Accepted Manuscript

Computational study on the drug resistance mechanism of HCV NS5B RNA-dependent RNA polymerase mutants V494I, V494A, M426A, and M423T toFilibuvir

Huiqun Wang, Chenchen Guo, Bo-Zhen Chen, Mingjuan Ji

PII: S0166-3542(14)00315-5DOI: http://dx.doi.org/10.1016/j.antiviral.2014.11.005Reference: AVR 3545

To appear in: Antiviral Research

Received Date: 3 June 2014Revised Date: 5 November 2014Accepted Date: 9 November 2014

Please cite this article as: Wang, H., Guo, C., Chen, B-Z., Ji, M., Computational study on the drug resistancemechanism of HCV NS5B RNA-dependent RNA polymerase mutants V494I, V494A, M426A, and M423T toFilibuvir, Antiviral Research (2014), doi: http://dx.doi.org/10.1016/j.antiviral.2014.11.005

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Computational study on the drug resistance mechanism of HCV NS5B RNA-dependent RNA polymerase

mutants V494I, V494A, M426A, and M423T to Filibuvir

Huiqun Wang, Chenchen Guo, Bo-Zhen Chen* and Mingjuan Ji

School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Yuquan Road 19A,

100049, Beijing, P. R. China.

Abstract

Filibuvir, a potent non-nucleoside inhibitor of the hepatitis C virus (HCV) NS5B RNA-dependent RNA

polymerase (RdRp), has shown great promise in phase IIb clinical trial. However, drug resistant mutations

towards Filibuvir have been identified. In the present study, the drug resistance mechanism of wild-type (WT)

and mutant NS5B polymerases (including V494I, V494A, M426A, and M423T) toward Filibuvir was

investigated by molecular modeling methods. The predicted binding free energy of these five complexes is

highly consistent with the experimental EC50 values of Filibuvir to the wild-type and mutant NS5B RdRps,

V494I < WT < V494A < M426A < M423T. Analysis of the individual energy terms indicates that the loss of

binding affinity is mainly attributed to the decrease in the van der Waals interaction contribution. Through

detailed analysis of the interaction between FBV and RdRpV494I, RdRpWT, RdRpV494A, RdRpM426A, and RdRpM423T,

several conclusions are made. Firstly, the smaller size of residue 494 side chain results in the smaller binding

affinity between FBV and RdRp. Secondly, the poor inhibition capacity of Filibuvir toward RdRpM426A is mainly

due to the decrease in the van der Walls interaction between Filibuvir and residue Leu-497M426A caused by the

spatial structure change of Ala-426M426A. Thirdly, the decrease in the binding affinity in mutation M423T is

attributed to the smaller binding cave and the cyclopentyl group of Filibuvir exposing outside the cave. Our

computational results will provide valuable information for developing more potent and selective inhibitors

toward HCV NS5B polymerase.

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Keywords Filibuvir · molecular dynamics (MD) · RNA-dependent RNA polymerase · mutation · drug resistance

mechanism

*Corresponding author: bozhenchen@hotmail.com. Tel.: +86 10 88256129; fax: +86 10 88256092.

1. Introduction

According to a data released from the World Health Organization, about three percent of the world’s

population had infected with the hepatitis C virus (HCV) (World Health Organization, 2012), and many of these

infected individuals are likely to develop serious HCV-related liver diseases, such as liver cirrhosis,

hepatocellular carcinoma (Leone et al., 2005). Therefore, HCV has become a great threat to human health.

HCV, a member of the Flaviviridae family, is a single-stranded, positive-sense RNA virus. Its genome

includes approximately 9600 nucleosides, and encodes a polyprotein precursor which is made up of the core

protein, envelope glycoproteins and the non-structural proteins (P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B)

(Tang et al., 2009). Among these proteins, NS5B polymerase is an important component of the viral replication

machinery as it encodes the viral RNA-dependent RNA polymerase (RdRp) which is the key enzyme for HCV

RNA synthesis (Behrens et al., 1996; Moradpour et al., 2004). Structurally, like other RdRps, NS5B RdRp

contains canonical thumb, finger, and palm domains resembling the human’s right hand. It has an encircled

enzyme active site, and its fingers and thumb subdomains interact with RNA (Lesburg et al., 1999). As one of the

viral RdRp, HCV NS5B RdRp has its own particular architecture that the fingers and thumb domains are

connected. This particularity makes it distinct from related mammalian DNA and RNA polymerases, hence using

NS5B RdRp as a drug target will greatly reduce the damage on human cells. Therefore, HCV NS5B RdRp has

represented an attractive drug target for the development of specific anti-HCV drugs and vaccines (Beaulieu et

al., 2002). Currently, a certain number of NS5B RdRp inhibitors have been reported and entered into clinical

trials. Based on their mode of action (Wang et al., 2012; Powdrill et al., 2010), these inhibitors can be broadly

categorized into nucleoside or nucleotide inhibitors (NIs) (Cole et al., 2009; Cretton-Scott et al., 2008),

non-nucleosides inhibitors (NNIs) (Bedard et al., 2009; Lazerwith et al., 2013) and pyrophosphate (PPi)

analogues (Koch et al., 2006; Summa et al., 2004). The NNIs bind at allosteric pockets of NS5B and prevent a

conformational change needed for initiation of RNA synthesis (Tomei et al., 2004, 2003; Gu et al., 2003).

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Filibuvir (FBV, Fig. 1) (Li et al., 2009) is a potent non-nucleoside inhibitor (NNI) developed by Pfizer in

2009 and shows significant promise in phase IIb clinical trial (Love et al., 2003; Shi et al., 2009). It binds to the

“Thumb II” allosteric pocket of NS5B RdRp and affects the formation of a productive replicase complex. In the

study by Shi et al. (2009), they described that FBV shows no cytotoxic effect in several human cell lines, up to

the highest concentration evaluated (320μmol/L). These properties of FBV support its use as a potential antiviral

agent to HCV-infected patients. Unfortunately, company Pfizer had discontinued the development of FBV after a

strategic review of its pipeline in Mar 11, 2013. But they also declared that this decision was not related to any

safety issue of FBV. Actually, the research of FBV is still ongoing (Jiao et al., 2014).

However, NS5B mutations that mediate resistance to FBV have been selected both in cell culture

(replicons) and in therapy studies. Experimental studies in cell culture with replicons (Troke et al., 2012)

demonstrated that the EC50 values of FBV to the WT, V494I, V494A, M426A, and M423T NS5B RdRps are 18,

13, 120, 172, and >11000 nmol/L, respectively. Thus, the treatment of chronic HCV infections with FBV still

faces a great challenge. So, it is necessary to identify the relationship between residue mutations of NS5B RdRp

and the inhibition capacity of FBV. At the present time, molecular modeling methods have been proved to be the

effective techniques for investigating the drug resistance mechanism of inhibitors. Several typical works aimed

at the drug resistance mechanism studies related to the HCV NS3/4A protease and NS5B polymerase have been

reported in the past few years (Davis and Thorpe, 2013; Guo et al., 2006; Klibanov et al., 2012; Welsch et al.,

2008, 2012; Xue et al., 2012, 2013, 2014; Guan et al., 2014; Jiao et al., 2014; Yi et al., 2012; Wang et al., 2014).

It is worth mentioning that in Davis and Thorpe’s study, they first demonstrated the evidence for a mechanistic

basis of allosteric inhibition in NS5B (Davis and Thorpe, 2013). And in the study by Xue et al. (2014), they

focused on the three representative mutations (M423T/V/I) in NS5B RdRp to explore the drug resistance

mechanism of HCV to FBV. Their results indicated that the threonine, isoleucine, and valine with a larger side

chain than methionine is the main reason leading to the decrease in the binding affinity between FBV and NS5B

RdRp.

In this work, to elucidate the drug resistance mechanism of FBV, we systemically investigate the

interaction mechanisms between FBV and the wild-type and mutant (V494I, V494A, M426A, and M423T)

NS5B RdRps (genotype 1) by using MD simulations, molecular mechanics/Poisson-Boltzmann surface area

(MM/PBSA) free energy calculations, and molecular mechanics/generalized born surface area (MM/GBSA) free

energy decomposition analysis (Wang and Kollman, 2000, 2001; Lee et al., 2000; Kuhn and Kollman, 2000; Hou

et al., 2002, 2003, 2006a, 2006b, 2008, 2010, 2011, 2012; Kollman et al., 2000; Wang et al., 2001; Lepšík et al.,

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2004; Weis et al., 2006; Hou and Yu, 2007; Luo et al., 2002；Gohlke and Case, 2004; Fang et al., 2008; Xu et al.,

2013; Sun et al., 2014). Based on the calculations, we will disclose the drug resistance mechanism of FBV

toward the wild-type and mutant NS5B RdRps.

2. Materials and methods

2.1. Structure of NS5B RdRp - FBV (RdRp/FBV) complex

The wild-type model of NS5B RdRp/FBV (RdRpWT/FBV) complex was derived from the crystal structure of the

complex of FBV with the wild-type NS5B RdRp (genotype 1) which was from the Research Collaboratory for

Structural Bioinformatics Brookhaven Protein Data Bank (PDB ID: 3FRZ) (Li et al., 2009). The models of

NS5B RdRpV494I/FBV, RdRpV494A/FBV, RdRpM426A/FBV, and RdRpM423T/FBV complexes were obtained by

substituting the residues Val-494, Val-494, Met-426, and Met-423 of the wild-type complex with residues Ile,

Ala, Ala, and Thr, respectively. Before the MD simulations were started, the missing hydrogen atoms of FBV

were added using SYBYL7.1 while the missing atoms of the wild-type NS5B RdRp were added using the tleap

program in AMBER11.0 (Case et al., 2005). FBV was minimized using the Hartree–Fock (HF)/3-21G

optimization calculations in Gaussian03 (Frisch et al., 2008), and the atom partial charges were generated by

fitting the electrostatic potentials derived by Gaussian via the restrained electrostatic potential (RESP) fitting

technique in AMBER11.0 (Bayly et al., 1993). The generation of the partial charges and the force field

parameters for FBV were accomplished by the antechamber program in AMBER11.0 (Wang et al., 2006). In the

following molecular mechanics (MM) minimizations and MD simulations, the general AMBER force field

(GAFF) (Wang et al., 2004) was used as the descriptive parameter for FBV, and the standard AMBER force field

(ff03) (Duan et al., 2003) was used to describe protein parameters. The appropriate number of chloride

counterions was added to neutralize the charge of the complex and then each system was immersed in a square

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periodic box of pre-equilibrated TIP3P (Jorgensen et al., 1983) water molecules with at least 10 Å distance

around the complex. This yielded a simulation box containing 9159 water molecules and the total number of

molecules of each system is 9736. Due to the mutations, the total number of atoms of each system is different

from each other and the values fluctuate from 66311 to 66320.

2.2. MD simulations

Precede to MD simulations, molecular mechanics (MM) optimizations were employed by the sander

program in AMBER11.0 (Case et al., 2005) to minimize the system via three steps: first, the water

molecules/ions were minimized by restraining the protein and ligand (4000 cycles of steepest descent and 2000

cycles of conjugate gradient minimizations); second, the side chains of the protein were minimized by

restraining the backbone of the protein (10000 cycles of steepest descent and 5000 cycles of conjugate gradient

minimizations); third, the whole system was minimized without any restraint (10000 cycles of steepest descent

and 5000 cycles of conjugate gradient minimizations).

In the MD simulations, the long-range electrostatic interactions were handled using the particle mesh Ewald

(PME) method (Darden et al., 1993). Periodic boundary conditions were applied to avoid edge effects in all

calculations. The SHAKE procedure was applied, and the time step was set to 2×10-6 ns (Ryckaert et al., 1977).

The systems were gradually heated in the NVT ensemble from 0 to 310 K via seven steps. Then, 20 ns MD

simulations were performed under the constant temperature of 310 K. During the sampling process, the

coordinates were saved every 2×10-4 ns, and the conformations generated from the simulations were used for

further binding free energy calculations and decomposition analysis.

2.3. MM/PBSA calculations

The binding free energies of all the systems were calculated by MM/PBSA procedure based on the

following equation (Kollman et al., 2000):

Δ = − − = Δ + Δ + Δ − Δbind complex protein ligand MM PB SAG G G G E G G T S

ΔEMM is the interaction energy between the receptor and the ligand in gas phase, which includes ΔEele

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(electrostatic energy), ΔEvdw (van der Waals energy), and ΔEint (internal energy); ΔGPB and ΔGSA are the

electrostatic and nonpolar contributions to desolvation upon inhibitor binding, respectively; –TΔS is the

contribution of the conformational entropy at temperature T.

The polar part ΔGPB could be calculated by solving the Poisson-Boltzmann (PB) equations (Rocchia et al.,

2001) for the MM/PBSA method or generalized Born (GB) model (Onufriev et al., 2004) for MM-GBSA method.

Here we used MM/PBSA method. In the PB calculations, the grid size that was used to solve the PB equation

was 2 Å, and the values of solvent and solute dielectric constants were set to 80 and 4, respectively. The

nonpolar solvation contribution was estimated by the LCPO method: SAΔG =0.00729ΔSASA (Weiser et

al., 1999), where SASA is the solvent accessible surface area determined with a solvent-probe radius of 1.4 Å.

The calculations of the binding free energy were accomplished by using the mm_pbsa program in AMBER11.0.

The receptor–ligand binding free energy was calculated based on 500 snapshots taken from 15 to 20 ns MD

simulation trajectories of each complex by using the mm_pbsa program in AMBER11.0. It is noted that the

conformational entropy (translation, rotation, and vibration) calculation is time consuming for the large system.

And considering the similarity of these five systems (same ligand binding to same protein with just a point

mutation), the calculated results of the entropic contribution for them should be similar to each other. In fact, the

previous studies (Cheng et al., 2011; Gao et al. 2011; Geng et al., 2013) have confirmed this statement. Therefore,

the entropic contribution is not computed in the present work, which may be no significant effect on the

conclusion.

2.4. MM/GBSA free energy decomposition analysis

Due to the high computational demand of the PB calculations, the interactions between FBV and each

residue of NS5B RdRp were computed using the MM/GBSA decomposition process by the mm_gbsa program in

AMBER11.0 (Gohlke et al., 2003). After the decomposition process, the energy contribution can be allocated to

each residue from the association of the receptor with the ligand, which includes three energy terms: van der

Waals contribution (ΔEvdw), electrostatic contribution (ΔEele), and solvation contribution (ΔGGB+ΔGSA). ΔEvdw and

ΔEele are van der Waals and electrostatic interactions between FBV and each protein residue that can be

computed by the sander program in AMBER11.0 (Case et al., 2005). The polar contribution of desolvation

(ΔGGB) was calculated using GB model, whose parameters were developed by Onufriev et al. (Onufriev et al.,

2004). The nonpolar contribution of desolvation (ΔGSA) was computed based on SASA determined with the

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ICOSA method (Case et al., 2010). All energy components were computed using 500 snapshots extracted from

the MD trajectory from 15 to 20 ns. The molecular surface and cartoon models were generated by PyMOL

(DeLano et al., 2002).

3. Results and discussion

3.1. The stability and flexibility of system simulations

We performed classical MD simulations on the RdRpWT/FBV, RdRpV494I/FBV, RdRpV494A/FBV,

RdRpM426A/FBV, and RdRpM423T/FBV complexes to predict the binding modes and explore the key residues

associated with the binding affinity. To obtain the stable systems and ensure the rationality of sampling strategy,

the 20 ns MD simulations on the five systems were carried out. The root-mean-square deviation (rmsd) values of

all the protein backbone atoms based on the respective crystal structures were calculated to analyze the stability

of the five systems, and the results were plotted in Fig. 2. It shows that the rmsd values of the backbone atoms in

all systems remain stable after the 10 ns MD simulation. And the average rmsd values of the five systems are

1.54, 1.81, 1.72, 1.80, and 1.57 Å, respectively. Moreover, the rmsd values of FBV in the five systems also

remain stable during the MD simulation, except for those in the RdRpV494A/FBV and RdRpM423T/FBV systems,

which have a fluctuation at 5.8 and 9.9 ns, respectively (see Fig. S1, Supplementary data). In addition, the

binding free energy values of the 500 snapshots extracted from the last 5 ns MD simulation of the five systems

are calculated and the results are displayed in Fig. S2 (Supplementary data). It shows that the binding free energy

values of the five systems are stable in the last 5 ns MD simulation, which further confirms the stability of our

systems and the rationality of our sampling strategy. These results indicate that, based on the snapshots extracted

from 15 to 20 ns, it is both reasonable and reliable to do the MM/PBSA binding free energy calculations and

MM/GBSA free energy decomposition analyses on these five systems.

3.2. Binding free energies predicted by MM/PBSA

Following the 20 ns MD simulations on the RdRpWT/FBV, RdRpV494I/FBV, RdRpV494A/FBV,

RdRpM426A/FBV, and RdRpM423T/FBV complexes, 500 snapshots extracted from 15 to 20 ns MD trajectory were

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used to calculate the binding free energy of these five complexes using MM/PBSA method. The predicted

binding free energies and the energy components, together with the experimentally derived EC50 values of the

five systems are displayed in Table 1. It shows that the predicted binding free energies of the RdRpV494I/FBV

complex is slightly smaller than that of the RdRpWT/FBV complex, which is consistent with the experimental

fact that the EC50 value of the former is marginally smaller than that of the latter. The predicted binding free

energies of the RdRpV494A/FBV, RdRpM426A/FBV, and RdRpM423T/FBV complexes are larger than that of the

RdRpWT/FBV complex and the order of them is in agreement with experimental EC50 values. In addition, to

determine which energy term has more impact on the binding free energy, the four individual energy components

(ΔEvdw, ΔEele, ΔGPB, and ΔGSA) are carefully compared and shown in Table 1. It shows that both the van der

Waals (ΔEvdw) and electrostatic (ΔEele) contributions are essential for the FBV binding to RdRp. However, the net

electrostatic contributions (ΔEele+ ΔGPB) of the NS5B RdRpWT/FBV, RdRpV494I/FBV, RdRpV494A/FBV,

RdRpM426A/FBV, and RdRpM423T/FBV complexes are 3.02, 2.69, 3.27, 3.02, and 2.52 kcal/mol (1 cal = 4.184 J),

respectively, showing a disadvantage for the binding of FBV with both wild-type and mutant RdRps. The

nonpolar desolvation energies (ΔGSA) of the five complexes are approximately the same. Based on aboveresults,

we conclude that the van der Waals is the most important energy term to distinguish the binding affinities among

these five systems.

3.3. Decomposition analysis of the binding free energies

To further understand the detailed interaction in the RdRp/FBV complex, we decompose the total binding

free energy into inhibitor-residue pairs by the MM/GBSA decomposition process. The quantitative information

of each residue’s contribution is extremely helpful to interpret the binding modes of FBV with RdRps. The

comparison of the key contributors of RdRp in the RdRpWT/FBV, RdRpV494I/FBV, RdRpV494A/FBV,

RdRpM426A/FBV, and RdRpM423T/FBV systems are shown in Fig. 3. Also, the structure of thumb subdomain of

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RdRp (residues from 371 to 562) contains all of the residues that contribute to the binding free energy of these

five systems (see Figs. 3 and 4). Thus, we only display this domain in Figs. 5, 8 and 11. These structures are

derived from the last snapshot of each system’s MD simulation. As described above, the van der Waals

contribution has important influence on the binding free energies. The van der Waals interactions between FBV

and key contributors of the wild-type and mutant complexes are calculated and the results are displayed in Fig.

S3 (Supplementary data). Moreover, we computed the visible percentages of hydrogen bonds between FBV and

RdRp during the MD simulations to study the influence of the mutation on the hydrogen bonding network, the

results of which were listed in Table 2. In the hydrogen bond calculation, a hydrogen bond was defined if the

donor-acceptor (H) distance was less than 3.5 Å and the donor-acceptor (H)-acceptor angle was larger than 120°.

Furthermore, the binding modes of the five systems with the key residues that have large contribution to the

binding affinities are displayed in Fig. 5.

3.4. The RdRpWT/FBV complex

In the RdRpWT/FBV complex, the thumb domain of RdRp has a long cleft which is approximately 30 Å

long, 10 Å wide, and 10 Å deep and FBV locates deeply in the cleft (see Fig. 4b). As shown in Fig. 3, residues

Leu-419, Arg-422, Met-423, His-475, Ser-476, Tyr-477, Ile-482, Leu-497, and Trp-528 are the residues having

major contributions to the binding free energy. Moreover, after analyzing the average structure of the last 5 ns

MD simulations, we find that there are mainly three important interactions between FBV and NS5B RdRp. The

first interaction is the key hydrophobic interactions between the cyclopentyl group of FBV and the hydrophobic

residues Leu-419, Arg-422, Met-423, Tyr-477, and Trp-528; and between the ethyl groups from the pyridine of

FBV and the hydrophobic residues Ile-482, Val-485, Ala-486, and Leu-497. The second interaction is the

hydrogen bond formed by the dihydropyrone carbonyl of FBV and residue Ser-476 ([N-H (Ser-476) ··· O3

(FBV)]). The third interaction is the π-π stacking interaction between the triazolopyrimidine of FBV and residue

His-475 (see Fig. S4, Supplementary data). These results are similar to the experimental analysis that these

residues have interaction with FBV (Li et al., 2009).

Considering mutations V494I, V494A, M426A, and M423T have no significant effects on the hydrogen

bonding network (see Table 2) and the π-π stacking interactions in all mutant systems, except RdRpM423T/FBV

system, have no distinct changes compared with RdRpWT/FBV system. Therefore, we focus on the interactions

of FBV with the hydrophobic residues to reveal drug resistance mechanism of FBV in the RdRpV494I/FBV,

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RdRpV494A/FBV, RdRpM426A/FBV, and RdRpM423T/FBV systems.

3.5. The RdRpV494I/FBV and RdRpV494A/FBV complexes

The key residues contributing to the binding free energy in the RdRpV494I/FBV complex are shown in Fig.

5b. Among these residues, only residue Lys-533 shows distinct difference in the contribution of the binding free

energy compared with RdRpWT/FBV complex (see Fig. 3a). As indicated in Fig. S3a (Supplementary data), the

difference of residue Lys-533 in the binding free energy contribution can be attributed to the variant contribution

of van der Waals interaction.

As shown in Fig. 4, the pyridine of FBV interacts with the front part of RdRp’s cleft (this part is abbreviated

as “front-cleft” for the convenience of description.). In the RdRpWT/FBV complex (Fig. 5a), residues Val-494

and Leu-489 are located at the bottom of front-cleft and residues Arg-490 and Pro-496 are located at both edges

of front-cleft. Therefore, the distances between residues Leu-489 and Pro-496, between residues Leu-489 and

Arg-490 stand for the depth of front-cleft. The distance between residues Arg-490 and Pro-496 represents the

width of front-cleft. We calculate the distances mentioned above in both RdRpWT/FBV and RdRpV494I/FBV

complexes during 20 ns MD simulation (see Fig. 6). The results show that residue Leu-489 moves close to

residue Pro-496 and residue Arg-490 leaves away from residue Pro-496 after the mutation V494I. While the

distance between residues Leu-489 and Arg-490 has no change. This observation indicates that front-cleft of

RdRpV494I/FBV complex is wider and shallower than that of RdRpWT/FBV complex (see Figs. 5a and 5b).

Detailed comparison of the residues in front-cleft in both RdRpV494I/FBV and RdRpWT/FBV complexes (see

Figs. 5a and 5b), reveals that the larger side chain of residue Ile-494V494I raises up the side chain of residue

Leu-489V494I, leading to the front-cleft of RdRpV494I/FBV complex shallow and residue Pro-496V494I leaving away

from residue Arg-490V494I which in consequence results in the front-cleft of RdRpV494I/FBV complex becoming

wider than that of RdRpWT/FBV complex (see Figs. 5a and 5b). These changes of front-cleft of RdRpV494I/FBV

complex make residue Lys-533V494I move towards FBVV494I. The distances between residue Lys-533 and FBV of

these two complexes obtained during the MD simulations confirm this change (see Fig. 7). The shorter distance

between residue Lys-533 and FBV in RdRpV494I/FBV complex results in a strong van der Waals interaction

between them, as confirmed by the result of energy decomposition analysis (see Fig. S3a, Supplementary data).

Therefore, the binding affinity between FBV and RdRpV494I increases.

Table 1 shows that the binding free energy of RdRpV494A/FBV complex is larger than that of RdRpWT/FBV

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(2.21 kcal/mol) and RdRpV494I/FBV (2.5 kcal/mol) complexes, which is consistent with the order of EC50 values

(Li et al., 2009). However，as shown in Figs. 3a, the energy decompose analysis indicates that the key residues

having contributions to the binding free energy in RdRpV494A/FBV complex are similar to those in RdRpWT/FBV

complex. And there are no residues in RdRpV494A/FBV complex showing distinct difference in the contributions

of the binding free energy. As V494A is also the mutation of the 494 site (similar to V494I), we calculate the

same distances as those in RdRpV494I/FBV complex. Since the distance between residues Leu-489V494A and

Pro-496V494A has a fluctuation from 15 to 20 ns, we extend the time of the MD simulation of RdRpV494A/FBV

complex to 25 ns and the result is displayed in Fig. S5 (Supplementary data). It shows that the positions of

residues Leu-489V494A, Arg-490V494A, Pro-496V494A, and Lys-533V494A have no significant change after residue

494 mutated from Val to Ala. Therefore, we can conclude that the smaller side chain of residue Ala-494V494A

leads to the smaller binding affinity between FBV and RdRpV494A, which is consistent with the previous result

that the larger side chain of residue 494 results in the larger binding affinity.

In order to further confirm our conclusion, we reduce the length of the side chain of residue 494 changing it

from Val to Gly, and conduct MD calculations on RdRpV494G/FBV complex as well as those of RdRpV494I/FBV

and RdRpV494A/FBV complexes. And the results are listed in the supplementary data. The rmsd values of the

FBV and the backbone atoms of RdRpV494G/FBV complex during the 20 ns simulation indicate that the system is

stable and the sampling from 15 to 20 ns is reliable (see Fig. S6, Supplementary data). Just as we expected, the

binding free energy of RdRpV494G/FBV complex is lager than that of RdRpV494A/FBV complex. And the binding

free energies of RdRpV494I/FBV, RdRpWT/FBV, RdRpV494A/FBV, and RdRpV494G/FBV complexes increases with

the size of residue 494 side chain (see Tables 1 and S1).

Therefore, based on above analysis, we can draw a conclusion that the side chain of residue 494 has

significant effect on the binding affinity between FBV and NS5B RdRp. When residue 494 changes from Val to

Ile, the side chain of Ile raises up the side chain of residue 489, resulting in front-cleft of RdRpV494I/FBV

complex becoming wider and shallower, and further increases the binding affinity between FBV and NS5B

RdRpV494I. While in the case of residue 494 changes from Val to Ala and Gly, the side chains of Ala and Gly are

too small to make the position of residue 489 side chain have significant change. Thus, RdRpV494A/FBV and

RdRpV494G/FBV complexes show non-distinct difference on the binding affinity compared with the RdRpWT/FBV

complex.

3.6. The RdRpM426A/FBV complex

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In RdRpM426A/FBV complex, it is found from Fig. 3b that residues Leu-419, Arg-422, Met-423, His-475,

Ser-476, Tyr-477, Ile-482, and Trp-528 have major contributions to the binding free energy, similar to

RdRpWT/FBV complex. The distinct difference of contribution to the binding free energy between

RdRpM426A/FBV and RdRpWT/FBV complexes mainly comes from residue Leu-497, which could be attributed to

the decrease in the van der Walls interaction between FBVM426A and Leu-497M426A (see Fig. S3, Supplementary

data). This suggests mutation M426A has great effect on the hydrophobic interaction between FBV and the

hydrophobic residue Leu-497.

Fig. 8 shows that residues Met-423WT, Met-426WT, and His-428WT are in a same α helix (defined as αA) and

residues Leu-497WT and Trp-500WT are in another α helix (defined as αB). By comparing Fig. 8a and Fig. 8b, we

find that the positions of the side chain of residues Met-423 and His-428 have a great change after residue

Met-426 mutated from Met to Ala. Figs. 8 and 9a shows that residue His-428 moves close to residue Trp-500 in

RdRpM426A/FBV complex, this suggests there is increase in the π-π stacking interaction between residues His-428

and Trp-500. Because of the increase in the π-π stacking interaction, the α helixes αA and αB move close to each

other, reducing the distance between residues Met-423 and Leu-497 (see Fig. 9b). As both residues Met-423 and

Leu-497 have a long branched chain, when these two residues move close to each other, the residue

Leu-497M426A side chain is pressed downward, leaving away from FBVM426A. The distance between residue

Met-423 and FBV, and distance between residue Leu-497 and FBV in RdRpM426A/FBV and RdRpWT/FBV

complexes are computed, and the results are shown in Fig. 10. It shows that the distance between residue

Met-423 and FBV of RdRpM426A/FBV complex has no significant change compared with that of RdRpWT/FBV

complex. Therefore, the interactions between residue Met-423 and FBV in RdRpM426A/FBV complex have no

change, which is also proved by the energy decomposition analysis (Fig. 3b). However, residue Leu-497 leaves

away from FBV, and the distance between residue Leu-497 and FBV in RdRpM426A/FBV complex stably

maintains at about 6.36 Å. While the corresponding distance in RdRpWT/FBV complex holds a stable value of

about 4.07 Å during the 20 ns MD simulation. The longer distance between residue Leu-497 and FBV indicates

13

that Leu-497 could hardly form van der Walls interaction with FBV in RdRpM426A/FBV complex, which also can

be observed in Fig. S3b. Hence, we infer that the decrease in the binding affinity of RdRpM426A/FBV complex

should be attributed to the fact that Ala-426 having a shorter side chain than Met-426.

To further prove above conclusions, we mutate residue 426 from Met to Ile having similar length to Met,

and perform 20 ns MD simulations on RdRpM426I/FBV complex. The related results are shown in Fig. S8 and

Table S1 (Supplementary data). Tables 1 and S1 show that the predicted binding free energy of RdRpM426I/FBV

complex is very similar to that of RdRpWT/FBV complex. Energy decomposition analysis of RdRpM426I/FBV

complex (see Fig. S9, Supplementary data) also confirms this result. Moreover, we also calculate the distances as

described in RdRpM426A/FBV complex and the results are displayed in Fig. S10 (Supplementary data). It shows

that except the distance between residues His-428 and Trp-500 of RdRpM426I/FBV complex being a little shorter

than that of RdRpWT/FBV complex other three distances of RdRpM426I/FBV complex are the same to the

corresponding distances of RdRpWT/FBV complex. Therefore, we can conclude that the replacement of the Met

with Ile has no significant effect on the binding affinity between FBV and RdRp. All above analysis further

confirm our conclusion that the decrease in the binding affinity of RdRpM426A/FBV complex is mainly attributed

to the shortening of the side chain of residue 426.

3.7. The RdRpM423T/FBV complex

The key residues having major contribution to the binding free energy in RdRpM423T/FBV complex are

shown in Fig. 5e. Among these residues, the interactions between FBV and the six residues Leu-419, Arg-422,

Thr-423, His-475, Leu-497, and Trp-528 are weaker than those in RdRpWT/FBV complex. However, the

interaction between FBV and residue Ser-476 in RdRpM423T/FBV complex is stronger than that in RdRpWT/FBV

complex. As shown in Figs. 3b, S3b, and S7 (Supplementary data), the decrease in interactions between FBV

and residues Leu-419, Arg-422, Thr-423, His-475, Leu-497, and Trp-528 in RdRpM423T/FBV complex is

attributed to the decrease in the van der Walls interaction.

14

The hydrophobic residues Leu-419, Arg-422, Met-423, Tyr-477, and Trp-528 form a hydrophobic cave that

has a great interaction with the cyclopentyl of FBV, in which residue Arg-422 locates at the bottom of the cave

and residue 423 is at the entrance of the cave. We compare this binding cave in RdRpM423T/FBV with that in

RdRpWT/FBV complexes, and find that as the spatial structure of the branched chain of Thr is larger than that of

Met, the cave becomes small after mutation M423T, which leads to the cyclopentyl of FBV exposing outside the

cave (see Fig. 11). These facts are also confirmed by the distance changes between residue Arg-422 and FBV,

between residues Arg-422 and Met-423 (see Fig. 12). Furthermore, the distances between FBV and residues

Leu-419, Thr-423, and Trp-528 in RdRpWT/FBV and RdRpM423T/FBV complexes during the 20 ns MD

simulations are also calculated, and the results are displayed in Fig. 13. From Figs. 12b and 13 we can see that

residues Leu-419, Arg-422, Thr-423, and Trp-528 leave far away from FBV after mutation M423T. The longer

distances between FBV and residues Leu-419M423T, Arg-422M423T, Thr-423M423T, and Trp-528M423T indicate that

van der Walls interactions between FBV and these residues decrease greatly, which is consistent with the results

of the energy decompose analysis (see Fig. S3b). Besides, the interactions between FBV and residues His-475

and Ser-476 are also affected by the change of the pocket as these two residues are close to the pocket. Fig. 14

shows that residue His-475 leaves away from FBV and residue Ser-476 moves close to FBV in RdRpM423T/FBV

complex, resulting in decrease in the π-π stacking interaction between His-475 and FBV and increase in the van

der Walls interaction between Ser-476 and FBV. This is in agreement with the results displayed in Fig. S3b. As

described in RdRpM426A/FBV complex, the spatial structure change of residue 423 has influence on residue

Leu-497. Therefore, we calculated the distance between FBV and residue Leu-497 (see Fig. 15). As shown in Fig.

15, the distance between FBV and residue Leu-497 in RdRpM423T/FBV complex is about 0.87 Å longer than that

in RdRpWT/FBV complex. Therefore, the van der Walls interaction between FBV and residue Leu-497 in

RdRpM423T/FBV complex is also smaller than that of RdRpWT/FBV complex (see Fig. S3b).

Based on above analysis, we can conclude that mutation M423T results in smaller hydrophobic cave and

further decreases the van der Walls interactions between FBV and the hydrophobic residues Leu-419, Arg-422,

Met-423, His-475, Leu-497, and Trp-528. These results are consistent with the study by Xue et al. (2014). In

order to confirm that the change of hydrophobic cave is resulted from the larger branched chain of Thr, we

remove the methyl of the branched chain of Thr and mutate residue 423 from Met to Ser, and perform 20 ns MD

simulations on RdRpM423S/FBV complex. Fig. S11 (Supplementary data) shows the rmsd values of the Filibuvir

and the backbone atoms of RdRpM423S/FBV complex during the 20 ns simulation. We can find that the rmsd

value of the backbone atoms of RdRpM423S/FBV complex holds a stable value at about 1.5 Å during the whole

15

MD simulation, while the rmsd values of Filibuvir has a large fluctuation at 9 ns and then remains at about 3.0 Å

to the end, which is within the acceptable scale. These results indicate that the system is stable and it is

reasonably and reliably sampling from 15 to 20 ns. The result of the binding free energy of RdRpM423S/FBV

complex is shown in Table S1. As shown in Tables 1 and S1, the binding free energy of RdRpM423S/FBV complex

is similar to that of RdRpWT/FBV complex, but much smaller than that of RdRpM423T/FBV complex. This

confirms our conclusion that the larger branched chain of residue 423 leads to the decrease in the binding affinity.

To determine whether mutation M423S has an effect on the hydrophobic pocket, we calculate the distances

between residues Arg-422 and Ser-423, between residue Arg-422 and FBV of RdRpM423S/FBV complex as well

as those in RdRpWT/FBV complex and the results are displayed in Fig. S12. It shows that mutation M423S has

no significant effect on the hydrophobic pocket.

Thus, we can draw a conclusion that the extra methyl in the branched chain of Thr-423 in RdRpM423T/FBV

complex is the main reason leading to the hydrophobic pocket becoming small and further results in the decrease

in the binding affinity between FBV and RdRpM423T.

4. Conclusion

To investigate the drug resistance mechanism of Filibuvir (FBV) over mutant NS5B polymerase, MD

simulations, MM/PBSA free energy calculations, and MM/GBSA free energy decomposition analysis have been

conducted in the present study. Our predicted results of binding free energies are highly consistent with the

experimental results. The energy decomposition analysis shows that the van der Waals contribution is the main

interaction between FBV and NS5B RdRp, and is the most important energy term to distinguish the binding

affinities of the five systems among the individual energy terms.

Compared RdRpWT/FBV complex with RdRpV494I/FBV, RdRpV494A/FBV, and RdRpV494G/FBV complexes,

we can draw a conclusion that the side chain of residue 494 has a great effect on the binding of FBV, and the

smaller side chain of residue 494 results in the smaller binding affinity between FBV and RdRp. As for mutation

M426A, after comparing the interactions of FBV with RdRpM426A and RdRpM426I, we observe that the shorter

side chain of Ala-426M426A compared to Met-426WT is the main reason for stronger interaction between FBV and

RdRpWT. For the RdRpM423T/FBV complex, we find that the extra methyl of the branched chain of residue

Thr-423M423T makes the hydrophobic cave smaller than that of RdRpWT/FBV and RdRpM423S/FBV complexes,

which also decreases the van der Walls interactions between FBV and residues His-475, Ser-476 and Leu-497.

16

The overall outcome is that the binding affinity between of RdRpM423T/FBV complex is much smaller than thatof

RdRpWT/FBV and RdRpM423S/FBV complexes.

To sum up, our study demonstrates the feasibility to investigate the drug resistance mechanism of FBV

toward NS5B RdRp using the molecular dynamics. We envision our work would provide some valuable clues

for structure-based design of novel inhibitors of NS5B RdRp in the near future.

Acknowledgments

We gratefully acknowledge the National Natural Science Foundation of China (Grant Nos. 21373217,

21173264) for supporting this work.

Appendix A. Supplementary data

Supplementary data associated with this manuscript can be found in the supplementary information file.

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Ketoamide resistance and hepatitis C virus fitness in val55 variants of the NS3 serine protease.

Antimicrobial agents and chemotherapy. 56, 1907-1915.

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methods. 3. The impact of force fields and ligand charge models. The Journal of Physical Chemistry B. 117,

8408-8421.

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NS3/4A protease inhibitors vaniprevir and MK-5172 by the combination use of molecular dynamics

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26

Computational study on the selectivity mechanism of HCV NS5B RNA-dependent RNA polymerase

mutants V494I, V494A, M426A and M423T to Filibuvir

Huiqun Wang, Chenchen Guo, Bo-Zhen Chen* and Mingjuan Ji

School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Yuquan Road

19A, 100049, Beijing, P. R. China.

*Corresponding author: bozhenchen@hotmail.com. Tel.: +86 10 88256129; fax: +86 10 88256129.

Fig. 1. Chemical structure of Filibuvir with atom notations.

27

Fig. 2. The root-mean-square-deviation (rmsd) of the backbone atoms (CA, N, C) of the five complexes relative

to the respective crystal structures.

28

Fig.3. The inhibitor-residue interaction spectrum of the wild-type and mutant RdRp/Filibuvir complexes

according to the MM/GBSA decomposition analysis. The X-axis represents the key residue numbers of the

NS5B RdRp. (a) WT, V494I, V494A; (b) WT, M426A, M423T. Compared with the RdRpWT/Filibuvir complex,

the residues having distinct difference in the contribution of the binding free energy in the mutant RdRp/Filibuvir

complexes are showed by arrows.

Fig. 4. Overall views of HCV NS5B polymerase with bound Filibuvir. The following colors highlight structural

features: blue, palm domain (residues 188 to 227 and 287 to 370); green, finger domain (residues 1 to 187 and

228 to 286); cyan, thumb domain (residues 371 to 562); magentas, Filibuvir. (a) cartoon model; (b) surface

model, rotated 90° from view in panel (a).

29

30

31

Fig. 5. Binding modes of NS5B RdRps/FBV complexes with key residues in the binding pocket. The NS5B

RdRps are shown as cartoon (in light pink) and surface (in light grey), the side chains of key residues in the

binding pocket are shown as sticks (in yellow), and FBV shown as sticks (in cyan). The mutant residues are

labeled by red word. The red dashed lines represent the hydrogen bonds. (a) WT, (b) V494I, (c) V494A, (d)

M426A, (e) M423T.

32

Fig. 6. Distance between atom CD2 (L489WT) and atom CG (P496WT) (a), between atom CD (R490WT) and atom

CG (P496WT) (b), between atom CB (R489WT) and atom CZ (R490WT) (c) in RdRpWT/FBV complex as well as

the corresponding distance in RdRpV494I/FBV complex during 20 ns MD simulation. The Δd in (a), (c) represents

the change of the width of the front-cleft; the Δd in (b) represents the change of the depth of the front-cleft after

mutation V494I.

Fig. 7. Distance between atom CE (K533WT) and atom C23 (FBVWT) in RdRpWT/FBV complex as well as the

corresponding distance in RdRpV494I/FBV complex during 20 ns MD simulation.

33

Fig. 8. Molecular cartoon of NS5B RdRpWT (in light-blue) (a) and RdRpM426A (in light-pink) (b) with key

residues presented as stick. These key residues in NS5B RdRps are colored. The red dashed lines represent the

shortest distances between these two residues.

Fig. 9. Distance between atom CG (H428WT) and atom CH2 (W500WT) (a), between atom CE (M423WT) and

atom CG (L497WT) (b) in RdRpWT/FBV complex as well as the corresponding distance in RdRpM426A/FBV

complex during 20 ns MD simulation. The Δd in (a) represents the change of the π-π stacking interaction

between His-428 and Trp-500 after mutation M426A.

34

Fig. 10. Distance between atom CE (M423WT) and atom C7 (FBV) (a), between atom CD2 (L497WT) and atom

C7 (FBV) (b) in RdRpWT/FBV complex as well as the corresponding distance in RdRpM426A/FBV complex

during 20 ns MD simulation.

Fig. 11. The binding pockets of FBV, which is shown in surface mode labeled with key residues of RdRpWT/FBV

(a) and RdRpM423T/FBV (b) complex.

Fig. 12. Distance between atom CZ (R422WT) and atom CG (M423WT) (a), between atom CB (R422WT) and atom

C15 (FBV) (b) in RdRpWT/FBV complex as well as the corresponding distance in RdRpM423T/FBV complex

during 20 ns MD simulation.

35

Fig. 13. Distance between atom CG (L419WT) and atom C7 (FBV) (a), between atom CB (M423WT) and atom

C16 (FBV) (b), between atom CD2 (W528WT) and atom C17 (FBV) (c) in RdRpWT/FBV complex as well as the

corresponding distance in RdRpM423T/FBV complex during 20 ns MD simulation.

Fig. 14. Distance between atom CB (H475WT) and atom C23 (FBV) (a), between atom CA (S476WT) and atom

C29 (FBV) (b) in RdRpWT/FBV complex as well as the corresponding distance in RdRpM423T/FBV complex

36

during 20 ns MD simulation. The Δd in (a) represents the change of the π-π stacking interaction between His-475

and FBV after mutation M423T.

Fig. 15. Distance between atom CB (L497WT) and atom C7 (FBV) in RdRpWT/FBV complex as well as the

corresponding distance in RdRpM423T/FBV complex during 20 ns MD simulation.

37

Supplementary data

Table S1 Binding free energies and individual energy terms of RdRp/Filibuvir (FBV) calculated by MM/PBSA

method (kcal/mol)

RdRps/FBV complex ΔEelea ΔEvdw

b ΔGPBc ΔGSA

d ΔHbinde

V494G -3.83+1.13 -45.94+4.54 6.55 +1.02 -6.36+0.35 -49.58+4.59

M426I -4.07+1.57 -48.73+3.98 6.99+1.23 -6.72+0.41 -52.53+4.03

M423S -6.60 +1.61 -48.94 +3.26 9.70+1.18 -7.03 +0.32 -52.86+3.33

a electrostatic contribution; b van der Waals contribution; c the polar contribution of desolvation; d the

nonpolar contribution of desolvation; e the binding free energy.

Fig. S1. The root-mean-square-deviation (rmsd) of Filibuvir in the five complexes relative to the respective

crystal structures.

38

Fig. S2. Fluctuation of the binding free energy of five NS5B RdRp/FBV complexes during 500 snapshots

extracted from the last 5 ns MD simulation.

Fig. S3. The inhibitor-residue van der Waals interaction spectrums of the wild-type and mutant complexes based

on the MM/GBSA decomposition analysis. The X-axis represents the key residue numbers of the NS5B RdRp.

39

(a) WT, V494I, V494A; (b) WT, M426A, M423T. Compared with the RdRpWT/Filibuvir complex, the residues

having distinct difference in the contribution of the van der Waals interaction in the mutant RdRp/Filibuvir

complexes are showed by arrows.

Fig. S4. The mainly three interaction areas between Filibuvir and NS5B RdRpWT complex.

40

Fig. S5. Distance between atom CD2 (L489WT) and atom CG (P496WT) (a), between atom CD (R490WT) and

atom CG (P496WT) (b), between atom CB (R489WT) and atom CZ (R490WT) (c), between atom CE (K533WT)

and atom C23 (FBVWT) (d) in RdRpWT/FBV complex during 20 ns MD simulation as well as the corresponding

distances in RdRpV494A/FBV complex during 25 ns MD simulation.

Fig. S6. The root-mean-square deviation (rmsd) of Filibuvir (a) and the backbone atoms (CA, N, C) of

RdRpV494G/FBV complex (b) relative to the crystal structure.

41

Fig. S7. The inhibitor-residue electrostatic interaction spectrums of the RdRpWT/FBV, RdRpM426A/FBV, and

RdRpM423T/FBV complexes based on the MM/GBSA decomposition analysis. The X-axis represents the key

residue numbers of the NS5B RdRp.

Fig. S8. The root-mean-square deviation (rmsd) of Filibuvir (a) and the backbone atoms (CA, N, C) of

RdRpM426I/FBV complex (b) relative to the crystal structure.

42

Fig. S9. The inhibitor-residue interaction spectrum of the RdRpWT/FBV, RdRpV494G/FBV, RdRpM426I/FBV, and

RdRpM423S/FBV complexes according to the MM/GBSA decomposition analysis. The X-axis represents the key

residue numbers of the NS5B RdRp.

43

Fig. S10. Distance between atom CG (H428WT) and atom CH2 (W500WT) (a), between atom CE (M423WT) and

atom CG (L497WT) (b), between atom CE (M423WT) and atom C7 (FBV) (c), between atom CD2 (L497WT) and

atom C7 (FBV) (d) in RdRpWT/FBV complex as well as the corresponding distance in RdRpM426I/FBV complex

during 20 ns MD simulation.

Fig. S11. The root-mean-square deviation (rmsd) of Filibuvir (a) and the backbone atoms (CA, N, C) of

RdRpM423S/FBV complex (b) relative to the crystal structure.

Fig. S12. Distance between atom CZ (R422WT) and atom CB (M423WT) (a), between atom CB (R422WT) and

atom C15 (FBV) (b) in RdRpWT/FBV complex as well as the corresponding distance in RdRpM423S/FBV

complex during 20 ns MD simulation.

44

Computational study on the selectivity mechanism of HCV NS5B

RNA-dependent RNA polymerase mutants V494I, V494A, M426A and

M423T to Filibuvir

Huiqun Wang, Chenchen Guo, Bo-Zhen Chen* and Mingjuan Ji

School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Yuquan Road

19A, 100049, Beijing, P. R. China.

*Corresponding author: bozhenchen@hotmail.com. Tel.: +86 10 88256129; fax: +86 10 88256129.

Table 1 Binding free energies and individual energy terms of RdRp/Filibuvir (FBV) calculated by MM/PBSA

method (kcal/mol)

RdRp/FBV

complex

ΔEelea ΔEvdw

b ΔGPBc ΔGSA

d ΔHbinde EC50

[Troke et al., 2012]

WT -4.33 +1.14 -50.15 +3.02 7.35 +0.99 -6.63 +0.30 -53.76 +3.10 18

V494I -7.63+1.78 -49.78+3.08 10.32 +1.15 -6.96+0.18 -54.05+3.02 13

V494A -1.37+1.03 -48.27+2.86 4.64 +1.03 -6.54+0.37 -51.55+2.86 120

M426A -3.07+1.12 -46.93+3.18 6.09+1.04 -6.39+0.35 -50.30+3.21 172

M423T -3.90 +1.56 -35.45 +2.92 6.42+1.41 -5.69 +0.38 -38.63+3.21 >11000

a electrostatic contribution; b van der Waals contribution; c the polar contribution of desolvation; d the nonpolar contribution of desolvation; e

the binding free energy.

45

Table 2 Visible percentage of hydrogen bonds during MD Simulations between the wild-type and five mutants

of NS5B polymerase and Filibuvir

Complexes Donor Accepter Occupied (%) Distance (Å) Angle (deg) f

RdRpWT/FBV :476@H:476@N :FBV @O3 94.41 2.953 160.64

RdRpV494I/FBV :476@H:476@N :FBV @O3 98.48 2.892 159.86

RdRpV494A/FBV :476@H:476@N :FBV @O3 95.55 2.908 158.68

RdRpM426A/FBV :476@H:476@N :FBV @O3 94.43 2.915 160.43

RdRpM423T/FBV :476@H:476@N :FBV @O3 84.04 2.864 155.75

f The angles are the real hydrogen bond angles rather than the stored angles obtained from the ptraj program of AMBER (the stored angles

are the supplementary angles of the real hydrogen bond angles).The definition of hydrogen bond uses the common convention.

46

Highlights

●Molecular modeling methods was used to study the selectivity mechanisms of Filibuvir toward wild-type

and mutant NS5B RdRp.

●We give a good explanation of the differences in curative efficacy of Filibuvir.

● The computational results could give valuable information for developing novel and safer HCV antiviral drugs.