 80.8% accuracy for the
the cells. Each c subunit contains four repeats of best poses (Wang et al. 2016). Following which, the
cystathionine beta-synthase (CBS) motifs. Although, protein was prepared and hydrogen atoms were added
site 4 is a non-exchangeable AMP binding site, sites 1 to the crystal structure by LePro tool. LeDock program
and 3 binds to nucleotide in a reversible manner (Chen is based on a combination of simulated annealing and
et al. 2013). Studies have shown that AMP could evolutionary optimization of the ligand pose and its
potentially change the proteins conformation in a way rotatable bonds (http://lephar.com). To investigate the
that dephosphorylation is blocked (Sanders et al. 2007). docking poses, LeDock score function was used. It uses
It has previously been investigated that drugs’ side- a physics/knowledge hybrid scoring scheme. Out of the
effect is due to the fact that they may have more than top 100 docked poses, the hit compounds were chosen
one target. Hence, drug repurposing has been devel- by visual inspection. In order to validate, the chosen hit
oped, which identifies new targets for existing drugs compounds, a different docking program with the
(Ma et al. 2013). Additionally, new target identifica- Lamarckian Genetic Algorithm (LGA), AutoDock
tion for existing drugs may be a useful method for Vina was used (Trott and Olson 2010).
saving money and time, because of complex and time-
consuming process of drug discovery (Gupta et al. Molecular dynamics simulation
2013). In silico drug repurposing is one of the widely
used novel methods (Cavalla 2013). It also grabs aging Among the various sampling algorithm for molecular
studies’ attention in recent years (Calvert et al. 2016). modeling, molecular dynamics is widely used in many
Here, we performed the structure-based virtual fields. In this algorithm, both ligand and protein are
screening (SBVS) to predict valuable activators for more efficiently flexible than others. In molecular
AMPK. Subsequently, molecular dynamics (MD) dynamics simulations, almost all of the conditions


Fig. 1 Validation of AMPK model: a Ramachandran plot, b verification of 3D analysis

Table 1 LeDock score and drug categorization of hit compounds with respect to c and ab binding sites
AMPK Generic name Ledock score Vina affinity Drug category
(binding (kcal/mol) (kcal/mol)

c Cangrelor - 14.1 - 9.3 Receptor antagonist which inhibits ADP platelet aggregation
Nacitentan - 10.9 - 9.3 Blocks the stimulation of vasculature hypertrophy, inflammation,
fibrosis, proliferation, and vasoconstriction
Levoleucovorin - 10.2 - 10.9 Active form of folinic acid
Glisoxepide - 10.2 - 10 Increase secretion of (pro)insulin
ab Ceftolozane - 12 - 7.8 Antibiotic
Ceftriaxone - 8.84 - 8.1 Treatment of infections
Levomefolic - 8.35 - 7.3 Primary biologically active form of folate

such as pressure, temperature, dielectric constant are under constant temperature and pressure (300 K and
present (Ganesan et al. 2017).All the MD simulations 1 atm respectively). The trajectories information was
of this study, took advantage of the GROMACS 5.1.2 analyzed using GROMACS utilities and VMD.
software (Pronk et al. 2013), using Amber99SB force
field (Hornak et al. 2006). Ligand topology files were Energy analysis via MM/GBSA approach
generated by ACPYPE tool. The protein–ligand
complexes were placed in the center of a triclinic The molecular mechanics/generalized born surface
box setting a minimal distance of 1 nm from all edges, area (MM/GBSA) calculations were performed by
which were solvated with TIP3P water molecules g_mmpbsa package (Kumari et al. 2014). In this
(Jorgensen et al. 1983). Before minimization, the method, the binding free energy of hit compounds was
systems were neutralized by adding Naþ and Cl at obtained from the following equation:
the physiological concentration of 150 mM NaCl. The 
DGbind ¼ Gcomplex  Gligand þ Greceptor
steepest descent algorithm was used for energy
minimization of all atoms, in which the minimization In the present study, a total of 100 snapshots were
has stopped when the Fmax was smaller than evenly taken in a time step of 200 frames. The binding
10 kJ/mol. Moreover, for equilibration of systems, free energy, the Van der Waals (VDW) energy,
100 ps NVT and 300 ps NPT were performed while electrostatic energy, polar solvation energy and
the atomic position of protein and ligands were
restrained. After all, 10 ns the simulations were done


eventually the solvent-accessible surface area (SASA)

energy were calculated in present study as described
previously (Kumari et al. 2014).
Additionally, to analyze the interactions between
residues and hit compounds, the MM/GBSA binding
energy decomposition analysis was performed (Tsui
and Case 2000). The binding interactions were
calculated by employing for the following compo-
nents: the van der Waals contribution (DGvdw), the
polar part of desolvation (DGGB), the electrostatic
contribution (DGele) and the non-polar part of desol-
vation (DGSA). Thus:

Fig. 2 RMSF values of the residues in the presence of hit

DG ¼ DGvdW þ DGele þ DGsol
compound during 10 ns of MD simulation at c binding site ¼ DGvdW þ DGele þ DGGB þ DGSA:

Fig. 3 Number of inter-

molecular hydrogen bonds
between hit compound and
the residues at c active site
of AMPK. a C2, b AMP,
c Cangrelor, d Nacitentan,
e Levoleucovorin and
f Glisoxepide


Fig. 4 Number of inter-

molecular hydrogen bonds
between hit compounds and
the residues at ab active site
of AMPK. a A769662,
b Ceftolozane,
c Ceftriaxone and
d Levomefolic acid

Results and discussion Among 100 top scores, 3 ligands for ab binding site
and 4 ligands for c binding site were chosen by
Model evaluation checking the types and number of bonds in pocket.
According to LeDock score, the reference ligands, C2,
After several rounds of energy minimization, the AMP and A769662 scored - 6.46, - 8.79 and
refined model was evaluated by using Ramachandran - 7.32 kcal/mol, respectively. However, the hit com-
plot. According to our results, 92.7% residues were in pounds scored higher than the above-stated values for
favored regions, 6.2% in the allowed regions and 1.1% the reference ligands (Table 1). Additionally, for
residues in the restricted regions. In general, well-built further validation of LeDock results, we redocked
stereochemical models implied a score close to 100%. and rescored top hit compounds by Vina. The
Therefore, the Ramachandran plot suggests that the reference ligands (C2, AMP and A769662 scored
model was of great quality. For further confirmation, - 7.8, - 8.5 and - 7.5 kcal/mol, respectively) and
the protein structure was also validated by Verify3D hit compounds scored approximately as same as
program. The Verify3D analysis is compatible for the LeDock score which could indicate the accuracy of
model via the amino acid sequence. In current study, our data.
all the amino acids showed an average 3D_ID score
above zero except 16 first amino acids of c subunit Stability of MD simulations
(Fig. 1).
Root mean square deviation (RMSD) is one of the
Virtual screening most important values to investigate the stability of a
system. The RMSD values of c and ab sites are shown
Molecular docking can be used to predict the ligands in Fig. S3. The backbone RMSDs for c site reached the
behavior in the binding site of protein. It consists of equilibrium state after about 2 ns simulation. Also, the
two basic steps: sampling the ligand pose (position ab site RMSD plot illustrated the stability for all
and orientation) and ranking the binding affinity by systems after about 5 ns. The lower value of RMSD
scoring function (Meng et al. 2011). Therefore, shows more stable system. Therefore, all the presently


Table 2 The p-effects, hydrophobic (alkyl) bonds and the number of hydrogen bonds between the hit compounds and at c and ab
binding sites as well
Binding site Compounds p-effects Hydrophobic (Alkyl) H-bonds

(c) C2 – – –
AMP R304, V281, 7
V302, I245 (Pi-alkyl)
Cangrelor H303, H156 (Pi–Pi, T-shaped) R75-LIGC25 5
R75 (Pi-cation) LLE93-LIGC28
F249 (Pi-alkyl)
Nacitentan R75 (Pi-cation) AlA232-LIGC31 4
H303, F278 (Pi–Pi stack) I245-LIGC31
H156, F249 (Pi–Pi T-shaped)
Levolcovorin I155 (Pi-alkyl) 7
M90 (Pi-alkyl)
A76 (Pi-alkyl)
Glisoxepide H156 (Pi–Pi T-shaped) I155-LIGC5 2
K248 (Pi-cation)
R304, ALA232 (Pi-alkyl)
(ab) A769662 F103 (Pi–Pi, T-shaped) V81-LIG565 1
V24- LIG565
Ceftolozane I59 (Pi-alkyl) R83-LIGC3 2
Ceftriaxone D33 (Pi-anion) 3
H109 (Pi–Pi stacked)
I59 (Pi-alkyl)
Levomefolic acid H109 (Pi–Pi T-shaped) 1

studied compounds, except Cangrelor showed the 0.1 nm. So, it seemed that the hit compounds and
most reliable stabilities in c binding site. reference ligands complexes had approximately the
The RMSF value of the residues with 10 ns MD same fluctuation for each residue in KD and AID
simulation of each system was analyzed by ‘‘gmx (Fig. 2).
rmsf’’ tool. In this way, the flexibility of each residue The RMSF value for ab binding site is presented in
in the presence of hit compounds was compared. In Fig. S6 in supporting information, which shows that
general, Fig. S4 depicts that the 4 activator-protein the C-terminals of c and b subunits are fluctuated at a
complexes posed similar distribution at residues higher rate. At the active site (residues 21–60 and
75–325 of the active site, which fluctuated less than 80–115 for a and b subunits, respectively), the hit
0.4 nm. Although C2 showed the most significant compounds fluctuated approximately at the same rate
fluctuation at KD domain (at residues 21–291 of a as AMPK. Among them Ceftriaxone had the most
subunit), its RMSF difference with other ligands was similar RMSF value to that of AMPK.
less than 0.1 nm. Besides, the AID part (residues
300–349) of a subunit illustrates this pattern as well. Protein–Ligand interactions
Even though AMPK (with no ligands) and Glisox-
epide showed the most significant fluctuation at the The hydrogen bond between hit compounds and
AID residues, they had an RMSF difference less than protein has been illustrated in Figs. 3 and 4. The c


Table 3 VDW, electrostatic, polar solvation, SASA and binding free energies of the hit compounds with respect to c and ab binding
AMPK (Binding Generic name van der Waal Electrostatic Polar solvation SASA energy Binding energy
sites) energy energy energy

(c) C2 - 90.20 ± 2 - 678.8 ± 7.7 849.6 ± 4.9 - 12.0 ± 0.0 68.7 ± 6

AMP - 167.2 ± 1.5 - 117.6 ± 1.3 203.7 ± 1.7 - 16.2 ± 0.06 - 97.2 ± 1.5
Cangrelor - 272.3 ± 22.4 - 541.3 ± 76.9 862.8 ± 81.2 - 30.0 ± 0.9 19 ± 49.1
Nacitentan - 236.8 ± 1.8 - 40.80 ± 1.8 157.8 ± 4.2 - 22.1 ± 0.1 - 142 ± 2
Levoleucovorin - 195.3 ± 2.3 - 668.3 ± 8.4 918.8 ± 7.4 - 22.1 ± 0.1 32.7 ± 5.6
Glisoxepide - 198.8 ± 2.4 - 120.9 ± 2.2 197.4 ± 5.1 - 22.4 ± 0.1 - 144.8 ± 2.6
(ab) A769662 - 144.7 ± 1.8 - 44.50 ± 6.1 31.60 ± 3.2 - 17.2 ± 0.1 - 174.7 ± 4.5
Ceftolozane - 210.7 ± 2.4 - 328.0 ± 11.5 485.9 ± 12 - 23.9 ± 0.2 - 76.8 ± 4.3
Ceftriaxone - 196.2 ± 2.6 - 191.0 ± 6.3 398.0 ± 7.6 - 20.2 ± 0.2 - 9.4 ± 3.9
Levomefolic - 99.00 ± 3.9 - 346.3 ± 19.3 671.9 ± 22.8 - 16.1 ± 0.4 210.7 ± 10.2
All values were calculated as kJ/mol

site analysis has revealed that Cangrelor and Levoleu- molecular docking (Hou et al. 2011).To further
covorin comprised about 10–11 hydrogen bonds the explore the interactions between AMPK and the
same as C2 (reference ligand), while Nacitentan and activators, the binding free energy decomposition
Glisoxepide showed less than 6 H-bonds. Addition- was also estimated. The binding free energies of the hit
ally, more information about hit compound-protein compounds in c docking site was encouraging because
interactions is presented in Table 2 and Fig. S6. We all of them had shown higher affinity as compared to
have found that Arg304, Arg75, Arg157 and Ser321 the reference (C2) compound. Table 3 illustrates that
were the key residues for H-bonds in C2complex. although the polar solvation energy was positively
Also, each of these residues could play a role in unfavorable for interactions, their SASA energy
binding of hit compounds via H-bond, p-effects or promoted the ligand binding capacity. Additionally,
hydrophobic interactions. It is obvious that Cangrelor it could be figured out that Van der Waals contribution
and Levoleucovorin have the most similarity in of hit compounds were much higher than C2, while
residues with the reference compound. their electrostatic contributions were weaker.
Figure 4 provides information about the number of MM/PBSA data revealed that binding free energies
H-bonds at ab binding site. Our results have shown of the hit compounds for the ab binding site were not
that Ceftolozane and Levomefolic acid had twice impressive compared to A769662 (Table 3). Among
hydrogen bonds than A769662; and Ceftriaxone them, Ceftolozane had the lowest binding free energy
comprised approximately same number of hydrogen (- 76.8 ± 4.3 kJ/mol) which is eventually a large
bonds as A769662. Furthermore, Table 2 and Fig. S7 amount compared to the reference compound. While,
illustrates that there was a rare similarity in the only Ceftolozane and Ceftriaxone had shown higher
residues between hit compounds and A769662. Van der Waals energy than A769662, all of them
exhibited lower value for electrostatic energy.
MM/PBSA and MM/GBSA data The binding free energy decomposition has revealed
the atomistic details of each residue in protein-activator
In spite of continuous effort for improving scoring interactions. As shown in Fig. 5, the common residues
functions, their precision for ranking the binding poses between C2 and hit compounds that contributed in the
and calculating binding free energies is still unsatis- interactions were Arg75, Arg157, Lys175, Lys248,
factory. Therefore, we recalculate the binding energy Arg304 (where Arg75, Arg157 and Arg304 were also
of top hit compounds by MM/GBSA, which is more mentioned in the Protein–Ligand interactions section).
rigorous than other available scoring function for It has been clarified from the present results that


Fig. 5 Decomposition of binding free energies on per-residue of c active site. a C2, b AMP, c Cangrelor, d Nacitentan,
e Levoleucovorin and f Glisoxepide

Levoleucovorin and Cangrelor interact with more Figure 6 provides information about free energy
similar residues compared to C2 than the other ligands. decomposition of ab binding site. According to the
Furthermore, the ligand-residue interaction energy was data, all the 3 hit compounds contributed to interaction
less for Nacitentan and Glisoxepide. with Gly41 and Val57 at subunit a and Arg108 at


Fig. 6 Decomposition of binding free energies on per-residue of ab active site of AMPK. a A769662, b Ceftolozane, c Ceftriaxone and
d levomefolic acid

compounds, Ceftolozane has shown the most similar

common residues with the reference compound.

KD-AID distance analysis

Under energy stress, AMP binds to c subunit, which

provides a more active conformation by pulling AID
away from KD domain. In this way, KD’s structure
switches to closed state, Hence, Thr172 is protected
from dephosphorylation (Xin et al. 2013). In order to
monitor the distance between the center of mass of KD
and AID during simulation, we used distance analyzes
from Gromacs utilities. As we expected, when AMP
Fig. 7 Average distance between the center of mass of KD and was bound to the binding site of the c subunit, the
AID at a subunit during simulation time distance between these two centers was at its maxi-
mum and in the presence of C2 in its binding site, the
subunit b. Arg108 is one of the most critical residues at distance was a bit lower than AMP (Fig. 7). All of the
ab binding site (Xiao et al. 2013). Among these 3 hit


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