title: The design and synthesis of potent inhibitors of hepatitis C virus NS3-4A proteinase.
abstract: Hepatitis C virus (HCV) is the cause of the majority of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV frequently leads to persistent infections that result in a range of clinical conditions including an asymptomatic carrier state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocellular carcinoma. The HCV genome consists of a single-stranded, positive sense RNA containing an open reading frame of approximately 9060 nucleotides. This is translated into a single polyprotein of approximately 3020 amino acids (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B), which in turn is processed by a series of host and viral proteinases into at least 10 cleavage products. The N-terminal portion of the NS3 protein encodes a serine proteinase that is responsible for the cleavage at the NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. The 54 amino acid NS4A protein is a cofactor that binds to the NS3 protein and enhances its proteolytic activity. This report describes the expression of a recombinant NS3-4A proteinase fusion protein in Escherichia coli and the in vitro characterization of the enzyme activity using synthetic peptide substrates. It then demonstrates how these results were employed to guide the design of potent inhibitors of this enzyme.
authors.affiliation: Department of Chemistry, Roche Discovery Welwyn, Welwyn Garden City, Hertfordshire, UK.
authors.forename: M R
authors.initials: MR
authors.lastname: Attwood
authors.forename: J M
authors.initials: JM
authors.lastname: Bennett
authors.forename: A D
authors.initials: AD
authors.lastname: Campbell
authors.forename: G G
authors.initials: GG
authors.lastname: Canning
authors.forename: M G
authors.initials: MG
authors.lastname: Carr
authors.forename: E
authors.initials: E
authors.lastname: Conway
authors.forename: R M
authors.initials: RM
authors.lastname: Dunsdon
authors.forename: J R
authors.initials: JR
authors.lastname: Greening
authors.forename: P S
authors.initials: PS
authors.lastname: Jones
authors.forename: P B
authors.initials: PB
authors.lastname: Kay
authors.forename: B K
authors.initials: BK
authors.lastname: Handa
authors.forename: D N
authors.initials: DN
authors.lastname: Hurst
authors.forename: N S
authors.initials: NS
authors.lastname: Jennings
authors.forename: S
authors.initials: S
authors.lastname: Jordan
authors.forename: E
authors.initials: E
authors.lastname: Keech
authors.forename: M A
authors.initials: MA
authors.lastname: O'Brien
authors.forename: H A
authors.initials: HA
authors.lastname: Overton
authors.forename: J
authors.initials: J
authors.lastname: King-Underwood
authors.forename: T M
authors.initials: TM
authors.lastname: Raynham
authors.forename: K P
authors.initials: KP
authors.lastname: Stenson
authors.forename: C S
authors.initials: CS
authors.lastname: Wilkinson
authors.forename: T C
authors.initials: TC
authors.lastname: Wilkinson
authors.forename: F X
authors.initials: FX
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D015195:Drug Design; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D010450:Endopeptidases; D010455:Peptides; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases