Hepatitis C virus (HCV) is a genetically diverse positive-stranded RNA virus of the Flaviviridae
family infecting an estimated 170 million people worldwide 
. Based on genetic diversity, HCV is divided into six major genotypes (genotypes 1–6) and numerous subtypes with different geographic distributions; genotypes 1 and 3 are the most prevalent worldwide 
. HCV infection is the leading cause of chronic liver disease that persists for decades and eventually progresses to cirrhosis, liver failure, or liver cancer 
. The current anti-HCV standard of care is a combination of pegylated interferon (Peg-IFN), ribavirin (RBV), and boceprevir or telaprevir, two recently approved antiviral agents targeting the viral NS3/4A protease 
. Sustained virologic response (SVR) –which is tantamount to cure–is achieved only in a subset of treated patients, depending on a combination of viral and host-cell genetic factors 
. For example, a human polymorphism at the IL28B gene is associated with poor interferon response 
. Most patients undergoing interferon-based therapies also experience significant adverse effects, including flu-like symptoms, anemia, and depression 
. Thus, current anti-HCV therapies are often not tolerated and ineffective for many patients, and novel direct-acting antiviral drugs are required for safer, more efficacious treatment.
Direct-acting antiviral agents have the potential to improve SVR rates and minimize treatment duration. The HCV NS3/4A protease – a chymotrypsin-like serine protease – is a prime therapeutic target that cleaves four known sites along the virally encoded polyprotein 
. The NS3/4A protease also hydrolyzes two human proteins, TRIF and MAVS, which are part of the innate immune system, thereby confounding the innate immune response to viral infection 
. Pharmaceutical companies have invested significant effort in developing NS3/4A protease inhibitors. Proof-of-concept of antiviral efficacy was first demonstrated in 2002 with the macrocyclic inhibitor BILN-2061 (ciluprevir) 
, which was later discontinued due to concerns about its cardiotoxicity 
. As noted above, boceprevir 
and telaprevir 
are two NS3/4A protease inhibitors recently approved by the Food and Drug Administration, marking an important milestone in anti-HCV research and drug development over the past two decades. Both boceprevir and telaprevir are linear ketoamide compounds that form a reversible, covalent bond with the catalytic serine of NS3/4A protease. Several non-covalent xprotease inhibitors have also advanced into human clinical trials; these inhibitors include both linear (BMS-650032 
, BI 201335 
) and macrocyclic compounds, containing either a P1–P3 (danoprevir 
, TMC435 
) or a P2–P4 (vaniprevir 
, MK-5172 
) macrocycle ().
The chemical structures of NS3/4A protease inhibitors.
The NS3/4A protease inhibitors rapidly reduce HCV RNA titers when administered as monotherapy 
and substantially improve SVR rates when given in combination with Peg-IFN and RBV 
. However, the high rate of HCV replication and poor fidelity of HCV's RNA-dependent RNA polymerase lead to heterogeneous virus populations in infected patients 
. These viral quasispecies exist at low levels in untreated patients, and resistant populations emerge under the selective pressure of direct-acting antiviral agents 
. In the majority of patients undergoing protease inhibitor therapy, resistance develops rapidly due to overlapping but distinct sets of NS3/4A mutations 
. In patients with genotype 1a, the R155K mutation causes resistance against nearly all inhibitors, but rarely occurs in genotype 1b patients 
. Instead, distinct resistance mutations arise in genotype 1b patients depending on the class of protease inhibitor used; A156 mutates in response to treatment with linear ketoamide protease inhibitors 
, while macrocyclic inhibitors more commonly select for D168A and R155K variants 
. Mutations at V36, T54, and V36+A155 are also associated with resistance to ketoamide inhibitors 
. Variations in the patterns of resistant mutations arise from the complex interplay between genotype, replication rates, mutation rates, and the resulting effect of mutations on viral fitness and drug potency. Clearly, despite the benefits of combination therapy in improving SVR rates, the emergence of resistance challenges the long-term efficacy of NS3/4A protease inhibitors.
Most primary drug-resistance mutations in NS3/4A protease occur around the active site in regions where drugs protrude from the substrate binding space, defined as the substrate envelope, because these changes can preferentially disrupt drug binding with minimal effect on substrate binding and viral fitness 
. The protease inhibitors danoprevir, TMC435, and boceprevir protrude from the substrate envelope in regions that correlate with known sites of resistance mutations. Notably, the large P2 moieties of danoprevir and TMC435 bind in the S2 subsite and extensively interact with residues R155, D168, and A156 
, which mutate to confer multi-drug resistance 
. These and other inhibitors with large P2 moieties derive much of their potency from binding in the S2 subsite 
, but how molecular changes at these residues selectively weaken inhibitor binding without compromising the binding of viral substrates is not clear. Elucidating the underlying molecular mechanisms of NS3/4A protease inhibitor resistance is therefore essential for developing new drugs that are less susceptible to resistance.
How single-site mutations at residues R155, A156 and D168 confer resistance against most protease inhibitors has not been elucidated in atomic detail. In this study, we report that four chemically representative protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – exhibit distinct susceptibilities to the protease variants R155K, A156T and D168A (). Sixteen high-resolution crystal structures of inhibitors in complexwith the wild-type protease and three drug resistant variants reveal the molecular basis underlying the unique resistance profiles of these inhibitors (). The P2 quinoxaline moiety of MK-5172 stacks against the protease catalytic triad in a novel conformation, explaining its retained potency against R155K and D168A. The flexible P2 isoindoline moiety of danoprevir containing a P1–P3 macrocycle packs against the mutated surfaces of A156T and D168A variants, explaining its relatively higher activity against both protease variants. However, the isoindoline moiety in vaniprevir is constrained due to the P2–P4 macrocycle, resulting in significantly lower activity against all three variants. Thus, incorporating either quinoxaline or flexible substituents at the P2 proline confers clear advantages. Taken together, these data highlight potential strategies for designing novel drugs that retain potency against a broader spectrum of resistant viral variants.
Drug susceptibilities against wild-type and resistant HCV clones and inhibitory activities against NS3/4A proteases.
X-ray data collection and crystallographic refinement statistics.