title: In vitro antiviral activity of SCH446211 (SCH6), a novel inhibitor of the hepatitis C virus NS3 serine protease.
abstract: BACKGROUND
  Current hepatitis C virus (HCV) therapies may cure approximately 60% of infections. They are often contraindicated or poorly tolerated, underscoring the need for safer and more effective drugs. A novel, alpha-ketoamide-derived, substrate-based inhibitor of the HCV serine protease (SCH446211) was developed. Compared with earlier reported inhibitors of similar chemical class, it has a P1'-P2' extension which provides extended interaction with the protease active site. The aim of this study was to evaluate the in vitro antiviral activity of SCH446211.
  
  
  METHODS
  Binding constant of SCH446211 to HCV NS3 protease was measured with the chromogenic substrate in vitro cleavage assay. Cell-based activity of SCH446211 was evaluated in replicon cells, which are Huh-7 hepatoma cells stably transfected with a subgenomic HCV RNA as reported previously. After 72 h of incubation with SCH446211, viral transcription and protein expression were measured by real-time RT-PCR (TaqMan), quantitative in situ hybridization, immunoblot and indirect immunofluorescence.
  
  
  RESULTS
  The binding constant of SCH446211 to HCV NS3 protease was 3.8 +/- 0.4 nM. HCV replication and protein expression were inhibited by SCH446211 in replicon cells as consistently shown by four techniques. In particular, based on quantitative real-time RT-PCR measurements, the IC50 and IC90 of SCH446211 were estimated to be 40 +/- 20 and 100 +/- 20 nM (n = 17), respectively. Long-term culture of replicon cells with SCH446211 reduced replicon RNA to <0.1 copy per cell. SCH446211 did not show cellular toxicity at concentrations up to 50 microM.
  
  
  CONCLUSIONS
  SCH446211 is a potent inhibitor of HCV protease in vitro. Its extended interaction with the HCV NS3 protease active site is associated with potent in vitro antiviral activity. This observation is potentially a useful guide for development of future potent inhibitors against HCV NS3 protease.
authors.affiliation: Department of Virology, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA. Rong.Liu@spcorp.com
authors.forename: Rong
authors.initials: R
authors.lastname: Liu
authors.forename: Karim
authors.initials: K
authors.lastname: Abid
authors.forename: John
authors.initials: J
authors.lastname: Pichardo
authors.forename: Valerio
authors.initials: V
authors.lastname: Pazienza
authors.forename: Paul
authors.initials: P
authors.lastname: Ingravallo
authors.forename: Rong
authors.initials: R
authors.lastname: Kong
authors.forename: Sony
authors.initials: S
authors.lastname: Agrawal
authors.forename: Stephane
authors.initials: S
authors.lastname: Bogen
authors.forename: Anil
authors.initials: A
authors.lastname: Saksena
authors.forename: Kuo-Chi
authors.initials: KC
authors.lastname: Cheng
authors.forename: Andrew
authors.initials: A
authors.lastname: Prongay
authors.forename: F George
authors.initials: FG
authors.lastname: Njoroge
authors.forename: Bahige M
authors.initials: BM
authors.lastname: Baroudy
authors.forename: Francesco
authors.initials: F
authors.lastname: Negro
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology*; D012367:RNA, Viral / Q000032:analysis; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D009842:Oligopeptides; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C510133:SCH6