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    • In fact most PIs are susceptible to

    2021-10-12

    In fact, most PIs are susceptible to substitutions at Asp168, which are often present in patients who fail therapy (Pawlotsky, 2016). Notably, the polymorphism Gln168 at this position underlies reduced efficacy of PIs against GT3 (Soumana et al., 2016a). Glecaprevir and voxilaprevir have improved resistance profiles against D168A and are active against GT3, but like grazoprevir are highly susceptible to substitutions at Ala156 (Ng et al., 2017) due to van der Waals clashes with their P2–P4 macrocycles (Ali et al., 2013, Soumana et al., 2016b). Unfortunately, even with the newest combinations some patients still fail therapy, with more than one RAS detected in the infecting viral neuraminidase inhibitor (Lawitz et al., 2014). The emergence of such double- and sometimes triple-site RAS variants in the clinic is threatening the effectiveness of current anti-HCV therapies (Gane et al., 2016). While the molecular basis of drug resistance caused by single-site RASs has been well characterized (Romano et al., 2010, Romano et al., 2012, Soumana et al., 2014, Soumana et al., 2016b), the impact of clinically relevant NS3/4A protease double substitutions on inhibitor binding and the mechanisms of drug resistance remain largely unexplored. One key clinically relevant protease variant is Y56H/D168A, often present in patients who fail therapy with the newer-generation PIs. Grazoprevir and paritaprevir are highly susceptible to this signature variant and exhibit over 500-fold loss in potency (Guo et al., 2017, Lontok et al., 2015, Zeuzem et al., 2014). Substitutions at Tyr56 rarely occur alone, but are becoming common in combination with substitutions at Asp168. These residues are not in physical contact, with Tyr56 located next to the catalytic His57, approximately 15 Å away from residue Asp168. Nevertheless, co-evolution of these two sites results in a detrimental loss of potency for all PIs. The molecular mechanism underlying high-level resistance of PIs against the Y56H/D168A double-substitution variant is unknown. To elucidate the molecular mechanism of resistance for PIs against the Y56H/D168A NS3/4A protease variant, we used a multi-disciplinary approach involving enzyme inhibition and antiviral assays, co-crystal structures, and molecular dynamics simulations. A panel of seven NS3/4A PIs (grazoprevir and four analogs, paritaprevir, and danoprevir) with varying macrocycle locations and P2 binding modes were tested for enzyme inhibition and antiviral potency. To tease out the impact of individual substitutions, we compared the double-substitution variant with both single substitutions and wild-type (WT) GT1a NS3/4A protease. While all inhibitors were 3–10 orders of magnitude less active against the Y56H/D168A NS3/4A protease variant, the potency loss was exacerbated for PIs that stack on the catalytic triad, including grazoprevir. Crystal structures and dynamic analysis of grazoprevir bound to protease variants revealed that this resistance is largely due to the Y56H substitution disrupting the favorable stacking interactions with the neighboring catalytic residue His57. Thus, in addition to the loss of the ionic network due to D168A substitution (O'Meara et al., 2013, Romano et al., 2012), decreased direct interactions with catalytic His57 underlie resistance against this double-substitution variant. For the prevention of such mechanisms of clinically relevant resistance, inhibitor design limiting interactions with Tyr56 while still maintaining stacking against the catalytic residues is warranted.
    Results
    Discussion Although drug resistance has been a major problem in the efficacy of anti-HCV therapeutics, especially for NS3/4A PIs, newer-generation inhibitors are robust against single-site substitutions that were once detrimental to PI clinical viability. More importantly, there are currently two all-oral regimens that have pan-genotypic HCV activity including against the evasive GT3 (Ng et al., 2017). Although much progress has been made in anti-HCV therapeutics, a new challenge that may threaten the success of PIs is the emergence of viral variants with more than one substitution in the protease (Gane et al., 2016, Ng et al., 2017). In this study we reveal the structural and dynamic mechanisms of drug resistance for the Y56H/D168A protease, a double-site RAS variant that has been identified in patients who failed therapy with PI-containing regimens (Gane et al., 2016).