High affinity peptide inhibitors of the hepatitis C virus NS3-4A protease refractory to common resistant mutants

Abstract

Hepatitis C virus (HCV) NS3-4A protease is essential for viral replication. All current small molecular weight drugs against NS3-4A are substrate peptidomimetics that have a similar binding and resistance profile. We developed inhibitory peptides (IPs) capping the active site and binding via a novel "tyrosine" finger at an alternative NS3-4A site that is of particular interest for further HCV drug development. The peptides are not cleaved due to a combination of geometrical constraints and impairment of the oxyanion hole function. Selection and optimization through combinatorial phagemid display, protein crystallography, and further modifications resulted in a 32-amino acid peptide with a K(i) of 0.53 nm. Inhibition of viral replication in cell culture was demonstrated by fusion to a cell-penetrating peptide. Negligible susceptibility to known (A156V and R155K) resistance mutations of the NS3-4A protease was observed. This work shows for the first time that antiviral peptides can target an intracellular site and reveals a novel druggable site on the HCV protease.

FIGURE 1.

Detailed views on the CP5-46-A/NS3-4A co-complex. A, overview on CP5-46-A (green) in complex with NS3-4A (blue/orange). Dashed red boxes indicate magnified views displayed in B–D. B, close up on NS3-4A active site (blue) with bound CP5-46-A (green). Catalytic triad residues Ser-139, His-57, and Asp-81 are colored in wheat, and other protease residues are colored in blue. A red dashed line shows the closest distance of Ser-139 and CP5-46-A backbone (Leu-9–Leu-10). H-bonds of an active site H₂O are drawn with black dashed lines. C, non-primed site of the protease, CP5-46-A main chain (green) forms an anti-parallel β-strand with the backbone of NS3-4A (blue). D, primed site of NS3-4A with accommodated tyrosine finger (aa ¹³PGYDP¹⁷). Coordinated water molecules are shown as red spheres. The NS4A peptide is colored orange. aa 10–12 and 18–19 of CP5-46-A are shown in line representation.

FIGURE 2.

A, structural effects upon binding of peptide CP5-46-A. CP5-46-A causes structural dislocation of a NS3-4A loop (aa Thr-38–Gln-41) close to the active site. Apo-NS3-4A protease with free active site is shown in gray (PDB code 1DXP). NS3-4A with bound peptide CP5-46-A (green) is shown in blue. B, N-terminal part of CP5-46-A binds to the non-prime (S) sites of NS3-4A. Locations of the protease subsites are labeled with semicircles (positions estimated from literature (47)). Peptide CP5-46-A is shown in stick representation with modeled surface. Salt bridge between His-19 and Asp-11 is indicated as a gray dashed line. C, comparison of the coordination of peptide CP5-46-A and the optimized peptide CP5-46A-4D5E. B-factors for the peptide residues are indicated by rainbow colors, whereas blue indicates a low and red a high B-factor (scale bar at bottom left corner). Peptides were superposed based on the NS3-4A backbone. D, illustration of the close coordination of the tyrosine finger motif. CP5-46-A residues are shown in gray, and NS3 residues are shown in orange. Inhibitory peptide residues 16–20 are not shown for the sake of clarity. Dashed lines indicate hydrogen bonds. Three coordinated water molecules close to Tyr-15 are displayed as red spheres.

FIGURE 3.

Binding of peptide CP5-46-A to NS3-4A compared with the natural substrate and small molecule inhibitors. A, binding of CP5-46-A (green) compared with the natural substrate 4A5B (orange, PDB code 3M5M). NS3 protease domain is shown in blue with modeled surface and side chains of active site residues in orange. B, binding of CP5-46-A (green) compared with small molecule inhibitor ITMN-191 (blue, PDB code 3M5L). C, binding of CP5-46-A (green) compared with small molecule inhibitor boceprevir (yellow, PDB code 2OC8). The side chains binding to the protease subsites are indicated (P1′–P6). Structures were aligned based on NS3/4 backbone residues.