Affinity of Rimantadine Enantiomers against Influenza A/M2 Protein Revisited

Article abstract of DOI:10.1021/acsmedchemlett.6b00311

Recent findings from solid state NMR (ssNMR) studies suggested that the (R)-enantiomer of rimantadine binds to the full M2 protein with higher affinity than the (S)-enantiomer. Intrigued by these findings, we applied functional assays, such as antiviral assay and electrophysiology (EP), to evaluate the binding affinity of rimantadine enantiomers to the M2 protein channel. Unexpectedly, no significant difference was found between the two enantiomers. Our experimental data based on the full M2 protein function were further supported by alchemical free energy calculations and isothermal titration calorimetry (ITC) allowing an evaluation of the binding affinity of rimantadine enantiomers to the M2TM pore. Both enantiomers have similar channel blockage, affinity, and antiviral potency.

Full text of DOI:10.1021/acsmedchemlett.6b00311

Letter
pubs.acs.org/acsmedchemlett
Affinity of Rimantadine Enantiomers against Influenza A/M2 Protein
Revisited
†
†
∥
‡
§
Antonios Drakopoulos, Christina Tzitzoglaki, Chulong Ma, Kathrin Freudenberger, Anja Hoffmann,
∥
‡
§
,∥
,†
Yanmei Hu, Gu
̈
nter Gauglitz, Michaela Schmidtke, Jun Wang,* and Antonios Kolocouris*
†
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, United States
∥
‡
§
Institut fu
Department of Virology and Antiviral Therapy, Jena University Hospital, Hans Knoell Str. 2, D-07745 Jena, Germany
S Supporting Information
̈ ̈
̈
r Physikalische und Theoretische Chemie, Eberhard-Karls-Universitat, D-72076 Tubingen, Germany
*
ABSTRACT: Recent findings from solid state NMR (ssNMR) studies
suggested that the (R)-enantiomer of rimantadine binds to the full M2
protein with higher affinity than the (S)-enantiomer. Intrigued by these
findings, we applied functional assays, such as antiviral assay and
electrophysiology (EP), to evaluate the binding affinity of rimantadine
enantiomers to the M2 protein channel. Unexpectedly, no significant
difference was found between the two enantiomers. Our experimental
data based on the full M2 protein function were further supported by
alchemical free energy calculations and isothermal titration calorimetry
(
ITC) allowing an evaluation of the binding affinity of rimantadine
enantiomers to the M2TM pore. Both enantiomers have similar channel
blockage, affinity, and antiviral potency.
KEYWORDS: Rimantadine enantiomers, isothermal titration calorimetry, free energy perturbation, Bennett’s acceptance ratio,
electrophysiology, synthesis, antiviral assay, membrane protein, influenza M2 pore
6
−9
mantadine (1) and rimantadine (2) (Scheme 1) are
channel blockers of proton transit by the influenza virus
occlusion of proton transit
and thereby preventing the viral
A
replication. The ssNMR results for 2 also demonstrated that
the ammonium group of the drug is pointing toward the four
H37 residues at the C-terminus. This orientation can be
9
Scheme 1. Structures of Studied Aminoadamantane
Derivatives Amantadine (1), Rimantadine (2, 2-R, and 2-S),
and Rimantadine Analogue 3
stabilized either through hydrogen bonds between the
ammonium group of the aminoadamantane ligand and water
molecules in the channel lumen which exist between the
13
imidazoles of H37 and the ligand, and/or with A30 carbonyls
1
4
9,14−16
in the vicinity, according to experimental
and MD
1
3,17−22
simulations data.
Provided that M2TM is a minimal
10
model for M2 binding, these high resolution structures can be
used for the development of new ligands which may bind more
effectively to the M2TM pore.
1
,2
M2 proton channel
and long used prophylactics and
3
The effect of ligand’s chirality in its binding with a chiral
receptor is of outstanding significance and the characterization
of protein−ligand interactions for each enantiomer separately
may identify potential stereospecific binding interactions to the
receptor. While rimantadine analogues are known antiviral
drugs for more than four decades, the relative potency of
rimantadine enantiomers has not been studied at the molecular
level. The binding affinity of each enantiomer results from
therapeutics against influenza A viruses. The primary binding
site of 1 and 2 is the lumen of the transmembrane domain of a
tetrameric M2 protein (M2TM: amino acids 22−46) that
4
forms the proton transit path.
Although 1 and 2 have been used as antivirals for decades, it
was only after 2008 that high resolution structures from X-ray
and ssNMR experiments unveiled the structures of M2TM in
5
−9
complex with 1 or 2.
According to these findings, the
M2TM protein channel is blocked by 1 or 2 via a pore-binding
6
−10
mechanism.
other aminoadamantane analogues,
all sides by V27 and A30 side chains, producing a steric
The adamantane cage in 1 or 2, as well as in
Received: August 12, 2016
Accepted: January 13, 2017
11−13
is tightly contacted on
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00311
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ACS Medicinal Chemistry Letters
Letter
chiral interactions with the binding area inside the 4-fold
symmetric M2 protein. Based on differences in isotropic
chemical shift changes measured using ssNMR and MD
simulations results, it has been recently suggested that 2-R
and 2-S have a strong but differential binding to full length M2,
Scheme 2. Synthetic Scheme for the Preparation of tert-Alkyl
Amine 3
23
i.e., that 2-R binds more tightly than 2-S. This was the first
state of the art ssNMR study of the full M2 protein and analysis
of the rimantadine enantiomers binding by ssNMR, but this
conclusion appears to be puzzling because: (1) 2-R and 2-S
have similar in vivo antiviral activity in protecting mice from
2
4
lethal influenza; (2) rimantadine was developed prior to the
992 FDA guidance on the development of stereoisomers. It
1
was approved as commercial drug in the US in 1993 containing
both enantiomers. To solve the controversy between the in
vitro binding assay, i.e., ssNMR results, and the in vivo efficacy
results, we hereby characterize the binding affinity of the two
rimantadine enantiomers and their antiviral efficacy using a
consortium of in vitro and cellular assays and biophysical/
computational studies.
Table 1. Antiviral Activity of Compounds 1−3 against
Influenza Virus A/Udorn/72 (H3N2) and A/WSN/33-M2-
N31S (H1N1) in Madin−Darby Canine Kidney Cells
a
a
compd IC50 (μM) (A/Udorn/72) IC50 (μM) (A/WSN/33-M2-N31S)
Taken together, our results demonstrate that rimantadine
enantiomers (2-R and 2-S) have equal potency. Since the
controversy aroused from previous ssNMR studies of 2-R, 2-S
bound to the full M2 protein, we first provided data including
the antiviral activity of 2-R, 2-S against amantadine-sensitive
influenza A virus strains and the blocking effect of the
enantiomers against M2 using EP. Then we investigated the
effect of the enantiomers to M2TM using biophysical
techniques. More specifically, the antiviral potency of
compounds 2-R, 2-S, and 3 against amantadine-sensitive A/
Udorn/72 and A/WSN/33-M2-N31S viruses was measured.
The blocking effect of 2-R, 2-S, and 3 against full length A/
Udorn/72 M2 protein (M2Udorn/72) and A/WSN/33-M2-N31S
protein (M2WSN/33‑N31S) conductance was measured using EP
via a two-electrode voltage clamp (TEVC) assay. Compound 3
is a nonchiral dimethyl analogue of rimantadine, and it was
designed and synthesized as a probe to independently validate
the biophysical assay results from 2-R and 2-S, as described
below. The ITC binding affinities of 2-R, 2-S, and 3 against the
M2TMUdorn/72 in its closed form at pH 8 were measured.
Following up, we applied a free energy perturbation coupled
with MD simulations (FEP/MD) scheme to calculate the
relative free energies of binding between the rimantadine
enantiomers (2-R and 2-S) as well as compound 3 to
M2TMUdorn/72. In particular, we calculated the relative free
energies of binding for the alchemical transformations of 3 to 2-
R and 3 to 2-S.
1
2
2
2
3
0.33 ± 0.04
0.05 ± 0.02
0.05 ± 0.01
0.06 ± 0.02
0.04 ± 0.01
0.48 ± 0.05
0.04 ± 0.02
0.04 ± 0.01
0.02 ± 0.01
0.03 ± 0.02
-R
-S
a
Mean and standard deviations of the 50% inhibitory concentration
IC ) of at least three independent measures.
(
50
28
protein as in a previous report. Specifically, the blocking effect
of the aminoadamantane derivatives against M2 was inves-
tigated with electrophysiology experiments using M2Udorn/72
(
Table 2). The potency of the inhibitors was expressed as the
Table 2. Block of Full-Length M2-Dependent Current by
Adamantane Analogues
a
M2Udorn/72
M2WSN/33‑N31S
% block after
2 min
% block after
5 min
% block after
2 min
% block after
5 min
compd
1
90 ± 2%
100 μM; 3)
95 ± 1%
(100 μM; 3)
(
2
96 ± 1%
100 μM; 3)
96 ± 1%
(100 μM; 3)
84 ± 1%
(100 μM; 3)
93 ± 0%
(100 μM; 3)
(
2
-R
93 ± 1%
(100 μM; 3)
95 ± 1%
(100 μM; 3)
71 ± 1%
(100 μM; 3)
90 ± 1%
(100 μM; 3)
2-S
95 ± 1%
100 μM; 3)
96 ± 1%
(100 μM; 3)
78 ± 1%
(100 μM; 4)
92 ± 0%
(100 μM; 4)
(
3
90 ± 2%
100 μM; 3)
96 ± 1%
(100 μM; 4)
56 ± 3%
(100 μM; 3)
80 ± 2%
(100 μM; 3)
(
For the synthesis of primary tert-alkyl amine 3 (AdC-
Me NH ), the tert-alkyl alcohol 5 was prepared according to
a
For each compound, percent block of pH-dependent M2 current at
listed concentrations (± SEM). Parentheses show number of
replicates.
2
2
Scheme 2 from the Grignard reaction between 1-adamantane
carbonyl chloride 4 and methylmagnesium iodide. Treatment
of tert-alkyl alcohol 5 with NaN /CF CO H in dichloro-
3
3
2
methane afforded azide 6 in high yield, which was further
inhibition percentage of the A/M2 current observed after 2 and
5 min of incubation with 100 μM compound. The electro-
physiology experiments against M2Udorn/72 showed that 2-R and
2-S block the M2 channel equally to amantadine (about 90%)
at a concentration of 100 μM. In addition, we also tested these
aminoadamantane analogues in inhibiting another amantadine-
sensitive M2 channel, M2WSN/33‑N31S, which contains the V28I
mutation in M2TM sequence, with an electrophysiology assay.
Again, no significant difference in channel blockage after 5 min
subjected to reduction through LiAlH to form tert-alkyl amine
4
3
in a good yield.
An antiviral assay was used
25,26
to compare the antiviral
activity of 1, 2, 2-R, 2-S, and 3 against two amantadine-sensitive
influenza A strains, A/Udorn/72 (H3N2), and A/WSN/33-
2
7
M2-N31S (H1N1) in MDCK cells. All compounds showed
submicromolar EC50 values against both influenza strains, and
there was no significant difference between the two rimantadine
enantiomers 2-R and 2-S (Table 1).
28
was found among 2-R, 2-S, and 3. As discussed previously,
The inhibitors were tested via TEVC assay using X. laevis
these measurements at 2 or 5 min are made prior to the
frog oocytes microinjected with RNA expressing the M2
establishment of equilibrium due to very slow on and off rates
B
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Letter
Table 3. Binding Constant, Free Energy, Enthalpy, and Entropy of Binding Derived from ITC Measurements for M2TMUdorn/72
a
b
c
,
d
c
,
e
c,f
ligand
Kd
ΔG
ΔH
−TΔS
1
2.17 ± 0.52
0.51 ± 0.26
0.32 ± 0.16
0.34 ± 0.12
0.13 ± 0.12
−7.77 ± 0.14
−8.64 ± 0.30
−8.97 ± 0.26
−8.88 ± 0.21
−9.30 ± 0.43
−6.66 ± 0.50
−7.60 ± 0.28
−7.54 ± 0.34
−7.73 ± 0.28
−1.11 ± 0.52
−1.04 ± 0.41
−1.42 ± 0.43
−1.15 ± 0.35
−5.12 ± 0.51
2
g
2
2
3
-R
g
-S
−4.19 ± 0.28
a
b
−1
−6
See Scheme 1. Binding constant K in μM calculated from measured K in M by K = 1/K × 10 and error in K in μM determined by K
(Ka, error/K ) × 10 (ITC measurements were performed in triplicate for each ligand to calculate means and standard deviations). In kcal mol .
Free energy of binding computed from K by ΔG = −RT ln(K /K ) with Kd = 1 M and T = 300 K and error in ΔG determined according to
d
a
d
a
d
d, error
2
a
−6
c
−1
=
d
ref
d
ref
d
d
2
RTKd,error
e
ΔGerror
=
with T = 300 K. Enthalpy of binding and error in the enthalpy of binding calculated from measured binding enthalpy and
(
Kd
)
measured error by ΔH = ΔH
(T/Tmeasured) with T = 300 K and the temperature at which the ITC measurements were performed Tmeasured =
measured
f
2
2
2
g
93.15 K. Entropy of binding calculated by ΔS = (−ΔG + ΔH)/T and error in ΔS computed by the equation ΔS
The purity of each enantiomer used was 90% for 2-R and 95% for 2-S; the enantiomeric excess (ee) of both 2-R and 2-S is 99% (Mosher’s method);
the purity of compound 3 used was >99%.
=
ΔG
+ ΔH
.
error
error
error
for entry of thedrugs into the constricted M2 channel and the
problems of maintaining cells at low pH for extended periods.
