Binding sensitivity of adefovir to the polymerase from different genotypes of HBV: molecular modeling, docking and dynamics simulation studies
Acta Pharmacologica Sinica
Binding sensitivity of adefovir to the polymerase from different genotypes of HBV: molecular modeling, docking and dynamics simulation studies
Jing LI 0
Yun DU 0
Xian LIU 0
Qian-cheng SHEN 0
Ai-long HUANG 1
Ming-yue ZHENG 0
Xiao-min LUO 0
Hua-liang JIANG 0
0 Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203 , China
1 Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing Medical University , Chongqing 400016 , China
Aim: To investigate the molecular mechanisms underlying the influence of DNA polymerase from different genotypes of hepatitis B virus (HBV) on the binding affinity of adefovir (ADV). Methods: Computational approaches, including homology modeling, docking, MD simulation and MM/PBSA free energy analyses were used. Results: Sequence analyses revealed that residue 238 near the binding pocket was not only a polymorphic site but also a genotypespecific site (His238 in genotype B; Asn238 in genotype C). The calculated binding free-energy supported the hypothesis that the polymerase from HBV genotype C was more sensitive to ADV than that from genotype B. By using MD simulation trajectory analysis, binding free energy decomposition and alanine scanning, some energy variation in the residues around the binding pocket was observed. Both the alanine mutations at residues 236 and 238 led to an increase of the energy difference between genotypes C and B (??GC-B), suggesting that these residues contributed to the genotype-associated antiviral variability with regard to the interaction with ADV. Conclusion: The results support the hypothesis that the HBV genotype C polymerase is more sensitive to ADV than that from genotype B. Moreover, residue N236 and the polymorphic site 238 play important roles in contributing to the higher sensitivity of genotype C over B in the interaction with ADV.
hepatitis; HBV; polymerase; genotype; adefovir; molecular dynamics simulation; free energy calculation
Hepatitis B virus (HBV) is a disease of global concern. This
virus has infected approximately 400 million people
worldwide, including 93 million people in China[
]. HBV is a
highly variable virus with mutations that occur because of
a shortage of proofreading activity of the DNA polymerase,
which results in the formation of different genotypes and
subtypes. There are eight hepatitis B virus genotypes (A-H)
throughout world. Genotypes B and C are the most common
genotypes in Asia[
]. Compared to genotype B, genotype C is
associated with later HBeAg seroconversion, more severe liver
disease and a higher incidence of hepatocellular carcinoma
Currently, the available antiviral drugs include
interferonalpha (IFN-?) and nucleotide analogue inhibitors of HBV
polymerase, among which lamivudine (3TC), telbivudine
(LdT), entecavir (ETV), adefovir (ADV) and tenofovir (TDV)
have been approved by the FDA[
] (Figure 1). ADV is an
acyclic nucleotide analog that inhibits the HBV DNA polymerase
and suppresses HBV replication at the level of DNA synthesis.
It is efficacious even for the treatment of lamivudine-resistant
chronic hepatitis B[
To treat patients with chronic hepatitis B, a personalized
approach allows for the treatment efficacy to be optimized,
which involves individualizing the therapy according to the
genetic differences of the infectious agents. Recently,
increasing evidence has shown that there are differences in the
antiviral response among HBV genotypes[
]. IFN-? therapy
was reported to be less effective in European patients infected
with genotype D compared to those infected with genotype
A, whereas in the Asian population, genotype C showed
cacy among different HBV genotypes. In this study, the
differences between genotypes B and C that influence the binding
affinity of ADV are studied using computational approaches.
Two 3D models were first constructed for the HBV
polymerase from genotypes B and C. Molecular docking,
dynamics simulations, and binding free-energy calculations were
then performed to explore the binding patterns of ADV and
provide insights into the difference in the binding free-energy
of genotypes B and C.
worse performance than genotype B. A clinical trial of 3TC
has demonstrated that patients infected with HBV genotype
A had higher DNA levels than those infected with genotype
]. For ADV therapy, the data associated with the
sensitivity among HBV genotypes are subtle, particularly between
the two predominant genotypes in Asia, ie, B and C. Zeng et
al identified a genotype-specific outcome, indicating that the
HBV genotype B shows a greater reduction of viral DNA than
genotype C (3.6 vs 3.1 logarithm copies/mL) at 48 weeks ADV
post-treatment in a Chinese Han population with chronic
]. However, another study of ADV therapy reported
that a difference in the level of viral DNA reduction (3.42 vs
3.65 logarithm copies/mL) was observed after 48 weeks of
treatment, although the association with HBV genotypes was
not statistically significant[
]. The differences in the average
reduction of viral DNA could partially reflect the difficulty
in analyzing the antiviral response to ADV therapy because
the different ethnicities, endpoint definitions, sample size and
whether the patients have been treated with other therapies
would all affect the results of the clinical trials[
Recently, a computational study of the possible mechanisms
of the different inhibitory effects of benzothiadiazine
molecules on HCV genotype-1a and genotype-1b NS5b polymerase
was reported, in which the ligand sensitivity was investigated
with homology modeling and molecular docking[
the structural information of the HBV polymerase from
genotypes B and C would also be helpful for investigating the
molecular basis of ADV sensitivity. Because the crystal
structure of the HBV polymerase is not available, homology models
based on the X-ray structure of HIV-1 RT have been reported
and utilized for determining the molecular mechanism of
inhibition and drug resistance[
]. Thus, no computational
study was reported regarding the molecular basis of drug
Homology modeling of HBV polymerase-DNA complex
The genotype consensus reference sequences reported by Rhee
] were used to represent the HBV polymerase genotypes
B and C. The sequence alignment between the two genotypes
was performed using the EMBOSS online program[
HIV-1 RT crystal structures are available in the PDB database.
Because the complexed ligand, TDV, is a methyl derivative
of ADV, the complexed structure of HIV-RT/TDV (PDB ID:
1T05) was selected as the template[
]. The recently published
alignment of HBV polymerase and HIV-1 RT that was
developed by Daga et al[
] was used as the reference template for
modeling the HBV polymerase domain. Following the
alignment, the homology models of genotypes B and C were built
using the automated modeling module, MODELLER[
], in the
Discovery Studio (DS) software package version 3.0 (Accelrys,
San Diego, CA, USA). The optimal level of the entire protein
and loops was set to high and medium, respectively. The
best structure for each genotype was then selected
according to the PDF (probability density function) scores and the
three-dimensional fit to the template protein. After the initial
model was prepared, the coordinates of the template-primer
DNA duplex (11/7), two Mg2+ cations and one original ligand
[tenofovir diphosphate (TDV-DP)] were transferred from
the template structure, 1T05, to form a quaternary complex.
After the energy minimization, the geometric quality of the
modeled structures was evaluated by PROCHECK[
protein structures were visualized with the PyMOL molecular
The molecular docking study was performed with the Glide
program (version 5.5)[
]. It should be noted that ADV is a
module of AMBER 9.0.
The binding free energy (?Gbind) of ADV-DP in the two
systems was calculated using the MM-PBSA method[
was computed as follows:
prodrug, and we modeled its bioactive form, ADV
diphosphate (ADV-DP), which is generated through stepwise
phosphorylation after the oral administration of ADV. The 3D
conformation of ADV-DP was constructed according to the
coordinates of the ligand, TDV-DP, which was deposited in
the template structure of HIV-RT, by deleting a methyl group.
After the ligand preparation, each modeled protein was
prepared with the protein preparation wizard in Maestro[
molecular docking was performed using standard precision
(SP) protocols with default parameters. The docking poses
were ranked by their glide scores, and the best predicted
conformation in each system was used for the subsequent
molecular dynamics simulation and binding energy analysis.
?Gbind=?Gcomplex ? (?Gprotein +?Gligand) (1)
?Gbind=?Egas +?Gsolv ?T?S (2)
?Egas =?Eint +?Eele +?Evdw (3)
?Gsolv =?GPB +?GSA (4)
?Egas, the sum of the molecular mechanical energies, is
composed of three parts: internal energy (?Eint), electrostatic
potential (?Eele) and van der Waals potential (?Evdw). The
solvation free energy (?Gsolv) can be divided into contributions
from the polar solvation free energy (?GPB) and the nonpolar
(?GSA). In the PB calculation, the solvent and solute
dielectric constant values were set to 80 and 2, respectively. The
] was used to estimate the non-polar
contribution as follows: ?GSA=0.0072??SASA, where a probe radium of
1.4 ? was set for the SASA (solvent-accessible surface area)
]. Because of the high computational demand for
large molecules, such as proteins, and the rough estimate of
the entropic contribution (-T?S)[
] and because we are
comparing the relative binding affinities of two similar systems (ie,
HBV polymerase genotypes B and C complexed with the same
inhibitor, ADV-DP), the entropic contribution of both systems
is disregarded in this study. The energy difference between
the two systems (??GC?B) is as follows:
C were retrieved using the National Center for Biotechnology
Information (NCBI) genotyping tool (Supplementary Information
Table S1). The sequence analyses of these reference sequences
revealed that amino acid (AA) 238 located near the ligand
binding pocket is different among the six consensus genotypic
reference sequences (ie, Gln/His/Asn is located at position
238 in genotype B and Asn is located at position 238 in
genotype C), and all the other polymorphic sites between the two
genotypes were found distant from the binding pocket.
Further statistical analyses of the published sequences reported
in NCBI confirmed the significant difference at position 238
between genotypes B and C. As shown in Table 1, the
occurrence rates of His and Asn at this position are 81% in genotype
B and 86% in genotype C, respectively.
Based on the above analyses, the amino acid at position
238 was set to His for genotype B and Asn for C. The overall
consensus reference genotype sequences used to construct
the homology models of the HBV polymerase genotype B
and genotype C share a high identity of 93.3% (Supplementary
Information Table S2). The sequence alignment of the HBV
polymerase with HIV-1 RT is shown in Figure 2. The final
homology models of the HBV polymerase genotypes B and
C are shown as ribbon diagrams in Figure 3. Similar to the
HIV-1 RT, the modeled structures of the HBV polymerase also
have three typical subdomains: finger (1 to 49 and 90 to 172),
palm (50 to 89 and 173 to 267) and thumb (268 to 344)[
As illustrated in Figure 3A, the two HBV polymerase
models and HIV-1 RT structure resemble to each other in the palm
and thumb regions; however, there is a major difference in the
finger subdomain. The HBV polymerase has a larger finger
region than the HIV RT and contains three long loops of 27,
11, and 9 residues in the HBV finger domain. Overall, the
high similarity can be observed between the HIV-1 RT X-ray
structure and the modeled HBV polymerase, particularly in
the conserved catalytic subdomains, A?G[
], which are
highlighted by different colors in Figure 3C. The structural
variations of those two genotypes existed primarily in the flexible
loop domain that is distant from ligand active site.
The homology models of the HBV polymerase genotypes B
and C were then evaluated by PROCHECK[
]. The overall G
factors were -0.2 and -0.22 for genotypes B and C, respectively,
indicating that the molecular geometries of the constructed
models are stereochemically acceptable. The Ramachandran
plots of the two models are shown in the supporting
information (Supplementary Information Figure S1). Of all the residues,
81.7% and 82.2% are located in the most favored regions for
genotypes B and C, respectively. Further analysis of the
models revealed that all the disallowed residues except Met204
located in the YMDD motif were located distant from the
putative ligand-binding site of the HBV polymerase.
Consistent with Daga et al[
], the corresponding Met residue was also
found in the disallowed region of many HIV-1 RT crystal
complexes, suggesting that the unusual configuration of Met204 is
a valid and conserved feature. These evaluation results show
that our homology models of HBV polymerase are of
Docking studies of ADV diphosphate (ADV-DP) with HBV polymerase genotypes B and C
The superposition of the two docked complexes with the
crystal protein, 1T05, suggests that the ADV-DP was docked
into the active site with favorable poses, both bearing a close
resemblance to the pose of the analogous ligand, TDV-DP,
in 1T05. The best possible binding modes of ADV-DP in the
HBV polymerase active sites of genotypes B and C are
illustrated in Figure 4. Both complexes are stabilized by extensive
hydrogen bonding networks. The nitrogen base of ADV-DP
displayed two hydrogen bonds with the complementary base
pair, dTMP, in the template chain. Moreover, it formed a ???
stacking interaction with the DNA base in the primer chain.
The ?-phosphate and ?-phosphate of the ligand formed a
hydrogen bond with the backbone amide NH of Ala86 and
Ala87. The side chains of Arg41 and Lys32 were involved
in ionic interactions with the phosphate group of ADV-DP.
Arg41, in the systems of genotypes B and C, formed three and
two hydrogen bonds with ADV-DP, respectively. The
carbonyl groups of Asp83, Asp205 and Val84 together with the
three phosphate groups of ADV-DP formed a metal chelating
interaction with the two Mg2+ ions that were present in the
active site. The docking scores of the two genotypes, B and
C, are also at the same level, with gscores of -10.25 and -10.07,
Assessing the binding mode of the different genotypes by MD simulation
To further validate our docked model, a molecular dynamics
simulation was preformed to obtain a more precise
ligandreceptor state that is close to the natural behavior of the
complex. The conformational stability of these two HBV
polymerase-ADV complexes was assessed by performing a 3.8
ns molecular dynamics simulation in an explicit water model
under the same conditions. The root mean square deviation
(RMSD) values with respect to the initial structures were
calculated along the 3.8 ns trajectories and are shown in Figure
5. The backbone RMSD values of the two systems tend to be
convergent after 2 ns of simulation, with fluctuations
approximately 4.5 ? and 4 ? for the structures of genotypes B and
C, respectively. The RMSD change of the conserved catalytic
subdomains, which is highlighted in Figure 3, exhibits a
similar tendency. Intuitively, the lower conserved subdomain
RMSD values of genotype C may suggest that the complex of
ADV-DP bound within the HBV polymerase genotype C is in
a more stable state.
Figure 6 summarizes distance fluctuations of the
corresponding atom pairs that are involved in the hydrogen bond
interactions. As shown, some significant hydrogen bond
interactions that were observed in the initial docking model (Figure
4) were maintained during the MD simulation. For example,
two hydrogen bonds between the 6-amino purine ring of
the ADV-DP and the paired base, dTMP, and the hydrogen
bond between Arg41 and the ?-phosphate of ADV-DP were
maintained through the entire simulation of both systems.
Moreover, we may also find a newly formed hydrogen bond
between the ?-phosphate of ADV-DP and the hydroxyl group
of Ser85 during the MD simulations.
The simulation system of genotype C showed superiority
during the shorter time needed to form a steady hydrogen
bonding interaction. Furthermore, the side-chain
conformation of Ser85 in the genotype C system was further stabilized
by a hydrogen bond with Asn236 in a loop region (Figure
6E), which was consistent with the binding pattern reported
by Langley et al[
]. However, in the genotype B system, the
hydrogen bonding interaction between Ser85 and Asn236 was
absent. The distance fluctuations that were observed from the
MD simulations suggest that the different AA compositions at
position 238 may affect the hydrogen bond formation between
Ser85 and Asn236.
MM/PBSA free energy analyses
The binding free energies obtained from the MM/PBSA
method are shown in Table 2. The table also lists the detailed
contribution of the various energy components. It is clear
from the table that the ADV-DP showed better binding
affinity towards HBV polymerase genotype C than genotype B
a Components: ?Eele, electrostatic energy; ?EvdW, van der Waals energy;
INT, internal energy arising from bond, angle and dihedral terms in the
MM force field; ?GSA, nonpolar contribution to the solvation free energy;
?GPBSA, solvation free energy, it is the sum of ?GPB and ?GSA; PBTOT, final
estimated binding free energy calculated from the terms above.
b Average energy over 100 snapshots in the last 800 ps of MD simulation.
c Standard error of mean values.
d The energy difference between genotype B and C (C?B).
To provide quantitative information regarding the detailed
contribution of the residues in the different HBV polymerase
genotypes, free energy decomposition analysis was utilized
to decompose the contributions to the binding energies on
a per-residue basis. Many residues were found to exhibit
influences on the binding to the inhibitor, ADV-DP. Several
amino acid residues, Lys32, Arg41, Asp83, Ser85, Ala86, Ala87,
Phe88, Asp205, and Asn236, and two DNA base pairs were
validated to have significant positive contributions with an
absolute energy greater than 1 kcal/mol. This is highlighted
in Figure 7. Notably, ADV-DP binds to similar key residues
in the two systems, which is consistent with the results of the
docking study and MD simulation. The favorable
contributions of Lys32, Arg41, Asp83, Ser85, Ala86, Ala87, and dTMP
are due to the hydrogen bonding interactions with ADV-DP.
The purine ring of ADV-DP formed a strong ?-? stacking
interaction with the primer DNA base, which also showed an
obvious hydrophobic interaction with Phe88. Phe88 is located
within the hydrophobic pocket comprised of Ala87, Phe88,
Ile180, and Met204. This finding is consistent with the report
by Daga et al[
]. In addition, for most of the key residues, a
slightly stronger binding energy contribution was found in the
genotype C system.
As observed in Figure 7, positive contributions were made
for all the key residues except Asp205, which possibly arises
from the repulsive interaction between the negatively charged
side chain of Asp205 and the negatively charged phosphate
group of ADV-DP. By contrast, the Mg2+ ions between Asp205
and the phosphate group of ADV in these two stable
complexes (Figure 4) mediate a favorable coordination linkage
between D205 and ADV-DP. The genotype-dependent,
polymorphic residue 238 shows less contribution to the binding
energy (?1.0 kcal/mol). A lower residue contribution to
genotype B than C was found at positions 236 and 238, which was
verified by the computational alanine scanning results (shown
in Table 3). Both the alanine mutations at residues 236 and
238 lead to losses of sensitivity, as reflected by an increase of
the binding free energy difference between genotypes C and
B (??GC?B). For the N236A mutant, the ??GC?B increased from
-20.96 (??GC?B, wild type) to -18.66 kcal/mol (??GC?B, N236A). For
the H/N238A mutant, the ??GC?B increased to -19.96 kcal/mol
(??GC?B, H/N238A). These results confirmed our hypothesis that
residues 236 and 238 contribute to the sensitivity of genotype
C over B regarding the interaction with ADV-DP. Moreover,
Asn236 is more important because the alanine mutation at this
residue site causes a more significant loss of sensitivity.
Hepatitis B is a growing problem worldwide. Genotypes B
and C are the two predominant types of HBV in Asia.
Clinical studies have been conducted to investigate the influence of
specific genotypes on ADV treatment; however, the results are
not yet conclusive. To further explore the underlying nature
of the sensitivity of different genotypes of HBV polymerase
to ADV, we used a computational protocol that combined
homology modeling and binding free-energy calculations with
For the homology modeling of different genotypes of HBV
polymerase, the representative sequence selection is of great
significance. The statistical sequence analyses shown in Table
1 reveal that residue 238 near the binding pocket is not only a
polymorphic site but is also a genotype-specific site (ie, His238
is present in genotype B and Asn238 is present in genotype
C). This observation is consistent with previously reported
sequence analysis results. An investigation of 192
treatmentna?ve Chinese patients showed that position 238 is a
genotypedependent polymorphic site, in which 52/55 genotype B
patients have a His at this position and 131/137 of genotype
C patients have an Asn[
]. Moreover, Rhee et al demonstrated
that the AA types His238 and Asn238 are typical for B and C
In the polymerase/RT region, the sequence identity among
various HBV strains is significant; however, the identity
between HBV and other viruses is relatively low. HIV-1 RT
shares approximately 25% sequence identity with HBV[
and has available crystal structures and is one of the
nearest relatives of HBV polymerase. Many of the functionally
important residues that are involved in the key protein-ligand
interactions in HIV-1 RT are conserved between the HIV-1 RT
structure and the HBV polymerase. These include the YMDD
motif, the MGY motif and the catalytic triad of aspartic acid
residues. For example, the catalytic triad Asp83, Asp205,
and Asp206 of HBV polymerase correspond to Asp110,
Asp185, and Asp186 in HIV RT (Figure 2). These three Asp
residues are responsible for the coordination of two Mg2+
ions, which are very important for the activity of the
polymerase. These data suggest that HIV-1 RT shares sufficient
structural and functional similarity with HBV polymerase to
serve as an excellent template to build a 3D structure for the
]. In our work, the sequence alignment
of our homology model referred to the alignment of the latest
sequence published by Daga et al. Based on this sequence, the
resulting homology model can match the results of the
experimental mutation studies of the HBV polymerase[
]. Only a
minor modification was made at site Asn236 according to the
work of Das et al[
], in which Asn236 of the HBV polymerase
was aligned with Pro217 of HIV-RT instead of the Thr216 as in
Daga?s work. Asn236 is an important residue in the binding
pocket of HBV polymerase and it is a primary drug resistance
mutation site to ADV-DP[
]. However, the alignment of Daga
et al would result in the Asn236 in the constructed structure
to be located too distant (~7 ?) from the nearest atom of the
ligand (CTP), as suggested by their docking study. Therefore,
for this residue, we chose the alignment of Das et al and used
residue Pro217. As a result, the position of Asn236 in the
constructed homology model is more feasible and is located in
close vicinity to the binding site of ADV-DP. Apart from this,
there are no other changes.
After the three-dimensional structure was successfully
constructed, the ADV-DP was docked into the HBV
polymeraseDNA complex. From the binding models, similar
hydrogenbond networks are observed for the ADV-DP in the HBV
polymerase genotypes B and C. In addition, the gscores of the
two systems are at the same level. All these indicate that the
binding affinity that was predicted by docking could not
discriminate between genotypes B and C. We must also note that
molecular docking cannot fully account for the impact of
protein flexibility, and a small change in receptor conformation
may lead to a significant variation of binding affinity. Because
identical structure templates were used in the homology
modeling of the HBV polymerase genotypes B and C, the similar
binding patterns of the two systems may simply be a result of
the similar static protein conformations that were modeled.
Therefore, to consider the flexibility of both the ligand, the
protein and also their interactions with the surrounding water
molecules, molecular dynamics simulations in combination
with MM/PBSA free energy calculations were performed.
In summary, the calculated results support the hypothesis
that the HBV polymerase genotype C is more sensitive to
ADV than genotype B. Moreover, residue N236 and the
polymorphic site 238 were further investigated and shown to play
an important role in contributing to the sensitivity of genotype
C over B in the interaction with ADV-DP. We believe that
this work should provide a basis for the further investigation
and understanding of the effect of polymerase genotypes on
ADV treatment. This allows for the optimization of individual
treatment and the minimization of side effects associated with
This work is supported by the State Key Laboratory of Drug
Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences. The authors gratefully acknowledge
financial support from the State Key Program of Basic Research of
China (No 2009CB918502), and the National Natural Science
Foundation of China (No 81001399).
Ming-yue ZHENG and Ai-long HUANG designed the study,
supervised the research and revised the manuscript. Jing LI
performed the research, analyzed the data and wrote the
manuscript. Yun DU, Xian LIU, Qian-cheng SHEN, Xiao-min LUO,
and Hua-liang JIANG discussed the results and commented
on the manuscript.
Supplementary Table and Figure are available at the Acta
Pharmacologica Sinica website.
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