Computational study on the binding and unbinding mechanism of HCV NS5B with the inhibitor GS-461203 and substrate using conventional and steered molecular dynamics simulations

    Computational study on the binding and unbinding mechanism of HCV NS5B with the inhibitor GS-461,203 and substrate using conventional and steered molecular dynamics simulations Dabo Pan, Yuzhen Niu, Lulu Ning, Yang Zhang, Huanxiang Liu, Xiaojun Yao PII: DOI: Reference:

S0169-7439(16)30124-1 doi: 10.1016/j.chemolab.2016.05.015 CHEMOM 3252

To appear in:

Chemometrics and Intelligent Laboratory Systems

Received date: Revised date: Accepted date:

14 March 2016 16 May 2016 20 May 2016

Please cite this article as: Dabo Pan, Yuzhen Niu, Lulu Ning, Yang Zhang, Huanxiang Liu, Xiaojun Yao, Computational study on the binding and unbinding mechanism of HCV NS5B with the inhibitor GS-461,203 and substrate using conventional and steered molecular dynamics simulations, Chemometrics and Intelligent Laboratory Systems (2016), doi: 10.1016/j.chemolab.2016.05.015

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ACCEPTED MANUSCRIPT Computational study on the binding and unbinding mechanism of HCV NS5B

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molecular dynamics simulations

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with the inhibitor GS-461203 and substrate using conventional and steered

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Dabo Pana, Yuzhen Niua, Lulu Ninga, Yang Zhangb, Huanxiang Liuc*, Xiaojun Yaoa*

State Key Laboratory of Applied Organic Chemistry, Department of Chemistry,

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Lanzhou University, Lanzhou 730000, China

School of Information Science & Engineering, Lanzhou University, Lanzhou, China

c

School of Pharmacy, Lanzhou University, Lanzhou 730000, China

*

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b

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Author to whom correspondence should be addressed.

Huanxiang Liu

School of Pharmacy, Lanzhou University, Lanzhou 730000, China E-mail: [email protected]. Tel: +86-931-891-5686 Fax: +86-931-891-5686 Xiaojun Yao Department of Chemistry, Lanzhou University, Lanzhou 730000, China E-mail: [email protected] Tel.: +86-931-891-2578 Fax: +86-931-891-2582 1

ACCEPTED MANUSCRIPT Abstract The active metabolite GS-461203 of hepatitis C virus (HCV) non-structural protein 5B (NS5B) inhibitor sofosbuvir can stall RNA synthesis or replication by

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competitively inhibiting the natural substrate nucleoside triphosphate like UTP. Unfortunately, S282T mutant can lead to the resistance to sofosbuvir. Here, the

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detailed binding mechanism and unbinding process of GS-461203 and UTP to HCV NS5B were unraveled by using conventional molecular dynamics (MD) simulation and steered molecular dynamics (SMD) simulation. Our simulation results

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demonstrate that both polar and nonpolar interactions are favorable for GS-461203 and UTP binding. Meanwhile, we also identified the key residues responsible for

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GS-461203 and UTP binding in NS5B-RNA together with the three unbinding process steps including translation, reversal of base and ribose and complete divorce.

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The 2’-fluoro-2’-C-methyl ribose of GS-461203 can form stronger polar and nonpolar

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interaction with residues S282 and I160 than UTP. The results can also explain the reason why GS-461203 can effectively incorporated into RNA synthesis or replication.

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In the S282T mutant system, the binding affinity attenuation of UTP relative to wild type HCV NS5B is less than that of GS-461203. The obtained binding and unbinding mechanism of HCV NS5B with the inhibitor GS-461203 and substrate in our work

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will provide useful guidance for the development of new and effective HCV NS5B inhibitors with low resistance.

Key words: Hepatitis C Virus NS5B RNA-dependent RNA polymerase; GS-461203; molecular dynamics simulation; binding free energy calculations

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ACCEPTED MANUSCRIPT Introduction The hepatitis C virus (HCV) remains a threat to public health since approximately 170 million individuals are infected worldwide. 80% of them can be progressed to chronic

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HCV infection. HCV is a member of the family Flaviviridae containing a positive-strand RNA genome[1, 2]. This genome encodes a polyprotein that is

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processed by host and viral proteases into 10 structural and nonstructural (NS) proteins which are required for viral replication. One of these proteins, non-structural protein 5B (NS5B) is an RNA-dependent RNA polymerase. This protein has a

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catalytic core with a classical right-hand structure consisting of finger, palm and thumb domains. The catalytic core precedes a linker and a C-terminal membrane

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insertion sequence[3-5]. NS5B is critical for synthesis of a minus-strand RNA, using the genome as a template. Then the subsequent synthesis of genomic plus-strand RNA

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is carried out with this minus-strand RNA as template. Therefore, NS5B is an

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attractive drug target for anti-HCV drug development[6]. Currently, there are two major classes of NS5B inhibitors: nonnucleoside inhibitors (NNIs), which bind to five

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allosteric sites on the catalytic domain and nucleoside inhibitors (NIs), which can be converted to their active triphosphates, acting as alternative substrates for the polymerase[7-9]. Compared to NNIs, NIs have broader genotype coverage and a

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higher barrier to viral resistance. Several NIs have been reported, such as sofosbuvir (GS-7977 and PSI-7977)[10], GS-0938

(PSI-352938),

TMC649128[11].

mericitabine

Among

these

(RG7128),

NIs,

ALS-2200

sofosbuvir

is

a

(VX-135)

and

prodrug

of

2’-deoxy-2’-fluoro-2’-C-methyluridine monophosphate and approved in the US Food and Drug Administration and the EU European Medicines Agency at the end of 2013 and at the beginning of 2014 [12, 13]. To inhibit HCV NS5B RNA-dependent RNA polymerase, sofosbuvir must be metabolized to its active triphosphate metabolite form GS-461203 (PSI-7409, 2’-deoxy-2’-fluoro-2’-C-methyluridine-triphosphate) with a serials of reactions[14, 15]. GS-461203 binds to NS5B RNA polymerase and is incorporated into the viral RNA, resulting in termination of viral RNA synthesis [16]. The NS5B elongation complex only moderately discriminates GS-461203, with Kd 3

ACCEPTED MANUSCRIPT (equilibrium constant) values of 113 μM compared to 490 μM for natural substrate UTP [8, 17]. However, the detailed competitive mechanisms between GS-461203 and UTP remain elusive. Compared to NNIs and NS3/4A protease inhibitors, the potent

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antiviral activity and high resistance barrier of sofosbuvir has been confirmed [13]. The S282T mutant, a primary mutation associated with resistance to sofosbuvir and

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other HCV NIs, has been identified and lead to a 4- to 24-fold decrease in susceptibility to sofosbuvir for all tested genotypes[18-20]. S282T mutation may discriminate against most 2′-C-methylated HCV NIs inhibitors. However, the detailed

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mechanism of drug resistance is still unclear [21, 22]. Exploring this drug resistance mechanism is important to the design and development of more effective HCV NIs.

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Understanding the binding mode of its substrate and inhibitor as well as drug resistance mechanism conferred by S282T mutant is therefore very useful to discover

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more efficacious, higher potency and higher barrier to resistance drug. Recently, the

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crystal structures of ternary complexes with NS5B-RNA-incoming nucleotides and NS5B-RNA-incoming inhibitor were determined [23]. These crystal structures are

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very useful to reveal key molecular interactions in the active site of the receptor and can provide a good starting point to study the different binding kinetic properties of GS-461203 and substrate UTP and also for reveal the detailed molecular mechanism

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about the unbinding process of the inhibitor and substrate [23]. Based on the crystal structures of the ligand and protein complex, molecular dynamics and free energy calculations are widely used to explore the protein-nucleic, protein-protein and protein-drug interaction and unbinding processes[24-31]. In addition to the thermodynamics, the binding kinetics between the drug and receptor is also important to assess the drug efficacy. The random acceleration molecular dynamics (RAMD) and SMD can explore the transition state about the binding and unbinding process of the drug-receptor interaction. Understanding the molecular interactions between drug and receptor at this transition state of drug unbinding process is central to the rational control of drug binding kinetics[32]. In this work, MD simulation and SMD simulation were employed to explore the competitive inhibition mechanism of the active form GS-461203 of sofosbuvir and 4

ACCEPTED MANUSCRIPT substrate UTP binding to NS5B-RNA complex. We also studied the drug resistance mechanism to GS-461203 conferred by S282T mutation based on. 200ns MD simulation. Binding free energy calculations by the molecular mechanics generalized

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Born surface area (MM-GBSA) method were employed to explore GS-461203 and natural substrate UTP binding mechanism in wild type and S282T mutant

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NS5B-RNA. The calculated binding free energies reveal that UTP has lower binding ability than GS-461203 in wild type NS5B. The S282T mutant causes lower binding ability for both GS-461203 and UTP. RAMD simulation[33] was employed to search

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possible ligand escaping pathways. SMD combined with Jarzynski’s equality[34] was used to construct potentials of mean force (PMFs) and free energy profiles of ligand

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dissociation reactions. Our results will be useful to understand competitive mechanisms between GS-461203 and UTP binding to NS5B-RNA complex and drug

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resistance mechanisms originating from S282T mutation.

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Materials and Methods

Construction of the Simulation System crystal

structures

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The

NS5B-RNA-incoming

of

HCV

nucleotides

(subtype UDP

and

2a)

ternary

complexes

NS5B-RNA-incoming

with

inhibitor

2′-F/2′-CH3-UDP (diphosphate metabolite of sofosbuvir) were extracted from the

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Protein Data Bank (PDB ID: 4WTF and 4WTG) [23]. To construct the ternary complexes of active triphosphate metabolite form GS-461203 (Fig. 1 and S1), we added the third phosphate group based on the crystal structure of UTP in HCV NS5B-UTP complex (PDB ID: 1GX6) [35]. The third phosphate group was also added to UDP in the ternary complexes with NS5B-RNA-incoming nucleotides UDP (Fig. S1). S282T mutant was obtained manually by using Pymol program[36]. Two Mn2+ ions around active site and the crystal water molecules were kept during the molecular dynamics simulation. Gaussian09 software [37] was used to perform geometric optimization of GS-461203 and UTP at the Hartree-Fock level with 6-31G* basis set. The restrained electrostatic potential (RESP) was used to describe the partial atomic charges. The general AMBER force field (GAFF) was used to describe the parameters of GS-461203 and UTP[38]. The standard AMBER force field for 5

ACCEPTED MANUSCRIPT bioorganic systems (ff99SB) [39]was employed to describe the protein. The counter ions Cl- were added to neutralize each system. Then the corresponding systems were solvated using atomistic TIP3P water[40] in a box with at least 10 Å distance around

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the complex. Molecular Dynamics Simulation

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All MD simulations were performed using AMBER10 package [41]. Initially, energy minimization was carried out for each solvated complex by three steps. Energy minimization was performed by the steepest descent method for the first 2500 steps

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and conjugated gradient method for the subsequent 2500 steps. During the energy minimization, harmonic restraints with a force constant of 10.0 kcal/(mol·Å2) was

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applied to NS5B-RNA-ligand complex and protein-RNA complex in the first two step. In the third step, all atoms were allowed to move freely. After energy minimization,

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all systems were gently annealed from 0 to 310.0 K over 100 ps in the NVT ensemble

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and equilibrated to adjust the solvent density under 1 atm pressure over 50 ps in the NPT ensemble simulation by restraining NS5B-RNA-ligand complex with a harmonic

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restraint weight of 10.0 kcal/(mol·Å2). An additional five MD equilibrations of 50 ps each were performed with the decreased restraint weights of 10.0, 5.0, 2.5, 1.0 and 0.1 kcal/(mol·Å2), respectively. These were followed by a last MD equilibration step of 50

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ps by releasing all the restraints. Afterward, 200ns production MD simulations were carried out without any restraint in the NPT ensemble at a temperature of 310.0 K and a pressure of 1 atm. An integration time step of 2 fs was used, and coordinate trajectory was recorded every 1 ps for all the equilibration and production runs. During the simulations, periodic boundary conditions were employed, and direct space interactions were truncated at a distance 12 Å with long-range contributions from the electrostatics included using the particle mesh Ewald (PME) approach[42]. Van der Waals contributions beyond the cutoff were included via the use of an isotropic long-range correction. Bond lengths involving bonds to hydrogen atoms were constrained using the SHAKE algorithm[43]. Thermodynamic Analysis Binding free energy for the active triphosphate metabolite form GS-461203 and 6

ACCEPTED MANUSCRIPT substrate UTP to HCV NS5B polymerase was calculated using the MM/GBSA method [44]. The MM/GBSA binding free energy consists of electrostatic (ΔEele), van der Waals(ΔEvdw), polar (ΔGsol-ele) and nonpolar (ΔGsol-np) component. The ΔEele and

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ΔEvdw contributions are computed using the molecular mechanics energy function in

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gas. The ΔGsol-ele contribution was calculated by solving the GB equation, with

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dielectric constants for solute and solvent set to 4 and 80, respectively. The ΔGsol-np contribution was estimated by the solvent-accessible surface area (SASA) determined using a water probe radius of 1.4 Å and the surface tension constant c was set to

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0.0072 kcal/(mol/Å2)[45]. MM/GBSA binding free energy and per-residue binding free energy decomposition were determined by extracting 500 snapshots extracted

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from the last 100 ns trajectory for each complex. Vibrational entropy contributions were estimated by normal mode analysis[46]. Because of the high computational

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demand, only 40 snapshots for ligand and the receptor binding region (protein

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residues 0-60, 135-399 and RNA) of NS5B-RNA were used in the normal mode analysis. Each snapshot was optimized for 100,000 steps using a distance-dependent

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dielectric of 4rij (rij is the distance between atoms i and j) until the root-mean-square of the gradient vector was less than 0.0001 kcal/(mol/Å2). Random Acceleration Molecular Dynamics (RAMD) Simulation

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In order to find the possible escaping pathway of ligand, RAMD simulation [33, 47] was employed. In this method, a randomly external forces was added to the ligand, The external force (Fext) applied to the ligand escaping from binding site is defined as 

Fext  f 0 v



, Where f0 and v are the constant force and the randomly oriented unit

vector, respectively. The velocity of ligand is expected to a certain threshold vmin as given in equation vmin = rmin/mΔt over m steps, where Δt is the time step (1 fs), and rmin is the specified minimum distance threshold before a direction change. The average velocity of the ligand over the previous m steps period (vmin) is the criterion to decide whether change the force vector: if the ligand’s velocity is lower than vmin, a new random force vector is reassigned, otherwise, the force vector is maintained. Here, the constant force was set to be 80, 70 and 60 kcal/mol·Å, respectively. The 7

ACCEPTED MANUSCRIPT value of m was chosen to be 50 and 30 steps for each constant force and rmin is 0.01 Å. Each constant force was performed with different steps for ten times of RAMD simulations. The RAMD simulations were implemented by using tclforces of the

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NAMD software [33, 48].

Steered Molecular Dynamics（SMD）Simulation and Potential of Mean Force

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（PMF）Construction

All the SMD simulations were applied using the NVT ensemble of the NAMD software package. The escaping pathway reaction coordinate was defined as the z axis

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distance between the Cα atom of W123 and the center of ligand heavy atom mass, based on the ligand escape pathway obtained by RAMD simulation. The constant

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velocity and the force constant were identified using the following stepwise optimization: the spring constant values were set to 40, 50, 60, 70 and 80 kcal/(mol·Å2)

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when a pulling velocity is 5 Å/ns. We found that the stiff spring approximation was

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satisfied when a spring constant was 70 kcal/(mol·Å2). Then, the pulling velocity values were set to 3, 4, 5, 6 Å/ns when keeping pulling velocity fixed at 70

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kcal/(mol·Å2). In order to keep the stiff spring approximation in all run time, the values of spring constant and pulling velocity were chosen 70 kcal/(mol·Å2) and 4 Å/ns, respectively. The value of the exerted force (Fex) was printed out every 1 ps (dt)

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in SMD, and the work done on the system (W) during SMD was calculated by numerical integration as shown in equation W[ x (t )]  

x (t )

0

Fex(t )dx(t ) . When The

stiff-spring approximation is used, F ( )   ( ) [49], where λ is correlated with the reaction coordinate. PMF along the escaping pathway reaction coordinate was computed from the SMD trajectories and using Jarzynski’s equality[34] as given in equation e F  e  w  , which relates the Helmholtz free energy (F) to W and

  (kBT )1  (0.6186 kcal / mol)1 . F  W  . The PMF was calculated using the first order of cumulant expansion approach. Results and Discussion The binding and unbinding mechanisms of natural substrate UTP to wild type 8

ACCEPTED MANUSCRIPT NS5B-RNA complex To assess the stability of studied systems during MD simulation, root mean square deviation (RMSD) of the heavy atoms of ligand (GS-461203 and substrate UTP) and

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the residues around 5 Å of the ligand relative to the starting structure were monitored (Fig. S2). We can see from Fig. S2 that each system tends to be stable after 100 ns.

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The trajectories of last 100 ns were used for the following analysis. To further assess the influence of sampling space on the binding free energy, two additional groups of snapshots for two different time-intervals 100–150 ns and 150–200 ns were extracted

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from the obtained trajectories. Binding free energy calculated by MM-GBSA method was used to assess the binding affinity of substrate UTP and GS-461203 to

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NS5B-RNA complex. The calculated results are shown in Table 1 and Tables S1–S2. It can be seen that the binding free energy calculated from different sampling space is

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similar to each other and the calculated binding free energies are reliable. The

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calculated binding free energy of substrate UTP binding to wild type NS5B-RNA is -29.41 kcal/mol. Both polar interaction (ΔGpolar = ΔEele + ΔGsol-ele) and nonpolar

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term (ΔGnonpolar = ΔEvdw + ΔGsol-np) are favorable to UTP binding. To obtain the detailed interaction profile between UTP and NS5B-RNA, MM-GBSA method was further used to decompose the interaction energies to the contribution of

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each residue. The per-residue contribution profile for the binding of UTP was shown in Fig. 2. There are 9 key residues of NS5B-RNA with more than 1 kcal/mol free energy contribution to the binding of UTP such as U6, R48, K51, K141, K155, R158, R222, H223 and F224. To disclose the contribution of these key residues from the structural perspective, clustering analysis was applied to extract the conformation that best represents the last 100 ns trajectory. The largest cluster was chosen and shown in Fig. 3a and Fig. S3a. It can be seen that the triphosphate moiety can be embedded well in the positive charge binding pocket formed by R158, R48, K51, K155, R158, R222, H223 and F224. The 2’-hydroxyl group of the ribose forms hydrogen bond with the side chain of D225. Compared with diphosphate group, triphosphate moiety has larger negative charge forcing the negative charge side chain of D225 far from its initial position in the NS5B-RNA-UDP complex [23]. 9

ACCEPTED MANUSCRIPT Furthermore, we studied the dissociation pathway of substrate UTP from NS5B-RNA complex by RAMD and SMD simulations. Firstly, the possible unbinding pathway of ligand was predicted using RAMD simulation method. The reaction coordinate was

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defined as the z axis between the Cα atom of W123 and the center of ligand heavy atom mass (Fig. 4). In order to receive the stiff spring approximation in SMD

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simulation, we assessed different spring constant values (40, 50, 60, 70 and 80 kcal/(mol·Å2)) and pulling velocity values (3, 4, 5, 6 Å/ns). The values of spring constant and pulling velocity was 70 kcal/(mol·Å2) and 4 Å/ns, respectively(Fig.

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S4-S7) and can satisfy the stiff spring approximation in SMD simulation. PMF calculated by applying Jarzynski’s equality along reaction coordinate was shown in

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Fig. 5 and Table 1. The PMF of UTP is 268.69 kcal/mol and is larger than that of experimental value because the polar interaction for nucleotide with four negative

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charges is difficult to be calculated accurately [50, 51]. Here, we only pay attention to

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the relative binding affinity and the unbinding processing of ligand rather than the absolute PMF value. To further character the dissociation pathway of UTP, the mean

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force profile and representative snapshots taken from the simulation trajectory of UTP exhibiting the lowest work value were shown in Fig. 6. The process of UTP unbinding from NS5A-RNA can be classified roughly into three steps: translation, reversal of

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base and ribose and complete divorce. Between 0 and 3 Å of reaction coordinate displacement, UTP gradually translates from binding site (Snapshot A and B), the second and third phosphate groups still insert into the positive charge binding pocket composed of side chains of R48, R158 and K51, hydrogen bonds between the side chain with negative charge of D225 and the 2’-hydroxyl group of the ribose form (snapshot A) and then disappear with the moving of the ribose, then two new hydrogen bonds form between 2’- hydroxyl group of the ribose and carboxylic acid group of D318(snapshot B). After that, the base and ribose motifs of UTP slightly rotate around triphosphate moiety (snapshot C-E). The base can form π-π stacking with phenyl group of F145 after the interaction involving the base motif of UTP and the side chain of K141 is lost, the base and ribose motifs of UTP reverse followed by the breakage of such interactions involving base and ribose motifs of UTP and 10

ACCEPTED MANUSCRIPT NS5B-RNA. Beyond 12 Å of reaction coordinate displacement, the very important polar interactions between triphosphate moiety of UTP and the side chains of R48, K51, R158, and K155 diminish or disappear (snapshot F). Only the side chain of

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K151 with positive charge can form hydrogen bonds with the negative triphosphate moiety of UTP that delay the further exit of UTP (snapshot G). The force profile is

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relatively flat until reaction coordinate is 26 Å, and UTP lies outside the binding site and completely divorce from NS5B-RNA complex (snapshot H). The binding and unbinding mechanisms of GS-461203 to wild type NS5B-RNA