Table 3 includes thermodynamic parameters of binding
against M2TMUdorn/72. Binding affinities were determined by
Table 4. Relative Binding Free Energies for Pairs of
Compounds Computed with the BAR Method for M2TM
Embedded in a DMPC Bilayer or Derived from
Experimental Binding Affinity Data in Table 3
2
9
ITC for M2TM-ligand systems in dodecylphosphocholine
DPC) micelles at pH 8, where M2TM fragments form stable
M2TMUdorn/72
(
3
0
a,
b
a,c
transformation
ΔΔGFEP
ΔΔGexp
tetramers. ITC measurements yield the enthalpy of binding
(
ΔH) as well as the dissociation constant (K ). From K , the
3 → 2-R
3 → 2-S
0.62 ± 0.14
0.68 ± 0.15
0.33 ± 0.50
0.42 ± 0.48
d
d
binding free energy (ΔG) is calculated (Table 3). The
estimation of the binding entropy is based on the difference
between ΔG and ΔH. Binding constants of 1, 2-R, 2-S, and
racemate 2 were measured in a previous work and were
included in Table 1. The enantiomers 2-R and 2-S may have a
different enthalpy of binding against M2TM protein since the
aIn kcal mol−1. Propagation error calculated according to the
bootstrap method. Difference in binding free energy calculated from
experimentally determined K
with T = 300 K and error calculated from individual experimental
b
32 c
13
A
B
values by ΔΔG = −RT ln (K /K )
d
d d
2
+ errorΔG,B2)
errors by error
=
(error
.
ΔΔG
ΔG,A
31
two complexes formed are diastereomers. As depicted in
Table 3, enantiomers 2-R and 2-S have the same K values
for retaining proper M2TM structure compared to other
35
d
against M2TMUdorn/72 (K = 0.34 and 0.32 μM respectively).
glycerophospholipids. We used an experimental structure
d
13
5,7
These values are close to the K = 0.51 μM of the racemic 2,
(PDB ID 2KQT ) determined at pH 7.5 in DMPC bilayers;
d
considering the errors of the measurements and that the
commercially available enantiomers have a lower chemical
purity compared to racemic 2 (see Table 3, note g). An effect
from the impurities on the ITC results of 2-R and 2-S cannot
be excluded. However, we do not expect large changes in the
the restraints of the apo-M2TM structure were originally
5
measured by the Cross group. This M2TM
structure is
Udorn/72
already adapted to these environmental conditions, and thus
only a short equilibration phase is required. The experimental
relative binding free energy values (ΔΔG ) for the trans-
exp
−1
measured K values for 2-R and 2-S, given also the very similar/
formations 3 → 2-R and 3 → 2-S were 0.33 and 0.42 kcal mol
d
identical affinity results of the two enantiomers from TEVC and
antiviral assays. Compound 3, having two methyl groups
(Table 4), favoring 3. The experimental relative binding free
energy values were quite close to calculated values (ΔΔGFEP) of
−1
instead of one methyl group in 2, has the smallest K (0.13
0.62 and 0.68 kcal mol , respectively (the accuracy of the
33,34
d
μM), i.e., the highest binding affinity of all studied amino-
adamantane compounds, suggesting that polar and lipophilic
characteristics are well balanced in its structure. All three
compounds (2-R, 2-S, and 3) were more potent than
amantadine (1).
calculations method is ∼1 kcal mol).
The calculations also
predict the experimental finding by ITC that 2-R and 2-S have
the same binding affinity against M2TMUdorn/72 (Table 3),
which is consistent with the results from aforementioned
antiviral and electrophysiology assays performed using full M2
protein (Tables 1 and 2). Thus, using the functional core of M2
(transmembrane domain) for binding studies is appropriate.
The simulated complexes of M2TMUdorn/72 with 2-R, 2-S,
and 3 showed that the height of the ligands inside the pore
differed only slightly, that is less than 0.3 Å toward the N-
terminus relatively to 1 (Table S1), and the orientation of 2-R
and 2-S in the pore differs only slightly, in accordance to their
induced similar ssNMR chemical shifts for S31 and G34 when
We then analyzed the binding properties of 2-R and 2-S by
1
3,32
performing alchemical free energy calculations
Bennett acceptance ratio (BAR) method.
using the
33,34
The calculations
for the alchemical transformations 3 → 2-R, 3 → 2-S were run,
and the results of the FEP/MD predictions were compared
with binding affinities measured by ITC (Table 4). The
computational predictions were set by employing a protocol
successfully benchmarked by our group in order to match
13
23
experimental conditions as closely as possible. M2TMUdorn/72
structure was simulated in the closed conformation found at
high pH and after assigning a neutral form for all H37.
M2TMUdorn/72-ligand complexes were simulated in DMPC
bilayers, which represent an optimal membrane mimetic system
complexed with M2. The center of mass between the four
V27 and the adamantyl ring of the ligand (V27-Ad) varies
between 4.0 and 4.5 Å on average (Table S1). For 2-R and 2-S
the average tilt angle was measured ∼14°, in accordance to
9
experimental ssNMR values, and for 3 the average tilt angle
C
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Figure 1. Representative replicas from the simulation (a) of 2-S and (b) of 2-R bound to M2TMUdorn/72. Seven and eight waters are shown between
the ligand and H37 residues, respectively. Three hydrogen bonds between the ammonium group of the ligand and three water molecules are shown.
Hydrogen bonding together with van der Waals interactions of the adamantane core with V27 and A30 stabilize the ligand inside the pore with its
ammonium group oriented toward the C-terminus.
was measured ∼5°. The angle between the pore axis or the
normal of the membrane and C−N bond vector was ∼11° for 1
and close to 50° for 2-R, 2-S, and 3. These angle values suggest
that the ammonium group of all aminoadamantane compounds
is oriented toward the C-terminus, in consistency with previous
geometric measures, which reflect van der Waals contacts, were
found to be similar for both enantiomers (see Table S1). These
measures are consistent with the calculated relative binding
affinities, which are in accordance to the ITC data and
functional assays described previously.
6
,7,9
13,32,36
experimental findings
and MD simulations
(Table
Our results demonstrated no difference in the binding affinity
S1). The distance between the adamantyl ring and the center of
between the two enantiomers, whereas the recent ssNMR
23
mass between the four A30 (Ad-A30) for 1−3 was measured
study concluded that 2-R is a stronger binder than 2-S.
According to our best understanding, the authors in ref 23
made the following important observations in the ssNMR
spectra regarding the relative binding of 2-R and 2-S. In the
presence of 2-R or 2-S a new resonance (i.e., one that was not
recorded in the spectrum of the apo-M2 protein) was observed
for S31 at 120/64 ppm in the 15N/ Ca spectrum, which had
strong intensity for both enantiomers. The G34 resonance had
also a stronger intensity for both enantiomers compared to the
spectrum of the apo-M2. The S31 and G34 resonance
frequencies were similar for both 2-R and 2-S. These results
provided an experimental evidence that both enantiomers bind
strongly and the binding site and orientation of the drug in the
pore are similar for the two enantiomers as mentioned in ref 23.
However, the authors reported the appearance of an additional
resonance of medium intensity for the 2-S enantiomer at 115/
63 ppm close to the frequencies of the S31 resonance of the
unbound M2 state at ∼114/62 ppm and suggested a weaker
∼
1 Å, and the distance CH (lig.)-G34Ca for 2-R, 2-S, and 3
3
was 2.9, 3.2 and 2 Å respectively, close to the REDOR
23
measurements for 2-R, 2-S. The adamantyl ring was embraced
by the V27 and A30 side chains, which defined the binding site
of the ligands. Compounds 1, 2-R, 2-S, and 3 form hydrogen
bonds (average of three hydrogen bonds) through the
ammonium group with neighboring water molecules which
are positioned between the ligand and H37 residues. In the area
located below the adamantyl ring toward the N-terminus no
13
1
3,22,32,36
waters were found,
which is consistent with the proton
blocking effect of 1 and other aminoadamantane deriva-
1
,12,28
tives.
A snapshot from the simulation of ligands 2-R and
2
-S is depicted in Figure 1. The CHCH fragment, which
3
includes the chiral carbon of 2-R, 2-S, fits into the cleft between
G34 and A30, with the later being a chiral amino acid that can
differentiate binding interactions between the two enantiomers.
However, the distance CH (lig.)-A30CH , which is similar for
3
3
both enantiomers (3.9 ± 0.3 and 3.5 ± 0.3 Å), suggests no
difference in their van der Waals interactions. The measures
suggest that hydrogen bonding interactions for 2-R, 2-S and
23
binding of 2-S. Possibly the results published by Wright et al.
are not in full agreement with those reported here as a
D
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ACS Medicinal Chemistry Letters
Letter
consequent of the different methodology they applied, i.e.,
ssNMR spectroscopy.
ACKNOWLEDGMENTS
■
We are grateful to Chiesi Hellas for supporting this research
and the Ph.D. work of C.T.; J.W. thanks the support from NIH
AI119187 and the PhRMA Foundation 2015 Research Starter
Grant in Pharmacology and Toxicology; A.H. and M.S. thank
Andreas Sauerbrei for continuous kind support.
In conclusion, our results showed that rimantadine
enantiomers (2-R and 2-S) bind equally well to the M2 proton
channel and have equal channel blockage and antiviral activity
against amantadine-sensitive M2 channels. This conclusion was
supported by a consortium of techniques including antiviral
assays, electrophysiology ITC, and FEP/MD. Our nonclinical
results support the previous use of rimantadine as a racemic
mixture drug for the prevention and treatment of influenza
virus infection. Further correlation of these results with the
pharmacokinetic and pharmacodynamic properties for each
enantiomer in humans would confirm these findings. For
example, although there are no significant differences in the
concentration−time profiles and disposition of 2-R and 2-S and
ABBREVIATIONS
■
M2TM, residues 22−46 of M2 protein comprising the
transmembrane domain; CPE assay, cytopathic effect assay;
ssNMR spectroscopy, solid state Nuclear Magnetic Resonance
spectroscopy; DMPC, 1,2-dimyristoyl-sn-glycero-3-phospho-
choline; FEP, free energy perturbation; MD, molecular
dynamics; REDOR experiment, rotational echo double
resonance experiment; ITC, isothermal titration calorimetry;
EP, electrophysiology; TEVC assay, two-electrode voltage
clamp assay; BAR, Bennett acceptance ratio; PDB, protein
data bank; IC50, 50% inhibitory concentration; ee, enantiomeric
excess
37
of the 3-hydroxyrimantadine metabolites, large stereospecific
differences in the disposition of their 4-hydroxyrimantadine
38
metabolites are observed. However, it should be noted that 3-
and 4-hydroxy metabolites, both of which are found in
rimantadine-treated patients, showed only modest inhibitory
activity against influenza A virus, i.e., they are modestly active
3
9
metabolites.
REFERENCES
■
(
1) Wang, C.; Takeuchi, K.; Pinto, L. H.; Lamb, R. A. Ion channel
activity of influenza A virus M2 protein: characterization of the
amantadine block. J. Virol. 1993, 67, 5585−94.
2) Chizhmakov, I. V.; Geraghty, F. M.; Ogden, D. C.; Hayhurst, A.;
ASSOCIATED CONTENT
Supporting Information
■
*
S
(
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00311.
Antoniou, M.; Hay, A. J. Selective proton permeability and pH
regulation of the influenza virus M2 channel expressed in mouse
erythroleukaemia cells. J. Physiol. 1996, 494, 329−36.
(
3) Hayden, F. G. Clinical applications of antiviral agents for
Supplementary material including computational and
experimental protocols, as well as one table with
measures from simulations, and references (PDF)
chemoprophylaxis and therapy of respiratory viral infections. Antiviral
Res. 1985, 5 (Suppl 1), 229−39.
(4) Wang, J.; Qiu, J. X.; Soto, C.; DeGrado, W. F. Structural and
dynamic mechanisms for the function and inhibition of the M2 proton
channel from influenza A virus. Curr. Opin. Struct. Biol. 2011, 21, 68−
80.
AUTHOR INFORMATION
■
(
5) Hu, J.; Asbury, T.; Achuthan, S.; Li, C.; Bertram, R.; Quine, J. R.;
Corresponding Authors
Fu, R.; Cross, T. A. Backbone structure of the amantadine-blocked
trans-membrane domain M2 proton channel from Influenza A virus.
Biophys. J. 2007, 92, 4335−43.
*
Tel: (+301) 210-7274834. Fax: (+301) 210 727 4747. E-mail:
ankol@pharm.uoa.gr.
Tel: 520-626-1366. Fax: 520-626-0749. E-mail: junwang@
*
(
6) Stouffer, A. L.; Acharya, R.; Salom, D.; Levine, A. S.; Di Costanzo,
pharmacy.arizona.edu.
L.; Soto, C. S.; Tereshko, V.; Nanda, V.; Stayrook, S.; DeGrado, W. F.
Structural basis for the function and inhibition of an influenza virus
proton channel. Nature 2008, 451, 596−9.
7) Cady, S. D.; Schmidt-Rohr, K.; Wang, J.; Soto, C. S.; Degrado, W.
F.; Hong, M. Structure of the amantadine binding site of influenza M2
ORCID
Jun Wang: 0000-0002-4845-4621
Antonios Kolocouris: 0000-0001-6110-1903
(
proton channels in lipid bilayers. Nature 2010, 463, 689−92.
Present Address
(
8) Pielak, R. M.; Oxenoid, K.; Chou, J. J. Structural investigation of
(
A.D.) Pharmaceutical and Medicinal Chemistry, Institute of
Pharmacy and Food Chemistry, Julius-Maximilians-Universitat
Wurzburg, Am Hubland, 97074 Wurzburg, Germany.
Author Contributions
rimantadine inhibition of the AM2-BM2 chimera channel of influenza
̈
viruses. Structure 2011, 19, 1655−1663.
̈
̈
(9) Cady, S. D.; Wang, J.; Wu, Y.; DeGrado, W. F.; Hong, M. Specific
binding of adamantane drugs and direction of their polar amines in the
pore of the influenza M2 transmembrane domain in lipid bilayers and
dodecylphosphocholine micelles determined by NMR spectroscopy. J.
Am. Chem. Soc. 2011, 133, 4274−84.
A.D. and C.T. contributed equally. This research includes part
of the Master thesis of A.D. and part of the Ph.D. work of C.T.
A.K. designed this research project. A.D. and C.T. both did FEP
calculations and ligand synthesis. C.M. and Y.H. in J.W. group
did the detailed EP experiments against the A/Udorn/72 and
A/WSN/33-M2-N31S M2 and CPE assay testing against A/
Udorn/72 virus. K.F. in G.G. group did the ITC measurements.
A.H. in M.S. group performed CPE inhibitory assays with A/
WSN/33-M2-N31S virus. A.K. wrote the manuscript, and J.W.,
M.S., A.H., and A.D. revised it.
