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Journal of Virology, May 2001, p. 4771-4779, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4771-4779.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Modeling and Biochemical Characterization
Reveal the Mechanism of Hepatitis B Virus Polymerase Resistance to
Lamivudine (3TC) and Emtricitabine (FTC)
Kalyan
Das,1
Xiaofeng
Xiong,2
Huiling
Yang,2
Christopher E.
Westland,2
Craig S.
Gibbs,2
Stefan G.
Sarafianos,1 and
Edward
Arnold1,*
Center for Advanced Biotechnology and
Medicine, Department of Chemistry, Rutgers University, Piscataway,
New Jersey,1 and Gilead Sciences, Foster
City, California2
Received 8 November 2000/Accepted 19 February 2001
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ABSTRACT |
Success in treating hepatitis B virus (HBV) infection with
nucleoside analog drugs like lamivudine is limited by the emergence of
drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second
mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val
mutations on the binding of HBV polymerase inhibitors and the natural
substrate dCTP were evaluated using an in vitro HBV polymerase assay.
Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine
triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate,
and lobucavir triphosphate was assessed by determination of inhibition
constants (Ki). Recognition of the natural
substrate, dCTP, was assessed by determination of Km values. The results from the in vitro
studies were as follows: (i) dCTP substrate binding was largely
unaffected by the mutations, with Km changing
moderately, only in a range of 0.6 to 2.6-fold; (ii)
Kis for 3TCTP and FTCTP against Met552Ile/Val
mutant HBV polymerases were increased 8- to 30-fold; and (iii) the
Leu528Met mutation had a modest effect on direct binding of these
-L-oxathiolane ring-containing nucleotide analogs. A
three-dimensional homology model of the catalytic core of HBV
polymerase was constructed via extrapolation from retroviral reverse
transcriptase structures. Molecular modeling studies using the HBV
polymerase homology model suggested that steric hindrance between the
mutant amino acid side chain and lamivudine or emtricitabine
could account for the resistance phenotype. Specifically, steric
conflict between the C
2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring (common
to both -L-nucleoside analogs lamivudine and
emtricitabine) is proposed to account for the resistance observed upon
Met552Ile/Val mutation. The effects of the Leu528Met mutation, which
also occurs near the HBV polymerase active site, appeared to be less
direct, potentially involving rearrangement of the deoxynucleoside
triphosphate-binding pocket residues. These modeling results suggest
that nucleotide analogs that are -D-enantiomers, that
have the sulfur replaced by a smaller atom, or that have modified or
acyclic ring systems may retain activity against lamivudine-resistant
mutants, consistent with the observed susceptibility of these mutants
to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.
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INTRODUCTION |
Hepatitis B virus (HBV) infection is
among the top 10 viral infections, affecting an estimated 300 million
people worldwide and over 1.5 million in the United States alone
(10, 24). Chronic HBV infection can lead to cirrhosis,
hepatocellular carcinoma, and liver failure. Treatment of chronically
HBV-infected patients with alpha interferon (28) is
limited by side effects, incomplete efficacy, restriction to patients
with compensated disease, and the requirement for parenteral
administration (8, 37). HBV, a hepadnavirus, replicates
through an intermediate reverse transcription step carried out by the
viral polymerase (19, 33), which is functionally and
structurally related to human immunodeficiency virus (HIV) reverse
transcriptase (RT). Some of the nucleoside analogs developed to treat
HIV infection are highly potent against HBV infection (4,
5) at concentrations below cytotoxic thresholds. Treatment of
chronically HBV-infected patients with nucleoside or nucleotide analogs
(Fig. 1), like lamivudine (3TC),
emtricitabine (FTC), famciclovir (the prodrug of penciclovir [PCV]),
adefovir dipivoxil (ADV [also called PMEA]), and lobucavir (LBV),
leads to significant decreases in serum virus levels (26).
Treatment with the nucleoside or nucleotide analogs has shown immediate clinical benefits such as reduced viral load, suppression of
progression of liver disease, and induction of immunological clearance
or seroconversion (6, 18). Drug-resistant strains of HBV
containing specific polymerase mutations emerge upon prolonged 3TC
treatment (14, 23; H. Fontaine, V. Thiers, and S. Pol,
Letter, Ann. Intern. Med. 131:716-717, 1999) and are the
primary cause of treatment failure. Treatment of HBV-infected patients
with 3TC in phase III clinical studies showed a sequential increase in
appearance of genotypic resistance in HBV patients: 24% in the first
year, 42% in the second year, 52% in the third year, and 67% in the
fourth year (N. W. Y. Leung, C. L. Lai, J. Dienstag, G. Schiff, J. Heathcote, M. Atkins, C. Marr, and W. C. Maddrey, presented at the Management of Hepatitis B Meeting, 8 to 10 September 2000).
As with other nucleotide polymerases, the triphosphates of the
nucleotide substrates or their analog inhibitors are the catalytically active forms for polymerization by HBV polymerase, and the
polymerization reaction has been shown to be Mg2+ ion
dependent (34). Two of three catalytically essential
aspartic acid residues are part of the highly conserved YMDD motif at
the active site of HBV polymerase and its close viral relatives,
including HIV type 1 (HIV-1) RT. The most common 3TC resistance
mutations, Met552Ile and Met552Val (Met552Ile/Val), appear at the Met
(M) position in the YMDD motif of the HBV polymerase, analogous to the
lamivudine resistance mutations Met184Val/Ile of HIV-1 RT. In a
departure from the pattern observed with HIV, 3TC-resistant HBV
frequently contains a second polymerase mutation, Leu528Met. Met552Ile/Val mutations alone and in combination with the Leu528Met mutation confer a high degree of resistance to 3TC triphosphate (3TCTP)
in vitro (Table 1). On the other hand,
ADV has been reported to be active against 3TC-resistant HBV in vitro
and in vivo (29, 30, 35). These data indicate
complementary drug resistance profiles for 3TC and ADV against HBV,
suggesting a potential advantage for combination therapy in treating
chronic HBV infection where the emergence of resistance to either agent
may be suppressed.
Knowledge of the structure of HBV polymerase would be valuable for
understanding the molecular basis of many of its properties, including
mechanisms of polymerization, inhibition, and drug resistance, and for
interpretation of clinical and biochemical data. Attempts to determine
the structure of HBV polymerase by various research groups have not yet
been successful, as they have been limited by failure to obtain
sufficient amounts of highly purified active protein.
The work presented here includes a molecular modeling study of HBV
polymerase based on available retroviral RT structures. The validity of
the model developed in the present study is supported by its ability to
explain some of the key biochemical data. The inhibition potencies of
3TCTP, FTCTP, ADV diphosphate (ADVDP), PCVTP, and LBVTP were evaluated
and compared with the Km for dCTP in in vitro
enzyme assays for wild type HBV polymerase and a Leu528Met mutant,
Met552Ile/Val mutants, and Leu528Met+Met552Ile/Val mutants. The results
were analyzed at the atomic level using the modeled three-dimensional
structure of HBV polymerase. dCTP, 3TCTP, FTCTP, and ADVDP were docked
into the modeled enzyme so that the differential effects of
Met552Ile/Val and Leu528Met mutations on different nucleotide analogs
could be examined. Possible effects of these mutations on some other
potent nucleotide inhibitors are addressed. Understanding of the roles
of these drug resistance mutations might be helpful in achieving the
broader goal of developing more effective antiviral strategies for the
treatment of chronic hepatitis B.
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MATERIALS AND METHODS |
Enzyme assay. (i) Inhibition of HBV polymerase.
Recombinant
HBV polymerases were overexpressed and partially purified from insect
cells as previously described (35). HBV polymerase
activity was monitored by measurement of the incorporation of
-32P-labeled deoxynucleoside triphosphate (dNTP) into
acid-precipitable products. Assays were performed in 40 µl of a
solution containing 100 mM Tris (pH 7.5), 10 mM MgCl2, 0.6 U of RNasin/ml, 5% glycerol, 0.2 µg of activated calf thymus
DNA/µl, 100 µM unlabeled dNTPs (e.g., dATP, dGTP, and dTTP),
various concentrations of a -32P-labeled dNTP (~500
Ci/mmol), and various concentrations of inhibitors. -32P-labeled dATP was used for the determination of the
inhibition constants for ADVDP -32P-labeled dGTP was
used for PCVTP and LBVTP, and -32P-labeled dCTP was used
for 3TCTP and FTCTP. HBV polymerase (5 µl, ~0.1 µg) was added to
start the reaction. Aliquots (12 µl) were taken at various time
points between 0 and 20 min and transferred onto 3MM paper disks. The
paper disks were washed three times in 5% trichloroacetic acid plus
1% sodium pyrophosphate and once in 95% ethanol. The incorporated
radioactivity was measured in a Beckman scintillation counter.
(ii) Enzyme kinetics.
Kinetic constants were determined by
fitting the initial rates to Lineweaver-Burk plots based on the
algorithms described by Cleland (3).
Molecular modeling. (i) Sequence alignments.
The protein
segment from position 354 to 694 of the polypeptide chain translated
from the HBV pol gene (Fig. 2)
is responsible for the RT activity of HBV. A model was generated for
amino acid residues 325 to 699 of the polypeptide chain, covering the
entire polymerase/RT region. The amino acid sequence identity is
significant among various HBV strains in the polymerase/RT region, but
there is relatively low sequence homology with other viral RTs and
polymerases. The nearest relatives of HBV polymerase, in terms of
sequence homology, for which crystal structures are available are HIV-1 RT and murine leukemia virus (MuLV) RT, both with less than 25% sequence identity. Our sequence alignment (Fig. 2), however, indicates that the functionally important amino acid residues are highly conserved among the polymerases of HBV, HIV-1, and MuLV. The sequence alignments allowed us to derive a three-dimensional structural model
for HBV polymerase from the known structures of HIV-1 RT and Moloney
MuLV (MMLV) RT.
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FIG. 2.
Schematic representation of the HBV pol gene,
an HBV polymerase homology model (amino acids 325 to 699), and the HBV
polymerase/HIV-1 RT sequence alignments used in constructing the model.
The HBV polymerase is shown as a ribbon diagram (2) with
the fingers (325 to 403 and 469 to 519), palm (404 to 440 and 520 to
613), and thumb (614 to 699) subdomains in blue, red, and green,
respectively. The bound double-stranded DNA template primer is shown as
a space-filled model in grey (with N and O atoms in blue and red,
respectively), and dCTP is in gold. The four proposed disulfide links
are represented by yellow lines. The HBV polymerase and HIV-1 RT
sequence alignments are also color coded by subdomains, and sequence
identities and amino acids functionally conserved between the two
enzymes are in cyan.
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(ii) Homology modeling, docking of substrates, and structure
analysis.
Crystal structures of HIV-1 RT (7, 11, 12)
and MuLV RT (9) were used as templates in the modeling of
the HBV polymerase domain. Multiple initial models for the HBV
polymerase were obtained using the amino acid sequence alignment-based
three-dimensional structure-generating program MODELLER-4
(31) and using the crystal structures of HIV-1 RT (PDB
codes: 1 RTD, 2HMI, and 1DLO) and of MULV RT (PDB code: 1MML) as
templates. The protein conformation of the model obtained by using the
HIV-1 RT-DNA-dNTP complex structure (1RTD) was used as the initial
scaffold for the HBV polymerase model. The less conserved regions,
insertions, and side chains were built by manual modeling using the
computer program O (15) and its reference to databases of
known main-chain conformations and preferred side-chain rotamers. The
other three models, derived from the HIV-1 RT-DNA-Fab complex (2HMI),
unliganded HIV-1 RT (1DLO), and MULV RT (1MML), were used as additional
guides in building the molecular model of HBV polymerase. The secondary structure for the model constructed as described above agreed very well
with a sequence-based secondary structure assignment for the region
using the program Homologue (21). Buried side chains were
manually oriented to have favorable interactions with each other. The
final model was minimized using the molecular graphics and simulation
program SYBYL, version 6.3 (Tripos, Inc.), and the quality of the
geometrical parameters of the model was evaluated by PROCHECK
(20); the overall G factor was 
0.22, indicating that the
molecular geometry is stereochemically reasonable. Amino acid residue
Met552 was modeled as part of an unusual type II' turn, as observed in
other RT structures. The main-chain conformations for all other amino
acids were within the favored regions of the Ramachandran plot. The
drug resistance mutations Met552Ile/Val and Leu528Met were modeled so
that (i) their side chains occupied positions that had minimal steric
conflict with neighboring amino acids; (ii) their side-chain torsion
angles fell within statistically favored ranges; and (iii) the side
chain of Val/Ile552 had an orientation similar to that of Ile184 in the
Met184Ile mutant HIV-1 RT-DNA structure (32). The
individual dNTP substrates and analog inhibitors were initially modeled
using as a guide the conformation of dTTP in the structure of the HIV-1
RT-DNA-dTTP complex (Fig. 3). After
energy minimization, the substrates and nucleotide analog inhibitors
were then docked into the active sites of the wild-type and mutant HBV
polymerase models using the program SYBYL. The validity of the final
model was further supported by the proximity of the positions of some
of the important drug resistance mutation sites with respect to
substrates (Table 2).
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FIG. 3.
Electrostatic-potential surface diagrams of the modeled
HBV polymerase (left) and of the HIV-1 RT-DNA-dNTP structure (right)
plotted using the program GRASP (27). Regions in red and
blue are charged negatively and positively, respectively. The locations
of amino acids interacting with the DNA are labeled.
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RESULTS |
In vitro assay.
The effects of Leu528Met and Met552Val/Ile
mutations on the binding of the natural substrate dCTP to HBV
polymerase were evaluated by comparing the Km
values for dCTP for the mutant enzymes to those for the wild-type
enzyme. The Km values (Table 1) for the substrate dCTP were 0.64- to 2.6-fold relative to those for the mutants
of HBV polymerase, indicating that the Leu528Met and Met552Val/Ile mutations do not significantly affect the binding of dCTP to HBV polymerase. In order to identify the desirable structural features for
anti-HBV agents to be used to treat or prevent the emergence of 3TC
resistance, five nucleotide analogs with different structural characteristics were tested for their sensitivities against
3TC-resistant HBV. ADVDP and PCVTP are acyclic nucleotide
analogs, 3TCTP and FTCTP are L-configuration
nucleotide analogs with a -oxathiolane ring, and LBVTP bears a
cyclobutyl replacement for the sugar moiety in the natural nucleotides.
All of the structures are shown in Fig. 1. Susceptibility to these
inhibitors was assessed by determining Ki in in
vitro polymerase assays using recombinant wild-type and mutant HBV polymerases.
The inhibition constants (
Ki) for 3TCTP, FTCTP,
ADVDP, PCVTP, and LBVTP against the Leu528Met mutant and Met552Val/Ile
mutants
are listed in Table
1. Our in vitro enzyme assay results showed
that a single mutation, M552V or M552I, in the YMDD motif caused
significant resistance to 3TCTP and FTCTP, with the inhibition
constants increased 8- to 30-fold compared to that for the wild-type
HBV polymerase. The acyclic nucleotides ADVDP and PCVTP and the
D-nucleotide LBVTP, however, remained active against all
3TC-resistant
mutant enzymes, with the inhibition constants increased
less than
3.1-fold (
36). A moderate (2.6-fold)
increase in
Ki for 3TCTP
and FTCTP against
Leu528Met HBV polymerase is indicative of a
minimal effect of the
single Leu528Met mutation on the nucleosides.
Similar observations were
reported for 3TC resistance in various
independent studies (
17,
22).
Overview of the model.
The final model (amino acids 325 to
699) of HBV polymerase is shown in Fig. 2. Like HIV-1 RT, the modeled
HBV polymerase has fingers (325 to 403 and 469 to 519), palm (404 to
440 and 520 to 613), and thumb (614 to 699) subdomains. The catalytic
triad residues Asp431, Asp553, and Asp554 of HBV polymerase correspond to Asp110, Asp185, and Asp186 in HIV-1 RT. Many of the key protein-DNA interactions and protein-dNTP interactions are conserved (Table 2)
between the HIV-1 RT structure and the modeled HBV polymerase. It is
intriguing that HBV polymerase probably contains an element analogous
to the "primer grip" of HIV-1 RT (13), including
residues Met598 and Gly599, which are equivalent to the conserved
residues Met230 and Gly231 of HIV-1 RT. Some major differences between the HBV polymerase model and HIV-1 RT structure include four modeled disulfide bonds in HBV polymerase compared to none in HIV-1 RT, and a
larger fingers region in HBV polymerase than in HIV-1 RT. The
differences in the fingers region between HBV polymerase and HIV-1 RT
may involve the different primers used by retroviral RTs and HBV
polymerase. Differences in the palm and thumb regions of the HBV
polymerase model and the HIV-1 RT structure are relatively small but
significant. The DNA-binding cleft in the HBV polymerase model (Fig. 3)
is well defined and more positively charged than the DNA-binding cleft
in the HIV-1 RT-DNA-dTTP complex structure (12). The
dNTP-binding region, between the palm and fingers subdomain, appears to
be partially filled by additional amino acids in the HBV model, with
the tip of its fingers touching the base of its thumb. This part of the
HBV polymerase model corresponds to the 3-4 region of the HIV-1
RT structure that contains some of the key HIV drug resistance mutation
sites, where mutations can confer resistance to nucleoside drugs like
zidovudine (AZT), dideoxyinosine (ddI), dideoxycytosine (ddC), and
stavudine (d4T). These antiviral drugs are not very potent against HBV.
Some of the HIV-1 RT mutations, conferring resistance to the above
drugs, are the natural amino acids in the wild-type HBV polymerase. The nucleoside resistance mutations Asp67Asn and Leu74Val of HIV-1 RT
correspond to Asn381 and Val391, respectively, of the HBV polymerase model. Two multidrug (AZT+d4T+ddI/ddC) HIV resistance mutations, Gln151Met and the insertion of three amino acids after Ser69, are found
in wild-type HBV. Positions 151 and 69 of HIV-1 RT correspond to the
positions of Met519 and Pro382, respectively, in the modeled HBV polymerase.
Positions of dNTP and nucleotide analog drugs.
In the modeled
HBV polymerase, the relative positions of the -, -, and
-phosphates of dCTP (and its analog inhibitors) with respect to the
catalytic triad were assumed to occupy positions very similar to those
of the dNTP in the crystal structure of the HIV-1 RT-DNA-dNTP complex
(12). The sugar and the base moieties of the dCTP were
oriented in their energy-minimized conformations, which are constrained
to base-pair with the first DNA template overhang. The YMDD motif of
the modeled enzyme interacts mostly with the sugar-phosphate portion of
the docked dCTP. The Met552 side chain points towards the deoxyribose
ring of dCTP. The position and orientation of this amino acid
correspond to those of Met184 in HIV-1 RT. Leu528 of HBV polymerase,
positionally equivalent to Phe160 of HIV-1 RT, is part
of a helix, and its side chain points to the space between
Met552 and Phe436. The aromatic ring of Phe436, positionally
equivalent to Tyr115 in HIV-1 RT, stacks almost in parallel
with the sugar ring of the substrate. Unlike Met552, Leu528 of
HBV polymerase does not have close interactions with the dNTP
substrate. Upon mutation, however, residue 528 has the
potential to affect the binding of dNTP (or its analog
inhibitor) by perturbing the side chains of surrounding amino acids,
particularly of Phe436 and Met552.
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DISCUSSION |
Effects of the Met552Ile/Val mutation.
The Met552Ile/Val
mutations in HBV polymerase, in both the presence and the absence of
the Leu528Met mutation, conferred resistance to 3TC and FTC, as
indicated by significant increases of Ki in in
vitro polymerase assays (Table 1).
In our molecular modeling studies, the docked dCTP substrate was
accommodated in a stereochemically feasible position and
orientation
(Fig.
4) in the wild-type HBV polymerase
model. Residue
Met552, which is part of the conserved YMDD motif in
RTs, is adjacent
to the bound nucleotide substrate. The accessible
surface area
(Fig.
4) of the YMDD region of HBV polymerase is
complementary
to the molecular surface of the dCTP. The Met552Ile/Val
mutation
limits the side-chain flexibility by introducing a branch,
methyl
group (C
2), to its C
atom. The most favorable conformation
for
a valine or an isoleucine at position 552 is with the C
2 atom
pointing towards the bound dNTP. The side chain of Ile184 in the
crystal structure of Met184Ile mutant HIV-1 RT-DNA (
32),
corresponding
to position 552 of the HBV polymerase model, also had a
similar
conformation. Molecular modeling of the Met552Val mutation
(Fig.
4) showed a decreased space between the protein and the
substrate.
Consistent with the small changes observed in kinetic
constants,
the Met552Ile/Val mutation does not appear to interfere
significantly
with the proposed binding of the dNTP in its
catalytically favorable
conformation, as shown in Fig.
4.
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FIG. 4.
The YMDD region of the modeled HBV polymerase with a
docked dCTP substrate. Amino acids Met552 and Leu528 are mutated to
confer resistance to 3TC and FTC. The orange molecular surface (left)
corresponds to the deoxyribose of the docked dCTP. The green molecular
surface of the protein around its YMDD region (left) indicated no
steric hindrance between the protein and the substrate. The space
between the two molecular surfaces is indicated by a white arrow. The
space between the protein and substrate is reduced upon
Met552Val+Leu528Met mutation (right).
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The nucleotide analog 3TCTP has an oxathiolane ring in a
-
L configuration, replacing the
-
D-deoxyribose ring of dCTP. Docking
of 3TCTP into the
active site of the wild-type HBV polymerase
model, with its
triphosphate and base oriented as in dCTP, showed
(Fig.
5) that the sulfur in the oxathiolane
ring points towards
the site of mutation (position 552). As a
consequence of the
-
L configuration of the oxathiolane
ring, which is inverted with
respect to the
-
D
configuration of the deoxyribose ring of a
natural substrate, the
docked 3TCTP occupies a larger volume extending
towards the side chain
of Met552. The Met552Ile/Val mutation adds
a methyl group at the C
2
position of the mutated amino acid,
pointing toward the sulfur atom of
the oxathiolane ring of 3TCTP
(Fig.
5). Our molecular modeling studies
suggest that steric hindrance
between the C
2-methyl group of
Ile/Val552 and the oxathiolane
ring of 3TCTP may result from binding of
3TCTP to the Met552Ile/Val
mutant HBV polymerase. A previous molecular
modeling study of
HBV polymerase (
1), based on less
detailed information about
HIV-1 RT structure, concluded that the
Met552Ile/Val mutation
leads to decreased protein-inhibitor
interactions. Subsequent
biochemical and structural data, however,
strongly support our
proposed mechanism involving steric hindrance with
the C
2-methyl
group of Ile/Val552. This mechanism may also apply
more broadly
to other
L-nucleoside analogs, like FTC, with
anti-HBV activity.
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FIG. 5.
Binding of 3TCTP to wild-type (left) and Met552Val
mutant (right) HBV polymerase. Molecular modeling suggests that steric
hindrance (right), between 3TCTP and the mutated amino acid, Val552, is
the primary cause of 3TCTP resistance. This steric conflict is not
observed in the binding of 3TCTP to the wild-type HBV polymerase.
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Effects of Leu528Met mutation.
In the wild-type HBV polymerase
model, Leu528 occupies a position between the side chains of Phe436 and
Met552. Although the Leu528 side chain points toward the sugar ring of
the docked dCTP, a shortest distance of about 4.5 Å between
them suggests that interactions between dCTP and Leu528 in wild-type
HBV polymerase are likely to be weak or indirect. The Leu528Met
mutation introduces a longer, yet more flexible, side chain. As a
consequence of this mutation, the side chain of Met528 may interact
directly with the docked 3TCTP or FTCTP but its greater flexibility,
unlike the Met552Ile/Val mutation, would disfavor steric conflict of Met528 with the nucleoside inhibitor. This hypothesis is in agreement with our results from in vitro studies on the effects of the Leu528Met mutation (Table 1) showing a modest increase in
Ki for both 3TCTP and FTCTP of only 2.6-fold at
the maximum.
A possible role of Leu528Met mutation would be a conformational
perturbation of the dNTP-binding region, in particular the
amino acids
Phe436 and Met552. Phe436, whose HIV-1 RT equivalent
is Tyr115, is
positioned below and stacked with the sugar ring
of dCTP or its analog
inhibitors. As discussed above, Met552Ile/Val
would introduce a rigid
side chain in the vicinity of the 3TCTP
oxathiolane ring. Our in vitro
assays showed a higher degree of
resistance of 3TCTP and FTCTP obtained
with the double mutations
Met552Ile/Val+Leu528Met than with the single
Met552Ile/Val mutation
(Table
1). A structural interpretation of this
enhanced effect
of the double mutation is the indirect involvement of
Leu528Met
mutation by reorienting the side chains of its surrounding
amino
acids, in particular of Phe436 and Ile/Val552. Such a
rearrangement
might also be responsible for compensating for the
reduction of
polymerase activity by the Met552Ile/Val mutation
(
22,
25).
Effects of Met552Ile/Val on other nucleoside inhibitors.
The
nucleoside and nucleotide analog inhibitors ADV and PCV show
complementary in vivo drug resistance profiles (16, 29, 35) with 3TC. An acyclic chain adds torsional flexibility to ADV
and PCV compared to 3TC or FTC, both of which contain a five-membered oxathiolane ring (Fig. 1). In addition, the chain connecting the base
and the -phosphonate group is shorter in ADV than in the oxathiolane
analogs. This disparity in length (and volume) is illustrated in
comparisons of molecular models of wild-type and Met552Val+Leu528Met
mutant HBV polymerases in complex with double-stranded DNA and
ADVDP. Our molecular modeling studies predict that smaller acyclic
nucleotide analogs can be accommodated more effectively than the
bulkier oxathiolanes in a more constrained and "crowded" dNTP-binding pocket containing the Met552Val+Leu528Met mutations. This
prediction is consistent with the resistance data that show that
combinations of Met552Val/Ile and Leu528Met mutations confer only 0.8- to 2.3-fold resistance to ADVDP (35) and 0.9- to 1.8-fold resistance to LBVTP. Docking of LBVTP onto the modeled HBV polymerase fragment suggested that the interaction between the inhibitor and the
mutating amino acids is qualitatively similar to that for ADVDP (Fig.
6). Furthermore, this model can explain
why 3TC-resistant HBV mutants still retain susceptibility to ADV, which
has thus far not been reported to select for resistance mutations in
HBV (5, 29).
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FIG. 6.
Both wild-type (left) and Met552Val+Leu528Met mutant
(right) HBV polymerase appear to have no steric conflict with a docked
ADVDP.
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Summary and implications for drug design.
Our molecular
modeling studies of HBV polymerase provide a plausible structural
basis for the effects of Met552Ile/Val and Leu528Met mutations on the
susceptibility of the enzyme to 3TCTP and FTCTP. Steric conflict
between the -branched mutant amino acid side chains and the sulfur
atom of the -L-oxathiolane ring of the inhibitor is
proposed as a structural explanation for 3TC resistance by the
Met552Ile/Val mutation in HBV polymerase. This explanation is in
agreement with the proposed effects of the YMDD mutation Met184Ile in
HIV-1 RT based on the comparison of the Met184Ile mutant HIV-1 RT-DNA
(32) with wild-type HIV-1 RT-DNA-dTTP (12)
and wild-type HIV-1 RT-DNA (7) structures. The Leu528Met mutation is proposed to have an indirect effect on substrate and inhibitor binding, potentially via rearrangement of its surrounding amino acids, particularly Phe436 and Met/Ile/Val552, although increased
interaction between the side chain of Met528 and an incoming nucleotide
cannot be ruled out. This mutation was reported to compensate for the
decreased polymerization by Met552Val (22, 25). The
presence of amino acids corresponding to drug resistance mutations in
HIV-1 RT at the equivalent positions in the wild-type HBV polymerase
model may explain the natural resistance of HBV to AZT and
dideoxynucleoside inhibitors.
The structural explanation of the effects of the Met552Ile/Val mutation
on inhibition by 3TCTP and FTCTP suggests that suitable
modifications
at the sugar ring of a dNTP analog could lead to
the design of
inhibitors with increased potency against the YMDD
mutant strain. A
similar suggestion was made for the design of
HIV-1 RT inhibitors with
reduced resistance due to a Met184Ile/Val
mutation in the YMDD motif
(
32). Our studies predicted stereochemically
feasible
binding of ADVDP at the active sites of both wild-type
and
Met552Ile/Val mutant HBV polymerase models, which is consistent
with
earlier favorable reports of the potency of ADV against Met552Ile
mutant HBV strains. Also, nucleoside analog inhibitors with a
smaller
sugar ring (e.g., LBV) or with different sugar ring conformational
preferences might lead to development of additional nucleotide
analogs
that would be effective against Met552Ile/Val mutant HBV
strains.
Differences in the mode of binding of nucleotide inhibitors
to the
dNTP-binding pocket of the HBV polymerase, as predicted
from the
current modeling studies, may account for the complementary
drug
resistance profiles seen for different nucleotide analogs
and support
the concept that combination therapy against HBV may
be more effective
than monotherapy through mutual suppression
of the emergence of
drug-resistant
variants.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge Gilead Sciences for support of this
work and an NIH MERIT award (R29 AI27690) from the National Institute
of Allergy and Infectious Diseases to Edward Arnold for support of
HIV-1 RT structural studies.
We thank Stephen Hughes for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CABM and Rutgers
University, 679 Hoes Ln., Piscataway, NJ 08854. Phone: (732) 235-5323. Fax: (732) 235-5788. E-mail: arnold{at}cabm.rutgers.edu.
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0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4771-4779.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
