Research Article 735

Drug design against a shifting target: a structural basis for

resistance to inhibitors in a variant of influenza virus
neuraminidase
Joseph N Varghese1*, Paul W Smith2, Steven L Sollis2, Tony J Blick1,
Anjali Sahasrabudhe1, Jennifer L McKimm-Breschkin1 and Peter M Colman1

Background: Inhibitors of the influenza virus neuraminidase have been shown Addresses: 1Biomolecular Research Institute, 343
to be effective antiviral agents in humans. Several studies have reported the Royal Parade, Parkville, 3052 Australia and 2Glaxo-
Wellcome Research and Development Limited,
selection of novel influenza strains when the virus is cultured with Stevenage, Herts, SG1 2NY, UK.
neuraminidase inhibitors in vitro. These resistant viruses have mutations either
in the neuraminidase or in the viral haemagglutinin. Inhibitors in which the *Corresponding author.
glycerol sidechain at position 6 of 2-deoxy-2,3-dehydro-N-acetylneuraminic E-mail: jose.varghese@bioresi.com.au
acid (Neu5Ac2en) has been replaced by carboxamide-linked hydrophobic Key words: antiviral, drug resistance, influenza,
substituents have recently been reported and shown to select neuraminidase inhibitors, neuraminidase
variants. This study seeks to clarify the structural and functional
consequences of replacing the glycerol sidechain of the inhibitor with other Received: 9 March 1998
Revisions requested: 25 March 1998
chemical constituents.
Revisions received: 6 April 1998
Accepted: 9 April 1998
Results: The neuraminidase variant Arg292→Lys is modified in one of three
arginine residues that encircle the carboxylate group of the substrate. The Structure 15 June 1998, 6:735–746
http://biomednet.com/elecref/0969212600600735
structure of this variant in complex with the carboxamide inhibitor used for its
selection, and with other Neu5Ac2en analogues, is reported here at high © Current Biology Ltd ISSN 0969-2126
resolution. The structural consequences of the mutation correlate with altered
inhibitory activity of the compounds compared with wild-type neuraminidase.

Conclusions: The Arg292→Lys variant of influenza neuraminidase affects

the binding of substrate by modification of the interaction with the substrate
carboxylate. This may be one of the structural correlates of the reduced
enzyme activity of the variant. Inhibitors that have replacements for the
glycerol at position 6 are further affected in the Arg292→Lys variant because
of structural changes in the binding site that apparently raise the energy
barrier for the conformational change in the enzyme required to
accommodate such inhibitors. These results provide evidence that a general
strategy for drug design when the target has a high mutation frequency is to
design the inhibitor to be as closely related as possible to the natural ligands
of the target.

Introduction via sialylated carbohydrates. It is also thought that NA facili-

Influenza is still a major disease of humans and some other
animals. The pathogen, an orthomyxovirus, has a seg-
mented negative-strand RNA genome that codes for two
surface glycoproteins [1], one of which is the enzyme neu-
raminidase (NA) [2,3]. NA cleaves terminal sialic acid 1
(N-acetyl neuraminic acid; Figure 1) from glycoconjugates
found on the surface of molecules on target cells in the
upper respiratory tract of susceptible mammals [4,5]. These
sialo-glycoconjugates are the receptors for influenza virus
which binds to them via the viral haemagglutinin (HA) [6],
tates passage of virus through the protective mucin covering
target cells by desialylation of the sialic acid rich mucin [8].
Following the determination of the X-ray structure of
influenza virus NA in Type A [9,10] and Type B strains
[11], there has been a renewed interest in using NA as a
molecular target for anti-influenza drug design. Earlier
attempts [12–15] to develop an NA inhibitor as an anti-
influenza drug, using a transition-state analogue Neu5Ac2en
2 (Figure 1), led to enzyme inhibition at micromolar levels.

the other surface glycoprotein on the virus particle. NA Drug design [3] based on the X-ray structure of influenza
destroys the HA receptors, allowing the elution of progeny virus NA [16] and its complex with sialic acid and
virus particles from infected cell surfaces [7] and preventing Neu5Ac2en [17] yielded two novel and potent NA
aggregation by HA of freshly synthesised viral glycoproteins inhibitors, 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en
736 Structure 1998, Vol 6 No 6
Figure 1
(3 and 4; Figure 1) [18,19], with Ki values in the nanomolar
range. These inhibitors have been shown to be effective
antiviral agents in cell culture against all type A and B
strains tested [18,20–22]. In addition, one of these inhibitors
4-guanidino-Neu5Ac2en 4 (Zanamivir, GG167; Figure 1)
has been shown to be effective in animal studies and clini-
cal trials for the treatment of influenza virus infections in
humans [23,24].
The chemical structures of influenza
neuraminidase inhibitors: 1, sialic acid (N-
acetylneuraminic acid (Neu5Ac)); 2, 2-deoxy-
2,3-dehydro-N-acetylneuraminic acid
(Neu5Ac2en); 3, 4-amino-Neu5Ac2en; 4,
Zanamivir, 4-guanidino-Neu5Ac2en; 5, 5-N-
acetyl-4-guanidino-6-methyl(propyl)
carboxamide-4,5-dihydro-2H-pyran-2-
carboxylic acid; 6, 5-N-acetyl-4-amino-6-
diethyl carboxamide-4,5-dihydro-2H-pyran-2-
carboxylic acid; and 7, GS4071, 4-N-acetyl-5-
amino-3-(1-ethylpropoxy)-1-cyclohexene-1-
carboxylic acid.
The broad spectrum efficacy of Zanamivir is believed to
derive from one of its design features, that is, that it
should (and does) interact only with residues in the
active site of NA that are conserved in all currently
known wild strains of influenza [16], dating back to the
isolation of the virus in 1933 [25]. The discovery of effec-
tive NA inhibitors marks a turning point in the evolution
of influenza virus because it is now possible to apply
 Research Article Inhibitor-resistant variant of neuraminidase Varghese et al.
selection pressure on these conserved active-site residues,
and some inhibitor- and drug-resistant mutants have
emerged during multiple in vitro passaging of virus in the
presence of drug [26–31]. An NA variant, E119G, that
has an active-site mutation of Glu119→Gly, has reduced
affinity to 4, and virus bearing this variant was able to
replicate in vitro in the presence of 4 [28,29]. Alterna-
tively, Zanamivir-resistant mutations were found to occur
in HA around the sialic acid binding sites [27]. These
mutations appear to lower the binding affinity of HA for
sialic acid receptors on cells. Mutant viruses are able to
elute from infected cells in the presence of 4 because of
a lowered affinity of HA for sialic acid [27]. To date,
however, no resistant virus has emerged in the course of
normal clinical trials. Experience with the amantadine
class of anti-influenza virus drugs has shown that viruses
resistant to these drugs arise rapidly [32] by mutations in
the haemagglutinin and in the ion channel protein M2.
For the human immunodeficiency virus (HIV), drug resis-
tance has been a severe problem, only recently addressed
by using multidrug therapy [33–35].
A new class of NA inhibitors, exemplified by 5-N-acetyl-
4-guanidino-6-methyl(propyl)carboxamide-4,5-dihydro-2H-
pyran-2-carboxylic acid (5; Figure 1) and 5-N-acetyl-4-
amino-6-diethyl carboxamide-4,5-dihydro-2H-pyran-2-car-
boxylic acid (6; Figure 1) has recently been reported, in
which hydrophobic substituents have replaced the glyc-
erol moiety at the 6-position [36–38]. A small conforma-
tional change in the active site of NA occurs to enable
these inhibitors to be accommodated. Glu276 changes its
position to form a salt link with Arg224, and thereby
creates the necessary hydrophobic pocket for the carbox-
amide substituents [37,38]. A carbocyclic analogue of
sialic acid 4-N-acetyl-5-amino-3-(1-ethylpropoxy)-1-cyclo-
hexene-1-carboxylic acid (GS4071) [39] (7; Figure 1)
which has a hydrophobic group attached to the 6-position
Figure 2
Stereo drawing of the local region around
residue 292 in the active site of R292K
(yellow) and wild-type (green) N9 NA. The
dotted lines represent noncovalent
interactions of residue 292 in the local
environment (yellow for the R292K mutant,
and green for the wild type). Two water N294
molecules, W1 and W2, that appear in the
mutant structure are also shown. These water
molecules are positioned near the locations of
the distal guanidinyl nitrogens of Arg292 in
the wild-type enzyme. The mainchain atoms
are represented by a tube.
737
via an ether link, has also been shown to inhibit NA and
virus replication in vitro.
Resistance to the carboxamide inhibitor 5 (Figure 1) has
been investigated in type A influenza with the N9 subtype
NA, and an active-site mutant NA, Arg292→Lys (R292K),
has been isolated [30]. The specific activity of the R292K
variant is only 20% of that of wild type, and virus bearing
this mutant NA shows decreased sensitivity to all NA
inhibitors [30]. The R292K mutant has also been reported
to arise in an avian N2 background after passaging in the
presence of Zanamivir [40]. Here we present structural
studies of this N9 mutant viral NA complexed with the
inhibitors 1–7. These studies suggest a structural basis for
the differential resistance of the R292K variant to all of
these inhibitors. They also provide evidence for a design
