Claims
- 1. A cell-based assay for identifying a compound that inhibits HCV RNA replication, comprising the steps of:
(a) providing a cell which expresses at least one enzyme associated with HCV RNA replication; (b) contacting said cell with a test compound; (c) determining whether said test compound inhibits HCV RNA replication; and (d) determining whether said test compound is cytotoxic to said cell.
- 2. The cell-based assay of claim 1, wherein said cell comprises a HCV replicon.
- 3. The cell-based assay of claim 2, wherein said HCV replicon comprises a polynucleotide having the nucleic acid sequence set forth in SEQ ID NO:1.
- 4. The cell-based assay of claim 2, wherein said HCV replicon encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:2.
- 5. The cell-based assay of claim 2, wherein said HCV replicon comprises the molecular construct set forth in FIG. 1.
- 6. The cell-based assay of claim 1, wherein said cell which expresses at least one enzyme associated with HCV RNA replication is a cell having ATCC Accession No. PTA-4583.
- 7. The cell-based assay of claim 1, wherein said assay is conducted in a single well.
- 8. The cell-based assay of claim 1, wherein said assay is conducted in two or more wells.
- 9. The cell-based assay of claim 1, wherein said enzyme is a protease.
- 10. The cell-based assay of claim 9, wherein said protease is a serine protease
- 11. The cell-based assay of claim 10, wherein said serine protease is NS3 protease.
- 12. The cell-based assay of claim 9, wherein said enzyme is NS4A.
- 13. The cell-based assay of claim 1, wherein said step of determining whether said test compound inhibits HCV RNA replication is conducted by contacting said cell with a fluorescence substrate.
- 14. The cell-based assay of claim 13, wherein said fluorescence substrate is a FRET peptide.
- 15. The cell-based assay of claim 1, wherein said step of determining whether said test compound is cytotoxic to said cell is conducted by contacting said cell with an Alamar Blue solution.
- 16. The cell-based assay of claim 1, wherein said cell-based assay is performed in a high-throughput manner.
- 17. A compound identified by the cell-based assay of claim 1.
- 18. A pharmaceutical composition comprising a compound of claim 17.
- 19. A method for treating hepatitis-C, comprising the step of administering to a mammalian species in need thereof a therapeutically effective amount of a compound of claim 17.
- 20. A cell-based assay for identifying a compound that inhibits HCV RNA replication, comprising the steps of:
(a) providing a cell which expresses at least one enzyme associated with HCV RNA replication; (b) contacting said cell with a test compound; (c) contacting said cell with a compound which permits the determination of whether said test compound inhibits HCV RNA replication; and (d) contacting said cell with an indicator solution which permits the determination of whether said test compound is cytotoxic to said cell.
- 21. The cell-based assay of claim 20, wherein said compound which permits the determination of whether said test compound inhibits HCV RNA replication is a FRET peptide.
- 22. The cell-based assay of claim 20, wherein said indicator solution which permits the determination of whether said test compound is cytotoxic to said cell is an Alamar Blue solution.
- 23. The cell-based assay of claim 20, wherein steps (a), (b), (c) and (d) are conducted in a single well.
- 24. A cell-based assay for identifying a compound that inhibits HCV RNA replication, comprising the steps of:
(a) providing a cell which expresses at least one enzyme associated with HCV RNA replication, said cell comprising a HCV replicon; (b) contacting said cell with a test compound; (c) contacting said cell with a FRET peptide for determining whether said test compound inhibits HCV RNA replication; and (d) contacting said cell with an indicator solution for determining whether said test compound is cytotoxic to said cell.
- 25. The cell-based assay of claim 24, wherein said indicator solution is an Alamar Blue solution.
- 26. The cell-based assay of claim 24, wherein said HCV replicon comprises a polynucleotide having the nucleic acid sequence set forth in SEQ ID NO:1.
- 27. The cell-based assay of claim 24, wherein said HCV replicon encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:2.
- 28. The cell-based assay of claim 24, wherein said HCV replicon comprises the molecular construct set forth in FIG. 1.
- 29. The cell-based assay of claim 24, wherein said cell which expresses at least one enzyme associated with HCV RNA replication is a cell having ATCC Accession No. PTA-4583.
- 30. A compound identified by the cell-based assay of claim 24.
- 31. A pharmaceutical composition comprising a compound of claim 24.
- 32. A method for treating hepatitis-C which comprises administering to a mammalian species in need thereof a therapeutically effective amount of a compound of claim 24.
- 33. A cell-based assay for identifying a compound that inhibits HCV RNA replication, comprising the steps of:
(a) providing a cell having ATCC Accession No. PTA-4583, said cell expressing at least one enzyme associated with HCV RNA replication; (b) contacting said cell with a test compound; (c) determining whether said test compound inhibits HCV RNA replication; and (d) determining whether said test compound is cytotoxic to said cell.
- 34. A reporter assay for identifying a compound that modulates that activity of a gene of interest, comprising the steps of:
(a) providing an expression system, said expression system comprising (i) a cell and (ii) a construct comprising a promoter region associated with said gene of interest operably linked to an enzyme associated with HCV RNA replication; (b) contacting said expression system with a test compound; and (c) contacting said expression system with a compound capable of detecting expression of said enzyme associated with HCV RNA replication.
- 35. The reporter assay of claim 34, wherein said enzyme associated with HCV RNA replication is NS3 protease.
- 36. The reporter assay of claim 34, wherein said compound capable of detecting expression of said enzyme associated with HCV RNA replication is a FRET peptide.
- 37. A cell having ATCC Accession No. PTA-4583.
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. Section 119(e) of U.S. Provisional Patent Application No. 60/402,661 filed Aug. 12, 2002.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60402661 |
Aug 2002 |
US |