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complex

As shown in Table 1, the binding free energy of GS-461203 with NS5B-RNA is

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-33.73 kcal/mol and is lower than that of UTP (-29.41 kcal/mol). Both the polar (-35.16 kcal/mol) and nonpolar (-18.87 kcal/mol) energy contributions are favorable

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to GS-461203 binding. Furthermore, 12 key residues with more than 1 kcal/mol

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energy contribution are A1, A2, U6, R48, K141, K155, R158, I160, R222, H223, F224 and S282 (Fig. 2b). Similarly to UTP, the triphosphate moiety of GS-461203

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embeds in the binding pocket composed by R48, K155, R158, R222, H223 and F224, the base of GS-461203 can forms several hydrogen bonds with A1, and K141. The interaction

with

U6,

I160,

D225,

S282can

stabilize

the

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2’-deoxy-2’-fluoro-2’-C-methyl ribose of GS-461203 (Fig. 3b and Fig. S3b). In order to further explore the difference of binding character between UTP and GS-461203, we decomposed the binding free energy to per residue. The contribution difference of per residue for GS-461203 and UTP binding to NS5B-RNA was shown in Fig. S8. There are five residues including A2, U6, I160, D225 and S282 with more than 0.5 kcal/mol free energy contribution difference These residues are more favorable for GS-461203 binding than UTP. Three residues K51, R158 and D318 have an opposite effect. By aligning the representative snapshot of GS-461203 and UTP (Fig. S8), we can see the triphosphate moiety of GS-461203 is moved outward the positive charge pocket relative to that of UTP. The interactions between negative charged side chains of K51 and R158 and GS-461203 is weakened because these side chains

are

far

from

the

third 11

phosphate

group.

Introducing

ACCEPTED MANUSCRIPT 2’-deoxy-2’-fluoro-2’-C-methyl ribose in GS-461203 can form a strong hydrogen bond with hydroxyl group of S282 and nonpolar interaction with I160 isopropyl group. These are the major reasons why the 2’-deoxy-2’-fluoro-2’-C-methyl ribose of

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GS-461203 is more favorable than the ribose motif for ligand binding.

The PMF of GS-461203 calculated along reaction coordinate is 290.62 kcal/mol and

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is higher than that of UTP (268.69 kcal/mol) (Table 1 and Fig. 5), This result is qualitatively in agreement with the experimental Kd values[8, 17] and binding free energy calculated by MM/GBSA method. Similar to UTP unbinding process,

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GS-461203 dissociation process contains three steps: translation, reversal of base and ribose and complete divorce (shown in Fig. 7). In the first translation step, the

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hydrogen bond between 2’-fluoro of ribose and hydroxyl group of S282 disappears but a new hydrogen bond between 3’-hydroxyl of ribose and guanidyl group of R48

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forms (snapshot A). 3’-hydroxyl group of the ribose forms two hydrogen bonds with

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the side chain of D225 and R48, respectively. The base is ordered by its interaction with the side chain of K141 and A1 though π-π stacking interaction between the base

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of U6 and GS-461203 is vanished (snapshot B). The triphosphate moiety moves out the positive charge pocket but only these hydrogen bonds between first phosphate group and side chain of R158 and R48 still remain. In the second reversal of the base

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and ribose step, GS-461203 stands up and occupies the unbinding channel. The interactions of base and ribose with NS5B-RNA vanish completely and the new hydrogen bond with R158 (snapshot C) then R386 (snapshot D) forms. GS-461203 adopted opposite conformation to initial binding conformation, the base turns toward outside the binding channel (snapshot E). The triphosphate moiety overcome the repulsive interaction originating from side chains with positive charge of D220, D318 and D319 and form a hydrogen bond with K151 (snapshot F and G). Lastly, when the reaction coordinate arrive 22 Å, GS-461203 escapes completely from NS5B-RNA complex

(snapshot

H).

Compared

with

UTP,

introducing

2’-deoxy-2’-fluoro-2’-C-methyl ribose in GS-461203 can form hydrogen bond with S282, R48 and D225 in unbinding pathway, which is the main reason why GS-461203 need overcome larger energy when it escapes from binding site. 12

ACCEPTED MANUSCRIPT Origins of binding affinity loss conferred by the S282T NS5B mutation From the above analysis, it can be seen that S282 plays an important role in the binding between GS-461203 and NS5B. To explore the origins of binding affinity loss

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conferred by the S282T NS5B mutation, we performed molecular dynamics simulation on several other systems including S282T NS5B mutation binding to UTP

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or GS-461203. 200 ns MD simulations and 10 parallel SMD simulations were performed for each system.

