title: Slow binding inhibition and mechanism of resistance of non-nucleoside polymerase inhibitors of hepatitis C virus.
abstract: The binding affinity of four palm and thumb site representative non-nucleoside inhibitors (NNIs) of HCV polymerase NS5B to wild-type and resistant NS5B polymerase proteins was determined, and the influence of RNA binding on NNI binding affinity was investigated. NNIs with high binding affinity potently inhibited HCV RNA polymerase activity and replicon replication. Among the compounds tested, HCV-796 showed slow binding kinetics to NS5B. The binding affinity of HCV-796 to NS5B increased 27-fold over a 3-h incubation period with an equilibrium Kd of 71 +/- 2 nm. Slow binding kinetics of HCV-796 was driven by slow dissociation from NS5B with a k(off) of 4.9 +/- 0.5 x 10(-4) s(-1). NS5B bound a long, 378-nucleotide HCV RNA oligonucleotide with high affinity (Kd = 6.9 +/- 0.3 nm), whereas the binding affinity was significantly lower for a short, 21-nucleotide RNA (Kd = 155.1 +/- 16.2 nm). The formation of the NS5B-HCV RNA complex did not affect the slow binding kinetics profile and only slightly reduced NS5B binding affinity of HCV-796. The magnitude of reduction of NNI binding affinity for the NS5B proteins with various resistance mutations in the palm and thumb binding sites correlated well with resistance -fold shifts in NS5B polymerase activity and replicon assays. Co-crystal structures of NS5B-Con1 and NS5B-BK with HCV-796 revealed a deep hydrophobic binding pocket at the palm region of NS5B. HCV-796 interaction with the induced binding pocket on NS5B is consistent with slow binding kinetics and loss of binding affinity with mutations at amino acid position 316.
authors.affiliation: Roche Palo Alto LLC, Palo Alto, California 94304, USA.
authors.forename: Julie Qi
authors.initials: JQ
authors.lastname: Hang
authors.forename: Yanli
authors.initials: Y
authors.lastname: Yang
authors.forename: Seth F
authors.initials: SF
authors.lastname: Harris
authors.forename: Vincent
authors.initials: V
authors.lastname: Leveque
authors.forename: Hannah J
authors.initials: HJ
authors.lastname: Whittington
authors.forename: Sonal
authors.initials: S
authors.lastname: Rajyaguru
authors.forename: Gloria
authors.initials: G
authors.lastname: Ao-Ieong
authors.forename: Matthew F
authors.initials: MF
authors.lastname: McCown
authors.forename: April
authors.initials: A
authors.lastname: Wong
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Giannetti
authors.forename: Sophie
authors.initials: S
authors.lastname: Le Pogam
authors.forename: Francisco
authors.initials: F
authors.lastname: Talamás
authors.forename: Nick
authors.initials: N
authors.lastname: Cammack
authors.forename: Isabel
authors.initials: I
authors.lastname: Nájera
authors.forename: Klaus
authors.initials: K
authors.lastname: Klumpp
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology; D001572:Benzofurans / Q000737:chemistry / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004279:DNA, Viral / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D001562:Benzimidazoles; D001572:Benzofurans; D004279:DNA, Viral; D009843:Oligoribonucleotides; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C031000:benzimidazole; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C105430:benzofuran