Investigation of the free energy profiles of amantadine and rimantadine in the AM2 binding pocket

Eur Biophys J
DOI 10.1007/s00249-015-1077-y

ORIGINAL PAPER

Investigation of the free energy profiles of amantadine
and rimantadine in the AM2 binding pocket
Hung Van Nguyen1 · Hieu Thanh Nguyen1 · Ly Thi Le1,2 

Received: 10 June 2015 / Revised: 20 August 2015 / Accepted: 30 August 2015
© European Biophysical Societies’ Association 2015

Abstract  The purpose of this work was to study the
mechanism of drug resistance of M2 channel proteins by
analyzing the interactions between the drugs amantadine
and rimantadine and M2 channel proteins (including the
wild type and the three mutants V27A, S31N, and G34A)
and the drug binding pathways, by use of a computational
approach. Our results showed that multiple drug-binding
sites were present in the M2 channel, and the trajectory of
the drugs through the M2 channel was determined. A novel
method was developed to investigate of free energy profiles
of the ligand–protein complexes. Our work provides a new
explanation of the large amount of experimental data on
drug efficacy.
Keywords  AM2 virus · Pathway docking · Amantadine ·
Rimantadine · M2 pocket

Introduction
The influenza A M2 channel protein is a target in antiinfluenza drug design because of its importance in viral
infection (Holsinger and Lamb 1991; Sugrue and Hay

1991; Takeda et al. 2002). The tetrameric structure of the
M2 protein forms a pH-dependent channel across the viral
H. Van Nguyen and H. T. Nguyen contributed equally to this
work.
* Ly Thi Le

1

Life Science Laboratory, Institute for Computational Science
and Technology, Ho Chi Minh City, Vietnam

2

School of Biotechnology of International University, Vietnam
National University, Ho Chi Minh City, Vietnam




membrane for control of proton conductance when the
virus penetrates cells (Pinto et al. 1992; Pielak and Chou
2010; Lin and Schroeder 2001). Acidification weakens
electrostatic interactions between matrix proteins and ribonucleoprotein (RNP) complexes, causing the disintegration
of the viral membrane and the movement of the uncoated
RNP from the cytosol to the nucleus. Because of its crucial
involvement in influenza viral pathogenesis, a variety of
M2 channel structures have been solved for structure-based
drug development (Tran et al. 2013) by use of different
techniques, for example site-directed infrared dichroism
(Kukol et al. 1999), UV resonance Raman spectroscopy

(Okada et al. 2001), electron spin resonance, and solidstate nuclear magnetic resonance (NMR) spectroscopy
(Nishimura et al. 2002; Kovacs et al. 2000; Tian et al. 2003;
Schnell and Chou 2008).
The M2 channel has four identical subunits and contains 97
residues per monomer (Lamb et al. 1985) in three main segments:
an extracellular N-terminal segment (residues 1–23), a transmembrane (TM) segment (residues 24–46), and an intracellular
C-terminal segment (residues 47–97) (Pielak and Chou 2010).
The transmembrane (TM) segment is the main region responsible
for proton conduction and inhibition of the channel. In investigations of the proton conductance of M2 channel proteins for drug
development, this segment has been studied in depth (Nishimura
et al. 2002; Hu et al. 2007; Stouffer et al. 2008; Cady and Hong
2008; Cady et al. 2010; Acharya et al. 2010). Residues 24–46 of
the four monomers form a TM helix bundle lined by the polar
residues Val27, Ser31, Gly34, His37, Trp41, Asp44, and Arg45.
The tetrameric His37–Trp41 cluster is the center of acid activation and proton conductance (Tang et al. 2002; Venkataraman
et al. 2005; Hu et al. 2006). The ionizable His37 is essential for
proton selectivity, and acts as a channel sensor (Wang et al. 1995),
whereas Trp41 is important for unidirectional conductance and
acts as a proton gate (Pielak and Chou 2010; Tang et al. 2002).

13




In addition to the Trp41 gate, it has been recently been proposed
that Val27, together with His37, forms a secondary gate (Yi et al.
2008; Nguyen and Le 2015).
Several M2 inhibitors have been approved by the FDA.
In particular, 1-aminoadamantane hydrochloride, known

as amantadine, is the first efficient drug in influenza therapeutics. Amantadine indirectly frustrates virus activity by
using a hydrophobic cage to prevent proton conductance by
the ion-channel. The other drug approved for treating influenza A, rimantadine (α-methyl-1-adamantane methylamine
hydrochloride), has efficacy comparable with that of amantadine but with a greater risk of adverse side effects (Stephenson and Nicholson 2001; Jefferson et al. 2004). The
WHO has, however, restricted the application of amantadine and rimantadine for treatment of influenza A because
of the rapidly increasing occurrence of resistant strains
(Tran et al. 2011; Le and Leluk 2011; Tran and Le 2014).
The sites of binding of amantadine and rimantadine on
the M2 channel have been a controversial issue for more
than 20 years, because of the location of drug-resistant
mutations at residue 26, 27, 30, 31, 34, and 38 (Hay et al.
1985; Wang et al. 1993). Interestingly, the side chains of
amino acids 27, 31, and 34 are predicted to face the channel interior, leading to a hypothesis that the drugs bind to
the inside of the channel (Pielak and Chou 2010). Schnell
and Chou (2008), however, by use of nuclear Overhauser
effect (NOE) experiments, detected four equivalent binding sites of rimantadine outside the channel. Rimantadine
acts as a link between the two adjoining helixes and indirectly keeps the channel gate closed. Although recent study
has suggested that binding positions of amantadine and its
derivatives inside the M2 channel are more energetically
favorable, the mechanism of the binding process remains
unclear (Jing et al. 2008; Ohigashi et al. 2009). For this reason, study of the molecular mechanism of binding of M2
inhibitors to their wild type and mutant targets will provide
important insight into the mechanism of M2 drug resistance.
In this research, we used a novel method, called “pathway docking”, to investigate the interaction between the
M2 channel and amantadine and rimantadine during their
entry into the channel pore of the wild type (WT) and three
drug-resistant mutants (V27A, S31 N and G34A), to gain
insight into the effects of mutations.

Eur Biophys J

Table 1  Adamantane-based inhibitors. The colored branches are
functional groups and their z box sizes are determined by the maximum length they can reach
Inhibitor

Amantadine

Rimantadine

5.89

7.39

Chemical structure

z (Å)

from complex 3C9J and rimantadine was constructed on
the basis of the amantadine structure by use of GaussView 5.0; the geometry of the drugs was then optimized
by use of Gaussian 09 (Table 1) (Hada et al. 2004). The
V27A, S31N, and G34A mutant models were generated by
use of the mutagenesis tool of Visual Molecular Dynamics
(VMD) (Humphrey et al. 1996). The free energies of binding between the inhibitors and the M2 channels were predicted by AutoDock Vina software (Trott and Olson 2010).
Finally, the conformation having the lowest energy and
smallest RMSD from docking output was chosen for subsequent analysis.
Free energy scanning method

Materials and methods

The basis of this method is movement of the grid box for
docking along the M2 channel from serine 22 to histidine

37 while the box size is modified to ensure the inhibitors
bind to the inside of the channel only (Fig. 1). The binding energies corresponding to each step were then assembled to produce the energy profile. In particular, the center
of mass of serine 22 and histidine 37 were chosen to form
a symmetric axis. This axis was then parallelized with the
z-axis by pivoting the protein, the ligands were docked into
it after the center of box had been moved 0.05 Å along the
z-axis from Ser22 to His37, or approximately 25 Å. At each
step, the x and y dimensions of the box were determined by
the difference between the respective maximum and minimum of the x and y coordinates of the protein’s atoms; their
z coordinates were the interval of z box size. The largest
length of the ligands was used to define the z size of the
box and only one z box size corresponded to each inhibitor
(Table 1).

Materials

Analytical methods

The 3D structure of the M2 channel was taken from Protein
Data Bank (PDB; entry 2L0J, strain A/Udon/307/1972).
It was derived from a complex embedded in DMPC
liposomes (Sharma et al. 2010). Amantadine was extracted

The free energies predicted for the interactions between
the inhibitors and the M2 channels were represented as a
function of the z-coordinate of the center of the box when
the docking process was complete. On the basis of these

13

Eur Biophys J
Fig. 1  The pathway docking
method moves the grid box
0.05 Å every docking step from
serine 22 to histidine 37 along a
symmetric axis (paralleled with
the z axis) of the M2 pore (a),
the z box size is determined by
the largest size of the inhibitor (b) and is kept constant
throughout the molecular docking process

free energy values, geometric features, as functions of z,
were displayed as significant when compounds “moved”
from outside to inside the pore. In the docking method,
the ligands were separated into two parts, i.e. “root” and
“branches”. Specifically, the geometric center of the adamantane cage represents the “root”, and that of each functional group represents a “branch” (Fig. 2). Besides the root
and branches, the center of the inhibitor was associated

z
Branch

Center

0
Root

x

o

y

Fig. 2  Simplified rimantadine geometric features represented by
the centers of the root (red), branch (yellow), and whole compound
(green), and the angles between the rotatable bond (blue) and the z
axis, θ, and the x axis, φ

with a relative binding position in the M2 pore. The orientation of each compound was represented the angle, θ,
between the rotatable bond and the z-axis, and the angle,
φ, between the rotatable bond and the x-axis. To determine
their differences, however, the Δθ and Δφ values were a
better choice for characterization of the change of state of
the ligands; these were defined as:

∆θi = θi+1 − θi

(1)

∆φi = φi+1 − φi

(2)

Results and discussion
Image of the penetration of amantadine
and rimantadine: the coexistence of two binding sites
of different energy inside the M2 channel and their
motion along the M2 pore
As shown in Figs. 3 and 4, the images of the penetration of
amantadine and rimantadine were characterized by profiles

of the binding free energies and by Δθ and Δφ . Here, the
free energy values, which were indicative of strong interaction of the inhibitors binding along the pore, and the differences between the angles indicated the stable structure
of M2 channels. This means that the positions at the bound
states were stable when the difference between the angles

13




Eur Biophys J

kCal/mol

A

0

His37

-4

A

∆E

Val27

-2

15

CEN
Root
Branch

Val27

12
∆θ

9

Z (Å)

120
0

Ser31

6
3

-120

Degree

21
18

Ser31

Gly34

-6

Degree

N

C

∆o

120

Gly34

0
-3

0

-6

-120

-9
-6

-3

0

3

6

9

12

15

18

21

-9

24

-6

-3

0

3

kCal/mol

B

2

∆E

0

Gly34

0
-120

21

12

15

18

21

Ser31

6
3
Gly34

0
-3

∆o

120

18

Val27

9

∆θ

120

15

CEN
Root
NH2
CH3

12

Ser31

Z (Å)

Degree

-6

Degree

15

Val27

-4

12

21
18

-2

9

Z (Å)

Z (Å)

B

6

-6

0

-9

-120

-9
-6

-3

0

3

6

9

12

15

18

21

24

-6

-3

0

3

6

9

Z (Å)

Z (Å)

Fig. 3  Free energy profile (top) and differences between the z angles
θ (middle) and x angles φ (bottom) along the channel from the C to
N-terminus (blue left–right arrow) were predicted by pathway docking for amantadine (a) and rimantadine (b)

was close to 0. To clarify the relationship between the position of the box and the positions of amantadine and rimantadine, the z coordinates of the root, the branches, and whole
molecule were represented as a function of the z coordinate
for the center of the box.
As is apparent from Figs. 3 and 4, the pathway along
which amantadine enters the pore (above His37) from outside
(Ser22) was described as follows. The drug was first isolated
by water molecules; it then interacted with residues 22–26.
Next, it stayed in front of the Val27 barrier (14–17 Å) until it
crossed the barrier and went deeper into the pore. There would

be a region between Val27 and Ser31 (8–10 Å) where the
binding energy gradually declined from the maximum close
to Val27 to a lower value at Ser31. When moving through

13

Fig. 4  The z position of the center of the roots, branches, and the
whole amantadine (a) and rimantadine (b) molecules were represented as a function of the z position in alignment with the center of
the docking box

this area, the order of the root, branches, and the center of the
drug were unchanged (Fig. 4a). Here, the result shows that the
region between Val27 and Ser31 has stronger binding affinity
for amantadine. After passing Val27, there were two positions
that amantadine had bound to, which ranged from −3 to 0 Å
and from 3 to 7 Å; they were separated by the Gly34 residue.
Between these two positions, the bound state at the Ser31 pore
was more stable and the free energy (−6.1 kCal/mol) was
higher than at the other (−6.0 kCal/mol). Finally, it stopped in
front of the sensor His37 and Trp41 gate. These findings confirmed previous results:
1. the existence of the secondary gate Val27 permeable
not only to water molecules but also to amantadine (Yi
et al. 2008);


Eur Biophys J

2. the region between Val27 and Ser31 could be a provisional binding position before amantadine reached the
most stable or the real bound state (Sansom and Kerr
1993); and

