Molecular Mechanism of Resistance in a Clinically Significant Double-Mutant Variant of HCV NS3/4A Protease

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology; Schiffer Lab; Graduate School of Biomedical Sciences, MD/PhD Program

Publication Date


Document Type



Biochemistry | Chemistry | Genetic Phenomena | Medicinal Chemistry and Pharmaceutics | Medicinal-Pharmaceutical Chemistry | Molecular Biology | Pharmacology | Structural Biology


Despite significant progress in hepatitis C virus (HCV) protease inhibitor (PI) drug design, resistance remains a problem causing treatment failure. Double-substitution variants, notably Y56H/D168A, have emerged in patients who fail therapy with a PI-containing regimen. The resistance conferred by Asp168 substitutions has been well characterized and avoided in newer inhibitors. However, an additional mutation at Tyr56 confers resistance to even the most robust inhibitors. Here, we elucidate the molecular mechanisms of resistance for the Y56H/D168A variant against grazoprevir (and four analogs), paritaprevir, and danoprevir through inhibition assays, co-crystal structures, and molecular dynamics simulations. The PIs' susceptibility to Y56H/D168A varies, with those stacking on the catalytic His57 losing the most potency. For such inhibitors, the Y56H substitution disrupts favorable stacking interactions with the neighboring catalytic His57. This indirect mechanism of resistance threatens to cause multi-PI failure as all HCV PIs in clinical development rely on interactions with the catalytic triad.


HCV, catalytic triad, crystal structure, double mutant, drug resistance, grazoprevir, molecular dynamics, protease inhibitor, resistance mechanism, resistance-associated substitution

DOI of Published Version



Structure. 2018 Oct 2;26(10):1360-1372.e5. doi: 10.1016/j.str.2018.07.004. Epub 2018 Aug 23. Link to article on publisher's site

Journal/Book/Conference Title

Structure (London, England : 1993)

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Link to Article in PubMed

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