Molecular dynamics simulation directed rational design of inhibitors targeting drug-resistant mutants of influenza A virus M2

Article abstract of DOI:10.1021/ja204969m

Influenza A virus M2 (A/M2) forms a homotetrameric proton selective channel in the viral membrane. It has been the drug target of antiviral drugs such as amantadine and rimantadine. However, most of the current virulent influenza A viruses carry drug-resistant mutations alongside the drug binding site, such as S31N, V27A, and L26F, etc., each of which might be dominant in a given flu season. Among these mutations, the V27A mutation was prevalent among transmissible viruses under drug selection pressure. Until now, V27A has not been successfully targeted by small molecule inhibitors, despite years of extensive medicinal chemistry research efforts and high throughput screening. Guided by molecular dynamics (MD) simulation of drug binding and the influence of drug binding on the dynamics of A/M2 from earlier experimental studies, we designed a series of potent spirane amine inhibitors targeting not only WT, but also both A/M2-27A and L26F mutants with IC₅₀s similar to that seen for amantadine's inhibition of the WT channel. The potencies of these inhibitors were further demonstrated in experimental binding and plaque reduction assays. These results demonstrate the power of MD simulations to probe the mechanism of drug binding as well as the ability to guide design of inhibitors of targets that had previously appeared to be undruggable.

Full text of DOI:10.1021/ja204969m

ARTICLE
pubs.acs.org/JACS
Molecular Dynamics Simulation Directed Rational Design of Inhibitors
Targeting Drug-Resistant Mutants of Influenza A Virus M2
Jun Wang,† Chunlong Ma,# Giacomo Fiorin,§ Vincenzo Carnevale,§ Tuo Wang,|| Fanghao Hu,||
Robert A. Lamb,^ Lawrence H. Pinto,# Mei Hong,|| Michael L. Klein,*,§ and William F. DeGrado*,†,‡
†Department of Chemistry, and ‡Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6059, United States
§Institute for Computational Molecular Science and Department of Chemistry, Temple University, Philadelphia, Pennsylvania
19122-6078, United States
Department of Chemistry, Iowa State University, Ames, Iowa 50011-3111, United States
^Howard Hughes Medical Institute and Department of Biochemistry, Molecular Biology and Cell Biology, and #Department of
Neurobiology and Physiology, Northwestern University, 2205 Tech Drive, Evanston, Illinois 60208-3500, United States
S Supporting Information
b
ABSTRACT: Influenza A virus M2 (A/M2) forms a homotetrameric proton selective
channel in the viral membrane. It has been the drug target of antiviral drugs such as
amantadine and rimantadine. However, most of the current virulent influenza A viruses
carry drug-resistant mutations alongside the drug binding site, such as S31N, V27A, and
L26F, etc., each of which might be dominant in a given flu season. Among these
mutations, the V27A mutation was prevalent among transmissible viruses under drug
selection pressure. Until now, V27A has not been successfully targeted by small molecule
inhibitors, despite years of extensive medicinal chemistry research efforts and high
throughput screening. Guided by molecular dynamics (MD) simulation of drug binding
and the influence of drug binding on the dynamics of A/M2 from earlier experimental
studies, we designed a series of potent spirane amine inhibitors targeting not only WT, but also both A/M2-27A and L26F mutants
with IC50s similar to that seen for amantadine’s inhibition of the WT channel. The potencies of these inhibitors were further
demonstrated in experimental binding and plaque reduction assays. These results demonstrate the power of MD simulations to
probe the mechanism of drug binding as well as the ability to guide design of inhibitors of targets that had previously appeared to be
undruggable.
