Kwon Hyock Joo, Xing Weimei, Chan Katie, Niedziela-Majka Anita, Brendza Katherine M, Kirschberg Thorsten, Kato Darryl, Link John O, Cheng Guofeng, Liu Xiaohong, Sakowicz Roman
Gilead Sciences, Inc., Foster City, California, United States of America.
PLoS One. 2015 Apr 9;10(4):e0122844. doi: 10.1371/journal.pone.0122844. eCollection 2015.
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.
来迪派韦是一种靶向丙型肝炎病毒NS5A蛋白的直接作用抗病毒药物（DAA），在复制子细胞中表现出皮摩尔活性。虽然其作用机制尚不清楚，但在HCV复制子细胞中赋予对来迪派韦耐药性的突变位于NS5A中，这表明NS5A是来迪派韦的直接靶点。迄今为止，在复制子或NS5A转染细胞中的共沉淀和交联实验尚未确凿地证明NS5A与来迪派韦之间存在直接、特异性的相互作用。使用重组全长NS5A，我们表明来迪派韦以高亲和力和特异性直接结合NS5A。来迪派韦与重组NS5A的结合是可饱和的，解离常数在低纳摩尔范围内。赋予对来迪派韦耐药性的NS5A突变形式（Y93H）与来迪派韦的结合减少。当前研究表明，来迪派韦通过直接结合抑制NS5A，而来迪派韦耐药性是与NS5A突变体结合亲和力降低的结果。
