Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Abstract

Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.

Fig. 1.

NS3/4A protease inhibitors and reported sites of drug resistance. (A) The leading protease inhibitors in development mimic the N-terminal side of the viral substrates. (B) The majority of reported drug resistance mutations cluster around the protease active site with the catalytic triad depicted in yellow.

Fig. 2.

Stereo view of N-terminal cleavage product binding to NS3/4A protease. N-terminal protease cleavage products (A) 3-4A, (B) 4A4B, (C) 4B5A, and (D) 5A5B are depicted as they bind to the protease active site. All conserved interactions are indicated by black dashes, while red lines depict interactions that are not present in all structures. The electrostatic network involving residues R123, D168, R155, and D81 is indicated by blue dashes.

Fig. 3.

Stereo view of the NS3/4A substrate envelope and protease inhibitors. (A) After active site superpositions, the overlapping van der Waals volume shared by any three of the four cleavage products defines the substrate envelope, depicted in blue. NS3/4A protease residues which mutate to confer drug resistance are shown in brown. (B) ITMN-191, (C) boceprevir and (D) TMC435 protrude from the substrate envelope at several locations, which correlate with known sites of drug-resistant mutations to each inhibitor, shown in red.