title: Peptidomimetic escape mechanisms arise via genetic diversity in the ligand-binding site of the hepatitis C virus NS3/4A serine protease.
abstract: OBJECTIVE
  It is a challenge to develop direct-acting antiviral agents that target the nonstructural protein 3/4A protease of hepatitis C virus because resistant variants develop. Ketoamide compounds, designed to mimic the natural protease substrate, have been developed as inhibitors. However, clinical trials have revealed rapid selection of resistant mutants, most of which are considered to be pre-existing variants.
  
  
  METHODS
  We identified residues near the ketoamide-binding site in x-ray structures of the genotype 1a protease, co-crystallized with boceprevir or a telaprevir-like ligand, and then identified variants at these positions in 219 genotype-1 sequences from a public database. We used side-chain modeling to assess the potential effects of these variants on the interaction between ketoamide and the protease, and compared these results with the phenotypic effects on ketoamide resistance, RNA replication capacity, and infectious virus yields in a cell culture model of infection.
  
  
  RESULTS
  Thirteen natural binding-site variants with potential for ketoamide resistance were identified at 10 residues in the protease, near the ketoamide binding site. Rotamer analysis of amino acid side-chain conformations indicated that 2 variants (R155K and D168G) could affect binding of telaprevir more than boceprevir. Measurements of antiviral susceptibility in cell-culture studies were consistent with this observation. Four variants (ie, Q41H, I132V, R155K, and D168G) caused low-to-moderate levels of ketoamide resistance; 3 of these were highly fit (Q41H, I132V, and R155K).
  
  
  CONCLUSIONS
  Using a comprehensive sequence and structure-based analysis, we showed how natural variation in the hepatitis C virus protease nonstructural protein 3/4A sequences might affect susceptibility to first-generation direct-acting antiviral agents. These findings increase our understanding of the molecular basis of ketoamide resistance among naturally existing viral variants.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Inflammatory Diseases Institute, Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599--7292, USA. christophwelsch@gmx.net
authors.forename: Christoph
authors.initials: C
authors.lastname: Welsch
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Shimakami
authors.forename: Christoph
authors.initials: C
authors.lastname: Hartmann
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.forename: Francisco S
authors.initials: FS
authors.lastname: Domingues
authors.forename: Thomas
authors.initials: T
authors.lastname: Lengauer
authors.forename: Stefan
authors.initials: S
authors.lastname: Zeuzem
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Lemon
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D015195:Drug Design; D024882:Drug Resistance, Viral / Q000235:genetics*; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D011110:Polymorphism, Genetic; D011392:Proline / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D013997:Time Factors; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D009842:Oligopeptides; D011392:Proline; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide