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  • The Y H D A variant was resistant to all

    2021-10-13

    The Y56H/D168A variant was resistant to all tested PIs, and the inhibitor binding mode determined the molecular mechanism of resistance. Prior to the development of grazoprevir, PIs typically contained large heterocyclic P2 moieties that strongly interacted with S2-subsite residues (Ali et al., 2013, Romano et al., 2012). This binding mode resulted in detrimental loss of potency against single-site RASs in the protease active site, especially at residues Arg155 and Asp168 (Ali et al., 2013, Romano et al., 2012). In the inhibitor-bound state, HCV NS3/4A protease has an extensive active-site electrostatic network that spans the catalytic triad residues His57 and Asp81 all the way to S2-subsite residues Arg155, Asp168, and Arg123. Residues Arg155 and Asp168, located beyond the substrate envelope, form a salt bridge that is critical to inhibitor binding and disrupted upon PFI-2 at either residue (Romano et al., 2010). For inhibitors with a binding mode similar to that of danoprevir, disruption of this electrostatic network due to an Asp168 substitution causes rearrangement of the Arg155 side chain, resulting in the loss of favorable cation-π interactions between the P2 isoindoline and Arg155 guanidinium group (O'Meara et al., 2013, Romano et al., 2012). For such inhibitors that do not stack on the catalytic residues and have no physical interactions with Tyr56, resistance to the double substitution is predominantly due to D168A. Thus the addition of Y56H substitution to D168A protease does not cause any further active-site changes or loss in potency. Grazoprevir and newer-generation inhibitors have a P2 quinoxaline moiety that makes extensive interactions with the catalytic triad, reducing susceptibility to single-site RASs at the S2 subsite (Soumana et al., 2016b). However, this binding mode causes vulnerability to substitutions that result in loss of critical π-π stacking interactions with the catalytic residue His57. The crystal structure of grazoprevir bound to the Y56H protease determined here reveals that this interaction is weakened when neighboring Tyr56 mutates to a PFI-2 smaller His. This loss is compounded in the double-substitution Y56H/D168A variant, thereby destabilizing the binding of grazoprevir. The alterations due to Y56H and the double-substitution Y56H/D168A are unlikely to affect the recognition and processing of the viral substrates (Table S1), as unlike grazoprevir the substrates do not stack on the aromatic surface of His57 (Romano et al., 2010, Romano et al., 2011, Romano et al., 2012). The substrates also make no direct contact with Tyr56, so substitutions at Tyr56 are unlikely to either directly or indirectly affect substrate turnover. As a general rule, the reliance of an inhibitor on interactions with the target that are not essential for biological function creates an opportunity for resistance causing substitutions (Romano et al., 2012, Soumana et al., 2016b, Yilmaz et al., 2016). When these interactions are within the substrate envelope or with catalytic residues, the chance of resistance emerging is minimized. Nevertheless, in this study we observe an indirect mechanism of resistance that disrupts interactions with the catalytic residue through substitution in a neighboring residue not involved in substrate recognition. Robustness of grazoprevir against single-site RASs has led to drug design efforts by pharmaceutical companies to pursue PIs with scaffolds similar to that of grazoprevir. Pan-genotypic PIs voxilaprevir and glecaprevir have a P2 quinoxaline and P2–P4 macrocycle as in grazoprevir. Considering the high similarity in the scaffolds of these latest-generation inhibitors, there is a danger that all PIs currently in the clinic might be susceptible to the same resistant variants, including Y56H/D168A. While these inhibitors have low susceptibility to single-site substitutions at residues Arg155 and Asp168, they have selected for double-substitution variants in in vitro studies (Ng et al., 2017). In fact, in vitro resistance testing of glecaprevir selected for resistance against GT3 double-substitution variant Y56H/Q168R, losing almost 1,400-fold in potency (Ng et al., 2017). This suggests that double-substitution variants containing Y56H may emerge in other genotypes and reduce the clinical effectiveness of PIs.