’ INTRODUCTION
receptor-mediated endocytosis, the low pH in the endosome
activates A/M2 and facilitates proton influx and disruption of the
interaction of the viral RNA with its matrix.13 Another function of
A/M2 is to equilibrate the pH across the Golgi to prevent the
premature conformational change of the hemagglutinin, which
acts as a pH-dependent fusogen.14ꢀ16
Although a variety of mutations can lead to amantadine-
resistance in vitro,17ꢀ19 only three mutants, S31N, V27A, and
L26F, are generally observed in transmissible viruses that infect
pigs, birds, and humans.20ꢀ22 These substitutions map alongside
the physiologically relevant drug binding site in the pore of the
channel.23 A very extensive survey of pore-lining mutants of the
M2 channel24 suggested a rationale for the surprising fitness of
these mutants relative to the many less transmissible drug-
resistant variants that are generated in patients during the course
of amantadine or rimantadine treatment.19,25 L26F, S31N, and
V27A are relatively unique in terms of retaining near-native
proton flux and pH activation curves,24 which appear to be
Influenza virus infections present a major health problem on
an annual basis, particularly in years of large-scale pandemics.
The approved classes of small molecule drugs for treatment of
influenza viral infections include neuraminidase inhibitors
(zanamivir and oseltamivir) and M2 channel blockers (aman-
tadine and rimantadine).1 Resistance to both classes is a pro-
blem: widespread resistance to the only orally bioavailable
neuraminidase inhibitor, oseltamivir (Tamiflu), was encountered
in the 2008ꢀ20092 and 2009ꢀ2010 flu seasons,3 and although
amantadine was successfully used for over three decades, resis-
tance is now so pervasive that the Centers for Disease Control and
Prevention (CDC) has advised against its continued use.4,5 Thus,
there is an urgent need to develop novel orally bioavailable
antivirals capable of targeting resistant strains of influenza A
viruses. While progress has been made in the area of neurami-
nidase inhibitors,6ꢀ8 the design of inhibitors that address highly
resistant forms of the M2 channel has proven to be challenging.1,9
Influenza A virus M2 proton channel (A/M2) forms a
homotetrameric channel in the viral membrane that selectively
conducts protons.10ꢀ12 Once viruses enter the infected cell by
Received: May 30, 2011
Published: July 11, 2011
r
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parameters finely tuned to respond to the properties of a given
virus’ hemagglutinin protein while minimizing toxicity for the
parent cell until viral production is complete. Although S31N is
the substitution found in current resistant strains, in other years
V27A has predominated.26 Recent studies showed that the
current predominance of S31N is not the result of drug selection
pressure, because S31N was prevalent before the introduction of
amantadine and has become widespread in regions where
amantadine was never used.20,22 Instead, V27A was identified
to be the major mutation emerging from drug selection pressure.
While the L26F and S31N mutation causes a 10ꢀ20-decrease in
the IC50s for amantadine inhibition, the corresponding V27A
mutation renders the channel entirely resistant to both amanta-
dine and rimantadine.27 We therefore focused on this particularly
challenging mutant.
Over the last four decades, systematic studies of amantadine
analogues and library screening have elucidated structureꢀactiv-
ity relationships and helped to identify potent channel-
blockers.1,9 However, there have been no confirmed reports of
small organic molecules that potently target highly amantadine-
resistant variants of A/M2. Here, we use a combination of
molecular dynamics (MD) and classical medicinal chemistry
approaches to design very potent inhibitors of V27A and L26F.
MD was used to explore the mechanism of binding of amanta-
dine to WT and of the designed inhibitor to V27A, thereby
informing the mechanism and potency of the designed com-
pounds. Potent inhibitors in general require an ammonium
group, which associates with discrete, water-lined sites that might
be hotspots for stabilizing a diffusing hydronium ion. These sites
appear to be retained in the L26F and V27A mutants and formed
the basis for the design of inhibitors that target these variants
while maintaining affinity for WT.
washed with cold ether again. Final peptide was dissolved in 50% B0
(59.9% isopropanol, 30% acetonitrile, 10% H2O, and 0.1% TFA)/50% A
(99.9% H2O, 0.1% TFA) and was purified by preparative C4 reverse
phase HPLC with a linear gradient of 70% B0 to 85% B0. The peptide was
eluted at 78% B0. The purity and identity of the peptide were confirmed
by analytical HPLC and MALDI-MS. Calculated MS, 2755.24; observed
MS, 2755.80.
Molecular Dynamics Simulations. All simulations in this work
were performed on the transmembrane region of the A/M2 bundle,
spanning residues 25ꢀ46 and identified in the following as M2TM. The
high-resolution structure of M2TM from Acharya et al.28 was used as
initial configuration. M2TM was embedded in a hydrated membrane of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) molecules, and
periodic boundary conditions were applied with a 80 Å ꢁ 80 Å ꢁ 77 Å
periodic box, corresponding to a 38 Å thick layer of water between two
periodic images of the bilayer. The hydrated bilayer was neutralized by
adding 27 K+ and 28 Clꢀ ions, respectively (KCl concentration of about
150 mM). The protein and the lipids were modeled using the
CHARMM27 force field,29 and water molecules were modeled using
the TIP3P force field.30 The electrostatic potential was solved by the
particle mesh Ewald (PME) method31 with an accuracy threshold of
10ꢀ6, a real space spherical cutoff of 12 Å, and a fast Fourier transform
(FFT) grid spacing of 0.8 Å. Lennard-Jones interactions were cut off at
12 Å, with a switching function starting from 10 Å. The equations of
motion were solved with the velocity Verlet integrator using a time step
of 1.5 fs. The lengths of all bonds involving hydrogen atoms were kept
constrained with the SHAKE method.32 The system was run at 310 K
and 1 atm using Langevin temperature33 and Langevin piston
pressure34,35 coupling schemes. Decay times for the thermostat and
barostat were chosen to be 1 and 0.1 ps, respectively. MD simulations
were performed with NAMD.36
Solid-State NMR (ssNMR). 1,2-Dimyristoyl-sn-glycero-3-phos-
phoch-line (DMPC) was used to reconstitute A27, V28, and G34
labeled A/M2(22-46)-V27A by detergent dialysis as described before.37
The dry lipid powder was suspended in 1 mL of pH 7.5 phosphate buffer
(10 mM NaH2PO4/Na2HPO4, 1 mM EDTA, and 0.01 mM NaN3),
votexed, and freezeꢀthawed eight times to create uniform lipid vesicles.
The peptide powder was codissolved with ∼20 mg of octyl-β-D-
glucopyranoside (OG) in 1 mL of the same buffer. The solution was
then mixed with 1 mL of lipid vesicle solution, votexed for 2 h, and
dialyzed with a 3.5 kDa molecular weight cutoff against 1 L of buffer at
4 ꢀC for 3 days. The buffer was changed every 8ꢀ12 h to remove the
detergent. The proteinꢀlipid precipitate appeared after one day. The
proteoliposome solution was centrifuged at 150 000g and 6 ꢀC for 4 h to
yield a membrane pellet with ∼40 wt % water. The final peptide:lipid
molar ratio was 1:8. Drugs were directly titrated into the membrane
pellet in the NMR rotor to a ratio of four per tetramer.
’ MATERIALS AND METHODS
Materials. All starting material chemicals were purchased from
commercial vendors and used without purification. Reactions were
carried out using HPLC grade solvents under N2 atmosphere. Com-
pounds were purified by silica gel flash column chromatography and
1
characterized by ESI-MS, H NMR, and 13C NMR. Details about the
inhibitor synthesis procedure and characterization can be found in the
Supporting Information.
Inhibitor Synthesis. Detailed synthesis procedure and compound
characterization can be found in the Supporting Information.
Two-Electrode Voltage Clamp (TEVC) Assay and Plaque
Reduction Assay. The inhibitors were tested via a two-electrode
patch clamp (TEVC) assay using Xenopus laevis frog oocytes micro-
injectected with RNA expressing the A/M2 protein as in a previous
report.27 The potency of the inhibitors was expressed as the percentage
inhibition of A/M2 current observed after 2 min of incubation with 100
μM compounds, and IC50 values were collected for selected potent
compounds.
