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New Antivirals and
Drug Resistance
Peter M. Colman
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria,
Australia 3050; email: [email protected]
Annu. Rev. Biochem. 2009. 78:95–118
Key Words
First published online as a Review in Advance on
March 2, 2009
antibody, HIV, influenza
The Annual Review of Biochemistry is online at
biochem.annualreviews.org
This article’s doi:
10.1146/annurev.biochem.78.082207.084029
c 2009 by Annual Reviews.
Copyright All rights reserved
0066-4154/09/0707-0095$20.00
Abstract
Progress in the discovery of new antiviral medicines is tempered by
the rapidity with which drug-resistant variants emerge. A review of
the resistance-suppressing properties of four classes of antivirals is
presented: influenza virus neuraminidase inhibitors, HIV protease inhibitors, antibodies, and protein-based fusion inhibitors. The analysis
supports the hypothesis that the more similar the drug is to the target’s
natural ligands, the higher the barrier to resistance. However, other factors, such as entropy compensation and solvent anchoring, might also
be exploited for improved drug design.
95
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Contents
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INTRODUCTION AND SCOPE . . . .
INFLUENZA VIRUS
NEURAMINIDASE . . . . . . . . . . . . . .
Neuraminidase Inhibitors . . . . . . . . . .
Resistance . . . . . . . . . . . . . . . . . . . . . . . . .
The N1 Subtype . . . . . . . . . . . . . . . . . . .
THE HIV PROTEASE . . . . . . . . . . . . . . .
Inhibitors and Drugs . . . . . . . . . . . . . . .
Prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . .
Off-Target Resistance . . . . . . . . . . . . . .
BIOLOGICALS AS ANTIVIRALS . . .
Neutralizing Antibodies . . . . . . . . . . . .
Inhibitors of Viral Membrane
Fusion. . . . . . . . . . . . . . . . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . .
96
96
97
99
101
102
102
105
106
107
107
109
112
INTRODUCTION AND SCOPE
Studies over the past one to two decades have
yielded many examples of new drugs and drug
candidates for treatment or prevention of viral infections. Most of these discoveries have
been aided, if not initiated, by knowledge of
the three-dimensional (3D) structure of the target. A large body of structural data accompanies these new medicines and informs an analysis of one of the major issues that bedevils the
management of infectious disease, namely the
rapid emergence of drug resistance. The differential capacity of drugs to suppress the selection of resistant virus is a property of both the
drug and its target. The discovery and clinical
experience of neuraminidase inhibitors for influenza and selected drugs for HIV are reviewed
in the context of drug resistance. Experience
with protein-based antivirals, including emerging structure-based approaches to vaccine design for HIV, are also discussed in this setting.
INFLUENZA VIRUS
NEURAMINIDASE
The neuraminidase inhibitors for treatment
and prevention of influenza virus infection are
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Colman
an early example of structure-based drug discovery. Two drugs are approved, a third is in
clinical evaluation, and a fourth remains undeveloped. Even though influenza is most commonly an acute infection, clinical drug resistance is observed. The two approved drugs have
very different resistance characteristics that can
be traced to the detail of their interaction with
the conserved catalytic machinery.
The neuraminidase of influenza virus (1) is
a homotetrameric glycoprotein anchored by a
fibrous stalk in the viral membrane. Its primary role in the infectious cycle is to liberate progeny from infected cells, but some roles
in mobilizing the virus through mucous secretions has also been described. The enzyme liberates N-acetyl neuraminic acid (Neu5Ac) from
α2-3 or α2-6 linkages to galactose, thereby destroying the receptor for the virus. All of the
enzymatic and antigenic properties of the protein are associated with the tetrameric globular
head, which is liberated when virus is treated
with proteolytic enzymes. Ten serologically distinct neuraminidases have been characterized
thus far, N1 through N9 associated with type A
influenza, and type B neuraminidase from type
B influenza viruses. The crystal structures of
representatives of the N2 (2, 3), N9 (4), and
type B (5) neuraminidases provided the platform for the discovery of the first examples of
the neuraminidase inhibitor drug class. Structures of compounds discussed below are depicted in Figure 1.
The sequence and structural invariance of
the enzyme active site, located on the axis of
a β-propeller fold, held the promise for drugs
effective against not only all antigenic strains
of a particular subtype but also all subtypes and
type B (1). The catalytic site has four invaginations accommodating substituents at C2 , C4 ,
C5 , and C6 on the substrate (6) (Figures 1a and
2a). A cluster of three arginyl residues (R118,
R292, R371) encircles the C2 -carboxylate of the
substrate, which binds in a twist-boat configuration. A tryptophan residue (W178) engages
the C5 -acetyl moiety. A network of hydrogen
bond donors and acceptors engage the C6 glycerol and C4 -hydroxyl, as commonly found
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in carbohydrate-binding proteins and enzymes.
Notable among these residues is E276, which
forms hydrogen bonds to the C8 and C9 hydroxyls from the glycerol moiety of the substrate.
The catalytic nucleophile is believed to be
Y406. These amino acids are “preset” for catalysis, because binding of Neu5Ac (or the transition state analog Neu5Ac2en) (Figure 1b) to
N2, N9, and subtype B neuraminidases causes
no conformational change in the enzyme. Such
conditions are likely to be optimal for designing
drugs that are truly substrate or transition state
mimetics.
a
HO
O
N
H
4 3
5
2
O
6
HO
HO
c
Neu5Ac2en
HO
O
OH O
O
N
H
1
O-
7 H
O
HO
O-
H
OH
8
OH
HO
9
4-Amino-Neu5Ac2en
d
Zanamivir
H2N
O
Neuraminidase Inhibitors
Replacement of the C4 -hydroxyl moiety of
Neu5Ac2en with amino- or guanidino substituents (Figures 1c,d and 2b), designed to
optimize chemical and shape complementarity
to the enzyme, resulted in approximately 100and 10,000-fold improvements, respectively, in
binding potency (7). Zanamivir (4-guanidinoNeu5Ac2en) binds in the active site almost exactly as predicted, but the guanidinium is not
hydrogen bonded to E119, rather it forms an
ionic contact with it through a stacking interaction (8). Although E119 is conserved in
wild influenza viruses, it is evidently not a catalytic residue because different amino acids are
found in its place in bacterial and mammalian
neuraminidases. Intranasal or inhaled dosing of
zanamivir is effective in treatment of influenza
in experimental animals (7) and in man (9), but
it is poorly distributed when taken orally, even
as an ester prodrug.
