Molecular basis of telaprevir resistance due to V36 and T54 mutations in the NS3-4A protease of the hepatitis C virus
- Christoph Welsch†1, 2, 3Email author,
- Francisco S Domingues†1,
- Simone Susser2, 3,
- Iris Antes1,
- Christoph Hartmann1,
- Gabriele Mayr1,
- Andreas Schlicker1,
- Christoph Sarrazin2, 3,
- Mario Albrecht1,
- Stefan Zeuzem2, 3 and
- Thomas Lengauer1
© Welsch et al.; licensee BioMed Central Ltd. 2008
Received: 17 July 2007
Accepted: 23 January 2008
Published: 23 January 2008
The inhibitor telaprevir (VX-950) of the hepatitis C virus (HCV) protease NS3-4A has been tested in a recent phase 1b clinical trial in patients infected with HCV genotype 1. This trial revealed residue mutations that confer varying degrees of drug resistance. In particular, two protease positions with the mutations V36A/G/L/M and T54A/S were associated with low to medium levels of drug resistance during viral breakthrough, together with only an intermediate reduction of viral replication fitness. These mutations are located in the protein interior and far away from the ligand binding pocket.
Based on the available experimental structures of NS3-4A, we analyze the binding mode of different ligands. We also investigate the binding mode of VX-950 by protein-ligand docking. A network of non-covalent interactions between amino acids of the protease structure and the interacting ligands is analyzed to discover possible mechanisms of drug resistance. We describe the potential impact of V36 and T54 mutants on the side chain and backbone conformations and on the non-covalent residue interactions. We propose possible explanations for their effects on the antiviral efficacy of drugs and viral fitness. Molecular dynamics simulations of T54A/S mutants and rotamer analysis of V36A/G/L/M side chains support our interpretations. Experimental data using an HCV V36G replicon assay corroborate our findings.
T54 mutants are expected to interfere with the catalytic triad and with the ligand binding site of the protease. Thus, the T54 mutants are assumed to affect the viral replication efficacy to a larger degree than V36 mutants. Mutations at V36 and/or T54 result in impaired interaction of the protease residues with the VX-950 cyclopropyl group, which explains the development of viral breakthrough variants.
More than 170 million people worldwide are chronically infected with the hepatitis C virus (HCV). Combination therapy with pegylated interferon-α plus ribavirin shows sustained virologic response rates of approximately 50% in HCV genotype 1 infected patients [1–3], which emphasizes the need for new antiviral drugs. The serine protease NS3-4A is a promising drug target for specific antiviral treatment. HCV genotypes exhibit about 80% sequence identity in NS3-4A, with highly conserved key residues . NS3-4A is bifunctional, possessing a protease as well as a helicase domain. Especially the protease domain is a target for rational drug design [5–8]. The serine protease has a chymotrypsin fold, which consists of the amino-terminal 181 amino acids of NS3. The three catalytic residues H57, D81 and S139 are located in a crevice between the two protease β-barrels [9–11]. The numbering used in the following is according to the structure 1DY8 taken from the Protein Data Bank (PDB) [13, 14]. The central region of NS4A is buried almost completely inside NS3 and serves as a cofactor for proper folding of NS3 .
The binding pocket of the protease is shallow, non-polar, and rather difficult to target. Therefore, the development of potent protease inhibitors has been a challenging task in the past. This is reflected by the variety of rational drug design approaches and drug candidates tested so far, for example, protease substrate or product analogs, serine-trap inhibitors, tripeptide inhibitors and de-novo peptidomimetics [6, 15]. Data for drug resistance and antiviral efficacy have been published for the protease inhibitors BILN-2061 (ciluprevir) [16, 17], VX-950 (telaprevir) [18–20], and SCH 503034 (boceprevir) [21, 22].
Enzymatic in vitro drug resistance data for telaprevir (VX-950)
IC50 mean (nM)
IC50 range (nM)
IC50 (fold changes)
HCV genotype 1a
The following sections describe the results of the analysis of the HCV protease structure of NS3-4A and the different ligand interaction modes using alternative experimental structure models. The ligand binding mode of the inhibitor VX-950 was investigated by computational protein-ligand docking. Structural changes in the binding pocket and the catalytic triad of the protease were characterized by molecular dynamics simulations of T54A/S mutants and rotamer analysis of V36A/G/L/M side chain conformations. A residue-based network of non-covalent interactions was constructed to investigate molecular mechanisms of drug resistance. Experimental data are provided for the V36G mutant to corroborate our findings. The last section comprises a sequence analysis of HCV genotypes and their polymorphisms with respect to the mutational sites discussed in this study.
Analysis of NS3-4A protease structures and ligand binding modes
Mutations at position T54
The same expectation holds for the conformation of the neighboring loop consisting of the residues V55, Y56, H57 and G58. T54 is located next to this loop structure (Figures 2 and 5b), which is involved in shaping the protease surface and the cavity accommodating the cyclopropyl group. Local conformational changes upon mutation at T54, particularly T54A, are expected to have an impact on the succeeding loop, affecting the cavity conformation and the residues Q41, F43 and H57 involved in direct interactions with the VX-950 cyclopropyl group (Figure 4).
We did not observe non-covalent interactions between T54 and the catalytic triad residues consisting of H57, D81 and S139. However, catalytic triad residues interact directly with residue V55, which follows T54. In addition, residues T54 and V55 interact via an H-bond (Figure 5c). Therefore, T54 interacts with each of the catalytic residues indirectly via V55. Together with the structural changes found in the ligand binding site (see 'Molecular dynamics simulations of T54 mutant structures' described below), a potential impact of the mutation T54A on catalytic residues might explain effects on the catalytic activity of the protease NS3-4A. We found no direct non-covalent interaction of T54 with G137, a residue of the oxyanion hole. Nevertheless, an indirect effect could occur via residue L44 and two edges (see network in Figure 4).
Molecular dynamics simulations of T54 mutant structures
In general, the surface and hydrophobic cavity are shallower in the mutant structure T54A than in the wild type, but this is not the case for T54S. Figure 6c illustrates the decreased volume of the cavity using the surface of the mutant structures, which covers the surface of the wild-type structure in the cyclopropyl binding pocket. In summary, the molecular dynamics simulations for T54A/S mutant structures corroborate the previous analysis of the residue interaction network. Both studies suggest a conformation change at the binding site for the T54 mutants.
Mutations at position V36
The network distance between V36 and the catalytic residues is larger than between T54 and the same residues. V36 interacts indirectly with S139 via a two-edge path including F43. At least three to four edges in the network need to be traversed to reach the other catalytic residues H57 or D81. No direct non-covalent interaction is present between V36 and any of the catalytic residues H57, D81 or S139. Similarly, there is no direct non-covalent interaction between V36 and the oxyanion hole at G137. An indirect interaction of V36 with G137 is possible via two edges (see network in Figure 4).
Rotamer analysis of V36 mutations
In vitroanalysis of the V36G resistance mutation using an HCV replicon-assay
Based upon our previous analysis, we performed a comparison of antiviral efficacies for the two protease inhibitors VX-950 and SCH 503034. Only the SCH 503034 inhibitor is lacking the cyclopropyl group (Figure 1). We used a wild-type HCV replicon assay (genotype 1b) and an assay harboring the V36G mutant for in vitro testing. Detailed information on experimental procedures is given in Materials and methods. We found that the SCH 503034 inhibitor is efficient on the V36G mutant with effective suppression of viral RNA titers and a mean IC50 value clearly below 5 μM. In contrast, VX-950 was less effective in the V36G mutant replicon assay, with an IC50 value of about 5 μM. In comparison to the wild-type replicon assay, viral suppression was considerably delayed only for VX-950 in the V36G mutant assay. SCH 503034 was nearly equally effective in viral suppression for both the V36G mutant assay and the wild-type assay (Figure S3 in Additional data file 1).
Comparison of HCV genotypes
Our results indicate that the cyclopropyl group of VX-950 is oriented towards a hydrophobic cavity in the binding pocket of the HCV protease NS3-4A. The cyclopropyl binding mode and the geometry of the cavity appear to play an essential role in the development of drug resistance by mutants at positions V36 and T54. The residue T54 lies in an anti-parallel β-sheet, which is followed by a loop structure involved in shaping the hydrophobic cavity. We expect a larger impact of T54A than T54S on the β-sheet conformation due to the affected H-bond formation.
