Hepatitis c virus assays
(19)AUSTRALIAN PATENT OFFICE (54) Title Hepatitis c virus assays (51)6 International Patent Classification(s) C12Q 001^70 (21) Application No: 2003256404 (22) Application Date: 2003.08.12 (87) WIPO No: WO04/015131 (30) Priority Data (31) Number (32) Date (33) Country 60/402,661 2002 .08 .12 US (43) Publication Date : 2004 .02.25 (43) Publication Journal Date : 2004 .04.08 (71) Applicant(s) BRISTOL-MYERS SQUIBB COMPANY (72) Inventor(s) Sun, Jin Hua; Gao, Min; O'Boylc, Donald R.; Rigat, Karen; Lemm, Julie A.; Nower, Peter (-1-1) Application NoAU2003256404 A8(19)AUSTRALIAN PATENT OFFICE (54) Title Hepatitis c virus assays (51)6 International Patent Classification(s) C12Q 001^70 (21) Application No: 2003256404 (22) Application Date: 2003.08.12 (87) WIPO No: WO04/015131 (30) Priority Data (31) Number (32) Date (33) Country 60/402,661 2002 .08 .12 US (43) Publication Date : 2004 .02.25 (43) Publication Journal Date : 2004 .04.08 (71) Applicant(s) BRISTOL-MYERS SQUIBB COMPANY (72) Inventor(s) Sun, Jin Hua; Gao, Min; O'Boylc, Donald R.; Rigat, Karen; Lemm, Julie A.; Nower, Peter-1- The present invention includes an assay useful for identifying inhibitors of Hepatitis C virus (HCV) activity. Particularly, the present invention is directed to a dual HCV assay useful for high throughput screening that quantifies both the amount of HCV RNA replication inhibitory activity associated with a test compound and the amount of cytotoxicity associated with that test compound. The present invention also includes compounds discovered using this assay, compositions containing such compounds and methods of treating Hepatitis C by the administration of such compounds. The present invention also includes reporter assays using enzymes associated with HCV RNA replication, as well as a cell line having ATTC Accession No. PTA-4583. WHAT IS CLAIMED IS: 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 Figure 1. <Desc/Clms Page number 26> 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. <Desc/Clms Page number 27> 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; <Desc/Clms Page number 28> (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 ceil 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 <Desc/Clms Page number 29> (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 Figure 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. <Desc/Clms Page number 30> 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. <Desc/Clms Page number 31> 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.