Peptide Synthesis. A/M2(22-46)-V27A peptide with A27, V28,
and G34 selectively 15N and 13C labeled was manually synthesized
using Fmoc chemistry at elevated temperature (75 ꢀC for both coupling
and deprotection) in a semiautomated Quest synthesizer using Rink
Amide Chemmatrix resin (Matrix Innovation Inc., Canada). Five
equivalents of amino acid, 5 equiv of HCTU, and 10 equiv of DIEA in
NMP were used for 5 min coupling. 5% piperazine and 0.1 M HOBt in
DMF were used as the deprotection solution to minimize aspartamide
formation. The peptide was cleaved from the resin using 95% TFA, 2.5%
Tris, and 2.5% H2O and was precipitated from ether after removal of
TFA. Ether was decanted after centrifugation, and the peptide was
Solid-state NMR experiments were carried out on a 400 MHz (9.4 T)
and a 600 MHz (14.1 T) Bruker AVANCE spectrometer using 4 mm
MAS probes. Typical radio frequency fields were 40ꢀ50 kHz for 13C and
15N and 70 kHz for 1H. 13C and 15N chemical shifts were referenced to
the R-Gly CO signal at 176.49 ppm on the TMS scale and the 15N signal
of N-acetyl-valine at 122 ppm on the liquid ammonia scale, respectively.
13
2D 15Nꢀ C correlation spectra were measured using a REDOR
sequence with a 0.8 ms mixing time for 13Cꢀ N coherence transfer.38
15
13
2D 13Cꢀ C correlation spectra were measured using a 1H-driven 13
C
spin diffusion sequence39 with a 40 ms 13C mixing time. All experiments
were performed at 273 K and under 7 kHz spinning.
’ RESULTS AND DISCUSSION
MD Simulations of Drug-Binding. A/M2 channel blockers
bind to a site within the N-terminal half of the pore, displacing
water from a pore that leads to the pH-sensing H37 residues.
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Figure 1. Layering of water molecules in the M2 channel pore. (a) Structure of the pore of A/M2 in the 1.65 Å X-ray structure (PDB: 3LBW) at
intermediate pH:28 the side chains Leu26, Val27, His37, and Trp41 are shown in brown, blue, orange, and purplish, respectively. Two layers of
crystallographically resolved water molecules are shown as red and purple spheres, while backbone carbonyls involved in hydrogen bonds with such
water molecules (Ala30 and Gly34) are in light blue. (b) Representative MD snapshot of amantadine (yellow) within the pore of M2, with water
molecules in red, purple, and pink. (c) Density map in cylindrical coordinates of the heavy atoms of M2 (green), water (red), and amantadine (black,
blue): oxygen atoms of residues 30 and 34 are in light blue. The density is averaged over the four monomers (see (f) for a graphical description of the
average over the azimuthal angle shown in the density maps) and over the entire MD trajectory: two spots of the water density are alternatively occupied,
and a dashed arrow shows the one not represented in (b). (d,e) MD snapshot and density map of M2 in complex with compound 1. Note that while
amantadine inserts its charged ammonium group into the outer layer of water molecules (b,c), larger molecules such as compound 1 can only be
accommodated when such water molecules are displaced (d,e).
Drugs bind most tightly when first incubated near neutral pH,
where the channel has been most extensively characterized in
bilayers and micelles by ssNMR,37,40ꢀ42 solution NMR,40,43and
X-ray crystallography.23,28 The transmembrane, TM, domain has
been studied with and without an additional C-terminal cyto-
plasmic helix, and in the presence and absence of drugs. Although
some details vary between structures, particularly in the cyto-
plasmic helices, the TM domain of all structures shows striking
similarities: the pore begins with a steric occlusion near the
exterior-facing V27 residue, the V27-valve, then widens to form
an approximately 12 Å N-terminal pore lined by small residues,
A30, S31, and G34. The N-terminal pore leads to the gate-
keeping H37 and W41 residues.11 Experimental studies, includ-
ing very recent solution and ssNMR measurements, place
amantadine in the N-terminal aqueous pore with its hydrophobic
adamantyl group docked against the V27 valve and the ammo-
nium projecting downward toward H37.40
To obtain additional insight into the mode of binding, we
performed classical MD simulations on A/M2 in complex with
amantadine, in a fully hydrated aqueous bilayer. The newly
available 1.65 Å crystal structure (PDB: 3LBW) was used as a
starting point (Figure 1a),28 and the drug was initially placed as in
the closely related ssNMR structure (PDB: 2KQT) of the
amantadine-A/M2 complex37 in phospholipids vesicles. Focus-
ing on the binding site, we analyzed the conformational distribu-
tion of the drug and water molecules. To examine the spatial
relationship of water, drug, and other atomic groups in the time-
averaged structure of the complex, we examined the density of
these atoms averaged over snapshots taken between 20 and 80 ns
of the simulations. To further facilitate analysis, two-dimensional
density maps were generated by rotational averaging about the
pseudo-4-fold axis of the channel (Figure 1c,e,f).
radial projections. On average, amantadine’s ammonium group is
hydrated by four water molecules (Figure 1b,c) in a square
pyramidal arrangement. This positively charged ammonium
hydrate is stabilized by forming hydrogen bonds with a carbonyl
box, formed by the four carbonyl groups of A30. Each carbonyl
group tilts slightly away from the R-helical axis to allow interac-
tion with water while retaining a helical hydrogen bond to an i+4
backbone amide, as is commonly observed in water-exposed sites
of helices.44,45 Interestingly, a similar situation is repeated one
helical turn down the channel, this time with a water molecule at
the apex of the pyramid; the four water molecules at the base
vertices of the square pyramid are stabilized by a carbonyl crown
formed by G34 and are strengthened by interactions with the
imidazole nitrogens from the H37-box. This “lower” tetrad of
water molecules is clearly seen in the electron density of the
crystal structure of the protein in the drug-free form, while only
diffuse density from solvent is seen near A30 in the absence of
drugs. MD simulations at room temperature of both the G34A
mutant of the crystal structure33 and the WT protein confirm that
the two tetrads are both mobile, but water molecules associated
to G34 and H37 are more stable than those associated to A30.
Thus, the upper site might be more easily displaced than the
lower subsite near G34/H37 by channel-blocking drugs.
We therefore examined whether longer and more extended
drugs might place their ammonium substituents deeper in the
channel, occupying the apical site of the lower water cluster
associated with G34 carbonyl box and H37-box. To test this idea,
we explored the consequences of systematically lengthening a
previously described series of spiro-bicyclic amine inhibitors,27
ultimately resulting in an elongated inhibitor with two spiro-
fused cycloheptane rings 1 (Figure 1d,e) as described in more
detail below. The computed geometry for this molecule bound to
WT is shown in Figure 1d,e. The site is constrained in one
direction by the V27 valve, forcing the polar end of the more
elongated inhibitor into the lower aqueous site (Figure 1d,e).
Thus, these calculations suggested that different hydrophobic
amine inhibitors might access one of two ammonium-binding
sites, depending on the steric properties of their aliphatic
substituents. This apparent flexibility provided a crucial insight
Amantadine remains stable during the simulations, primarily
rotating about its symmetry axis, with its apolar adamantyl group
snugly bound against a constricted, hydrophobic V27 valve, and
its ammonium group projecting downward toward the water
cluster. The water molecules show nanosecond fluctuations in
which they hop between preferred (but not invariant) positions,
observable as regions of high density in the two-dimensional
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Figure 2. Shape modulation of the drug-binding pocket in the pore of A/M2 channel. A cartoon representation of the A/M2-TM helix bundle is shown
for WT (a,b), V27A (c,d), and L26F (e,f). The molecular surface of the channel pore is highlighted, and pore-lining residues Leu/Phe26 (brown),
Val/Ala27 (blue), His37 (orange), and Trp41 (violet) are shown as sticks. Pore water molecules and amantadine are shown for wt as red spheres and
sticks, respectively. The positions of water oxygens and amantadine are obtained from the crystal structure28 (PDB: 3LBW) and the ssNMR structure of
the A/M2-amantadine complex29 (PDB: 2KQT), respectively. For each structure, the radius of the pore (computed using Hole47) is plotted as a
function of the displacement along the channel axis in the region between His37 and Val-Ala27; the range of values corresponding to the entry region of
the channel pore is highlighted in pink.
Table 1. Effect of Extending the Length of the 6,6-Spirane
to enable design of dual-specificity inhibitors for WT as well
as V27A.
Core on Potency against WT A/M2 and A/M2-V27Aa
The V27A and L26F Mutants Have Larger, More Solvent-
Exposed N-Terminal Pores. To investigate how V27A and
L26F mutations affect the size and shape of the binding site,
we built theoretical models of these mutants in the drug-free
form using the recently solved high resolution structure of A/
M228 (PDB: 3LBW) as an initial configuration. Initial models
were obtained by replacing the side chains of V27 and L26 with
Ala and Phe, respectively, and refined by performing classical MD
simulations in a bilayer of 1,2-dimyristoyl-sn-glycero-3-phospho-
choline (DMPC) lipids hydrated by explicit solvent molecules.
After a relaxation phase of several nanoseconds with the protein
gradually released, the structures of the mutants were observed to
be stable over a timespan of at least several tens of nanoseconds
(backbone rmsd of the order of 1 Å). As expected, the mutation
of the valines into alanines at position 27 does not perturb
significantly the overall structure of the bundle. Therefore, the
effect of the mutation can be easily rationalized by observing that
replacing the bulky Val side chain with a small Ala methyl moiety
results in an expansion of the pore radius near the top of the
amantadine binding site (Figure 2c,d). A solution NMR structure
of this mutant,46 which was published subsequent to the com-
pletion of these calculations, is in agreement with this conclusion
and helps validate the modeling protocol. Particularly note-
worthy and somewhat less anticipated is the fact that a similar
net effect is observed upon mutation of L26, which is not pore-
lining, into phenylalanine (Figure 2e,f). In this case, the intro-
duction of the phenyl groups results in less efficient packing near
the N-terminus of the helix. As a result, the entry region of the
pore is more disordered and features, on average, a larger radius.
Most importantly, while these mutations resulted in large changes in
the upper region of the pore, the structure of the channel and the water
appeared largely unaffected near A30, G34, and H37. Thus, the
challenge was to design molecules that were larger than amantadine
and able to fill the increased volume of the upper pore created by V27A
and L26F mutant, while still being accommodated within the WT
structure. The ability of the pore to accommodate alkyl-ammonium
hydrates near either A30 or G34’s carbonyl groups provided flexibility
in the design of molecules that could penetrate different depths
depending on the volume of the upper pore of the mutants.
a IC50 values were measured versus A/M2 from the Udorn strain of the
virus, or the V27A mutant expressed in Xenopus laevis oocytes. All
compounds are tested in hydrochloride form. The proton currents and
extent of inhibition in the presence of various concentrations of drug
were measured as described in the Materials and Methods.
Design and Synthesis of Inhibitors Targeting A/M2-V27A.
Previously, we discovered a potent inhibitor of WT A/M2 (2;
IC50 = 0.9 ( 0.1 μM) (Table 1) while investigating structure
ꢀactivity relationships of a random screen hit (BL-174348).
Although compound 2 was ineffective against V27A,27,49 ssNMR
characterization of this compound in complex with WT A/M2 in
bilayers showed that this spiro-piperidine 2 had a greater impact
on the dynamics and magnetic environment of the pore than
amantadine, and that it interacted over a more extended site
within the channel. Thus, it appeared to be an attractive scaffold
upon which to build functional groups to fill in the more spacious
vestibule created by the V27A mutation. Indeed, a one carbon
extension of the spiro-piperidine 2 gave spirane amine 3, which
was the first molecule to show weak but saturable inhibition of
V27A (IC50 = 84.9 ( 13.6 μM).27 Encouraged by this result, the
length of 3 was further extended by inserting a second carbon
spacer, via a simple methylene in 4 and its methyl-substituted
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analogue, 5. Their ability to inhibit proton currents was measured
in Xenopus laevis oocytes expressing either WT A/M2 or A/M2-
V27A. Encouragingly, 4 and 5 had potencies similar to that of
amantadine against WT; more importantly, they had similar low
IC50 values against V27A, which amantadine is completely
unable to inhibit.
Similar potency increases could be achieved by homologating
the six-membered rings of 3. The potency increased as the fully
aliphatic ring was expanded from six to seven carbon atoms (7)
(Table 2), and the same trend was observed when the amine-
containing ring was expanded from six to seven carbon atoms
(8). Combining these substitutions in the bis-cycloheptyl-spir-
ane amine, 1, resulted in a compound with a 2-fold higher
potency than amantadine against WT A/M2, and great potency
against the amantadine-insensitive mutant V27A with IC50 of
11.3 ( 0.7 μM. As a negative control, the six-membered aliphatic
ring of compound 3 was converted to the five-membered ring in
6, which only showed minimal inhibition against V27A mutant.
Design of Potent Spiro-adamantane Inhibitor of A/M2-
V27A and L26F. MD simulations were used to further probe the
mode of binding of amantadine and the potent bis-cycloheptyl-
spirane amine 1 to both WT and V27A. Amantadine failed to
bind a unique site when placed within V27A, and instead bound
with its ammonium occupying either the upper or the lower
aqueous sites. Moreover, its apolar adamantane cage was sig-
nificantly less well dehydrated in V27A versus WT, explaining the
loss in potency for the mutant. This behavior contrasts with that
seen for the bis-cycloheptyl-spirane amine 1. As expected, the
drug shifted its position upward in V27A, to allow its alkyl group
to fill the larger cavity near the channel entrance (Figure 3e). Its
ammonium group occupies the lower aqueous site in WT, where
it forms solvent-mediated hydrogen bonds with the carbonyl of
G34, versus A30 in V27A (Figure 3d,e). Although its apolar
portion is more effectively dehydrated than amanatadine in
V27A, the diffuse density of the upper ring of 1, when bound
to V27A (Figure 3d), indicates that it averages between multiple
orientations and does not fully fill the cavity. Thus, we suspected
that its affinity might be further increased by modulating the
steric bulk of its apolar substituent. We first consider converting
the “upper” cyclohexane ring of 3 to a bicyclo[3.3.1]nonane
(intermediate I, Figure 4a), which can be further constrained
by a methylene linkage to give spiroadamantane 9 (Figure 4a).
This inhibitor was simulated against WT, V27A, and L26F
(Figure 3f,g). It was found that 9 had a pose similar to 1 in
V27A with the hydrophobic adamantane filling in the extra space
near A27. In the case of WT and L26F, compound 9 was pushed
lower toward H37 and forms water-mediated hydrogen bonding
with H37. It is noted that high density of 9 was observed in all
three variants (including L26F, not shown), suggesting tight
binding with each mutant.
Table 2. Effect of Ring Size on Potency of Spirane Amine
Inhibitors against WT A/M2 and A/M2-V27Aa
Compound 9 was then synthesized using a Robinson annula-
tion reaction starting from 2-adamantanecarboaldehyde (Sup-
porting Information Scheme 2), and its structure was confirmed
by X-ray crystallography (Figure 4b, Cambridge Crystallographic
Data Centre deposition number: 824269). Spiroadamantane 9
proved to be the most potent V27A inhibitor, showing more than
280-fold lower IC50 for V27A than 3 (Figure 4c). Furthermore,
this inhibitor was also highly active against another amantadine-
resistant mutant L26F (Figure 4c), consistent with homology
a IC50 values were measured versus A/M2 from the Udorn strain of the
virus, or the V27A mutant expressed in Xenopus laevis oocytes. All
compounds were tested in hydrochloride salt form. The proton currents
and extent of inhibition in the presence of various concentrations of drug
were measured as described in the Materials and Methods.
Figure 3. Density profiles for amantadine, 1, and 9 for WT and V27A. (a) Graphical description of the average over the azimuthal angle shown the
density maps. (bꢀg) Density of protein (green), water (red), and drug (blue, black) heavy atoms, computed from MD simulations, and averaged over
the four monomers (see illustration in (a)). Carbonyl groups from A30 and G34 are shown in light blue. Panels (b) and (c) show densities for the
complex between amantadine and WT, A/M2-V27A, respectively, (d) and (e) between 1 and WT, A/M2-V27A, respectively, and (f) and (g) between 9
and WT, A/M2-V27A, respectively.
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Figure 4. Spiroadamantane inhibitor 9 design and its dose response
curve on WT, V27A, L26F, and S31N inhibition. (a) Structure-based
design of spiroadamantane 9. (b) X-ray crystal structure of spiroada-
mantane 9. (c) Dose response curve of 9 against WT, V27A, and S31N
A/M2 inhibition.
13
Figure 6. 2D 15Nꢀ C correlation spectra of A/M2-V27A in DMPC
bilayers without and with spirane amine 9 and 1 bound. (a) Drug-free
peptide. (b) With 9 bound. (c) With 1 bound. Four drugs per tetramer
were used in (b,c). (dꢀf) 1D 15N cross sections without drug (top) and
with spirane amine 9 (bottom). (d) A27. (e) V28. (f) G34. (g) 13C and
15N chemical shifts (ppm) of A/M2-V27A without drug and with drugs
9 and 1. Where two peaks are observed per site, intensities are labeled as
strong (s), weak (w), or medium (m).
inhibition of A/M2-V27A virus replication even at 50 μM
concentration. Thus, the inhibitory potency of the compounds
against amantadine-resistant A/M2-V27A mutant channels seen
in electrophysiology assays results in potent inhibition of the
replication of influenza A virus.
Direct Binding of Spirane Amine to the TM Domain of
V27A. Solid-state NMR (ssNMR) spectroscopy provides a
powerful probe of the structure of proteins in phospholipid
bilayer environments. We synthesized A/M2-V27A TM peptide
(22ꢀ46, M2TM) containing uniformly 15N, 13C-labeled A27,
V28, and G34 (Supporting Information). The labeled pep-
tide was reconstituted into DMPC bilayers, and 13C and 15N
chemical shifts were measured using 2D magic-angle-spinning
correlation experiments to examine drug-induced perturbation.
Figure 6a and b shows that the V28 15N resonance was sharpened
and shifted downfield by 2 ppm upon incubation with excess 9.
By contrast, incubation of A/M2TM-V27A with the weaker-
binding 1 resulted in two partially overlapping peaks: the less
intense peak has a chemical shift close to that seen in the absence
of the inhibitor, indicative of either less complete binding or a
minor conformation that is similar to the one found in the
uncomplexed form. The more intense resonance in the pre-
sence of 1 has a strongly perturbed chemical shift, although the
Figure 5. Plaque reduction assay of spirane amines on A/M2-V27A
mutant virus. Effects of compounds on influenza A virus (A/Udorn/72)
V27A mutant were evaluated by plaque formation on MDCK cells in the
presence or absence of the compounds (10 μM or dose dependent) as
described previously.27 9 is able to significantly reduce A/M2-V27A
replication in as low as 5 μM concentration. As control, amantadine has
no effect on virus replication at up to 50 μM.
modeling and MD results showing that L26F and V27A have
similarly expanded central cavities.
Activities in Plaque Reduction Assay. The inhibitory effect
of four of the most potent inhibitors on A/M2-V27A mutant was
confirmed by plaque reduction assay of influenza A virus. Both
the size and the number of plaque formation were significantly
reduced by all four inhibitors at 10 μM concentration (Figure 5).
Consistent with the electrophysiology result, 9 was most potent
and inhibited plaque formation dramatically at concentrations
as low as 1 μM. As a negative control, amantadine showed no
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perturbation was less pronounced than that occasioned by the
binding of 9. Furthermore, the 15N and 13C chemical shifts of
G34 have previously been shown to be broad, heterogeneous,
and sensitive to drug-binding in WT A/M2 (Figure 6a).50 As was
the case for binding spirane inhibitors to WT M2TM,49 incuba-
tion of V27A with 9 or 1 sharpened the peak, altered the intensity
ratios, and shifted the chemical shift positions for the resonances
associated with G34 in V27A (Figure 6b,c). Finally, compound 9
caused greater peak sharpening to the A27 peak than 1. These
findings indicate a direct interaction of the drug with V27A and
suggest that the 10-fold higher potency of 9 as compared to 1 is a
result of a tighter and more extended interaction between the
drug and the channel.
’ AUTHOR INFORMATION
Corresponding Author
mlklein@temple.edu; wdegrado@mail.med.upenn.edu
’ ACKNOWLEDGMENT
This work was supported by NIH grants GM56423 and
AI74571 to W.F.D. and GM088204 to M.H. J.W. thanks
Dr. Patrick J. Carroll (University of Pennsylvania) for assistance
in obtaining X-ray crystallographic data.
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Despite extensive efforts, there have been no well-documen-
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