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Molecular dynamics study on drug resistance

mechanism of HCV NS3/4A protease inhibitor:
BI201335
a a
Jianjian Fu & Jing Wei
a
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of
Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, P.R. China
Published online: 27 May 2014.

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To cite this article: Jianjian Fu & Jing Wei (2014): Molecular dynamics study on drug resistance mechanism of HCV NS3/4A
protease inhibitor: BI201335, Molecular Simulation, DOI: 10.1080/08927022.2014.917298

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 Molecular Simulation, 2014
http://dx.doi.org/10.1080/08927022.2014.917298

Molecular dynamics study on drug resistance mechanism of HCV NS3/4A protease

inhibitor: BI201335
Jianjian Fu and Jing Wei*
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin
University, Tianjin 300072, P.R. China
(Received 28 November 2013; final version received 15 April 2014)

Hepatitis C virus (HCV) infection is a serious threat to global health. NS3 serine protease is one of the most advanced HCV
drug targets. However, the high mutation rate makes many protease inhibitors ineffective and allows viral replication to
continue. To investigate the structural basis of the molecular mechanism of HCV resistance to inhibitors, molecular
dynamics and molecular mechanics Poisson Boltzmann/surface area calculations were carried out on HCV NS3 serine
protease BI201335 complexes. The drug resistance to BI201335 is explained by the fact that seven single mutations
weaken the biological activity by lessening the sum of electrostatic interactions in the gas phase and polar solvation. The
computational results demonstrate that the mutations affect the BI201335 binding through direct and indirect mechanisms.
Downloaded by [Nipissing University] at 20:44 13 October 2014

Seven single mutations lead to significant changes in the conformation, such as the shifts of the side chain of His57 and
Lys136 and the movement of the P2 group of BI201335 towards the solvent. Furthermore, the contributions of Lys136
significantly decrease, which is the most major binding attraction. The shifts of the side chain of His57 induce the lack of
hydrogen bond between His57 with Asp81 expert for D168G mutation. Detailing the molecular mechanisms of BI201335
drug resistance provides some helpful insights into the nature of mutational effect and aid the rational design of potent
inhibitors combating HCV.
Keywords: HCV NS3 serine protease; BI201335; drug resistance mechanism; MM-PBSA

1. Introduction improved tolerance. Second-wave protease inhibitors offer

Estimates of the global prevalence of hepatitis C virus
(HCV) reveal that 170 million people worldwide are
infected with HCV, representing , 3% of the worlds
population.[1] HCV is recognised as a major cause of
chronic liver disease, cirrhosis, hepatocellular carcinomas,
as well as liver transplantations in developed countries.
[2,3] More than 350,000 people die from hepatitis C-
related liver diseases every year.[1] However until now,
there is no effective protective vaccine to fight HCV
infection owing to its high mutation rate.
For the past decade, the standard treatment of HCV
infection is a combination of pegylated interferon a and
ribavirin (Peg-IFN/RBV). Unfortunately, Peg-IFN/RBV
produces sustained virological response in only 45% of
genotype 1-infected patients and is limited by substantial
side effects.[4,5] In 2011, the treatment of HCV infection
has been revolutionised by the approval of two ketoamide
inhibitors boceprevir and telaprevir, which are the first
direct-acting antiviral agents against the NS3/4A serine
protease-inhibiting intracellular viral replication.[6 8]
However, these first-generation drugs also have significant
limitations due to a thrice-daily dosing (with food),
extensive drug drug interactions and low barriers to
resistance. Thus, there is an urgent need to develop the
virus-specific drugs that have promising efficacy and
improved pharmacokinetics and tolerable side effect
profiles, such as BI201335, simprevir and asunaprevir,
which are in phase III trials.[9,10]
BI201335, developed by Boehringer Ingelheim Incor-
poration, is a promising drug candidate that targets the
viral NS3/NS4A serine protease.[11] Recently, the crystal
structure of BI201335 bound to NS3/4A was reported,[12]
which shows that BI201335 adopts an extended confor-
mation, similar to other protease inhibitors, to interact with
the enzymes substrate-binding site (Figure 1). However,
unlike other inhibitors, BI201335 possesses a C-terminal
carboxylic acid that non-covalently binds to the active site,
and a bromo-quinoline substitution on its proline residue
that provides significant potency to the viral protease.[12]
As patients begin treatment, some mutations arise as a
consequence of the selective pressures of antiviral drugs
and result in drug-resistant variants. To unravel the atomic
mechanism of drug resistance, it is essential to develop
specific and effective drugs against HCV. Several
computational studies have been performed to understand
the drug resistance mechanism of NS3/4A mutations: the
free energy perturbation method and molecular mechan-
ism Poisson Boltzmann surface area (MM-PBSA)
method.[13 19] Molecular mechanics (MM) force field
is one of the most popular approaches to estimate the

*Corresponding author. Email: jingwei_201311@163.com

q 2014 Taylor & Francis
 2 J. Fu and J. Wei
Figure 1. (Colour online) The ligand binding modes of BI201335 in WT NS3/4A. For clarity, only interacting residues are displayed. (a)
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Molecular surface representation of the active site of the WT NS3/4A protease (PDB ID: 3P8N) with BI201335 and the selected sub-sites
are indicated (H-bond, hydrophobic and mild polar are coloured in magenta, green and blue, separately). (b) 2D chemical structure of
BI201335 and the schematic representation of the hydrogen bond interaction with NS3/4A protease (ligand exposure is coloured in
purple.
affinity of protein ligand binding in rational drug
discovery.
The high-resolution structure of the NS3/4A
BI201335 complex can be a starting point to understand
the binding and resistance mechanism due to point
mutations. In this study, on the basis of the structure of
BI201335 in complex with the wild-type (WT) NS3/4A
and its seven single-site mutations, molecular dynamics
(MD) simulation, binding free energy calculation and free
energy decomposition were performed to explore the
binding and resistance mechanism of NS3/4A to
BI201335. The binding free energies were calculated by
using MM, polar solvation energy from Generalized Born
(GB)/Poisson Boltzmann (PB) continuum solvation, and
non-polar solvation energy from a surface area model,
referred to as MM-GBSA/MM-PBSA.[20,21]
2. Materials and methods
2.1 Preparation of the system
The initial crystal structures of the WT HCV NS3/4A
serine protease complexed with the drug BI201335 (PDB
code: 3P8N) with a resolution of 1.9 A [12] was extracted
from atomic coordinate entries available in the Protein
Date Bank. An Na ion was removed for the reason that it
is far from the active site of the protease.
As the experimentally determined structures of the
mutant proteins were not available, Modeller software [22]
was used to generate seven mutant structures (R155K/Q,
A156V/T, D168G/A/V) in complex with BI201335 by
substituting specific residues using the WT structure as the
template. The procedure involved the mutated residue to
the desired residue followed by MD simulated annealing
optimisation.
2.2 MD simulations
MD simulations of the eight systems were performed
using the SANDER module, implemented in the Amber
10.0 package.[23] The Amber parm99SB [24] force field
and the general AMBER force field [25] were,
respectively, used for the protein and the ligand. The
force field parameters of BI201335 were prepared with the
Antechamber module.[26] Counter ions (Cl2) were added
to neutralise the charge of each inhibitor-bound system.
The complex was solved in the TIP3P water box,[27] with
the truncated octahedron periodic box of size 8 A under the
explicit waters. The particle mesh Ewald method [28] was
used to treat long-range electrostatic interactions. The cut-
off distance for the long-range electrostatic and the van der
Waals energy terms were set at 8.0 A. The SHAKE
algorithm [29] was applied to fix all covalent bonds
containing a hydrogen atom. The solvated system was
energy-minimised in three steps prior to the MD
simulation. At first, the protein ligand was frozen while
the solvent water molecules and counterions were allowed
to move. Next, the ligand and the side chain of the receptor
residues were allowed to move in addition to the water
molecules. Finally, all atoms were permitted to move
freely. Subsequently, the complex of the minimised
structures was heated from 0 to 300 K for 100 ps
calculation, the temperature was maintained at a
temperature of 300 K with a coupling coefficient of
2.0 fs. After heating calculation, each system was
equilibrated in the canonical ensemble of density
 Molecular Simulation 3

calculation for 100 ps and thus for 0.5 ns without restraint.
Finally, the systems were subjected to a 30- ns MD
simulation at 300 K and 1 atm pressure with an integration
step of 2 fs and NPT as an ensemble type. The MD
simulation was performed with a periodic boundary
condition. Atom coordinates were collected to analyse the
structure in detail using the PTRAJ modules. The
convergence of energies, the H-bond, the distance and
the atomic root-mean-square deviation (RMSD) of the
stability of the systems were analysed based on the above-
mentioned MD simulations.
2.3 MM-PBSA calculations
MM-PBSA method in AMBER10 was implemented to
calculate the binding free energies between BI201335 and
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energies in several earlier studies applying the same
approximation.[32,33] In our calculations, the energy
contribution from entropy changes on ligand binding was
not included.
2.4 Residue inhibitor interaction decomposition
On account of the huge demand of computational
resources for PB calculations, the interaction between
inhibitors and each residue in protease was analysed using
the MM-GBSA module in AMBER 10.[34] Energy
decomposition calculation analyses same snapshots as
the free energy calculation. The binding interaction of
each inhibitor residue pair includes four terms:
DGinhibitor2residues DGvdw DGele DGgb DGnp : 5

the protease.[20,21,23] For each system, a total number of

where van der Waals contribution (DGvdw) and electro-
4000 snapshots were extracted from the last 20-ns MD
static contribution (DGele) can be calculated by the Sander
simulation. In general, the binding free energies in
program in Amber 10.0. The polar solvation contribution
condensed phase can be calculated according to the
(DGgb) is computed using the GB module.
following equations.
The total free energy of binding was composed of the
following terms:
3. Results and discussion
DGb DEMM DGsol 2 TDS; 1 3.1 Stability and flexibility of the system simulations
To explore the effect of mutations on the conformational
DEMM DEele DEvdw ; 2 stability of NS3/4A BI201335 complexes, the RMSD of
backbone of the protease and inhibitor to their original
conformations during the 30 ns production phase were
DGsol DGpb DGnp ; 3 calculated and are depicted in Figure 2. The RMSD plots
indicate that the conformations of the WT and seven
where DGb is the binding free energy in solution; DEMM is single-mutant NS3/4A BI201335 complexes became
the MM free energy in gas phase, comprising electrostatic
(DEele) and van der Waals (DEvdw) interactions between
the ligand and the protease. The salvation free energy
(DGsol) is further divided into two components, where
DGsol is the solvation free energy, and DGpb and DGnp are
the polar and non-polar contributions to the solvation free
energy, respectively.[30,31] DGpb is determined by solving
the PB equations numerically and calculates the electro-
static energy according to the electrostatic potential,[31]
and the non-polar contribution is estimated by the solvent-
accessibility surface area (SASA) method, where the
values for g and b were set to 0.00542 kcal/(mol A2) and
0.92 kcal/mol, respectively [30]:

DGnp gSASA b: 4

The contribution of entropy (TDS) to binding free energies

is an important contribution in enzyme ligand bindings.
However, a number of studies published by Kollman and
Kuhn and Warshels group showed that entropy does not
contribute much to the relative binding free energies of the
ligands to the same protein and the good agreement
between the calculated and experimental relative binding
Figure 2. RMSDs, all Ca atoms o the NS3/4A BI201335
complex during the MD simulations complexes during the
production phases relative to the initial minimised structures. (a)
Time evolution of the RMSD of all protein backbone atoms. (b)
Time evolution of the RMSD of heavy atoms for the inhibitors.
 4 J. Fu and J. Wei

Table 1. Binding free energy components for the NS3/4A BI201335 complexes by using the MM-PBSA method (kcal/mol).

DEvdw DEele DEMM DGnp DGpb DGsol

Complex DEele DGpb DGb Resistance level [35]

WT 244.50 2282.24 2 326.75 2 6.76 281.22 1.02 274.46 252.30 1
A156V 246.63 2219.09 2 265.72 2 6.91 225.25 6.16 218.35 247.38 150 ^ 10
A156T 241.19 2240.20 2 281.39 2 5.72 242.59 2.39 236.87 244.53 270 ^ 98
R155Q 247.75 2219.37 2 267.12 2 6.56 227.96 8.59 221.40 245.72 60 ^ 9
R155K 244.69 2246.14 2 290.83 2 6.09 251.13 4.99 245.04 245.79 350 ^ 14
D168G 252.33 2228.14 2 280.47 2 7.45 250.64 21.50 243.19 237.29 80 ^ 30
D168A 249.44 2225.75 2 275.19 2 7.10 243.94 18.19 236.84 238.35 690 ^ 332
D168V 237.71 2266.93 2 304.74 2 5.80 265.66 21.27 259.86 244.88 970 ^ 256

equilibrated and stable after 10 ns. The RMSD of all eight 245.72, 237.29, 247.38, 244.53, 245.79, 238.35 and
complexes exhibit low backbone RMSD values with mean 244.88 kcal/mol, respectively.
values around 0.86 1.31 A for the backbone and around Figure 3 depicts the insignificant changes in the
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1.80 2.66 A for inhibitor, respectively. The trajectory of
A156V mutant/BI201335 complex fluctuates more than
other trajectories, which implies that the A156V mutant
complex undergoes larger changes in conformation than
other seven complexes (1.31 A for the backbone and
2.66 A for inhibitor, respectively). This result suggests that
the conformations are similar to the starting structures, and
the stabilities of the dynamic equilibriums for eight
complexes are reliable. Considering these, the confor-
mations of the complexes after equilibrium were chosen to
assess the quality of MD simulations, energetic and
structural properties.
3.2 Analyses of binding free energies
To gain insight into the forces involved in substrate binding,
total binding free energy and individual energy components
for each system were obtained by using the MM/PBSA
method based on these trajectories. Table 1 lists the van der
Waals (DEvdw), electrostatic (DEele), non-polar solvation
(DGnp) and electrostatic solvation (DGpb) for the eight
proteaseinhibitor complexes. As shown in Table 1, the
predicted binding free energies of BI201335 to WT, R155Q,
D168G, A156V, A156T, R155K, D168A and D168V
mutants of HCV NS3/4A serine protease are 252.30,
energetic profile. In all eight protease inhibitor com-
plexes, the MM free energies are the main driving force of
energies to the inhibitor binding. In particular, electrostatic
interactions in the gas phase provide extraordinarily
significant contribution to the binding free energies.
However, their favourable effects are offset by polar
solvation terms that are positive and unfavourable for the
binding free energies. The resulting balance of the two
contributions, the electrostatic interactions in the gas phase
and the polar solvation terms, is slightly unfavourable to
binding interactions in all eight systems. In eight systems,
van der Waals energies are about seven times stronger than
non-polar solvation energies. Thus, it can be concluded
that van der Waals energy is also the driving force for the
binding of BI201335 NS3/4A complexes. Non-polar
solvation terms, which correspond to the burial of SASA
upon binding, contribute slightly favourably.
Compared with the WT, the binding affinity of each
single mutation complex decreases by 4.92 15.01 kcal/
mol, which elucidates that the mutant NS3/4A binds
weakly to BI201335 than the WT NS3/4A. This also
explains the fact that mutated NS3/4A proteases exhibit
drug resistance to BI201335. It is very encouraging that
the resistance levels of available experiment [35] of the
BI201335 mutant NS3/4A complexes are consistent with

Figure 3. Profiles of various binding energy components (kcal/mol) for the WT and mutant NS3/4A BI201335 complexes.

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Figure 4. Per-residue interaction spectrum of NS3/4A serine protease with BI201335. (a) WT, (b) A156T/V, (c) R155K/Q, (d) D168A/
G/V.
our predictions. The differences of the non-polar solvation
energies among the eight systems are very small, which
indicates that the better cavity packing is maintained in the
mutated complexes. The decrease of the sum of
electrostatic interactions in the gas phase and the polar
solvation terms should be essential to the resistance
relative to the WT.
3.3 Free energy decomposition analysis
To locate the residues, which contribute to BI201335
interaction, and understand the mutation mechanisms,
absolute binding free energies are calculated for eight
complexes by MM/GBSA method. The binding free
energy is decomposed into inhibitor residue pairs in order
to generate an inhibitor residue interaction spectrum.
Figure 4 illustrates the decomposition of DGb values on a
per-residue basis for eight complexes.
For WT systems, the major binding attractions come
from the residues Lys136, Gly137, Ser138, Ser139,
Arg155 and Ala156, suggesting that they play important
roles in the binding. The favourable forces of these six
residues are electrostatic energies, which is in accordance
with the H-binding network from the experiment. Lys136
is the most important amino acid that contributes a lot to
the binding affinities (2 6.79 kcal/mol). Seven single
Molecular Simulation 5
mutations have obvious effect on the interaction between
Lys136 and BI201335, where DGb values of Lys136 drop
to some extent (ranging from 2 1.11 to 2 5.16 kcal/mol).
The decrease in the contribution of Lys136 can explain the
drug resistance to BI201335. For all eight systems, the
main force for amino acid His57 is van der Waals energy.
The contribution of His57 to the binding affinity is
stronger in D168A, R155Q/K and A156V/T than WT.
Interestingly, the decompositions of DGb values of Asp81
residues have unfavourable contribution of 0.69
1.22 kcal/mol except for D168A mutation that shows a
favourable contribution of 2 1.50 kcal/mol.
3.4 Intermolecular hydrogen bonds analysis
Hydrogen bonds play an essential role in stabilising
protein ligand complexes. To understand the effects of
mutations on the inter-molecular interactions, we
investigated the geometry and stability of the hydrogen-
bond network formed by the inhibitor in the binding
pocket. In Table 2, the protease BI201335 H-bond
contacts are characterised in terms of distances between
heavy atoms and their percentage of occurrence on the
basis of the trajectories of the MD simulations (4000
snapshots). For the identification of hydrogen bonds, the
distance between donor and acceptor heavy atom was less
 6 J. Fu and J. Wei

Table 2. The occupancy (%) of hydrogen bonds.

Donor Acceptor Acceptor WT A156V A156T R155Q R155K D168G D168A D168V
1@O18 157@H 157@N 99.56 96.22 99.83 99.90 99.68 99.23 91.16 90.78
1@O3 137@H 137@N 98.43 96.11 98.57 99.41 98.38 10.27 62.33 87.94
1@O1 139@HG 139@OG 93.68 94.22 97.65 97.05 97.59 14.45 29.44 73.27
157@O 1@H24 1@N24 98.14 95.30 99.24 98.44 99.06 89.71 89.92 89.46
81@OD2 j 57@H 57@N 50.65 0.19 2.08 1.00 2.33 47.42 8.27 3.17
80@O 155@HH11 155@NH2 64.17 0.10 72.19 60.70 30.68 16.41
155@O 1@H9 1@N9 72.84 33.70 73.02 73.40 45.24 81.40 85.16 72.92
168@OD2 155@HH21 155@NH2 87.44 78.62 85.33
168@OD1 155@HH21 155@NH2 71.29 55.40 78.95
168@OD2 155@HE 155@NE 63.100 71.00 72.76

than 3.5 A, and the D H A angle was greater than or
equal to 1208.
BI201335 has a peptidic backbone so that it can form
extended, substrate-like interactions with an exposed b-
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binding affinity by only about 270 ^ 98-versus 150 ^ 10-
fold change.[35]
We can also observe that the non-bonded electrostatic
energy plays a major role in the ligand binding of

barrel of the enzymes substrate-binding site. BI201335
forms five hydrogen bonds with WT protease that plays
an important role in ligand binding (Figure 1), including
between the P1 P2 amide N9 and Arg155: O, between
the carbonyl O18 of the P2 P3 amide and Ala157: N,
between the carbamate nitrogen of the N-terminal
capping group and Ala157: O, between the oxygen
atom O1 of carboxyl group and Ser139OG and between
the oxygen atom O3 of carboxyl group and Gly137N.
These five hydrogen bonds are preserved with occurrence
frequencies of 72.84%, 99.56%, 98.14%, 93.68% and
98.43%, respectively. These two H-bonds between
BI201335 and Ala157 are stable in the seven mutant
inhibitor complexes which have maintained about 90%
occupancy during the MD simulation. However, the
hydrogen bonds between carboxyl group and Gly137 and
Ser139 are weaker in D168G/A than other complexes,
especially D168G with lower occupancies of 10.27% and
14.45%, respectively. The canonical substrate-like
intermolecular hydrogen bond between the P1 P2
amide N9 and Arg155: O is broken in A156V and
R155K mutations.
3.5 The drug resistance mechanism of the studied
mutants to BI201335
3.5.1 A156V/A156T mutation
A156 is located on the S2 strand in close contact with both
trans-hydroxyproline and bromo-quinoline P2 part of
BI201335. The presence of P2-proline motif in all reported
inhibitors has been shown to cause increased susceptibility
against mutations at the A156 position.[17]
The total binding free energies of the mutant A156V
and the mutant A156T are 2 47.38 and 2 44.53 kcal/mol
(Table 1), which agrees well with the experimental data
that the A156T mutation affects the binding of BI201335
to a greater extent than A156V mutation, decreasing the
BI201335 HCV NS3/4A WT and A156V/A156T mutant
complexes. Although there is a decreased non-bonded
electrostatic energy in both the mutant complexes, A156V
complex has a larger decrease than A156T mutation
(2 219.09 kcal/mol vs. 2 240.20 kcal/mol). However, the
solvent shielding of this interaction would make its
contribution by the displacement of P2 and P3 moieties.
As shown in Table 1, a relatively higher de-solvation cost
was noticed for A156T (236.87 kcal/mol) than A156V
(218.35 kcal/mol) which along with a decrease in DEvdw
made it more resistant than A156V mutation
(2 41.19 kcal/mol for A156T vs. 2 46.63 kcal/mol for
A156V).
Furthermore, we give a detailed picture of the effects
of these changes in the binding pocket of BI201335.
Compared with the WT, we monitored the structural
changes (shown in Figure 5(b)). Ala156 is mutated in a
more bulky residue Val/Thr resulting in a steric hindrance
to BI201335. The crowding in the region makes the
quinolone-P2 substituent of BI201335 to move away from
the extended S2 sub-site. The BI201335 motion further
induces the shift of the side chain of Lys136 and His57,
resulting in their contribution change.
The shift of key residue Lys136 affects severely the
interaction with BI201335 which its contribution drops to
2 2.35/2 2.96 kcal/mol for A156V/A156T mutation while
2 6.79 kcal/mol for WT, mainly from the decrease of
electrostatic interaction.
The shift of His57 leads to the loss of hydrogen bond
between His57 and Asp81, which makes the main chain of
Gln80 and Asp81 more flexible. Especially, the hydrogen
bond between Gln80 and Arg155 is broken in the A156V
mutant, which results in the decrease of the contribution of
Arg155 (2 0.75 kcal/mol). To profoundly analyse this
phenomenon, the hydrogen bond distances (defined by the
distance of Gln80O and Arg155NH2) were monitored
over the all-time simulation, and the plots of distance
 Molecular Simulation 7

versus the simulation time are shown in Figure 6. The plots
suggest that the hydrogen bond interaction between Gln80
and Arg155 disappears completely throughout the whole
simulation for A156V mutation. Interestingly, the
contributions of His57 increase from 2 0.77 kcal/mol in
WT NS3/4A compared with 2 3.02 kcal/mol in A156V
mutation and 2 1.78 kcal/mol in A156T mutation,
respectively, which indicates that the former is main
driving force of the van der Waals interaction and the latter
is main driving force of the electrostatic interaction.
3.5.2 R155Q/R155K mutation
To survey the changes in conformation, we superimposed
the structure of NS3/4A BI201335 complexes based on
the coordinates of the protease backbone atoms (shown in
Figure 5(c)). The side chain of Arg155 locates its
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guanidine at the bottom of the S2 pocket and adopts an
extended conformation to bind with the large bromo-
quinoline P2 substituent of BI201335. A salt bridge
formed between Asp168 with Arg155 and a hydrogen
bond between Gln80 and Arg155 play important roles in
the stabilisation of NS3/4A BI201335 complex. Thus,
substitutions at this position are the major causes of
clinical resistance to BI201335, often leading to a large
decrease in ligand binding.
The main factor contributing to the resistance level of
R155Q/K was the decrease in the electrostatic interaction
(2 219.37 kcal/mol for R155Q and 2 246.14 kcal/mol for
R155K). Both R155K and R155Q substitutions result in
the loss of salt bridge interactions with Asp168 and lack
hydrogen bond with Gln80, which makes Gln80 and
Asp81 more flexible. Furthermore, the hydrogen bond
between His57 and Asp81 is lost and a significant shift in

Figure 5. (Colour online) The superposition of the binding of BI201335 to WT and mutant NS3/4A. The proteins are shown in stick
model and the inhibitors are shown in wire model. WT NS3/4A BI201335 complex is colour-coded: magenta. (a) WT NS3/4A
BI201335 complex and NS3/4A BI201335 complex. (b) The superposition of the binding of BI201335 to WT, A156V mutant (green)
and A156T mutant (orange). (c) The superposition of the binding of BI201335 to WT and R155Q (green), R155K (orange). (d) The
superposition of the binding of BI201335 to WT and D168G (cyan), D168A (green) and D168V (orange).
 8 J. Fu and J. Wei
Figure 6. The monitoring for the hydrogen bond between Q80
and R155 during the all-time simulations. Plots show the time
evolution of hydrogen bond distance of Gln80O and Arg155NH2
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for WT NS3/4A BI201335 complex, A156V mutant NS3/4A
BI201335 complex and A156T mutant NS3/4A BI201335
complex.
the positions of the side chain of His57 was observed.
However, the contribution of His57 becomes stronger due
to electrostatic and van der Waals interaction energies
(Supplementary Table 1). In addition, His57 bonds with
Lys155 through the hydrogen bond interaction with
occurrence frequency of 92.54% in R155K mutation. The
P2 group of the inhibitor has extended away from the S2
sub-site and more exposed to the solvent. Furthermore, the
side chain of Lys136 shifts to avoid crowding resulting
from the movement of P2 group. This explains the reason
for the decrease in the contribution of Lys136.
When Arg155 is mutated to Lys155, the electrostatic
interactions between Lys155 and the P1 P2 amide N9
thus weaken. Compared with WT with occupancy of
72.84%, the R155K mutation does have an obvious effect
on the hydrogen bond between residue 155 and BI201335,
where the hydrogen bond occupancies are reduced to
45.24%. The above-mentioned result might be the
explanation for the higher resistance level for R155K
than R155Q.
3.5.3 D168G/D168A/D168V mutations
During the interaction of NS3/4A and BI201335, Asp168
does not directly participate in the anchoring of BI201335.
However, Asp168 is indirectly stabilising the confor-
mation of Arg155 residue by a salt bridge, which is very
important to orient Arg155 in a favourable conformation
to BI201335 binding.
For all the three mutations, D168G/A/V, significant
efforts were focused on BI201335 because of its
sensitivity to these mutations (80- to 970-fold resistance
level). Computational modelling indicates that the
decrease in binding of D168G/A/V variant proteases
with BI201335 is due to the indirect interaction for two
main reasons. First, the mutations of Asp168 prevent the
formation of the salt bridge between Arg155 and residue
168. Thus, Arg155 looses its S2-anchoring property
towards the P2 quinoline moiety of BI201335. Second, the
side chain of glycine/alanine/valine is shorter than the side
chain of aspartic acid, causing the shift of BI201335
towards the residue 168. In turn, the backbone of
BI201335 moves away from the original conformations
and the extended P2 group is further exposed to the
solvent.
The D168G mutation results in a strong 80-fold
resistance. However, the D168G mutation does not have
any direct impact on BI201335, which the decomposition
of the binding free energy of residue 168 has no significant
change. The Gly168 side chain is too short to provide more
interaction with the P2 group. Thus, D168G directly alters
the conformation of the Arg155 side chain and indirectly
alters the conformation of the complex between BI201335
and NS3/4A (Figure 5(d)). The P2 group adopts an
extended conformation in the solvent-exposed domain.
The movement of BI201335 disrupts the hydrogen bonds
with both Ser139 and Gly137 and results in the shift of the
side chain of His57 and Lys136. The contributions of
Lys136, Ser138 and Ser139 are weaker in D168G than the
WT NS3/4A complex. Interestingly, an increase in the
interaction energy is observed for Arg155 with 0.83 kcal/
mol in D168G complexes compared with the WT
complex, which is mainly from electrostatic contribution.
The D168A and D168V mutations, even though with a
higher 690-fold and 970-fold resistance, share the same
molecular mechanism. However, the changes in confor-
mations disrupt the hydrogen bonds between His57 and
Asp81 and between Gln80 and Arg155. The contributions
of Gly137, Ser138, Ser139, Arg155 and Ala156 become
weaker in D168A mutations than in D168G mutations,
which explains its stronger resistance. Interestingly, the
D168A mutation induced some additional favourable
interactions with Asp81, as compared with the unfavour-
able interactions for the other seven systems.
4. Conclusions
In our work, 30-ns MD simulations combined with the
calculation of the binding free energy by using MM-PBSA
have been performed to investigate the mechanism of the
drug resistance to BI201335. The results have shown that
the rank of calculated absolute binding free energy is in
good agreement with the experimental result. The decrease
in the electrostatic energy in the gas phase definitely drives
the drug resistance to BI201335. The mutations of A156,
R155 and D168 seriously affect the conformation of
the side chain of Lys136 and His57, leading to the
unfavourable induced-fit binding with BI201335. The

mutations induce the movement of the large P2 moiety of
BI201335 away from the extended S2 sub-site. These
detailed insights into the mechanism of BI201335
resistance will be very useful in the structure-based
designing of novel inhibitors that will be less susceptible
to resistance.
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