Molecular dynamics simulations of T54A/S mutant structures support our interpretation. We observed more pronounced structural changes in the case of T54A compared to T54S, which impact the binding pocket, particularly at the hydrophobic cavity that accommodates the cyclopropyl group. We also observed a reduced depth of the cyclopropyl binding cavity for the T54A mutant structure. In vitro data for T54A revealed an 11.7-fold increase of IC50, whereas T54S showed only a minimal level of drug resistance, with a 1.9-fold increase in IC50 (Table 1) [19, 26–28]. We suppose that the minor impact on the protease structure and the less compromised VX-950 binding in the case of T54S results in low-level drug resistance, in contrast to T54A with higher drug resistance levels. Furthermore, we analyzed potential molecular mechanisms affecting catalytic residues of the NS3-4A protease and the implications for viral replication efficacy. A network of non-covalent residue interactions demonstrated possible effects of T54 mutants not only on the ligand binding site, but also on the catalytic residues. This is in agreement with results of molecular dynamics simulations upon T54A/S mutation and underlines the considerable negative influence of T54 mutants on the protease catalytic activity.
We found V36 to be located farther away from the hydrophobic cavity than T54, both in the three-dimensional structure and in the residue interaction network derived from the NS3 protease structure. We observed non-covalent interactions of the wild-type V36 with a residue that shapes the hydrophobic cavity. The mutations V36A/G/L/M allow a displacement of the side chain of this residue, thereby changing the shape of the cavity. Thus, the V36 mutants affect only the shape of the cyclopropyl binding cavity, which is in agreement with the corresponding low-level drug resistance and weak IC50 fold changes of only 1.7 to 6.9 (Table 1) for V36A/L/M single mutations [19, 26–28]. We conjecture that the binding affinities of the VX-950 compound are modified only marginally, which is consistent with the low-level drug resistance. The residue V36 and its mutants are only of minor relevance for the protease catalytic activity. In comparison with T54, we observed lower network connectivity and larger distance from catalytic triad residues in the network for the V36 node. This may explain why V36 mutants have been observed in all breakthrough patients and more frequently in follow-up sequencing data than T54 mutants, which indicates greater protease enzymatic activities and better viral replication efficacies [18, 19, 26–28]. After withdrawal of VX-950, V36 mutants remained at a fairly steady frequency in HCV quasispecies populations, most probably due to an only slightly decreased viral replication rate and a low-level drug resistance [18, 19, 26–28].
Moreover, we performed a comprehensive comparison of NS3 protease sequences for all HCV genotypes. We found only minor variability at the mutational sites and residue positions investigated in this study. The clinically most relevant HCV genotypes 1, 2 and 3 are particularly similar in contrast to other genotypes. Altogether, we assume closely related molecular resistance mechanisms for all HCV genotypes when treated with VX-950 or compounds with a similar scaffold.
We identified a narrow hydrophobic cavity in the binding pocket of the protease NS3-4A accommodating the cyclopropyl group of VX-950 (telaprevir). Mutations at V36 and T54 are expected to affect local conformation and the geometry of this cavity, which explains the observed drug resistance. We used a structural network of non-covalent interactions between NS3 protease residues to investigate molecular effects underlying drug resistance. Notably, this novel methodological approach is of general applicability for many studies of protein structure and function. In our work, the residue interaction network allowed the identification of key mechanisms responsible for conformational changes in the ligand binding pocket and hydrophobic cavity as well as for functional effects on the protease catalytic residues. Molecular dynamics simulations and rotamer analysis support our findings well. Additionally, we performed experimental inhibitor studies with VX-950 and SCH 503034 in a mutant HCV replicon assay, which corroborated our results.
Based on the present work, we conclude that add-on or switch to complementary protease inhibitors, possessing no cyclopropyl or similar group in an equivalent position as in VX-950, might help to avoid cross-resistance during viral breakthrough and follow-up. Therefore, we suggest further experiments to examine our observations. NS3 protease mutants could be tested for their antiviral efficacy and compromised viral replication. Based upon our findings, it would be of interest to compare the efficacy of VX-950 against that of SCH 503034 for other V36 and T54 mutants. Apart from that, crystal structure information would be desirable for mutant structures with co-complexed drugs like VX-950 to confirm our computational analysis.
Materials and methods
Analysis of experimental structural models of NS3-4A
Alternative experimental structure models of the HCV protease NS3 were compared based on the differences of the intramolecular distances using the backbone carbon alpha (Cα) atoms and the geometric centers of the side chain atoms . In total, 37 experimental models available in the PDB [13, 14] were analyzed, including five structure models lacking NS4A. The 32 different structure models of the NS3-4A protease were superimposed for further analysis, excluding the five models without NS4A due to major conformational differences. Invariant structural regions were identified and superimposed . Multiple structure models determined by X-ray crystallography are normally available from each PDB entry because it includes more than one protease domain in the asymmetric unit. We used PyMOL  for the visualization of protein structure images. Chimera  was used for the calculation of buried cavities within the NS3-4A protease (PDB entry 2FM2) and the derived mutant structures.
The protein-ligand docking of VX-950 was performed using PDB entry 1RTL of the protease NS3-4A. The binding pocket was defined as a subset of all residues that have at least one atom closer than 6.5 Å to any atom of the 1RTL ligand. The ScreenScore  parameterization of the docking program FlexX  was applied to account for the mainly hydrophobic nature of VX-950 and to compensate small-scale induced-fit effects because ScreenScore uses a softer consensus scoring function than the standard FlexX does. The chemical structure of the ligand VX-950 (Figure 1) was drawn with MDL ISIS/Draw . The three-dimensional structure was derived by energy minimization with MMFF94. The cyclopropyl group of VX-950 was selected as the base fragment of FlexX to achieve a high sampling rate on this group. First, VX-950 was docked into the binding pocket without specifying a covalent bond. FlexX automatically places VX-950 in a non-covalent binding mode so that the cyclopropyl group is placed in the same hydrophobic region as the CPX ligand in 1RTL and so the ketone oxygen is nearby the S139 side chain. Next, we fixed the covalent bond and relaxed the structure of VX-950 using a 100-step energy minimization. We chose this two-step setup to ensure that the docking is not biased by the geometrical constraints of a predefined covalent bond. The covalent bonding was observed for analogous ketoamide inhibitors  and ketoacid inhibitors . We used MOE (Molecular Operating Environment)  to visualize ligand interaction diagrams for 1RTL and 2FM2 ligands and PyMOL  for the visualization of the VX-950 docking results.
Network of non-covalent interactions
In the following, the term interaction denotes non-covalent interactions. The non-covalent H-bond and van der Waals interactions between amino acids were identified in PDB entry 1RTL and represented as a two-dimensional network. We used the WHAT IF web interface [42, 43] to identify H-bonds and van der Waals interactions between residues. The network was visualized in Cytoscape . LIGPLOT  was additionally applied to identify H-bonds and van der Waals interactions of the ligand with amino acids of the protease NS3-4A. The local connectivity of each residue was calculated as the number of its interactions using the Cytoscape plugin NetworkAnalyzer [46, 47], which suggested residues of functional importance in the interaction network. Distances between residue nodes were computed by NetworkAnalyzer as the minimum number of interaction edges connecting two nodes.
Rotamer analysis and side chain orientation
We predicted side chain conformations of the mutated residues V36A/G/L/M with the tool IRECS (Iterated Restriction of Conformational Space) . IRECS analyses ensembles of possible rotameric states of side chains and subsequently filters out states with unlikely interaction patterns with the backbone and other side chains.
Molecular dynamics simulations
Molecular dynamics (MD) simulations were performed for the wild-type structure of the NS3-4A protease and the mutants T54A and T54S using the program GROMACS3.3 . Regarding the mutant structures, the wild-type structure (PDB entry 2FM2) was used and the corresponding side chain (T54) was mutated using the tool IRECS . The original ligand SCH 446211 from 2FM2 was used for the simulations. This choice was based on the fact that the goal of the simulations was to evaluate the influence of the mutations on the protein structure in general, which should be the same regardless of the bound ligand and should not depend on the binding of a specific ligand. Thus, we used the original experimental ligand SCH 446211 in order to avoid potential artifacts originating from docking inaccuracies in our simulation. The analysis of the simulation results was based on the final structures of the simulations after equilibration. For the simulations, the GROMOS96 force field  and the SPC water model were used, applying periodic boundary conditions. The long-range non-bonded interactions were treated by particle-mesh Ewald summation, and a time step of 2 fs was used. Throughout the simulations, the bond lengths were constrained to ideal values using the LINCS procedure . The system was heated from 0 to 300 K over 120 ps, and the simulations were then continued at 300 K and at a constant pressure of 1 atm for 2 ns. The temperature and pressure were maintained by weak coupling to an external bath with a temperature coupling relaxation time of 0.1 ps and a compressibility of 4.5·10-5 . For the analysis of MD simulation results, the average structures of the protease-inhibitor complexes of the last 100 ps of the simulations were used, superimposing backbone Cα atoms. On the basis of these structures, the conformations of the residues in the binding pocket and the pocket surface were analyzed. The tool VMD  was applied for the visualization of simulated protease-ligand structures.
Multiple sequence analysis
Sequences of the different HCV variants of the NS3-4A protease were retrieved from the UniProtKB database [53, 54]. HCV genotypes are named according to a recent consensus proposal for a unified system of HCV genotype nomenclature . The UniProtKB accession numbers of the sequences reported in this paper are given in Table S1 in Additional data file 1. A multiple sequence alignment (Figure 9) of the NS3-4A protease domain was computed using MUSCLE  and subsequently improved by minor manual modifications using the SEAVIEW alignment editor . The secondary structure assignment to the PDB structure 1DY8 was taken from the DSSP database [57, 58]. The sequence alignment figure was illustrated using GeneDoc . Residue numbering in the manuscript is according to HCV genotype 1b, PDB entry 1DY8.
In vitro IC50determination of mutant NS3-4A proteases
VX-950 was synthesized by the European Network of Excellence for Viral Resistance in Hepatitis C (viRgil, Drugpharm), dissolved in dimethyl sulfoxide as a 6.6 mM solution. SCH 503034 was synthesized by Schering-Plough Corporation (Kenilworth, NJ, USA), dissolved in dimethyl sulfoxide as a 19 mM solution. Both compounds were stored at 4°C.
Plasmids and in vitroRNA transcription
The plasmid pFKI389neo/NS3-3'/ET contains HCV subgenomic replicon sequences derived from HCV genotype 1b and an upstream T7 promoter for in vitro RNA synthesis. The point mutation was generated with the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The plasmid was linearized with ScaI and purified by phenol chloroform extraction. The linearized and purified plasmid was transcribed by using a T7 RNA polymerase (Promega, Madison, WI, USA) according to the manufacturer's instructions. All of the plasmids and RNAs were checked for purity and integrity by standard procedures.
Generation of HCV replicon cell lines
Huh-7.5 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; PAA Laboratories GmbH, Pasching, Germany) and 2 mM L-glutamine. The cells were transfected with an in vitro-transcribed subgenomic HCV replicon RNA. The wild-type sequence was identical to that of the pFKI389neo/NS3-3'/ET replicon . Stable cells containing the self-replicating HCV replicon were selected and maintained in the presence of 750 μg of G418 (Invitrogen) per ml and were used for HCV replicon assays.
Two-day HCV replicon assay
HCV replicon cells were plated in a 6-well plate at a density of 2 × 105 cells per well in DMEM with 10% FBS. On the following day (24 h later), the culture medium was replaced with DMEM containing either no compound as a control or compounds serially diluted in the presence of 10% FBS and 750 μg/ml G418. After the cells were incubated with the compounds for 48 h, the intracellular RNA was extracted with an RNeasy kit (Qiagen, Valencia, CA, USA). The level of HCV RNA was determined by a real-time quantitative reverse transcription-PCR (RT-PCR) assay (Taqman) with a pair of HCV-specific primers (5'-ACG CAG AAA GCG TCT AGC CAT-3' and 5'-TAC TCA CCG GTT CCG CAG A-3'), an HCV-specific probe (5'-6FAM-TCC TGG AGG CTG CAC GAC ACT CA XT-PH-3), and an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). The IC50 was defined as the concentration of compound at which the HCV RNA level in the replicon cells was reduced by 50%.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 includes supplementary figures and table. Figure S1 illustrates NS3-4A protease-ligand interactions. Figure S2 shows the complete network of non-covalent, H-bond and van der Waals interactions of the NS3-4A protease for the PDB entry 1RTL. Figure S3 gives results of SCH 503034 and VX-950 inhibitor studies using an HCV V36G mutant replicon assay. Table S1 lists HCV genotypes included into the multiple sequence alignment of Figure 9.
Dulbecco's modified Eagle's medium
fetal bovine serum
hepatitis C virus
inhibitory concentration 50%
Protein Data Bank
We are grateful to Dr Ann D Kwong for helpful discussion. We thank Dr Johan Neyts and Dr Piet Herdewijn (Katholieke Universiteit Leuven, Netherlands) and the viRgil Drugpharm for generously providing VX-950. The present study was supported by a DFG grant to CW, CS, MA, SZ and TL (Klinische Forschergruppe, KFO 129/1-1, TP2, TP3, TP6) and two European 6th framework Networks of Excellence, viRgil (LSHM-CT-2004-503359) and BioSapiens (LSHG-CT-2003-503265), funded by the European Commission.
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