During the interaction of NS5B-RNA with substrate UTP, S282 does not play a direct

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role in UTP binding. However, when S282 is mutated into T282, the additional methyl group occupies the position between residue 282 and UTP, which disturbs the

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initial reasonable binding model in wild type. The binding free energy of UTP binding to S282T NS5B-RNA calculated by MM-GBSA (Table 1) and PMF of UTP calculated

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by applying Jarzynski’s equality along reaction coordinate (Table 1 and Fig. 5) are

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-27.13 kcal/mol and 250.85 kcal/mol, respectively. S282T leads to the binding affinity of UTP attenuating which is well consistent with the experimental result that S282T

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causes the reduction of HCV replication[52]. Furthermore, we compared the energy and structure characteristics of UTP binding to wild type and S282T mutant. The energy difference of each residue contribution in the S282T NS5B-RNA relative to

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wild type one. The representative structure were shown in Fig. 2c, 3c and S2c. Compared with wild type system, UTP moves slightly far from A1 and the interaction between triphosphate moiety. The negative side chain of R48, K51, H223 is weakened though T282 contacts with UTP. The unbinding process of UTP in S282T is similar to that in wild type because residue 282 does not locate in UTP unbinding pathway. The slighter loss binding affinity caused by S282T mutation induces UTP easily escaping from binding site (Fig. S10). The NS5B S282T mutation exhibits decreased susceptibility to most 2′-C-methylated HCV NIs, including sofosbuvir. This mutation may cause a steric clash with the 2′-C-methyl motif of inhibitors [21, 53]. From Table 1 and Fig. 4, it can be seen that the binding free energies of GS-461203 to wild type, S282T NS5B-RNA are -33.73 kcal/mol and -24.97 kcal/mol, respectively. The corresponding PMF values based on 13

ACCEPTED MANUSCRIPT SMD simulation are 290.62 kcal/mol and 239.07 kcal/mol, respectively. It indicates that S282T has a lower binding affinity compared with wild type, which is well consistent with the experimental result S282T causes a 4- to 24-fold decrease in the

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susceptibility to sofosbuvir for all tested genotypes[18-20]. From the perspective of energy, the reduced energy contribution of residues A1, U6, R222 and H223 should be

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responsible for the weaker binding affinity of GS-461203 to NS5B-RNA S282T mutant compared with that in wild type NS5B-RNA(shown in Fig. 2d). Compared with wild type NS5B, the hydrogen bond between 2’-fluoro of ribose and side chain

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hydroxyl group of residue 282 vanishes and the interactions between diphosphate group and negative side chain of R222 and H223 decrease largely. The base of

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nucleotide A1 moves upward slightly and can not form base pairing with the base of GS-461302 due to the large side chain of residue T282 disturbs the initial position of

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the base of nucleotide A1 (Fig. 2d and 3d). This is consistent with the crystallographic

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data, the proximity of S282 to the conserved I160 and G283 interacts with the ultimate base pair, and involve a steric conflict between S282T and the incoming

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nucleotide[23, 54, 55]. It can be seen from the unbinding process (Fig. S11) that GS-461203 has a similar dissociation mechanism to wild type NS5B. The loss of interaction above mentioned makes GS-461203 easily translate and the reversing of

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base and ribose though residue 282 is far from the GS-461203 dissociation pathway. Conclusions

In this study, we performed MD and SMD simulations to explore the interaction mode between NS5B-RNA and its substrate UTP as well as the inhibitor GS-461203. The simulation results suggest that the triphosphate moiety is well inserted into positive binding pocket, the base group can form base pairing with A1. The polar interaction between 2’-fluoro of ribose and hydroxyl group of S282, the nonpolar interaction between 2’-C-methyl ribose and isopropyl group of I160 are major reason why GS-461203 can incorporate effectively into the substrate UTP binding site. Their unbinding process contains three steps including translation, reversal of base and ribose and complete escape. We also studied the origins of binding affinity loss caused by the S282T mutation in NS5B. The MM-GBSA calculations based on the MD 14

ACCEPTED MANUSCRIPT simulation trajectories and PMF analysis based on SMD simulation indicate that the S282T mutated NS5B-RNA had a lower binding affinity to substrate UTP or active metabolize GS-461203 of inhibitor sofosbuvir than that in wild type. This result is

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consistent with the experimental result that the S282T mutated NS5B cause drug resistance and RNA replication attenuation. S282T mutant introduces an additional

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methyl group and disturbs the initial binding mechanism which is responsible for binding affinity decrease. The obtained results of this study have allowed a deeper understanding of the competitive mechanism between natural substrate UTP and

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active metabolite GS-461203 of inhibitor sofosbuvir and origins of binding affinity loss caused by the S282T mutation. The results will be valuable to the structure-based

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design of more potent inhibitors targeting HCV RNA replication process.

