Molecular basis of telaprevir resistance due to V36 and T54 mutations in the NS3-4A protease of the hepatitis C virus

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Molecular structures of the NS3-4A serine protease inhibitors VX-950 (telaprevir) and SCH 503034 (boceprevir) as well as of the co-crystallized protease ligands CPX and SCH 446211. The P₁ to P₄ and P'₁ to P'₂ groups are numbered according to the nomenclature of Schechter and Berger [61]. Residues surrounding a cleavage site are designated from the amino- to carboxyl-terminus, that is, P₄-P₃-P₂-P₁ P'₁-P'₂-P'₃-P'₄, with the scissile bond between P₁ and P'₁. They are annotated only for SCH 446211.

Figure 2

NS3-4A protease domain of PDB structure 1RTL with co-crystallized ligand CPX (yellow) [32] and a second ligand, SCH 446211 (light blue), taken from the superimposed PDB structure 2FM2 [30]. The protease binding pocket from structure 1RTL is shown as a transparent surface patch. The residues V36 and T54 are depicted as stick-and-ball models, located in the parallel β-strands β1 and β3 of an anti-parallel β-sheet (dark blue).

Figure 3

VX-950 protein-ligand binding. (a) Surface representation of the NS3-4A protease binding pocket (PDB entry 1RTL) with the docked VX-950 compound. VX-950 is covalently bound to S139. The cyclopropyl group is oriented towards a hydrophobic cavity. The surface of the protein was colored with the vacuum electrostatics function of PyMOL. Charges are computed with the Amber 99 force field and projected on the protein surface, whereas colored patches (red = positive, blue = negative) denote polar regions and white patches apolar protein regions. (b) MOE plot for interactions of the protease with the VX-950 compound. The legend is at the bottom.

Figure 4

Network of non-covalent residue interactions for the NS3-4A protease and the corresponding list of protein-ligand interactions. (a) Network analysis of non-covalent residue interactions for the NS3-4A protease (PDB entry 1RTL). Nodes represent residues and colored edges represent different types of interactions: van der Waals interactions, backbone-side chain (blue), side chain-side chain (red); H-bond interactions, backbone-side chain (green), side chain-side chain (orange). Protein-ligand interactions for the 1RTL and 2FM2 ligands CPX and SCH 446211, respectively, as well as for VX-950 are tagged by brown Arabic numerals above each residue node (see (b)). Catalytic residues are yellow and the mutated residues are blue (V36), red (T54) and grey (R155, A156). (b) List of van der Waals interactions (vdW), H-bonds (HB) and covalent bonds (CB) for the 1RTL and 2FM2 ligands CPX and SCH 446211, respectively, and the VX-950 ligand docking result. Each dot or square represents one interaction of the ligand with an amino acid of the NS3-4A protease, and dots indicate interactions with the cyclopropyl group. Brown Arabic numerals refer to protein-ligand interactions in the network of non-covalent interactions (a).

Figure 5

Structure and network analysis of non-covalent residue interactions for T54. Left column: visualization of the NS3-4A protease structure and surface of the binding pocket of 1RTL with co-crystallized ligands taken from two superimposed PDB structures: 1RTL with ligand CPX (yellow) and 2FM2 with ligand SCH 446211 (light blue). Right column: corresponding network analysis of non-covalent residue interactions for T54 mutants. Residues presumed to interact with the cyclopropyl group of VX-950 are indicated by black dots. Nodes represent residues and colored edges represent different types of interactions (see Figure 4): van der Waals interactions, backbone-side chain (blue), side chain-side chain (red); H-bond interactions, backbone-side chain (green), side chain-side chain (orange). (a) Anti-parallel β-sheet and H-bond interactions of T54 with L44 and V55 (yellow). H-bonds are shown as cyan dotted lines and corresponding distances printed in cyan. (b) Loop-forming residues (orange) and hydrophobic pocket conformation. (c) Impact of T54 mutants on the catalytic triad via the node V55 (purple).

Figure 6

MD simulations of T54 mutants. (a) Three-dimensional structure analysis of the NS3-4A protease binding pocket taken from the equilibrated structures of the MD simulations of the wild-type (PDB entry 2FM2) and the T54A and T54S mutants (all with ligand SCH 446211). The wild-type protease structure is shown in green and its ligand in yellow. The mutants T54A and T54S are colored red and blue, respectively, and the corresponding ligands white. Part of the surface of the T54A mutant structure is shown in wireframe representation. For the sake of clarity, we have not included the surfaces of the wild-type protein and the T54S mutant structure. The side chains of the residues H57 and S139 in the wild-type structure extend out of the surface of the mutant structure. One of the hydroxyl groups of the SCH 446211 ligand in the wild-type structure clashes with the mutant's surface. Thus, in the mutated structures, this group is rotated by about 90 degrees to the upper right of the inhibitor. (b) Structural changes observed by MD simulations are reflected by the corresponding network of non-covalent residue interactions. Important conformational changes occur at L44 and V55, which do not directly interact with the ligand. Only H57 and S139 contact the ligand, and H57 forms direct interactions with the cyclopropyl group of VX-950. Nodes in the network are highlighted according to the MD simulation results (for details, see legend of Figure 5). (c) Protein surface of the binding pocket for the wild-type protease structure (green) with the ligand SCH 446211 (yellow) in comparison to the T54A mutant structure (red). The changes in the surface (circled in black) correspond to considerable side chain movements as discussed in (a). (d) Protein surface of the binding pocket for the wild-type protease structure (green) with the ligand SCH 446211 (yellow) in comparison to the T54S mutant structure (blue). The changes in the surface area (circled in black) correspond to considerable side chain movements as described in (a).

Figure 7

Visualization of the NS3-4A protease binding pocket (left) and the corresponding network of non-covalent residue interactions in the neighborhood of F43 (right). For details, see the legend of Figure 5.

Figure 8

Rotameric states and conformational differences for V36 mutants A/G/L/M computed with IRECS using the PDB entry 1RTL. The figure illustrates the relative position of mutant side chains (light grey) and the wild-type residue V36 (blue). The important carbon atoms of the side chains are indicated as C_α, C_β and C_γ. Protein backbone changes are depicted in black. The contribution of F43 to the hydrophobic cavity conformation and the cyclopropyl binding pocket is illustrated by means of a transparent surface patch.

Figure 9

Multiple alignment of NS3 protease sequences for different HCV genotypes. UniProtKB accession numbers are given in Table S1 in Additional data file 1. The aligned sequences contain amino acids 16 to 176 according to the PDB entry 1DY8 (UniProtKB accession number P26662). The DSSP secondary structure assignment for 1DY8 is illustrated at the top of the alignment with curled lines for α-helices and arrows for β-strands. The catalytic triad consisting of H57, D81 and S139 and a zinc finger formed by C97, C99 and C145 is indicated at the bottom of the alignment by orange and yellow squares, respectively. The binding cavity for the cyclopropyl group of the VX-950 compound and CPX ligand is marked by purple squares. Text labels annotate different sites of drug resistance mutations (V36, T54, R155, A156) for VX-950. Amino acids are shaded in different grey levels according to their physicochemical properties: aliphatic (A/C/G/I/L/M/N/Q/V), black (white letters); aromatic (F/W/Y), white (black letters); cyclic (P), light grey (white letters); basic (H/K/R), grey (white letters); acidic (D/E), dark grey (white letters). Amino acids with conformational changes described in the paper are framed by green boxes.