Various attempts to improve the pharmacological properties of zanamivir focused on
replacing the glycerol moiety with hydrophobic substituents linked through a carboxamide
(10, 11). This study revealed that, although
the catalytic machinery of the type A and B
neuraminidases appeared almost identical, subtle but significant differences exist in the immediately surrounding structure. These differences manifest themselves as selective binding
of Neu5Ac2en analogs, in this case preferentially to type A (12). An example of such a
b
Neu5Ac
+H N
3
O
NH2
+HN
O
O
N
H
O
HO
O-
N
H
H
O
HO
OH
O-
H
OH
HO
HO
e
Diethyl-carboxamide
analog of part c
O
f
+H N
3
O
N
H
O
Oseltamivir carboxylate
O
O-
+H
3N
O
N
H
O-
O
O
N
Figure 1
Chemical structures of neuraminidase ligands.
glycerol replacement (Figure 1e) is illustrated
(Figure 2c). One arm of the diethyl moiety of
the carboxamide side chain buries itself in a
hydrophobic pocket created by the movement
of E276. However, amino acid sequence differences between type A and B neuraminidases
conspire to present a higher energy barrier to
this modest structural transition in type B than
in N2 or N9 neuraminidases. A series of carboxamide analogs of Neu5Ac2en show similar selectivity for type A viruses over type B,
making them unattractive clinical candidates
(12).
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a
b
R224
H274
D151
E276
E119
R292
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R118
R371
c
d
R224
R224
E276
E276
Figure 2
(a) Neu5Ac binds to neuraminidase in a twist-boat configuration. The C2 -carboxylate is surrounded (left to
right) by R292, R371, and R118. E119 and D151 are close to the C4 -hydroxyl of the sugar. The
C6 -glycerol-binding site includes E276 and R224. H274 is at the far left. The enzyme structure does not
change when Neu5Ac binds [Protein Data Bank (PDB) code 1MWE] (124). Molecular figures prepared with
PyMOL (125). (b) Zanamivir bound to N9 neuraminidase (PDB code 1NNC). Neu5Ac2en is a transition
state analog, and zanamivir is the 4-guanidino derivative of Neu5Ac2en. The protein surface is shaded gray.
(c) A carboxamide analog of 4-amino Neu5Ac2en introduces a change in the conformation of the active site
at E276, which is now engaged with R224, to create a hydrophobic pocket for the “deep” ethyl group (PDB
code 2QWJ). (d ) Oseltamivir carboxylate induces the same conformational change seen in c (PDB code
2QWK).
A more radical redesign of the pyran to a
carbocyclic scaffold (Figure 1f ) led to the discovery of the orally active neuraminidase inhibitor oseltamivir (13). The active ingredient is
generated by the removal of the carboxylic acid
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Colman
ester by host enzymes. The pentyl ether (which
now replaces the glycerol) induces similar but
subtly different conformational changes in the
enzyme to those observed in the Neu5Ac2en
carboxamides (Figure 2d ), but now binding
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to type B neuraminidases is less compromised
and leaves open a therapeutic window. A more
comprehensive account of the discovery of
the neuraminidase inhibitor class is available
(14).
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Resistance
The earliest attempts to culture neuraminidase
inhibitor-resistant influenza viruses with
Neu5Ac2en analogs resulted in viruses with
off-target mutations in the receptor-binding
site of the hemagglutinin (15), revealing
an essential balance between the affinity of
the hemagglutinin for the receptor and the
efficiency of destruction of the receptor by the
neuraminidase. In separate experiments, variants in the catalytic site of the neuraminidase
were observed (16). Zanamivir selected viruses
with E119G, rationalized by the absence of the
ionic interaction with the novel 4-substituent
of the drug. E119G has the same enzyme
activity as the wild type but is less stable than
wild type (17). Other variants at that position,
a
selected in tissue culture by zanamivir, are
E119A and E119D. The glycine and alanine
mutants cause loss of binding of zanamivir by
up to 1000-fold, and the aspartic acid mutant
by up to 2500-fold (18).
A resistant virus selected with one of the carboxamide analogs (19) revealed a mutation in
one of the three argininyl residues that characterize all known neuraminidases, R292. Substitution to K292 results in some loss of enzyme activity but a large (250- to 1000-fold) loss
of binding potency to carboxamide analogs of
Neu5Ac2en. Structural studies of carboxamides
bound to the R292K variant reveal that E276
does change conformation to allow binding into
the hydrophobic pocket (20). The R292K variant most likely acquires resistance by introducing a barrier to the structural transition through
which E276 engages R224 and creates the novel
hydrophobic binding pocket (Figure 3a). That
barrier is a hydrogen bond between E276 and
K292, an interaction revealed in the crystal
structure of the unliganded R292K variant and
not accessible in the wild-type structure.
b
Carboxamide
Oseltamivir carboxylate
Wild type
Mutant
Figure 3
(a) Overlays of the carboxamide of Figure 2c with oseltamivir carboxylate bound to the R292K mutant N9
neuraminidase. Binding to both compounds is compromised by the mutation. A structural rationale is that
reduction of carboxamide binding is due to the penalty of inducing the conformational change, whereas for
oseltamivir carboxylate, it is due to the failure to create the hydrophobic binding site (PDB codes 2QWG,
2QWH). (b) Overlay of oseltamivir carboxylate bound to wild-type and H274Y mutant N1 neuraminidase.
The larger tyrosyl residue in the mutant seems to prevent the movement of E276 that is needed for
high-affinity binding (PDB codes 2HU4 and 3C10).
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The loss of inhibitory potency toward
R292K N9 neuraminidase of a panel of
neuraminidase inhibitors, including Neu5Ac,
Neu5Ac2en, 4-amino-Neu5Ac2en, zanamivir,
two Neu5Ac2en carboxamides, and oseltamivir
carboxylate, is greater the less the inhibitor resembles the substrate (20). The outlier in similarity to the substrate is oseltamivir carboxylate,
for which a 6500-fold loss of binding was determined in the structural background of N9. This
analysis supports the principle that drugs that
more closely resemble natural substrates or ligands are better equipped to suppress the emergence of a drug-resistant virus. Suppression of
such a virus demands a minimal loss of inhibitory activity toward the mutant and a minimal loss of biological activity by the mutant.
A drug-resistant virus must be able to maintain
its capacity to bind to and (if necessary) process its natural substrates while discriminating
between them and the drug.
Interestingly, oseltamivir carboxylate does
not induce the conformational switch in E276
in R292K mutant N9 neuraminidases (20), unlike the analogous carboxamides (Figure 3a).
Oseltamivir carboxylate does not properly engage with the R292K enzyme, the pentyl ether
resting “uncomfortably” over the hydrophilic
binding site used by the glycerol moiety of the
substrate. It may be that the same subtleties that
allow oseltamivir carboxylate to retain binding
efficacy to wild-type type B neuraminidases are
at play. In any event, loss of binding potency of
oseltamivir carboxylate to the N2 subtype neuraminidases has been estimated to be as high as
30,000-fold (21).
Although experiments in tissue culture are
informative, selection pressures in infected patients on drug therapy are very different and
can lead to quite different outcomes compared
to in vitro studies. Different dosing regimes and
routes of administration result in different levels of drug at the site of infection (21a, 21b), and
natural clearance mechanisms may be at play in
clearing even drug-resistant viruses. A head-tohead comparison of zanamivir and oseltamivir
is compromised because zanamivir has been far
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Colman
less widely used in the treatment of seasonal
influenza than has oseltamivir.
Both zanamivir (oral inhalation) and oseltamivir (oral) are prescribed for acute 5day treatment of infected patients. Analysis of
matched pairs of virus from 41 patients treated
with zanamivir did not reveal any examples of
drug-resistant virus (22). Similar studies, with
much larger patient numbers, reveal that two
percent of oseltamivir-treated individuals shed
drug-resistant virus (23). That number is as
high as 18% in one pediatric study wherein the
dosing is thought to have been suboptimal (24).
The R292K mutation is frequently seen in these
studies. Other mutations detected in these patients are E119V and N294S. The appearance
of drug-resistant viruses in these treated patients does not noticeably compromise the efficacy of treatment, although it may render prophylaxis to immediate contacts ineffective. This
may be the way in which amantadine-resistant
influenza viruses have taken hold (25, 26).
In the N1 antigenic background, the H274Y
mutant is selected by oseltamivir and reduces
sensitivity to the drug by several hundredfold
(27). Unlike a number of other active-site variants, the H274Y virus is transmissible in ferrets (28). Furthermore, this mutant was found
in early 2008 in H1N1 human influenza viruses
in Europe (29). Of 437 viruses tested, 59 were
resistant to oseltamivir by assay or by identification of H274Y, yet there is no evidence that
any of the patients from whom samples were obtained had either been treated with oseltamivir
or exposed to individuals who had. This observation is causing concern lest this mutation become associated with an avian H5N1
strain and render stockpiled oseltamivir ineffective against an H5N1 pandemic avian influenza. The molecular basis for resistance to
H274Y in N1 neuraminidase was described following a structural study of the mutant enzyme bound to both zanamivir and oseltamivir
carboxylate (30). Y274 in the mutant prevents
E276 from adopting the necessary “switched”
conformation demanded by the pentyl ether
side chain, whereas substrate and zanamivir are
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still accommodated (Figure 3b). The 265-fold
loss of potency in H274Y (N1) for oseltamivir
carboxylate is due to a 10-fold slower association rate and a 25-fold faster dissociation
rate. In comparison, the kinetics for binding
to zanamivir are essentially the same as for
the wild-type H274 (30). Another N1 mutant,
N294S, is also resistant to oseltamivir carboxylate by virtue of hydrogen bonding to E276
and restraining the necessary conformational
change associated with drug binding (30).
Baseline sensitivity of type B viruses to both
zanamivir and oseltamivir is lower than for
type A viruses, and the emergence in Japan
of wild influenza B strains with further reduced sensitivity to neuraminidase inhibitors is
of some concern (31, 32). These viruses have
been isolated from patients not undergoing
treatment with neuraminidase inhibitors, and
transmission between individuals seems highly
likely. Three such mutants were reported with
the following mean IC50 (nM) values against
zanamivir and oseltamivir, respectively, D198N
(50, 240), I222T (25, 480), and S250G (190,
50). One additional mutant, G402S (50, 280),
appeared during treatment with oseltamivir.
Wild-type sensitivity (IC50 ) in this study was
6 nM for zanamivir and 72 nM for oseltamivir.
Oseltamivir was some 90 times more widely
used in Japan than zanamivir in 2004/2005, and
it is possible that this has led to the altered drug
sensitivity in the communities studied (31).
Testing the sensitivity of clade 1 and clade
2 H5N1 neuraminidases to neuraminidase inhibitors has revealed that clade 2 viruses isolated
from Indonesia in 2005 are six- to sevenfold less
sensitive to oseltamivir than clade 1 viruses (33).
It is important to determine if this difference
extends to clade 2 viruses isolated elsewehere
and to identify its underlying molecular basis,
which remains equivocal on the basis of current
sequence analysis. No difference in sensitivity
to zanamivir was detected in this study.
A comparison of resistance properties of oseltamivir and zanamivir must recognize the vast
excess of use of the one over the other. A numerical excess of reports of drug resistance to
oseltamivir is expected. The most troubling
development in resistance is the H274Y mutation in H1N1 viruses from patients apparently
naive to any inhibitor.
The N1 Subtype
Neuraminidase subtypes commonly found in
human influenza viruses are N1, N2, and type
B. Other subtypes occur in wild and domestic
bird populations. H5N1 viruses are the cause of
the contemporary highly pathogenic avian influenza, a virus which has prompted many governments around the world to stockpile neuraminidase inhibitors as a first line of defense
against its spread in humans. The 3D structure
of the N1 subtype is therefore of great interest,
even though there were no grounds (on the basis of its amino acid sequence) for believing that
it would depart from the structures displayed by
N2, N9, and type B neuraminidase.
However, the recently described structure of
the N1 neuraminidase subtype, together with
that of the N4 and N8 subtypes, reveals a new
subsite within the enzyme active site (34). As a
result of a novel fold for a short loop segment of
the structure, amino acids 147–152 in N2 numbering, the pocket adjacent to the C4 -hydroxyl
moiety of Neu5Ac is further opened up, inviting exploration by medicinal chemists. Amino
acid residues affected by the altered loop conformation include E119 and D151; the former
is one of the amino acids targeted by both the
guanidinium group of zanamivir and the amino
group of oseltamivir carboxylate, and the latter is a putative catalytic residue whose precise function is unknown. The signature amino
acid sequence in N1, N4, and N8 that produces
this novel architecture has not been identified
but seems to include residues beyond the loop
itself.
When inhibitors were introduced to N1 or
N8, either at low inhibitor concentration or
with short incubation time, binding was observed in the active site with no change in
conformation, i.e., with the enlarged C4 subsite still intact (34). However, higher concentrations of ligand caused the loop to close in
on the catalytic site, yielding a structure that
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appears indistinguishable from those reported
earlier for N2, N9, and type B neuraminidase
inhibitor complexes. The inhibitory concentrations of zanamivir and oseltamivir carboxylate
for N1 are not appreciably weaker than the
∼1 nM values observed for N2 and N9, suggesting that the conformational rearrangement
associated with closure of the C4 subsite in N1
comes with little energetic penalty. No function
has yet been ascribed to the enlarged C4 subsite of N1. It seems unlikely to be of catalytic
significance because of the structural similarity of all complexes of influenza neuraminidases
with Neu5Ac2en. Other possible functions for
an enlarged catalytic site are altered substrate
specificity for the enzyme and even a role in receptor binding, as in the parainfluenza viruses
wherein a single active site in the HN protein
supports both receptor-binding and catalytic
functions (35). No current data support any of
these possibilities.
In the event that the new site lacks any biological function, targeting it for new antiviral
discovery may be of limited therapeutic benefit
should resistant viruses rapidly emerge. Nevertheless, exploiting this feature in the design
of new neuraminidase inhibitors would further
test the hypothesis that greater similarity between a drug and a natural ligand leads to better
suppression of resistant virus.
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ANRV378-BI78-05
THE HIV PROTEASE
Although enzymes are generally viewed as favorable drug targets, the protease of HIV poses
special problems owing to the dynamic properties of the proteins that control access of substrates to the active site. Nevertheless, protease
inhibitors have become an integral component
of antiretroviral therapy (36, 37).
The cleavage of the Gag and Pol polyproteins of HIV by the viral protease is an essential step in maturation of the virus. The
protease is a symmetric dimer with a pair of
active-site aspartyl residues (D25, D25 ) in close
proximity to the symmetry axes and the scissile bond of the substrate (38–41). The 10 natural polyprotein cleavage sites are remarkably
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Colman
diverse, including at the P1 (four occurrences
of F, three of L, and one of Y, M, and N) and
P1 (three P, two F, W, Y, L, M, and A) sites
(42). Even a palindromic sequence of amino
acids is stereochemically asymmetric, and association of the protease dimer with its various substrates is inherently asymmetric (43).
Furthermore, access of substrate to the active site requires a movement of the so-called
flaps, which are open and loosely structured in
the apoenzyme (39, 44) and close over bound
substrates and inhibitors (Figures 4 and 5a,b).
These features contrast starkly with the influenza neuraminidase wherein the specificity
for terminal Neu5Ac is paramount and little or
no structural rearrangement of the enzyme occurs during binding. Thus, in addition to the
catalytic machinery, functional domains of the
protease include those involved in its essential
dynamic properties and in its dimerization, and
these represent legitimate, if difficult, drug targets. A further distinguishing feature of the protease from the neuraminidase is that its substrates are also the products of a viral gene and
hence susceptible to selection pressure. Indeed,
protease drug-resistant variants with mutations
in both the enzyme active site and in the substrate have been described.
Inhibitors and Drugs
All but one of the currently registered HIV
protease inhibitors are peptidomimetics (37).
A vast literature has accumulated on drug
resistance-associated mutations, revealing that
substitutions in the substrate-binding cleft are
often associated with substitutions that determine movement of the flaps, the former
compromising substrate (and drug) binding
and the latter providing compensating enhanced catalytic efficiency. Thus, the double
mutant V82T/I84V directly impacts substrate
(Figure 5a) and inhibitor (45) (Figure 5b) interactions in the P1 and P1 sites, and the double mutant M46I/L63P is rate enhancing for
many of the natural cleavage events (46). These
four mutations alone confer broad resistance
to the first generation of protease inhibitors.
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b
Amprenavir
H
N
O
O
N
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O
H
O
O
OH
S
O
H
N
N
S
O
H O
O
H
O
O
O
Brecanavir
NH2
OH
N
S
O
O
d
CF3
H
N
O
O
Darunavir
H
Tipranavir
NH2
OH
O
c
10:11
H O
H
N
O
OH
N
O
S
O
H O
O
N
O
S
e
f
TMC-126
O
H
O
H O
H O
H
N
R=H, GS-8373; R=Et, GS-8374
O
OH
N
O
S
O
O
H
O
H O
H O
H
N
O
OH
N
S
O
O
O
OR
P OR
O
Figure 4
Chemical structures of protease inhibitors.
Computational studies support the notion that
the compensatory mutation M46I alters the dynamic properties of the flaps (46a). The structural basis for other compensatory mutants
(e.g., L90M, which is spatially adjacent to the
catalytic aspartate D25) is more subtle. In that
case, a computational simulation (46b) fails to
capture the experimentally determined poise of
nelfinavir bound to the mutant enzyme (46c).
Currently, mutations at some 15 residues are
characterized in the Stanford HIV database (47,
48) as major resistance mutations for one or
more of the approved protease inhibitor drugs.
Attention has recently become focused
on strategies for improving the performance
of the drug class both at inhibiting known
drug-resistant strains and at suppressing the
emergence of future drug-resistant strains. A
structural study of six of the natural substrates,
complexed with an inactive (D25N) mutant
protease, has led to the idea that specificity is determined primarily by substrate shape (42) and
that the envelope defined by this common shape
should guide the design of future inhibitors (49,
50). Analysis of the loss of inhibitory potency of
five first-generation protease inhibitors to 13
different protease mutants shows some correlation with the volume of the inhibitor lying
outside the substrate envelope (50). This study,
which formally takes no account of the chemical
similarity between the inhibitor and the substrate, is for five peptidomimetic drugs, each
with a noncleavable hydroxyethylene linkage in
place of the scissile peptide bond (Figure 5b).
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a
P1' pocket residues
Catalytic
HIV protease dimer
aspartates
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ARVLAEAM
b
P1 pocket residues
c
Protease surface
Protease
GS-8373
Core of peptide
(VLAE)
Amprenavir
Figure 5
(a) The HIV protease dimer showing the two catalytic aspartates (mutated to N25 in this structure), the P1,
and P1 pocket residues 82 and 84, and the peptide substrate, ARVLAEAM (PDB code 1F7A). (b) Overlay of
amprenavir bound to the protease (PDB code 1HPV) with the core of the peptide (VLAE) from panel a.
(c) GS-8373, a darunavir analog, modifed with a phosphonate at P1, bound to the protease (trace in green),
illustrating the exposure of the phosphonate at the protease surface (shaded green) (PDB code 2I4D).
Thus, chemical similarity (as well as shape similarity) to substrate is apparent in all these drugs,
and a strong chemical similarity to substrate has
previously been qualitatively scored to amprenavir (Figure 4a) (51), the drug favored by the
substrate envelope analysis.
Tipranavir (Figure 4b), the only nonpeptidomimetic compound (52) currently licensed,
also selects resistant variants, albeit slowly
and requiring multiple sequence changes (53).
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Colman
Unlike other inhibitors, binding of tipranavir to
the protease is almost entirely entropy driven
(H -0.7 Kcal/mol; -TS -13.9 Kcal/mol)
(54). The mechanism by which it maintains
binding affinity to four different drug-resistant
mutants, including one selected by tipranavir
itself, has been analyzed crystallographically
and thermodynamically, showing that the entropic losses that accompany binding to certain
mutants are usually either offset by enthalpic
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gains or, at worst, associated with minimal
enthalpic losses. In contrast, in cases where
measurements are available for peptidomimetic
inhibitors, loss of binding to drug-resistant
mutants is largely accounted for by enthalpic
losses. Although the structural correlates of this
anomalous behavior of tipranavir remain poorly
understood, tipranavir binding to the enzyme
is characterized by seven direct (as opposed
to water-mediated) hydrogen bonds to “conserved” active-site elements (54). The rather
different resistance profile of tipranavir to other
inhibitors advocates its use in combination therapies. There is not yet any evidence to suggest
that tipranavir as a single agent is any more or
less effective in suppressing resistance than are
the peptidomimetics.
The second generation of peptidomimetic
protease inhibitors is exemplified by darunavir
(Figure 4c), an analog of amprenavir modified in the P2 site (55, 56). Structural studies of darunavir and amprenavir bound to
wild-type protease and to a triple mutant
(L63P/V82T/I84V) reveal additional interactions between darunavir and the enzyme; these
interactions explain its 100-fold tighter binding
than amprenavir to wild-type protease. Against
the triple mutant, darunavir retains a 33-fold
advantage in potency over amprenavir. One feature of the interactions between darunavir and
the protease is the extent to which backbone
atoms of the protease are engaged, including
by the novel bis-tetrahydrofuranylurethane P2
moiety (57). Because of the widely conserved
conformation of the protein backbone across
diverse protease mutants, a strategy of targeting
the backbone promises to suppress drug resistance. Backbone hydrogen bonds to substrate
are a feature of the six substrate complexes described above (42), and in this sense, targeting the backbone demands substrate similarity.
However, darunavir does make an H-bond to
the backbone amide of D30, an interaction not
shared with substrates. Darunavir is marginally
more effective in inhibiting flap mutants (at positions 48, 50, and 54) than is saquinavir (58).
It will be especially interesting to observe the
performance of darunavir in the clinic in suppressing drug resistance.
Brecanavir (Figure 4d ) is also a derivative of
amprenavir/darunavir. It has a P1 site extension
terminating with a thiazole, which structurally
closely overlays with the phosphonate of GS8374 (see below) (59). Brecanavir also retains
potency against a broad spectrum of protease
inhibitor-resistant viruses. In vitro brecanavir
selects mutants in the protease active site, although high-level resistance was only observed
in the context of A28S, a mutation that reduces
viral replication efficacy (60). Despite this encouraging profile with respect to potency and
resistance, the development of brecanavir was
halted in December 2006 because of formulation problems, another reminder of the many
competing properties that are sought in drug
candidates.
Prodrugs
One way to improve the therapeutic profile of
a drug is to alter its distribution in favor of
the site of action of the drug target, in this
case inside an infected cell. Increasing the effective concentration of the drug might suppress mutants that would otherwise be viable.
Thus, attachment to a protease inhibitor scaffold of a charged phosphonate, masked by a
chemical moiety that is selectively removed by
intracellular enzymes, should improve the retention of the compound inside cells (61). Pursuit of this strategy, based on derivitizing the P1
position of TMC-126 (Figure 4e) (an analog
of amprenavir and darunavir), led to the unexpected finding that a phosphonate-containing
inhibitor, GS-8373 (Figure 4f ), has an improved binding profile to a broad range of
inhibitor-resistant protease variants. Crystallographic analysis reveals that the phosphonate
moiety of GS-8373 projects into the solvent
(Figure 5c), on a track adjacent to but not
overlapping with the path used by substrates,
where it is loosely ordered with few if any specific interactions with the protease. The diethyl ester prodrug (GS-8374) (Figure 4f ) is
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an equally potent inhibitor in vitro, supporting
the conclusion that the phosphonate makes no
specific interactions with the protease. The performance of the two compounds in antiviral assays is as predicted: The charged phosphonate
is inactive, and the neutral form shows equal
or better activity to TMC-126. Isothermal
titration calorimetry of GS-8373, GS-8374,
TMC-126, and amprenavir to two mutant
proteases (M46I/I47V/I50V and I84V/L90M)
showed that the phosphonates retain activity to the mutants, whereas the nonphosphonates suffer a 10- to 40-fold loss of activity. A
similar trend was observed for phosphonatederivatized atazanavir. In all cases studied, the
calorimetric data show enthalpic losses in the
binding interaction to mutants compared with
wild type, but entropic compensation for these
losses is always greater in the phosphonatecontaining inhibitors thereby minimizing the
loss of binding potency to the mutants. A detailed structure analysis shows that the poise of
the TMC-126 core is identical in wild type and
in the I84V/L90M mutant. In contrast, a small
but significant difference is observed for GS8374 in wild type compared to mutant, suggesting it is more adaptable to its binding site than
is TMC-126 and could be considered anchored
by the solvent (61).
This approach is suggested as a way forward for suppression of drug-resistant virus
(61, 62). Attempts to culture virus in the
presence of various phosphonate-containing
protease inhibitors were unsuccessful, but
parallel studies with TMC-126 generated an
L10F/A28S/M46I/I50V mutant. These results
seem at odds with the substrate similarity
principle for suppression of drug resistance.
The phosphonate moiety fails the chemicalsimilarity-to-substrate test and occupies a
volume beyond the substrate envelope, as
defined above (50). Of course, the natural
substrate is the polyprotein, which does extend
beyond the conventionally defined substratebinding cleft. Perhaps the solvent-anchored
moiety is mimicking these distal elements of
the substrate and/or targeting regions of the
protease structure that are essential for the
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Colman
dynamic incorporation of substrate into the active site, in which case resistant mutants may be
unviable. Its near neighbors on the protein are
G52, F53, and P81, and mutation at G52 or P81
has not yet been reported (47). If these residues
are somehow functionally important, then the
approach for HIV holds great promise. Exemplifying the generalization of this approach to
other targets will be watched with great interest.
Off-Target Resistance
Off-target resistance to protease inhibitors has
also been reported. Mutations in cleavage sites
of the Gag polyprotein have previously been
viewed as “rescue mutants” in the context of
protease mutations that directly affect drug
binding (63). However, some studies have detected resistance to protease inhibitors without concomitant mutations in the protease that
affect drug binding (64), suggesting off-target
mutations. More recently in vitro selection with
the protease inhibitor RO033-4649 has resulted in drug-resistant variants possessing Gag
mutations with no protease mutations (65); the
Gag mutants were associated with increased
processing efficiency for one of the intermediate products (NCp6). It was also shown that the
NC/p1 cleavage site mutant A431V could confer fourfold resistance to ritonavir. Evidently,
increasing the susceptibility of the polyprotein
to protease processing is a novel and bona fide
mechanism by which HIV acquires resistance
to protease inhibitors (65). Longer-term culturing of virus with GS-8374 led to a 15-fold
loss of drug sensitivity owing to mutations in
the Gag polyprotein. The only protease mutant detected was K41R, a mutation outside
the peptide-binding tunnel but close to the
peptide’s entry and exit points. However, this
mutation alone does not alter drug sensitivity.
The observed 15-fold loss of efficacy could be
mapped entirely to the Gag mutations (66).
One unmistakable trend in current protease inhibitor drug discovery is toward compounds with improved resistance properties.
Molecules with increased substrate similarity,
both in shape and chemistry, have improved
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performance in inhibiting extant drug-resistant
strains and in suppressing the emergence of new
ones (50, 51). Nevertheless, inhibitors, such
as GS-8374 (61) and tipranavir (54), that display obvious departures from substrateness also
score well in resistance properties. It remains
to be seen whether GS-8374 is additionally targeting some other essential functionality of the
protease, such as its dynamic properties. The
appearance of off-target resistance may pose an
ultimate barrier to the degree to which a protease inhibitor can truly suppress the selection
of drug-resistant virus.
BIOLOGICALS AS ANTIVIRALS
There are few examples of registered proteinbased antivirals, but more are expected in future. Here the resistance-suppressing prospects
of such antiviral agents are considered.
Neutralizing Antibodies
Neutralizing antibodies, raised through
vaccination or prior infection, are highly
successful antiviral agents. Of course, viral
strain variation can give rise to resistance to
antibody. Following the early structural studies
of the structure and antigenicity of influenza
virus antigens (2, 67) and human rhinoviruses
(68), a large body of data has accumulated
around the question of antigenic escape from
neutralizing antibody. Landmark studies, using
monoclonal antibodies to select “monoclonal”
variants (69), showed that single amino acid
sequence changes sufficed to abolish binding
of the antigen to the antibody, and subsequent
structural studies showed that the structural
consequences of these single amino acid
sequence changes were indeed only local (70,
71). Later, it was realized that certain amino
acid sequence substitutions could be tolerated
within the antibody-antigen interface (72),
that two different antibodies could engage a
common epitope in chemically unrelated ways
(73), and that mutations in an epitope could
have quite different effects on the binding of
two antibodies sharing that epitope (74).
Functionally, essential sites of viruses appear susceptible to antibody surveillance, but
a number of mechanisms operate to avoid detection. In the case of the influenza antigens,
the enzyme site of the neuraminidase presents
a surface that is smaller than a typical footprint
of an antibody-binding site (2, 75). Carbohydrate masking and conformational gymnastics
feature as protection mechanisms of HIV gp160
(76). Even when a functional site is exposed, a
thought experiment suggests that only a special subset of antibodies, i.e., those that see the
site in the same way as does the natural ligand,
would be truly broadly neutralizing (77).
Vaccination against influenza virus requires
annual matching of vaccine strains to circulating viral strains. Despite an improved understanding of global circulation and evolution
of type A influenza viruses (78), mismatches
still occur (79). For other viral diseases (smallpox, measles, mumps, hepatitis B), vaccination is extraordinarily successful. For HIV, the
large number of recorded strains suggests that
vaccination to raise neutralizing antibody will
fail to protect against nonhomologous strains.
However, the characterization of a number of
broadly neutralizing antibodies for HIV (80)
has raised the prospect of attempting to elicit
just such a response via vaccination. Four such
antibodies have now been well characterized:
b12 binds to the CD4-binding site on gp120,
2G12 binds a cluster of oligomannose residues,
and 2F5 and 4E10 bind to membrane proximal
sites on gp41. The International AIDS Vaccine
Initiative has established a Neutralizing Antibody Consortium, which is pursuing the crystal
structures of antibody complexes with components of gp160 as a basis for rational vaccine
design (81). Already, crystal structures for these
four broadly neutralizing antibodies have been
determined.
Antibody b12 is one of the few full-length
immunoglobulin structures to have yielded to
crystallographic analysis. It has an elongated
CDR H3 loop, which is thought to insert into
the recessed CD4-binding site on gp120 (82). A
structure of the Fab in complex with a peptide
designed to mimic the discontinuous epitope
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of b12 disproved the design hypothesis, indicating how difficult is the design of immunogens (83). Antibody 2G12 has a unique domain
swap generating a dimeric structure with two
normal antigen-binding sites, (each comprising
one VL and one VH domain) flanking one unusual binding site at the interface of the two VH
domains (84). Cocrystallization with mannosecontaining carbohydrates confirms that the
normal sites each bind one oligosaccharide and
that the unusual site binds two. Oligomannose
occurs on gp160 of most HIV strains. Structural studies of antibody 2F5 complexed with
cognate peptides from the membrane proximal
region of gp41 (85, 86) reveal that the apex of
the CDR H3 loop of the antibody is formed
by four hydrophobic residues, L, F, V, and I.
These amino acids play little part in binding
the peptide, and they are exposed in a way that
may promote association with a membranetethered peptide. Nonconservative mutation of
the phenylalanine residue in the apex decreases
binding to both the peptide and to gp41 by up
to 10-fold, but severely (100-fold) compromises
the neutralizing effect of 2F5 against otherwise
sensitive HIV strains (87). Antibody 4E10 also
has a long CDR H3 loop, which extends beyond the contacting interface with a 13-residue
helical peptide (88). Here, the apical CDR H3
residue is W, again so oriented that it could contact the viral membrane when 4E10 binds to
virus. Crystal structures of 4E10 Fab, bound to
three related designed peptides, have led to a
better definition of the epitope and of ways of
stabilizing its structure for incorporation into a
candidate immunogen (89). Collectively, these
structural data provide a good launching pad for
designing structures that might elicit broadly
neutralizing antibodies.
However, the critically important structure
of the natural immunogen, gp160, still remains
unknown. The Neutralizing Antibody Consortium’s strategy is to extend the panel of known
broadly neutralizing antibodies and characterize their epitopes, determine the 3D structure
of functional HIV glycoproteins with and without these antibodies bound, and use this information to rationally design immunogens (90).
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The recent NIAID HIV Vaccine Summit in
March 2008 listed as two of its nine highest research priorities the determination of the 3D
structure of the envelope trimer and finding
a way to elicit broadly neutralizing antibody
(91).
In combating a virus where strain variation
arises readily through error-prone replication,
an antibody that is capable of binding to all
functional strains of the virus would need to
have the very special properties of binding exclusively to a functional site and engaging that
site in a chemically similar way to the functional
binding partner of the virus (77). This goal
might be achieved with an appropriate polyclonal response to an epitope representing, say,
the receptor-binding site on a virus or the viral
fusion machinery.
Currently only one monoclonal antibody is
registered as an antiviral agent. Palivizumab
(Synagis®) is a humanized mouse monoclonal
antibody approved for antiviral prophylaxis in
pediatric populations susceptible to respiratory syncitial virus (RSV). Antibody-resistant
viruses have been selected in tissue culture and
then tested for prophylactic efficacy in cotton
rats (92). Three variants in the RSV Fusion (F)
protein were selected in vitro, K272M, K272Q,
and N268I. The structure of the RSV F protein
is known only by homology with the paramyxovirus F proteins (93). Interestingly, in both the
pre- and postfusion structures of the F protein (94–96), these amino acids are located in
the surface between two helical segments. Infection of animals with N268I virus was still
blocked by palivizumab, but with K272Q, the
virus was not. The F protein mutant K272Q
was also selected in vivo (97), and in tissue culture, the K272M virus grows less well than the
N268I virus (98). An affinity-matured form of
palivizumab is in development. It is not known
whether clinical failures of palivizumab prohylaxis in premature infants (99) were the result
of infection by antibody-resistant RSV strains.
There is considerable interest in the use
of monoclonal antibody therapy for hepatitis
C virus, especially in liver transplant patients
(99a). These developments stand to benefit
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from experiences in other settings of viral
infection.
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Inhibitors of Viral Membrane Fusion
Current understanding of the molecular basis
for the fusion of a viral membrane with a target cell membrane stems from the description
of the structure of the influenza virus hemagglutinin in its pre- and postfusion conformations (100). In one other system, the paramyxoviruses, comparable information is available
on the structures of the pre- and postfusion
forms of the fusion protein (96, 101). For HIV,
a structure for gp160 remains elusive, but numerous studies of the postfusion conformation
of gp41 have informed the exploitation of the
fusion machinery as a drug target. Early studies
on the antiviral properties of peptides, spanning
putative helical regions of HIV gp41(102–104),
have their basis in the observation that membrane fusion follows the formation of a structure often referred to as a six-helix bundle (105,
106). (Figure 6a). This helical bundle is believed to be formed via a pathway in which one
intermediate is a trimeric coiled-coil structure
comprising helical region 1 (or HR1), which
can associate with the target membrane via a
“fusion peptide” at its N-terminal end. A second helical region (HR2) from the C terminus
of the fusion protein engages with the trimeric
coiled coil in an antiparallel complex such that
its C terminus, inserted in the viral membrane,
is at the same end of the helical bundle as the
target membrane. Thus, the putative intermediate structure, the trimeric coiled coil of HR1
peptides, has been viewed as a potential drug
target for disruption of the fusion-competent
conformer.
Enfuvirtide (T-20) is the first drug to validate this approach. It is an oligopeptide, comprising residues 127–162 of the HIV fusion
protein gp41 (Figure 6b). This sequence overlaps with HR2 (residues 117–154) as defined
in structural studies (106), and its mode of action is generally believed to be prevention of the
formation of the helical bundle by competition
with an identical sequence on the virus. Such a
drug seems to meet the requirement of closely
resembling the natural substrate, and yet the
barrier to emergence of enfuvirtide-resistant
virus in the clinic is low (107). The earliest
studies of resistance to enfuvirtide in tissue culture (108) revealed drug-resistant mutations in
HR1, in the peptide sequence G36-I37-V38,
but no compensating mutations were observed
in HR2, despite the fact that G36 and V38 face
residues K144 and N145 in the outer HR2 helix
(106). Other studies suggest that compensating
mutations might occur elsewhere in the envelope protein (109). Resistant variants selected
in the clinic map to this same region (36–45) of
HR1 (107). Common resistance mutations are
G36 to D, S, E, and V and V38 to A, G, E,
and M. Loss of binding of enfuvirtide to corresponding mutants displayed in the HR1 region
of gp41 correlates with loss of antiviral activity
(110), supporting the proposed mode of action
(Figure 6c).
However, other studies show that enfuvirtide only weakly inhibits formation of the sixhelix bundle, at least when compared with peptides representing different segments of HR2
(111), suggesting that the mode of action of enfuvirtide may be more complex than previously
thought. The structure of the HR1 trimeric
coiled coil presents a prominent pocket-shaped
feature into which amino acids W117, W120,
D121, and I124 from HR2 are inserted when
the helical bundle is formed (Figure 6c). Such
a cavity is an enticing target for drug discovery,
and several attempts are underway to exploit
it, including the use of D-peptides (112, 113).
A discussion of organic antagonists of fusion
is beyond the scope of this article. The cavity on the HR1 trimer is toward its C-terminal
end (near G61), and the residues of HR2 that
insert into it are not present in enfuvirtide
(Figure 6b). Considering the HR1 coiled coil
as the drug target, at its N-terminal end are
the mutations conferring resistance to enfuvirtide, and at its C-teminal end is the cavity
(Figure 6c). A study of the antiviral properties of a number of overlapping HR2 peptides
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a
HR2
HR1
HR1
HR2
HR1
HR2
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b
77
HR1
30
KLYREVALIRAQLQKIGWVTLQLLHQQAEIARLLNNQQQVIGSLLQRA — fusion-peptide
117
154
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDK WASLWNWFNISNWLWYIR
HR2
Links HR2 to membrane anchor
Enfuvirtide
Literature
peptides
T-20
T-1249
T-2635
SFT
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF
TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL
WIEWEREISNYTNQIYEILTESQNQQDRNEKDLLE
c
G36 mutation site
V38 mutation site
Figure 6
(a) The postfusion state of the assembly of helical regions one and two (HR1 and HR2) in gp41 (PDB code 1ENV). The trimeric
nature of the assembly is illustrated by its coloring ( green, cyan, and magenta), and the ribbon drawing of the magenta subunit is
overlayed with the atomic structure in stick format. The N-terminal end of HR1 and the C-terminal end of HR2 are to the right as in
panel b. (b) The first and second lines are the amino acid sequences of HR1 and HR2, with their approximate structural alignment from
PDB code 1ENV. (c) The six-helix bundle of gp41 with surfaces colored as in (a) and with HR2 of the magenta subunit shown as a coil
and highlighting ( yellow surface) the enfuvirtide-resistant mutation sites G36 and V38. Also shown in stick format is the pocket-binding
motif at the N-terminal end of HR2 (PDB code 1ENV).
including 127–162 (enfuvirtide), 122–157, and
117–152 has recently shown that the middle sequence is some 100-fold less potent than either
of the other two (111). This suggests that the
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Colman
HR2 helix has distinct domains of interaction
with HR1, including with the cavity at one end
and with lipids or the fusion peptide at the other
end.
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T-1249 is a second generation peptide, again
derived from amino acid sequences comprising
and adjacent to HR2 (114). The sequence is a
chimera derived from HIV-1, HIV-2, and SIV
and displays a pocket-binding motif (see above)
at its N-terminal end, although the location of
this motif is displaced by one heptad from its position in the gp41 sequence (Figure 6b). Drugresistant virus has been selected both in vitro
and in vivo, displaying HR1 mutants (residues
37 and 38) that are cross-resistant to T-20
(115). A subsequent study in patients already
treated with enfuvirtide generated mutants in
both HR1 and HR2 (116). Clinical development of T-1249 has been discontinued (117).
A third-generation peptide, T-2635
(Figure 6b), has also been reported with a
span similar to the HR2 helix but containing
engineered sequences to enhance helicity and
stability (118). Curiously, V38 mutants selected
with enfuvirtide or T-1249 remain susceptible
(some even more susceptible than wild type)
to T-2635 (119), even though the sequences
on the peptides adjacent to V38 are essentially
identical. The structural basis for these observations is unknown. Perhaps the modes of
interaction of enfuvirtide, T-1249, and T-2635
with HR1 are not identical. Perhaps the
additional interaction energy deriving from the
pocket-binding motif at the N-terminal end of
T-2635 allows efficacious binding even in the
face of V38 mutations. It will be interesting to
see whether the nonnative sequences in T-2635
give rise to a novel panel of resistant viruses.
Sifuvirtide, another third-generation peptide
(Figure 6b), is similar in span and sequence
to T-2635 and contains engineered elements
for stability, potency, and pharmacokinetics
(120). It lacks the C-terminal decapeptide of
enfuvirtide, which may explain its failure to
interact with lipid membranes. Like T-2635,
it prevents replication of enfuvirtide-resistant
HIV strains, and it was safe and well-tolerated
in a Phase 1a study (120). Its profile in suppressing the emergence of drug-resistant virus
is yet unknown.
A complementary drug target is HR2 itself, but until recently, attempts to exploit this
target with peptides related to HR1 have been
frustrated by poor solubility of the relevant sequences. In isolation, HR1 is expected to exist
as a trimeric coiled coil. Commencing with an
HR1 sequence including residues 29 through
79 (T-865), an engineered peptide, which includes the core “a” and “d” residues of the heptad from HR1 of RSV, has improved stability
and antiviral activity (118). Passaging virus in
the presence of a related HR1 trimeric sequence
selected variants with mutations in HR2, supporting the model for the mode of action of
the HR1 peptides. Once again, it will be interesting to see whether the use of nonnative sequences in HR1 lowers the barrier for drug resistance to emerge. A novel way to target HR2
and to overcome some of the solubility issues
with HR1 trimers is with the peptide known
as 5-helix (121). This construct contains the sequence HR1-HR2-HR1-HR2-HR1 connected
with linkers and assembles into a 5-helix bundle (122), presenting a binding site for one HR2
helix (Figure 6c). 5-helix is inhibitory of a number of HIV variants at nanomolar levels (121).
Kinetic studies suggest that, during viral infection, the HR2 helix is accessible for only a few
seconds, underscoring the importance of the association kinetics of any agent that seeks to capture it (123). The resistance profile of 5-helix is
unknown.
The early structural insights into viral membrane fusion are the foundation for the fusioninhibitor class of HIV drugs. The pattern of
drug-resistant mutations generally fits a model
with the mode of action through disruption of
formation of the fusion-competent conformer
of gp41. Nevertheless, a molecular interpretation of the enfuvirtide-selected resistance mutations still lacks a sound structural basis. No
examples of structures of drug bound to its
target, nor to a drug-resistant target, are currently available. That aside, we remain confronted with the fact that the native sequence
from HR2 and the adjacent membrane proximal region represented in the drug select for
resistance. Features of enfuvirtide that are departures from the “natural ligand” include that
it is monomeric, is not covalently associated
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with HR1, and is not representative of the entire
binding surface to the HR1 coiled coil. Future
analysis of the second- and third-generation
HR2-based peptides and of the emerging opportunities with HR1-based peptides will further inform approaches to raising the resistance
barrier to this important drug class.
SUMMARY
Annu. Rev. Biochem. 2009.78:95-118. Downloaded from arjournals.annualreviews.org
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Target sites for antivirals should be functional sites. The failure of antibody-mediated
immunity to combat viral strain variation is
usually a failure of the antibody to target
a functional site. Notwithstanding the enormous impact that protease inhibitors have made
as part of the anti-HIV armamentarium, a
second-order preference for the target might
be that its function should be directed at host
molecules and not at virus-encoded molecules,
which are themselves subject to selection.
Minimizing the chemical differences between
the target’s natural ligands and the drug will
raise the barrier to the emergence of mutated targets that can distinguish between the
two.
Multicomponent therapy will continue as
a prominent strategy for suppressing the
emergence of drug-resistant variants, but increased attention will be paid to optimizing
the resistance-suppressing characteristics of the
individual components. The principles discussed are clearly not restricted to antiviral drug
discovery.
SUMMARY POINTS
1. The barrier to emergent resistance is a property of the drug and its target.
2. Very subtle chemical changes in the drug can radically alter that barrier.
3. The drug-binding site is best confined to functionally essential elements.
4. Stereochemical similarity between the drug and natural ligands is desirable.
FUTURE ISSUES
1. Further review is needed of extant resistance data for generic trends to inform new drug
discovery.
2. Monitoring the performance of new drugs with purportedly higher barriers to resistance
should continue.
DISCLOSURE STATEMENT
The author owns shares in Biota Holdings Ltd., which receives royalties from GlaxoSmithKline
on sales of Relenza.
ACKNOWLEDGMENTS
I thank Brian Smith for helpful discussions and the NHMRC (Australia) for support.
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