3. two positions, Ser31–Gly34 (SG) (Cady et al. 2009)
and Gly34–His37 (GH) (Gandhi et al. 1999), which
were potential binding sites of amantadine.
New information from this representation were also valuable: although the plots of energies and angles indicated
that amantadine should be at pore SG, a minor difference
in energies suggests that it was also binding to the GH site.
In other words, there could be two significant binding sites,
resulting in a high possibility that amantadine could move
from one binding site to the other as a result of thermal
motion.
When amantadine penetrated the M2 channel via the pathway with the lowest energy, its orientation along the path
had rotated the hydrophobic cage and the hydrophilic head
(Fig. 4a). In front of residue Val27, its root—the adamantane
cage and its branch—the primary amine group, was horizontal
to the z-axis or at the same z position (12–18 Å). After crossing the barrier, they had turned vertically, the branch pointed
to Ser31 and the root pointed to Val27 (9–12 Å). This orientation was maintained for a short time before the root turned
back and pointed toward the C-terminal (7–9 Å). Next, its
branch turned back and pointed toward Gly34 whereas the
root pointed at Ser31 (0–7 Å). In addition, the primary amine
group bound at that position until the root crossed Gly34 and
pointed to His37 (−2 to 0 Å). The sequential orientation above
was considered reasonable only if the root and the branch of
amantadine exchanged their role as a “hook”. The cage would
hook around Val27 (11 Å) and the primary amine group
rotated around it. It then held at Ser31 (6.3 Å) and the cage
rotated around it. Finally, the primary amine group hooked to
Gly34 (0.7 Å) and the Adamantane cage could stay above it
in the most stable bound state or rotated around it to move to
the second stable state. This explains why experimental structures of the complex of amantadine and the M2 channel have
different positions (Stouffer et al. 2008; Yi et al. 2008; Cady

et al. 2009; Gandhi et al. 1999) and orientations (Stouffer et al.
2008; Cady et al. 2009; Gandhi et al. 1999). They were simply positions in the “walking” process of the inhibitors. The
“walking” process of amantadine is shown in Fig. 5. These
results explain why the resistant mutations span more than
three helical turns, whereas amantadine has a diameter of only
5 Å and cannot interact with the entire N-terminal half of the
M2 channel (Pielak and Chou 2010).
For rimantadine, the Val27, Ser31, and Gly34 residues
were still used as crucial factors for analysis of the process
of penetration of the inhibitor into the M2 pore (Fig. 4b).
Compared with amantadine, the free energy values and
binding sites were very different:

Root

Branch

Val27

Ser31

Gly34

His37

Fig. 5  Illustration of the walking process of amantadine in the M2
pore

1. Most of the points in the free energy plots for rimantadine were substantially lower than for amantadine,
and the plots of the angles for rimantadine also fluctuated less than for amantadine (Fig. 3). This means

rimantadine bound more strongly than amantadine, in
good agreement with the experimental observation that
rimantadine is more efficient than amantadine for treatment of influenza A infection (Stephenson and Nicholson 2001; Jefferson et al. 2004).
2. The binding sites of rimantadine were also shrunk
and were more specific than for amantadine in the SG
and GH regions, except for the area in front of Val27
(Fig.  3). After addition of a methyl group, the hydrophobicity of the functional group including the primary
amine group, had substantially increased. Rimantadine
bound closer to Val27 (a hydrophobic side chain) or
at the Ser22–Val27 (SV) region than amantadine. The
binding site at SG and GH was narrower and focused
on Ser31 and Gly34 (a polar and hydrophobic side
chain) for the same purpose. Therefore, the difference
between the energy of the bound state for SG or GH
was larger for rimantadine than for amantadine. In
other words, rimantadine bound strongly at S31 resi-

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Eur Biophys J

due, and, as a result, the probability of transition from
SG to the GH bound state was limited.
Effects of mutations on penetration: loss of the
inhibitor trap Val27, the unstable binding place
Asn31, and the third gate Ala34 blocked the channel
to amantadine and rimantadine

The major importance of the Val27, Ser31, and Gly34
residues and their mutants V27A, S31N, and G34A was
strongly confirmed by the mechanism of resistance to
amantadine (Wang et al. 1995; Hay et al. 1985). In this part
of our work the mechanisms of drug resistance of mutants

In more detail, the results showed that:

3
2

WT
S31N

Val27

-3
-8

∆E (kCal/mol)

1. V27A: the barrier at residue 27 had vanished;
2. S31N: the binding free energies in the SG region were
wider and deeper; and
3. G34A: when mutation occurred at the Gly34 residue (low barrier) to become Ala34 (steep barrier), the
inhibitors needed more energy to cross the barrier,
because the steep barrier of the Ala34 residue was
higher than that of Val27 residue and even higher than
that of the His37 residue.

A
S31N
WT

120

Ser31

Gly34

-6
3

∆θ

A

of the M2 channel were determined from the energy profile
or from the process of penetration of the inhibitors.
On the basis of the energy profile, we found that mutated
residues led to different effects of amantadine and rimantadine on the M2 channel (Fig. 6). It was found that:

V27A

2
-3

0
-120

-8
-6
3

120

G34A

2

o

-3

0

-8
-6

-120

-6

-4

2

4

0

6

13

19

15

21

24
-6

Z (Å)
WT
S31N

2

Gly34

∆E (kCal/mol)

3

6

9

12

15

18

21

9

12

15

18

21

B
120

Val27
Ser31

0

-6

0

Z (Å)

B
-4

-3

0

V27A

2

-120

-4
-6

120
G34A

o

2

0

-4
-120

-6
-6

-4

2

4

6

0

13

19

15

21

24

Z (Å)

Fig. 6  Energy profiles for amantadine (a) and rimantadine (b) in the
WT and three M2 channel variants: S31N, V27A, and G34A

13

-6

-3

0

3

6

Z (Å)

Fig. 7  Angle profiles of amantadine (a) and rimantadine (b) in the
WT and S31N mutant


Eur Biophys J

1. The unequal energies at the two sides of the secondary gate, Val27 (Fig. 3), created a gate trap for the
inhibitors. It was, therefore, easier for amantadine or
rimantadine to penetrate the pore than to move in the
opposite direction. The loss of the secondary gate was
caused by V27A involved vanishing of the trap, and
the inhibitors could not stably bind inside the V27A
mutant pore. Furthermore, the absence of the gate
Val27 increased water flux in the pore (Yi et al. 2008)
and indirectly reduced inhibition by the Adamantane
cage.
2. The deeper energy around the 31st mutant position
proved that both amantadine and rimantadine were

sufficiently bound. But, as is apparent from Fig. 7,
the large width led to unstable binding of amantadine
and rimantadine (0–10 Å). Minor differences between
the profiles for the mutant S31N and wild type were
revealed in our virtual experiments.
3. The mutant G34A contained in a third gate, which prevented deeper penetration of the inhibitors.

Each mutation has different effect on the inhibitor, but
the mechanism of drug-resistance of the M2 channel was
not apparent from our pathway docking results. One explanation could be that the virus replaced the Val27 gate,
which could longer prevent passage of the inhibitors getting through, by a new higher-energy gate at Gly34 and
mutated residues in front of the third gate (S31N is typical mutant), which have strong affinity for amantadine and
rimantadine.

Conclusions
To depict penetration of the M2 channel by amantadine and
rimantadine, the inhibitors were docked 500 times into the
wild type and its mutants (V27A, S31N, and G34A) by use
of pathway docking. At every docking step, the grid box
was moved 0.05 Å, in the direction from the N-terminus to
the C-terminus, and the box size was adjusted to ensure the
inhibitors were inside the channel. The energy and angle
profiles led to several significant findings.
1. There was not only one binding site for amantadine
and rimantadine in the M2 channel but two positions,
at Ser31 (SG region) and Gly34 (GH region). The
inhibitors bound at each position with different energies or probabilities, and the binding energy in the SG
region was higher than that in the other. The binding
energy difference was not large (0.1 kCal/mol) for
amantadine, which led to ease of transition between the

two positions. However, it was different for rimanta-

dine with the added methyl group. The binding affinity along the channel for rimantadine was higher and
more concentrated than for amantadine, at Ser31 and
Gly34; this is explained by the lower hydrophilicity of
the functional groups.
2. The hydrophobic cage and the primary amine group of
amantadine and rimantadine may act as hooks by use
of which the ligands step inside the channel at Val27,
Ser31, and Gly34. This explains the many empirical binding positions and mutations spanning the M2
channel.
3. The mechanism of drug resistance of M2 is probably
a result of three features of the mutants. First, residues
which interact strongly with the primary amine group
of the inhibitors (Ser31 in our work) are replaced by
residues with lower affinities. Second, water flux is
increased by loss of the secondary gate Val27 in the
V27A mutation. Finally, a replacement gate G34A is
created to prevent deeper penetration of the inhibitors
and to replace Val27 in control of water flux inside the
channel. These processes cause unstable binding of
inhibitors, thereby reducing inhibition by the hydrophobic cage of the drugs and preserving the normal
activity of the M2 channel.
Acknowledgments  The work was funded by the Vietnam National
Foundation for Science and Technology Development (NAFOSTED)
under grant number 106.01-2012.66. Computing resources and support provided by the Institute for Computational Science and Technology, Ho Chi Minh City, are gratefully acknowledged. We would
like to thank Professors Thanh Truong and Mai Suan Ly for valuable
advice.

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