̈
(10) Ma, C.; Polishchuk, A. L.; Ohigashi, Y.; Stouffer, A. L.; Schon,
A.; Magavern, E.; Jing, X.; Lear, J. D.; Freire, E.; Lamb, R. A.;
DeGrado, W. F.; Pinto, L. H. Identification of the functional core of
the influenza A virus A/M2 proton-selective ion channel. Proc. Natl.
Acad. Sci. U. S. A. 2009, 106, 12283−12288.
(11) Kolocouris, N.; Foscolos, G. B.; Kolocouris, A.; Marakos, P.;
Pouli, N.; Fytas, G.; Ikeda, S.; De Clercq, E. Synthesis and antiviral
activity evaluation of some aminoadamantane derivatives. J. Med.
Chem. 1994, 37, 2896−2902.
Notes
(12) Kolocouris, A.; Tzitzoglaki, C.; Johnson, F. B.; Zell, R.; Wright,
The authors declare no competing financial interest.
A. K.; Cross, T. A.; Tietjen, I.; Fedida, D.; Busath, D. D.
E
DOI: 10.1021/acsmedchemlett.6b00311
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Aminoadamantanes with persistent in vitro efficacy against H1N1
(29) Chaires, J. B. Calorimetry and thermodynamics in drug design.
Annu. Rev. Biophys. 2008, 37, 135−151.
(30) Salom, D.; Hill, B. R.; Lear, J. D.; DeGrado, W. F. pH-
Dependent tetramerization and amantadine binding of the trans-
membrane helix of M2 from the influenza A virus. Biochemistry 2000,
39, 14160−14170.
(31) Fokkens, J.; Klebe, G. A simple protocol to estimate differences
in protein binding affinity for enantiomers without prior resolution of
racemates. Angew. Chem., Int. Ed. 2006, 45, 985−989.
(32) Gkeka, P.; Eleftheratos, S.; Kolocouris, A.; Cournia, Z. Free
energy calculations reveal the origin of binding preference for
aminoadamantane blockers of influenza A/M2TM pore. J. Chem.
Theory Comput. 2013, 9, 1272−1281.
(
2009) influenza A. J. Med. Chem. 2014, 57, 4629−39.
(
13) Ioannidis, H.; Drakopoulos, A.; Tzitzoglaki, C.; Homeyer, N.;
Kolarov, F.; Gkeka, P.; Freudenberger, K.; Liolios, C.; Gauglitz, G.;
Cournia, Z.; Gohlke, H.; Kolocouris, A. Alchemical free energy
calculations and isothermal titration calorimetry measurements of
aminoadamantanes bound to the closed state of influenza A/M2TM. J.
Chem. Inf. Model. 2016, 56, 862−876.
(
14) Andreas, L. B.; Barnes, A. B.; Corzilius, B.; Chou, J. J.; Miller, E.
A.; Caporini, M.; Rosay, M.; Griffin, R. G. Dynamic nuclear
polarization study of inhibitor binding to the M2(18−60) proton
transporter from influenza A. Biochemistry 2013, 52, 2774−82.
(
15) Yi, M.; Cross, T. A.; Zhou, H.-X. Conformational heterogeneity
of the M2 proton channel and a structural model for channel
(33) Bennett, C. H. Efficient estimation of free energy differences
from Monte Carlo data. J. Comput. Phys. 1976, 22, 245−268.
(34) Chipot, C., Pohorille, A., Eds. Free Energy Calculations. Theory
and Applications in Chemistry and Biology; Springer Verlag: Berlin, NY,
2007.
activation. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 13311−13316.
(
16) Luo, W.; Hong, M. Conformational changes of an ion channel
detected through water-protein interactions using solid-state NMR
spectroscopy. J. Am. Chem. Soc. 2010, 132, 2378−84.
(35) Cady, S.; Wang, T.; Hong, M. Membrane-dependent effects of a
(
17) Intharathep, P.; Laohpongspaisan, C.; Rungrotmongkol, T.;
cytoplasmic helix on the structure and drug binding of the influenza
Loisruangsin, A.; Malaisree, M.; Decha, P.; Aruksakunwong, O.;
Chuenpennit, K.; Kaiyawet, N.; Sompornpisut, P.; Pianwanit, S.;
Hannongbua, S. How amantadine and rimantadine inhibit proton
transport in the M2 protein channel. J. Mol. Graphics Modell. 2008, 27,
virus M2 Protein. J. Am. Chem. Soc. 2011, 133, 11572−11579.
(36) Wang, J.; Ma, C.; Fiorin, G.; Carnevale, V.; Wang, T.; Hu, F.;
Lamb, R. A.; Pinto, L. H.; Hong, M.; Klein, M. L.; DeGrado, W. F.