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Acknowledgement

This work was supported by the National Natural Science Foundation of China (Grant

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No. 21475054), the Fundamental Research Funds for the Central Universities (Grant

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No. lzujbky-2014-191).

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Table 1. Binding free energies and energy components calculated by MM/GBSA method and the PMF values (kcal/ mol). contributiona

GS-461203 _WT

ΔEele

-425.47 ± 0.22

-417.13 ± 0.20

-409.57 ± 0.27

ΔEvdw

-12.84 ± 0.15

-10.05 ± 0.16

-16.27 ± 0.16

-12.09 ± 0.16

ΔEMM

-438.32 ± 0.22

-427.19 ± 0.21

-425.84 ± 0.29

-426.14 ± 0.24

ΔGsol-np

-6.03 ± 0.03

-5.96 ± 0.02

-5.71 ± 0.03

-6.13 ± 0.03

ΔGsol-ele

390.32 ± 0.21

385.90 ± 0.19

380.96 ± 0.26

384.04 ± 0.22

ΔGsol

384.28 ± 0.21

379.94 ± 0.19

375.25 ± 0.26

377.91 ± 0.22

-35.16 ± 0.11

-31.23 ± 0.10

-28.61 ± 0.10

-30.01 ± 0.12

-18.87 ± 0.15

-16.01 ± 0.15

-21.98 ± 0.16

-18.22 ± 0.16

ΔHbind

-54.03 ± 0.14

-47.24 ± 0.14

-50.59 ± 0.17

-48.23 ± 0.15

TΔS

-20.30 ± 0.60

-22.27 ± 0.54

-21.18 ± 0.52

-21.10 ± 0.55

ΔGbind

-33.73 ±0.62

-24.97 ± 0.56

-29.41 ± 0.55

-27.13 ± 0.57

d

290.62 ± 2.08

239.07 ± 2.23

268.69 ± 1.28

250.85 ± 1.48

PMF

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ΔGnonpolar

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ΔGpolar

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GS-461203 _S282T

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Note: a mean ± standard errors(SE); bΔGpolar = ΔEele + ΔGsol-ele;

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ΔGsol-np; dPMF: mean of PMF ± SD.

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c

-414.06 ± 0.24

ΔGnonpolar = ΔEvdw +

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primer, and nucleotides/inhibitor. (red dot, RNA primer; cyan dot, RNA template;

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stick, inhibitor GS-461203; sphere, S282 residue; yellow cartoon, NS5B)

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Fig. 2. The residue contributions of NS5B to ligand binding: (A) UTP_WT; (B) GS-461203_WT; (C)-(D) the energy difference of each residue contribution for the S282T mutated HCV NS5B relative to the wild type one. (C) UTP; (D) GS-461203.

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Fig. 3. The interaction between GS-461203 or UTP and HCV NS5B in wild type and S282T mutant.

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Fig. 4. Unbinding pathways of GS-461203 (pink stick) and UTP (green stick) escaped from NS5B on RAMD simulations

Fig. 5. PMF profiles of GS-461203 and UTP.

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Fig. 6. (top) Representative frames from the simulation of the NS5B-RNA/UTP

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complex exhibiting the lowest work value. (bottom) the mean force profile of UTP as a function of the reaction coordinate.

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Fig. 7. (top) Representative frames from the simulation of the NS5B-RNA/GS461203 complex exhibiting the lowest work value. (bottom) the mean force profile of UTP as

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a function of the reaction coordinate.

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Fig. 7.

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ACCEPTED MANUSCRIPT Highlights  The competitive mechanism between GS-461203 and UTP to NS5B was studied.

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 The resistance mechanism to GS-461203 conferred by S282T mutant was

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explored.

 The unbinding pathway of GS-461203 to NS5B was studied using random acceleration molecular dynamics simulation

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molecular dynamics simulation

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 The PMF profile of GS-461203 to NS5B was calculated based on steered

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