principle for drugs directed at moving targets.
Results
The three-dimensional structures of 1–7 complexed with
wild-type N9 NA and the R292K variant of N9 NA have
been determined by X-ray crystallography. For the com-
plexes reported here, inhibitors were soaked into crystals of
wild type or R292K NA at 18°C and flash frozen to –166°C
in a cold nitrogen stream (except for the complex of 5 with
wild type, which was collected at 18°C). Details of the
experimental conditions and determination of the atomic
structure of these complexes are given in the Materials and
methods section.
The R292K variant structure
The mutation of Arg292 to lysine results in a very local
change of structure when compared to the wild-type
enzyme (Figure 2). In the wild-type NA, Arg292, together
with Arg118 and Arg371, engages the 2-carboxylate group
of sialic acid in the active site and is partly responsible
for distorting the pyranose ring from a chair to a boat
E276 E276
N294
E277 E277
K292 K292
W2 W2
W1 W1
Y406 Y406
R371 R371 Structure
738 Structure 1998, Vol 6 No 6
conformation [17]. The distal primary guanidinyl amide
of Arg292 interacts with Glu277 and the tyrosyl oxygen
of Tyr406 (Figure 2), and the other two guanidinyl nitro-
gens interact with Asn294. The guanidinyl group of
Arg292 is parallel with the peptide plane of mainchain
atoms of residues 348–349. Two water molecules in the
active-site cavity form hydrogen-bond interactions to the
primary amides of Arg292 before the entry of substrate
into the active site.
In the mutant structure, the amino group of Lys292
forms an ionic interaction with Glu276. In wild-type N9
NA this interaction is insignificant, as the guanidinyl
nitrogens of Arg292 are some 4 Å distant. In the variant,
the distal guanidinyl nitrogens in the wild-type Arg292
are replaced by two water molecules (W1 and W2 in
Figure 2) about 1 Å from these nitrogen atoms. W1 con-
nects the lysyl amino to Tyr406, Glu277 and Arg371 via
hydrogen bonds, and W2 mediates the interaction
between Asn294, the carbonyl oxygen of residue 348 and
the amino group of Lys292. In this conformation the dis-
tance of the lysine nitrogen to the Oδ oxygen of Asn294
increases by about 0.8 Å, and the distance from the car-
boxylate oxygen of Glu277 increases by about 0.3 Å when
compared with the guanidinyl nitrogens of the wild-type
structure. Some of the minor changes in the conforma-
tion of the local region around Lys292 are a 0.6 Å move-
ment of the hydroxyl of Tyr406 away from Lys292, a
0.3 Å movement of the mainchain oxygen of Gly348
closer to the nitrogen of Lys292, and a 0.4 Å movement
of the carboxylate oxygen of Glu276 towards Lys292.
Figure 3
(a) A246 A246
R3
R224 R224
H274 H274
E276 E276
Y Y
E277
(b)
The loop around residue 246 has a slight movement
(0.2 Å) away from Lys292, and the water molecules in
the neighbourhood of Lys292 are perturbed by about
0.5 Å from their positions in the wild-type NA.
Complexes of R292K with compounds 2–4
When complexed with the R292K N9 NA, Neu5Ac2en,
4-amino-Neu5Ac2en and Zanamivir (2–4) are positioned
almost identically in the active-site pocket. Interactions
with NA are similar to those in complexes with wild-type
enzyme [41], especially for the C4 and C5 substituents,
with some differences around the 2-carboxylate where a
water molecule is now interposed between that group
and Lys292. Small differences are also observed at the
site of interaction of the 6-glycerol group with Glu276,
all attributable to the Lys292–Glu276 salt link in the
variant. In the wild type, the glycerol group binds deeper
into the pocket, moving about 0.3 Å towards the floor in
the direction of the Arg292 and Glu276 residues. In the
mutant enzyme, the Lys292 amine nitrogen directly
interacts with the O8 hydroxyl of the 6-glycerol group
and a carboxylate oxygen of Glu276 (which moves 1.1 Å
from the position in the wild-type enzyme, while
keeping the other carboxylate oxygen approximately in
the same position interacting with the O9 hydroxyl of the
glycerol) preventing the glycerol from penetrating as
deeply as in the wild type (see 3 in Figure 3a). The glyc-
erol group of Neu5Ac2en in the mutant, however, binds
about halfway between its position in the wild-type
enzyme and its location in the complexes of 3 and 4 in
the mutant enzyme (Figure 3b).
The binding of sialic acid and O4-substituted
Neu5Ac2en analogues in the active sites of
the wild-type and R292K mutant of N9 NA.
(a) Stereo drawing of the 4-amino-
R37 Neu5Ac2en inhibitor complexed with the wild-
type (yellow) and the R292K mutant of N9 NA
(blue), superimposed on each other in the
region of the inhibitor-binding pocket. Dotted
green lines represent the nonbonded
E277 interactions of the 6-triol of 4-amino-
Neu5Ac2en and the active-site residues in the
mutant complex. (b) Stereo drawing of the
superposition of sialic acid, Neu5Ac2en, 4-
amino- and 4-guanidino-Neu5Ac2en
complexed in the wild-type N9 NA (yellow),
and in the R292K mutant (red). The
Neu5Ac2en–R292K N9 NA complex is
shown in green; atoms are in standard
colours. The classes of binding are evident. All
of the compounds fail to penetrate the active
site of the mutant as deeply as they do that of
the wild type.
Structure
 Research Article Inhibitor-resistant variant of neuraminidase Varghese et al. 739

Figure 4

Stereo view superimposition of the

carboxamide inhibitor 5 in the active site of R224 R224
the R292K mutant NA structure (blue) and the A246 A246
uncomplexed R292K mutant NA structure H274 R152 H274 R152
(yellow). Note the movement of the
carboxylate of Glu276, from pointing into the E276 E276
active-site cavity to forming a salt bridge with
Arg224 in the complex, and the hydrophobic N294 N294
pocket with which the 6-carboxamide group
interacts. Hydrogen bonds are shown as
K292 E277 D151 K292 E277 D151
green dotted lines.
W2 W2

W1 W1
R118 R118
R371 Y406 R371 Y406
Structure

Complexes of R292K with compounds 5 and 6
The inhibitor 5, which was used to select R292K, binds in
a similar manner to the variant as do other carboxamides to
wild type [37,38]. The 4-guanidino group of 5 interacts
with Glu119, Glu227 and Asp151, together with main-
chain carbonyl oxygens of residues 178 and 151. The nitro-
gen of the 5-N-acetyl group interacts with a water molecule,
the carbonyl oxygen with Arg152, and the methyl group
with Trp178. All of these interactions are isosteric with
those of Zanamivir in complex with the R292K variant
(see above).
The carboxamide sidechain of 5 forms different interac-
tions with the variant protein than does the glycerol of
the natural substrate, however. The propyl group of 5
displaces three water molecules in the uncomplexed
enzyme, and the methyl group forms a hydrophobic
interaction with Glu276. A hydrophobic pocket is created
by the formation of an internal salt bridge between
Glu276 and Arg224 (Figure 4). This allows the methyl
and propyl groups of the carboxamide respectively to
enter the cis and trans hydrophobic pockets bounded by
the Glu276–Arg224 salt link, Ile222 and Ala246. In the
uncomplexed enzyme, Lys292 forms an ionic interaction
with a carboxylate oxygen of Glu276 (3.1 Å), the other
carboxylate oxygen forming a hydrogen bond (2.8 Å) with
a primary guanidinyl nitrogen of Arg224. This lysine–glu-
tamate interaction is broken in the complex, whereas the
carboxylate oxygen forms an ionic interaction with the
secondary guanidinyl nitrogen of Arg224 and the Nε2 of
His274. This involves a movement of 2.5 Å of this car-
boxylate oxygen on forming the complex. The other car-
boxylate oxygen of Glu276 retains its interaction with a
primary guanidinyl nitrogen of Arg224.
Compound 6 is some ten times less effective than 5 as an
inhibitor of wild-type NA (Table 1). It nevertheless forms
very similar complexes with both wild type and R292K to
those observed with 5. The salt link between Glu276
and Arg224 is formed and the ethyl substituents enter the

Table 1

Resistance indices for inhibitor binding to the R292K variant versus wild type.

Inhibitor* Ki (mM) Ki resistance IC50 (mM) IC50 resistance

wild type/variant wild type/variant

1 55/1820 33 nd nd
2 2.64/280 106 20/410 20
3 0.148/14 95 0.075/2.5† 33†
4 0.002/0.033 16 0.002/0.11† 55†
5 0.004/2.160 540 0.02/5.26† 263†
6 nd nd 0.23/230 1000
7 nd nd 0.002/13 6500

*Inhibitors are as defined in Figure 1. The index is defined as the ratio which have been determined according to methods described in [30].
of the Ki (or IC50) for the variant to the Ki (or IC50) for the wild type. Ki nd, not determined.
and IC50 values from [30], except for the values marked with a dagger
740 Structure 1998, Vol 6 No 6
newly created hydrophobic environment. The weaker
binding of 6 compared with 5 may relate to energy penal-
ties associated with accommodating the bulkier ethyl
group in the cis pocket. In all complexes the plane of the
carboxamide is normal to the plane of the pyranose, and
this preferred orientation restricts the location of both the
cis and trans substituents of the carboxamide.
Complexes of R292K and wild-type NA with compound 7
The X-ray structure of a complex of GS4071 (7) and N9
NA has recently been determined at 2.8 Å resolution
[39]. We have extended the resolution to 2 Å and note
that 7, a carbocyclic sialic acid analogue, binds to the active
site of wild-type NA in a similar way to the carboxamide
inhibitor 6. The hydrophobic pentyl ether group of 7
binds in the hydrophobic pocket created by the rotation
of Glu276 to form the salt link with Arg224. This result is
different from the previous structure [39] in that the ori-
entation of the carboxylate of Glu276 is reported to be
slightly different, and the distances of the pentyl group
from Glu276 are longer, probably a result of the lower
resolution of that structure. In wild type, 6 and 7 display
significantly different structures as a consequence of the
planar carboxamide in 6 and its preferred orientation
with respect to the ring. Thus one of the ethyl groups of
6 is more deeply buried than its counterpart in 7. Fur-
thermore, the two ring structures are slightly displaced
from each other (Figure 5).
In the mutant enzyme, 7 fails to induce the formation of
the internal salt link between Glu276 and Arg224. The
hydrophobic pocket that receives the pentyl ether group
in the wild type is thus not established in the mutant.
This might be attributed to Glu276 being more tightly
anchored to the mutant Lys292 than to the wild-type
Arg292. As a consequence of the hydrophobic pocket not
being formed, the pentyl ether group fails to enter the site
Figure 5
A246 R152 A246
R224
H274 H274
N294 N294
E276 D15 E276
E277
R371 R118
E E
Y406
Structure
as deeply as it does in the wild type, but interactions with
the carboxylate, the amino group and the acetamido group
of the inhibitor with the enzyme are retained (Figure 6).
The comparison with 6 bound to the mutant further illus-
trates the subtlety of the system, because in this complex
the internal salt link is still formed, despite the interaction
of Lys292 with Glu276. One is left to argue the importance
of the carboxamide as a linking structure which, because
of its preferred orientation normal to the pyranose ring,
can ‘force’ the entry of one of the ethyl groups into its
hydrophobic pocket.
Differential resistance of inhibitors to R292K
Table 1 summarises the published data [30] on the
inhibitory properties of the compounds in Figure 1 against
wild-type and variant (R292K) NA activity. These data are
either in the form of IC50 values or Ki values. We define a
‘resistance index’ as the ratio of the Ki (or IC50) for the
variant to that for the wild type, and observe that resis-
tance increases through the series of compounds 1–7, in
accordance with their decreasing similarity to the transi-
tion-state analogue 2. Thus influenza viruses bearing the
NA mutant R292K can be expected to have higher ‘resis-
tance’ to 7 than to other inhibitors studied here.
The complex of R292K N9 NA with compound 1
X-ray diffraction analysis of crystals, flash frozen to –166°C,
of the R292K N9 NA with 20 mM Neu5Ac (1) soaked for
4 h at room temperature (18°C), revealed two sialic acid
moieties bound to the NA. The first is in the conserved
active site in a twisted boat conformation as reported previ-
ously [17], except that the interactions between the R292K
enzyme and the carboxylate are as described for inhibitors
2–5; that is, a water molecule has been introduced between
Lys292 and the carboxylate, compared with the direct inter-
action with Arg292 in the wild type.
Stereo view overlay of the wild-type
complexes with 6 (blue) and 7 (yellow). Note
R152 that the orientation of the carboxamide in 6
results in one of its ethyl groups penetrating
R224
the pocket more deeply than its counterpart in
7. Dotted red lines represent hydrogen bonds;
green dotted lines represent noncovalent
interactions.
D151
R371 E277 R118
Y406

Figure 6
Stereo drawing of inhibitor 7 complexed with
the wild-type (yellow) and the R292K mutant
(blue) of tern N9 NA. The structures were
superimposed on each other in the region of
the inhibitor-binding pocket. Interactions of 7
with the wild-type active-site residues are H274
shown as green dotted lines, and interactions
N294
of Glu276 in the R292K mutant are shown as
red dotted lines.
Structure
The second sialic acid binding site is observed in a chair
conformation similar to that found in the sialic acid binding
sites of HA [42]. The location of this second site is the same
as that observed in wild type [43] soaked in 1 for 4 h at
4°C. This site is unoccupied in the room temperature soak
in the wild-type enzyme. The location of this site had been
predicted from the properties of antibody-selected vari-
ants [44] in wild-type A/tern/Australia/G70C/75 N9 NA
[45], and in A/FPV/Rostock/34 N1 NA [46]. The structure
of the wild-type NA–Neu5Ac complex at the second site
of NA for the 4°C soak is similar to the structure of the
R292K N9 NA–Neu5Ac complex in the 18°C soak (root
mean square deviation [rmsd] of 0.12 Å over residues 367
to 373, 400 to 403 and 432 in the neighbourhood of the
second site). However, four additional water molecules
were found interacting with 1 in the second site in the
wild type, and there was tighter electron density of the
Neu5Ac moiety as a result of the higher resolution of the
wild-type N9 NA–Neu5Ac complex.
This observation is not central to the current findings on
drug-resistant variants, but does again illustrate that subtle
effects accompany mutations. The Arg292→Lys substi-
tution is 14 Å away from the second binding site and yet
it modulates its affinity for sialic acid such that room
temperature (18°C) binding is now observed, whereas in
the wild type low temperature (4°C) is required to capture
sialic acid.
Discussion
The active site of influenza virus NA has been conserved
in all currently known field strains of the virus [10,16],
including the recently described avian influenza in
humans [47]. Evidently, it is not necessary to alter the
active-site residues to produce variants that can escape
antibody binding. It has been proposed [2] that antibodies
are unable to exert selection pressure on the active site
because an antibody footprint is typically larger than the
Research Article Inhibitor-resistant variant of neuraminidase Varghese et al. 741
A246 R152 A246 R152
R224 R224
H274
N294
D15 D151
E276 E276
E277
R371 R118 R371 E277 R118
E E
Y406 Y406
active site, and mutations occurring outside the active site
are sufficient to abolish binding [48–51]. It has also been
suggested [41] that the highly conserved active site is a
result of the virus optimising its ability to spread in the
environment of the upper respiratory tract. This involves
overcoming immobilisation in the protective mucin layer,
and facilitation of the elution of progeny from infected
cells. This would require a balance of the rate of desialyla-
tion by NA and the rate of attachment by HA to sialylated
glycoconjugates, implying that alterations in the enzyme
characteristics of NA could be critical to the viability of
the virus; hence the highly conserved nature of the
enzyme active site. The selection in vitro by a NA
inhibitor of virus with HA mutations and decreased recep-
tor-binding affinities of HA [27] is further indication of the
interplay between HA binding and NA activity.
The NA inhibitors Zanamivir 4 [18] and 5 [36–38] do
apply selection pressure in vitro on the enzyme active-
site residues. The mutant Glu119→Gly (E119G) was
selected after serial passage of the virus in the presence
of Zanamivir. This variant confers resistance by affecting
interactions of the 4-guanidinium group of Zanamivir
with Glu119 [29] while at the same time retaining the
interactions with the natural substrate sialic acid. E119G
has the catalytic activity of wild type but its stability is
compromised [52].
The R292K mutant has arisen after passage of virus in 5
[30]. It has also been selected by 4 in a different genetic
background (H4N2) [40]. This indicates that the R292K
mutation successfully reduces the binding of both 4 and 5
in the mutant enzymes, implying that the mutation of
Arg292→Lys alters the binding not only of the 6-carbox-
amide substituent but also of other elements of 5 and 4,
respectively. In fact, compared with wild-type NA, the
R292K variant exhibited reduced binding to all of the
inhibitors studied here (see Table 1). For the purposes of
742 Structure 1998, Vol 6 No 6
this discussion, the inhibitors can be classified into one of
three groups depending on the nature of the substituent at
position C6: the first class have a glycerol sidechain at posi-
tion C6 (1, 2, 3 and 4); the second a carboxamide derivative
(5 and 6); and the third an ether-linked derivative (7). The
consequences of the mutation at Arg292 for inhibitor
binding appear to fall into two categories.
Firstly, and common to all inhibitor groups, is the effect of
the substitution on interactions with the inhibitor carboxy-
late. The hydrogen bonding of Arg292 in the wild type to
the inhibitor carboxylate is replaced for all inhibitors by
hydrogen bonding of Lys292 via a bound water molecule
in the variant. This water molecule (W2) is also a struc-
tural feature of the unliganded variant. This suggests that
the common effect for all inhibitors is that the energy of
the interaction between the inhibitor carboxylate and the
protein is smaller in the variant than in the wild type. This
may, at least in part, also explain the decrease in the spe-
cific enzyme activity to 20% of wild-type values [30].
Secondly, but differently for each of the three groups,
is the effect of altering the interactions in the pocket
which accommodates the sidechain at C6. For the first
inhibitor group (1–4), interactions between Lys292 and
the C8-hydroxyl prevent the inhibitors from settling as
deeply into the binding site as they do in the wild type.
Estimating the energetic effect of this is difficult because
of the balancing effect of somewhat higher solvent expo-
sure of the inhibitor with the enthalpic gain of an addi-
tional hydrogen bond (to Lys292) of the C8-hydroxyl. The
net effect of the mutation on the glycerol-containing
inhibitors is to reduce binding by one to two orders of
magnitude (see Table 1). For the second inhibitor group
(5,6), the conformation and position of the carboxamide is
identical for wild type and variant, but in the case of the
variant, the salt link between Lys292 and Glu276 must be
broken in order to create the hydrophobic binding pocket
for the carboxamide substituents. Glu276 undergoes a
similar conformational change in wild type, but its move-
ment is not constrained as it is in the variant because
Arg292 does not interact directly with Glu276. The
penalty associated with breaking the Lys292–Glu276
interaction and loss of hydrogen-bond interactions by
Glu276 and Lys292 with the glycerol compared with only
van der Waals interaction with the carboxamide group,
may contribute to an additional loss of binding of 5 and 6
to the variant when compared with the group 1 inhibitors;
that is, two to three orders of magnitude compared with
one to two orders of magnitude (see Table 1). Finally, the
C6 substituent of 7 does not bind in the same position, or
with the same conformation, to wild type and variant. In
the wild type, the conformational change of Glu276 occurs
unimpeded but, in the variant, the interaction between
Glu276 and Lys292 is not disrupted and the binding
pocket for the pentyl ether group is not created. This
manifests itself as a further compromise to binding (see
Table 1), as 7 is unable to cause the necessary conforma-
tional changes to accommodate the pentyl ether group.
There is a qualitative correlation between the effect of
the Arg292→lysine substitution on the structure of com-
plexes with inhibitors and on the binding properties of
the inhibitors. The largest structural consequences occur
with the inhibitors whose binding is most severely com-
promised, that is 7 and then 6 and 5. Smaller structural
alterations are associated with inhibitors that retain the
glycerol sidechain, and these compounds are only mar-
ginally impaired as inhibitors as a consequence of the
Arg292 replacement by lysine.
Even more interesting is the qualitative correlation evident
in Table 1 between the degree of resistance of the variant
over wild type and the degree of structural dissimilarity of
the inhibitor to the transition-state analogue 2. Structural
similarity is a complex issue and can be measured in many
ways. Here we note without quantification that glycerol-
containing inhibitors are less compromised in their binding
to R292K than are nonglycerol-containing inhibitors. Insofar
as Lys292 interacts directly with the glycerol moiety, or
with C6 substituents of inhibitors, there is some structural
logic to this observation as well as some implication for
drug design against rapidly mutating targets.
Microorganisms develop resistance to drugs in a variety
of ways, only the simplest of which we consider here, and
that is by the selection of point mutations. Even in such
cases, unexpected complexities arise such as the appear-
ance of a mutation in a protein that is not the drug target,
for example the HA variants selected with the NA
inhibitors [27]. Thus, our discussion of drug resistance,
and how it might be addressed at the level of drug design,
is limited to resistance arising through point mutations in
the target.
In this restricted context, successful drug-resistant variants
are characterised by a high resistance index (as defined in
Table 1) for the drug, with retention of the essential func-
tional attributes of the wild type. The R292K variant has
retained only 20% of wild-type enzyme activity, but its
high resistance to 7, 6 and 5 suggests that this mutant will
be more successful against these inhibitors than against
those that retain a glycerol moiety at C6.
We have also examined the resistance index of the
inhibitors in Figure 1 for the NA variant E119G, which
was selected with 4, and find that only compounds 4 and 5
are significantly resistant (approximately 200-fold) for this
variant. Clearly, resistance is a function of both the
inhibitor and the variant. The fact that only inhibitors
with a guanidinium substitution at C4 are resistant to a
variant at position 119 that interacts directly with the
 Research Article Inhibitor-resistant variant of neuraminidase Varghese et al.
guanidinium is consistent with the principle that inhibitors
that are more closely related to the natural substrate (or
transition-state analogue) will be less resistant than others.
With respect to the E119G variant, alterations to the glyc-
erol pocket have little effect on resistance.
The introduction of new drugs to combat infectious micro-
organisms often results in the emergence of drug-resistant
variants, and in some cases the point mutations that give
rise to the resistant phenotype are well documented but
poorly understood. For influenza virus and the drug aman-
tidine, there is no known correlation between the drug and
any natural ligand for the sites to which it binds. Variants
arise rapidly, both in vivo and in vitro [32]. Inhibitors of the
HIV reverse transcriptase are of two types, nucleoside and
non-nucleoside analogues, which bind respectively in the
dNTP-binding site and remotely to that site [53]. Analysis
of inhibitor-resistant mutations is complicated by incom-
plete understanding of the interactions with natural sub-
strates. HIV protease inhibitors also lead to drug resistance
with quite complex results, affecting either the binding of
the inhibitor or the inherent catalytic rate of the enzyme
[54]. These consequences can be compensatory. Compari-
son of the drugs for similarity with substrate is complicated
by the promiscuity of the cleavage-site specificity [55,56]
and also the dynamics of the system, which requires large
flap movements of the protease to allow substrate entry to
the active site.
None of the complicating features found in these other
systems, where there is nevertheless a wealth of drug-
resistance data, is present in the system we describe here.
Thus, although the design principle of minimal departure
from the natural ligand is, in retrospect, self-evident, we
believe these data provide the first experimental demon-
stration of its validity. Design features that will conflict
with this principle include the desirability of creating
ligands with higher affinity for the target than the natural
ligand and in some cases the requirement for selectivity of
a drug for a microbial target over the homologous protein
in the host.
In conclusion, both the structural and binding effects of
the Arg292→Lys mutation are most pronounced on
inhibitors that least resemble the natural ligand. This sug-
gests that inhibitor design, at least for targets that have
high mutation frequency, should aim to retain as many
features as practicable of the natural ligand. The principle
behind such a design strategy is simple — variants in the
natural ligand binding site must retain some binding to
that ligand to preserve the protein function.
Biological implications
Understanding the ability of a virus to become resistant
to drugs that interfere with its biological activity is of
great importance to the development of sustainable
743
antiviral drug therapy. The RNA genome of influenza
virus encodes two surface glycoproteins: neuraminidase
(NA) and haemagglutinin (HA). NA cleaves terminal
sialic acid from glycoconjugates found on the surface of
target cells. These sialo-glycoconjugates are the recep-
tors for influenza virus which binds to them via the viral
HA. Several anti-influenza drugs, for example Zana-
mavir, have been designed to inhibit viral NA. Two
drug-resistant mutations (Glu→Gly and Arg→Lys) in
the conserved active site of NA, have been observed after
multiple passages of the virus in vitro in the presence of
these inhibitors. These mutations have resulted in lower
thermostability, in the case of the E119G mutation, and
altered NA activity in the R292K mutation. This raises
the question as to whether drug-resistant mutations can
arise that leave the virus viability unchanged, and why
only a few mutations have occurred to date. As no field
strain is known to have mutated in the active-site pocket
for the past 65 years, it could be inferred that selection
pressure is operating to balance the rate of desialylation
of the viral environment compared with the rate of
attachment of the HA to sialic acid containing receptors.
This balance is altered by NA inhibitors, and consequent
selection pressure on the virus leads to the emergence of
resistant strains in vitro.
Viable variants, selected under drug pressure, will gener-
ally retain wild-type function. This requires retention of
binding interactions with the functional ligands at the
same time as loss of binding interactions with the drug:
such an outcome will be more likely if the drug and the
natural ligand bind to the target in chemically different
ways. The molecular interplay between the target site, the
natural ligand, and the inhibitor or drug, is reminiscent of
interactions between macromolecules in which two differ-
ent proteins can form complexes with a common target
site on a third protein [51]. The determinants of binding
between macromolecules, and between macromolecule
and small ligand, are chemically degenerate. The data and
argument presented here suggest that an optimum strat-
egy for drug design in the face of high mutation frequency
of the target is a minimal strategy that preserves as many
structural features of the natural ligand as practicable. It
is not sufficient to target evolutionarily conserved residues
in the ligand-binding pocket. This work provides evidence
that one should aim to target drugs via chemical interac-
tions that resemble, as closely as possible, those used by
the natural ligand. Such a strategy minimises the possibil-
ity of mutations, selectively abrogating interactions with
the drug while retaining interactions with natural ligands
that are required for protein function.
Materials and methods
The R292K N9 NA mutant of the NWS/G70C virus was isolated by
serial passage of virus in the presence of 5 [30] in MDCK cells. The N9
NA from influenza virus A/NWS/G70C and the R292K N9 NA mutant
virus were purified as described previously [57] and crystallised by
744 Structure 1998, Vol 6 No 6

Table 2

Data collection conditions and statistics for X-ray diffraction from R292K (mt) and from mutant and wild-type (wt) N9 NA
complexes with inhibitor.

Crystal/ Number of Unique Resolution Cell Rsym Rmerge Soaking

inhibitor* observations data (Å) edge (Å) conditions

mt 156,483 51,358 1.7 181.37 0.074 0.076 None

mt/1 163,196 35,098 1.8 180.73 0.099 0.100 20 mM 4 h
mt/2 250,274 48,031 1.6 180.79 0.079 0.089 20 mM 4 h
mt/3 187,582 37,126 2.0 180.91 0.077 0.089 20 mM 18 h
mt/4 227,793 29,925 2.0 180.85 0.084 0.099 20 mM 3 h
mt/5 133,428 36,084 1.9 181.41 0.075 0.077 10 mM 18 h
wt/5* 111,968 28,605 2.0 182.80 0.060 0.087 10 mM 18 h
mt/6 330,993 45,702 1.8 180.89 0.074 0.073 10 mM 1.5 h
wt/6 106,146 32,767 2.0 180.86 0.121 0.125 10 mM 1 h
mt/7 170,194 33,424 1.8 180.98 0.076 0.081 20 mM 1 h
wt/7 154,336 37,216 1.8 180.95 0.088 0.092 10 mM 1 h

*Inhibitors 1–7 are as defined in Figure 1; mt, mutant, wt, wild type. All flash frozen to –166°C. The data set was collected at the Photon
soaks were carried out at 18°C, and data were collected from crystals Factory (Tsukuba, Japan) at 18°C.

established procedures [45]. The crystals were transferred to 20%
glycerol while maintaining the concentration of the phosphate buffer
prior to freezing in a cold stream of nitrogen gas at –166°C. X-ray dif-
fraction data were collected on a Rigaku R-axis II Image Plate X-ray
detector mounted on a MAC Science SRA M18XH1 rotating anode
X-ray generator, operating at 47kV and 60mA with focussing mirrors.
One dataset, compound 5 complexed with wild type, was collected at
room temperature on beamline 6A2 at the Photon Factory. The soaking
conditions of the inhibitor–NA complexes are given on Table 2,
together with the X-ray data collection statistics.
The structure of the R292K mutant NA was determined by examination
of the difference Fourier of the X-ray data from crystals of R292K
mutant and wild-type N9 using phases obtained from the refined N9
structure [29,58,59]. This resulted in the largest negative peak
(–10.3σ) at the guanidinium group of Arg292 and the largest positive
peak (+8.3σ) near Arg292 corresponding to the Nζ of a lysine substi-
tuted for Arg292. This was consistent with a mutation of R292K. Apart
from some water molecules near the lysine mutation, no other large dif-
ferences were noted, indicating that this mutation was the only one in
the N9 NA head of the mutant NWS/G70C virus. The structure was
then refined to 2 Å resolution using the wildtype structure modified by
the lysine substitution at position 292, and the water molecules in the
active site removed, as a starting atomic model, using the crystallo-
graphic refinement program X-PLOR [60]. Water molecules in the
active site and elsewhere were added by examining the difference
Fourier peaks of observed and calculated structure factors of the
atomic model that were greater than 5σ. The final refinement statistics
are given in Table 3.
The structure of the inhibitor–R292K mutant NA complexes were deter-
mined by examining the difference Fourier of structure factors from the
inhibitor complexes and the uncomplexed R292K mutant NA crystals,
using the phases of the uncomplexed refined structure with active-site
water molecules removed. The inhibitors appeared as a large positive
feature in the difference Fourier (> 5σ) which clearly showed the location
of all the atoms in the inhibitor, and active-site water molecules. Atomic
models were built from these difference Fourier features, and refined by
X-PLOR. Additional waters and/or modifications to sidechain orientations
were carried out in the atomic models as in the refinement of the uncom-
plexed structure described above, and the structures refined to 2 Å reso-
lution or higher. Refinement statistics of these inhibitor complexes are
given on Table 3. In all the above X-PLOR refinements all charges in
charged amino acids were set to zero, as well as charged groups on the
inhibitors. The stereochemical restraints used were of Engh and Huber
[61], and all the structures retained good stereogeometry (see Table 2).

Table 3

Structure refinement statistics for R292K (mt) and wild-type (wt) N9 NA and inhibitor complexes.

Structure* Resolution Selected Final Rms† deviations from ideal

(Å) reflections R factor Bonds (Å) Angles (°)

mt 6–1.7 47,115 0.171 0.013 1.82

mt/1 6–2.0 28,729 0.166 0.013 1.91
mt/2 6–1.6 46,535 0.173 0.014 1.81
mt/3 6–2.0 30,018 0.165 0.013 1.87
mt/4 6–2.0 25,513 0.149 0.013 1.86
mt/5 6–1.9 34,738 0.167 0.014 1.89
wt/5 6–2.0 25,550 0.184 0.015 1.96
mt/6 6–1.8 39,147 0.183 0.013 1.93
wt/6 6–2.0 27,445 0.177 0.014 2.00
mt/7 6–1.8 31,985 0.172 0.013 1.93
wt/7 6–2.0 30,431 0.172 0.014 1.97

*Inhibitors 1–7 are as defined in Figure 1; mt, mutant, wt, wild type. †Rms, root mean square.
 Research Article Inhibitor-resistant variant of neuraminidase Varghese et al.
Figure 7
1.0
0.9
0.8 Completeness
0.7
0.6
R or C
0.5
0.4
0.25
0.3 R factor
0.20
0.2 0.15
0.10
0.1
0 1/d
0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
Structure
Completeness of X-ray data and the final R factors of the refined
atomic models as a function of resolution 1/d (where d is the
resolution in Å) of the R292K mutant NA and complexes with inhibitors
(solid lines). The traces are colour-coded: native, black; 1, Neu5Ac
grey; 2, Neu5Ac2en blue; 3, 4-amino-Neu5Ac2en orange; 4, Zanamivir
red; 5, green; 6, purple; and 7, yellow. Inhibitor–wild-type complexes
are represented as dashed lines. The blue dashed lines represent the
Luzzati error contours from 0.1 to 0.3 Å in steps of 0.05 Å.
The completeness of the data and the final R factor as a function of reso-
lution is given in Figure 7.
Accession numbers
Coordinates for the 11 structures have been deposited with the
Brookhaven Protein Data Bank (accession numbers 2QWA, 2QWB,
2QWC, 2QWD, 2QWE, 2QWF, 2QWG, 2QWH, 2QWI, 2QWJ,
2QWK).
Acknowledgements
We thank Bert van Donkelaar and Pat Pilling for technical assistance, N
Sakabe for assistance at the Photon Factory, Tsukuba, Japan, the Australian
National Beam Line Program for travel support and Biota Holdings Ltd and
Glaxo-Wellcome for support.
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