Molecular dynamics simulation directed rational design of inhibitors
targeting drug-resistant mutants of influenza A virus M2. J. Am. Chem.
Soc. 2011, 133, 12834−12841.
3
(
42−348.
18) Khurana, E.; Devane, R. H.; Dal Peraro, M.; Klein, M. L.
Computational study of drug binding to the membrane-bound
tetrameric M2 peptide bundle from influenza A virus. Biochim.
Biophys. Acta, Biomembr. 2011, 1808, 530−537.
(37) Miwa, B. J.; Choma, N.; Brown, S. Y.; Keigher, N.; Garland, W.;
Fukuda, E. K. Quantitation of the enantiomers of rimantadine in
human plasma and urine by gas chromatography-mass spectrometry. J.
Chromatogr., Biomed. Appl. 1988, 431, 343−352.
(
19) Leonov, H.; Astrahan, P.; Krugliak, M.; Arkin, I. T. How Do
aminoadamantanes block the influenza M2 channel, and how does
(38) Choma, N.; Davis, P. P.; Edorn, R. W.; Fukuda, E. K.
resistance develop? J. Am. Chem. Soc. 2011, 133, 9903−9911.
Quantitation of the enantiomers of rimantadine and its hydroxylated
metabolites in human plasma by gas chromatography/mass spectrom-
etry. Biomed. Chromatogr. 1992, 6, 12−15.
(
20) Gianti, E.; Carnevale, V.; DeGrado, W. F.; Klein, M. L.; Fiorin,
G. Hydrogen-bonded water molecules in the M2 channel of the
influenza a virus guide the binding preferences of ammonium-based
inhibitors. J. Phys. Chem. B 2015, 119, 1173−83.
(39) Manchand, P. S.; Cerutti, R. L.; Martin, J. A.; Hill, C. H.;
Merrett, E. K.; Keech, E.; Belshe, R. B.; Connell, E. V.; Sim, I. S.
Synthesis and antiviral activity of metabolite of rimantadine. J. Med.
Chem. 1990, 33, 1992−1995.
(
21) Wang, J.; Ma, C.; Fiorin, G.; Carnevale, V.; Wang, T.; Hu, F.;
Lamb, R. A.; Pinto, L. H.; Hong, M.; Klein, M. L.; DeGrado, W. F.
Molecular dynamics simulation directed rational design of inhibitors
targeting drug-resistant mutants of influenza A virus M2. J. Am. Chem.
Soc. 2011, 133, 12834−12841.
(
22) Gleed, M. L.; Ioannidis, H.; Kolocouris, A.; Busath, D. D.
Resistance-mutation (N31) effects on drug orientation and channel
hydration in amantadine-bound influenza A M2. J. Phys. Chem. B 2015,
1
(
19, 11548−59.
23) Wright, A. K.; Batsomboon, P.; Dai, J.; Hung, I.; Zhou, H.-X.;
Dudley, G. B.; Cross, T. A. Differential binding of rimantadine
enantiomers to influenza A M2 proton channel. J. Am. Chem. Soc.
2
(
016, 138, 1506−1509.
24) Aldrich, P. E.; Hermann, E. C.; Meier, W. E.; Paulshock, M.;
Prichard, W. W.; Snyder, J. A.; Watts, J. C. Antiviral agents. 2.
Structure-activity relationships of compounds related to 1-adamantan-
amine. J. Med. Chem. 1971, 14, 535−543.
(
25) Torres, E.; Duque, M. D.; Vanderlinden, E.; Ma, C.; Pinto, L.
H.; Camps, P.; Froeyen, M.; Vazquez, S.; Naesens, L. Role of the viral
́
hemagglutinin in the anti-influenza virus activity of newly synthesized
polycyclic amine compounds. Antiviral Res. 2013, 99, 281.
(
26) Schmidtke, M.; Schnittler, U.; Jahn, B.; Dahse, H.-M.; Stelzner,
A. A rapid assay for evaluation of antiviral activity against coxsackie
virus B3, influenza virus A, and herpes simplex virus type 1. J. Virol.
Methods 2001, 95, 133−143.
(
̈
27) Schade, D.; Kotthaus, J.; Riebling, L.; Kotthaus, J.; Muller-
Fielitz, H.; Raasch, W.; Hoffmann, A.; Schmidtke, M.; Clement, B.
Zanamivir amidoxime- and N-hydroxyguanidine-based prodrug
approaches to tackle poor oral bioavailability. J. Pharm. Sci. 2015,
1
(
04, 3208−3219.
28) Balannik, V.; Wang, J.; Ohigashi, Y.; Jing, X.; Magavern, E.;
Lamb, R. A.; DeGrado, W. F.; Pinto, L. H. Design and pharmacological
characterization of inhibitors of amantadine-resistant mutants of the
M2 ion channel of influenza A virus. Biochemistry 2009, 48, 11872.
F
DOI: 10.1021/acsmedchemlett.6b00311
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX