title: Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.
abstract: Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants.
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Hyock Joo
authors: HJ
authors: Kwon
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Weimei
authors: W
authors: Xing
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Katie
authors: K
authors: Chan
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Anita
authors: A
authors: Niedziela-Majka
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Katherine M
authors: KM
authors: Brendza
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Thorsten
authors: T
authors: Kirschberg
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Darryl
authors: D
authors: Kato
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: John O
authors: JO
authors: Link
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Guofeng
authors: G
authors: Cheng
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Xiaohong
authors: X
authors: Liu
authors: Gilead Sciences, Inc., Foster City, California, United States of America.
authors: Roman
authors: R
authors: Sakowicz
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral / Q000235:genetics*; D005449:Fluorenes / Q000737:chemistry / Q000494:pharmacology*; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000473:pathology / Q000821:virology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D001562:Benzimidazoles; D005449:Fluorenes; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C586541:ledipasvir; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase